Lecture Manual 313 3r3x6w

LECTURE MANUAL

Crop Protection 313 BENEFICIAL ARTHROPODS AND MICROORGANISMS

PURIFICACION ORODIO CAHATIAN AND EVELYN PULIDO ESTEBAN

2011

TRANSMITTAL

This manual attached hereto entitled, LECTURE MANUAL OF BENFICIAL

ARTHROPODS

AND

MICROOGANISMS,

prepared

and

submitted by PURIFICACION O. CAHATIAN AND EVELYN P. ESTEBAN, is hereby accepted as guide/reference material for teaching BSA, BSFS, OPPT, RT, MS and PhD Crop Protection at the College of Agriculture, University of Southern Mindanao, Kabacan, Cotabato.

ADEFLOR G. GARCIA Ph D Dean, College of Agriculture

Date

ANTONIO N. TACARDON Ph D Vice President for Academic Affairs

Date

UNIVERSITY OF SOUTHERN MINDANAO College of Agriculture DEPARTMENT OF ENTOMOLOGY SYLLABUS I.

Course Number Descriptive Title Credit Unit Hour Prerequisite

: Crop Prot 313 : Beneficial Arthropods and Microorganisms : 3 units : 2 hours lecture and 3 hours laboratory per week : Crop Prot 2 or Consent of Instructor

II. III.

Course Description Statement of Goals National Goals

: Biology and ecology of beneficial arthropods and microorganisms

Regional Goals Institutional Goal IV.

V.

Course Objective

: 1. Develop the students‟ full potential as a person. : 2. Develop the students‟ personal discipline, cultural, spiritual and moral values. : 3. Acquire entrepreneurial and technological skills for peace and economic prosperity. : 4. Show consciousness and responsibility for environmental issues and concerns. : 5. Develop critical awareness and appreciation of the scientific and technological advancements for the students to bridge the gap between and among cultural communities. At the end of the semester, the students must be able to do the following with 90% accuracy:

Cognitive

1. Discuss the concepts and principles of biological control of arthropods pests of agricultural plants. 2. Characterize factors affecting the growth and survival of beneficial arthropods and microorganisms

Affective

3. Appreciate the importance of the nature, mechanism and interactions involved among the host and parasite system.

Psychomotor

4. Outline and discuss the various methods of mass production, application and evaluation of effectiveness of beneficial arthropods and microorganisms

Rationale

One of the tools for insect control found in today‟s arsenal is biological control. Under natural conditions, insect populations are kept in check by the influences of multitude of environmental factors. Given proper attention, these factors, in concert, bring about the “natural control” of a given arthropod pest population.

VI. Grading System

: The grading system of the department shall be computed using the following formula: FIRST TERM GRADE (First half of the semester): Midterm Grade (MGT) = Quizzes & Other Requirements + Pre-midterm + Midterm Exam 3 SECOND TERM GRADE (Second half of the semester): Second Term Grade (SGT) = Quizzes + Final Exam 3 TENTATIVE FINAL GRADE: Tentative Final Grade (TFG) = MGT + STG (2) 3 FINAL GRADE: Final Grade = TFG (2) + Laboratory Grade 3

COURSE OUTLINE

ORIENTATION OF THEFOLLOWING: a. Grading System b. Course Requirements c. School Policies on:  Attendance and Punctuality  Overloading  Cheating and Stealing, etc.  Student Classification and Retention d. USM Vision, Mission and Goals e. USM Core Values (GREAT) God centered Responsive Excellent Assertive for Truth f. CA Goal and Objectives g. Syllabus of the Course h. Values for Integration in the BSA Curriculum i. Subject Professor‟s Classroom Mgt Policies

TIME ALLOTMENT Lec (HRS) Lab

2

SPECIFIC OBJECTIVES

METHODOLOGY

At the end of the period the students shall be able to:

Lecture

a. internalize the grading system and explain the same to their parents b. identify the course requirements c. explain the school policies to their peers and parents d. memorize the USM vision & Mission e. memorize the USM Core Values f. memorize the CA Goal and Objectives g. memorize the chapters of the course syllabus h. identify and explain the values for integration in the BSA Curriculum i. recite classroom policies

Open forum

Group discussion

SUPPLEMENTARY ACTIVITIES/ COURSE REQUIREMENTS

INTRUCTIONAL MATERIALS/ EQUIPMENT

METHODS OF EVALUATION

Photocopy the course syllabus and the values for integration in the BSA Curriculum

USM Student Code

Check if they have discussed with their parents the grading system and the school policies

Laptop DLP

Memorize and internalize the USM Vision, Mission and Core Values Explain to their parents the grading system, school policies discussed, USM Vision/Mission and CA Goal and Objectives

Course Syllabus Chalkboard/ Whiteboard Pieces of Chalk/ Whiteboard pen

Quiz on the USM Vision/Mission CA Goal and Objectives and Values for Integration in the BSA Curriculum

REFERENCES

USM Code

Student

CA Bulletin Handouts on the Values for Integration in the BSA Curriculum Revised Code

USM

COURSE OUTLINE


I. Introduction A. The biological word its balancing mechanisms

2

3

and

SPECIFIC OBJECTIVES

At the end of the unit, the students are expected to be able to:

B. Review of ecological concepts and principlesbasis of biological control

a. discuss the concepts of the biological world and its balancing mechanisms

C. Review of basic definition of pests and their characteristics

b. explain the ecological concepts as bases for biological control

D. Factors that determine the existence of an organism in an ecosystem

c. explain the basic explanation of pests and their characteristics

METHODOLOGY

Lecture

SUPPLEMENTARY ACTIVITIES/ COURSE REQUIRMENTS

INTRUCTIONAL MATERIALS/ COURSE REQUIRMENTS

Discussion of results of field survey

References/ Textbooks

Literature search discussion filed survey

Laptop

Discussion Laboratory


Short quiz Attendance

to of

Field survey, collection and identification of arthropod pests and their natural enemies

DLP

Graded recitation

Stereoscope

d. identify the factors that determine the existence of organisms in an ecosystem II. Concepts of Biological Control of Agricultural Pests

4

3

a. state the concepts biocon

of

Lecture

Library work on definition of

References/ textbooks

Laboratory exercise

Blackboard

Discussion A.

B.

C.

Definition and concepts of biological control Unique characteristics of Pests as it affect biological control strategies The host plant – an important component of biocon/types of host plant resistance affecting insect

b. explain the unique characteristics of pests as it affects biocon strategies c. explain the importance of host plant resistance in biocon

Attendance

Library method Chalk Field survey of insect population

Short quiz

Overhead/slide projectors transparencies Stereoscope Specimens

Graded recitation Laboratory output

COURSE OUTLINE


III. Nature of Biological Control Agents

4

6


A. Parasitoids, Pathogens, and Predators of Insects and Vertebrate Pest

Control

METHODOLOGY

of b. Discuss the antagonists of pathogens and biocon of weeds


Lecture

Library work

Discussion

Internet surfing


References/ Textbooks


Short quizzes Attendance

Laptop Library method

a. Enumerate the natural enemies of insects and vertebrate

B. Antagonist of Pathogen C. Biological Weeds

SPECIFIC OBJECTIVES

Laboratory exercise

Graded recitation DLP

Laboratory method

Laboratory output

Interactive learning Computer assisted teaching Learning visits

IV. Assessment, Production and Releases

Mass Field

4

6


A. Assessment of Parasitism B. Nutrition Production agents

and of

Mass biocon

a. assess parasitism in the laboratory and in the field b. discuss the nutrition and mass production techniques for biocon agents

Lecture

Library work

Discussion

Internet surfing

Library method

Laboratory exercise

Laboratory method Interactive learning Computer assisted teaching Learning visits

References/ Textbooks Blackboard Chalk Overhead/slide Projectors Transparencies Slide film Stereoscope Specimens Eraser Laboratory manual Lecture manual

Short quizzes Attendance Graded recitation Unit exam

COURSE OUTLINE

V. Methods and Approaches to Biological Control A. Quarantine and exclusion B. Use of resistant host plant C. Destruction by cultural management or infected/affected host D. Conservation and Augmentation E. Integration with other control tactics for pest management


4

9

SPECIFIC OBJECTIVES

METHODOLOGY




At the end of the unit, the students must be able to:

Lecture

Library work on

References

Short quizzes

Discussion

Laboratory exercise

Textbooks

Attendance

a. Discuss the importance of quarantine and exclusion in biocon

Laboratory method

Blackboard

Graded recitation

Chalk

Mid-term exam

Project method

Overhead/slide projector

Laboratory output

b. Discuss the importance of using resistant host plants in biocon

Library method

Transparences Slide film

c. Explain the importance of destruction of infected/affected host d. Explain conservation and augmentation of biocon agents e. Discuss the integration of biocon w/ other control tactics for IPM

Stereoscope specimens

References

Arun, PR. 2000. Seasonality and Abundance of Insects. Bhaharathiar University, Coimbatore Campbell, NA; JB Reece; LG Mitchell and MR Taylor. 2003. Biology: Concepts and Connections. 4 th e. Benjamin Cumings. N.Y. 781 pp. DeBach, P., 1991, Biological Control by Natural Enemies, 2nd edition, Cambridge University Press, Cambridge, MA. Goggle.2011http://extension.entom.purdue.edu/landscape/notes295N1/Lecture_links/Lecture_9_Pesticides_files /frame.htm Romoser, WS & JG Stoffolano.1994.The Science of Entomology 3rd ed. WBC,England Stoner, K.2004. Approaches to Biological Control of Insect Pests. Coop Ext. Pub,.Univ of Maine Vreysen, MBJ,AS Robinson and J. Hendrich.2007. Area-wide control of insects pests. Springer, The Netherlands

PREPARED BY: PURIFICACION O. CAHATIAN Professor

EVELYN P. ESTEBAN Professor

CHECKED BY: PURIFICACION O. CAHATIAN Department Chairperson

APPROVED BY: ADEFLOR G. GARCIA Dean

USM VISION

Quality and relevant education for its clientele to be globally competitive, culture-sensitive, and morally responsive human resources for sustainable development.

USM MISSION

Help accelerate the socio-economic development, promote harmony among diverse communities, and improve the quality of life through instruction, research, extension and resource generation in Southern Philippines.

COLLEGE OF AGRICULTURE GOAL

Assumes the prime role in pursuing a culture of excellence in agricultural instruction, research, extension and production activities in undergraduate and graduate degree programs. The College of Agriculture endeavors to train morally upright men and women who will be competent leaders in various fields of agricultural education, science and technology.

OBJECTIVES OF THE COLLEGE OF AGRICULTURE Produce graduates who are locally and globally competitive; develop and train manpower resources who will provide moral leadership in the agriindustrial and socio-economic development of Southern Philippines in of national development endeavors. Specifically, the CA aims to: 1. Produce BS, MS and PH D degree holders who are: a. well trained and skilled in the production and management of all kinds of crops and livestock; b. competent and proficient agricultural extension specialists and vocational teachers in agriculture; c. expert crop protection managers and farming systems specialists with management strategies built on the premise of sound and sustainable agricultural practices; d. competent plant breeders and tissue culture experts; e. skilled soil scientists and soil fertility management experts; f. proficient in communication skills g. equipped with excellent entrepreneurial capabilities

TABLE OF CONTENTS Chapter 1

Topic Introduction The Biological World and Its Balancing Mechanisms Review of Ecological Concepts Definition of Pest Factors that Determine the Existence of an Organism in an Ecosystem Concepts of Biological Control of Agricultural Pests A. Definition and Concepts of Biocon B. Unique Characteristics of Pests Nature of Biocon Agents A. Parasitoid, Pathogens and Predators B. Antagonists of Plant Pathogens C. Biocon Agents of Weeds Assessment, Mass Production and Field Releases A. Assessment of Effectiveness B. Nutrition and Mass Production Methods and Approaches to Biocon A. Quarantine and Exclusion B. Use of Resistant Host Plant C. Conservation by Cultural Management D. Conservation and Augmentation E. Integrated with Other Control Tactics A. B. C. D.

2

3

4

5

Page 1 1 7 9 10 15 15 17 20 20 46 52 60 60 60 86 86 89 93 94

LIST OF FIGURES

Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Title Food Chain Food Web Locusts Functional Response Parasitoid Lady Beetle Lady Beetle Larva Green Lacewing Adult Green Lacewing Larva Syrphid Fly Adult Syrphid Fly Larva Hunting Wasp Crab Spider Tachinid Fly Egg Tachinid Fly Adult NPV Bt Fungi Microsporidian Refuge for Dermaptera

Page 5 6 9 13 21 21 21 22 22 23 23 23 23 23 23 29 33 41 44 93

Chapter I

INTRODUCTION

A. The Biological World and Its Balancing Mechanism The term habitat refers to the kind of place where an organism normally lives. In contrast, a niche is the “occupation” of an organism. It defines the role of an organism in an ecosystem, such as a “fish-eating wader” for a heron, or a “plant-juice-sipping summer buzzer” for a cicada. An organism‟s niche may change during different life stages. For example, a tadpole typically lives in the water and eats plant material, while the adult frog may catch insects from the shore. The source of energy for all life on Earth is the sun. Green plants (and some bacteria) are the only organisms that can directly capture the sun‟s energy and change it into a form that other organisms can use. Through the process of photosynthesis, plants use sunlight to change carbon dioxide and water into sugar and oxygen. The oxygen is given off into the air, where it is available to other organisms including humans. Simple sugar molecules make energy available to plants and, by forming the basic units of complex carbohydrates, contribute to plant structure. Other organisms then eat the plants, or eat organisms that eat plants, and in doing so indirectly gain the benefit of the sun‟s energy to run their bodies. The flow of sunlight energy is therefore ed from procedures (green plants) to primary consumers (animals that eat plants, such a leafhoppers) to secondary consumers (animals that eat other animals, such as birds); this sequence is known as a food chain. As energy is ed along the food chain, much is used up at each level as it works to run each organism. This energy is given off as heat and results in less energy being available at each stage along the food chain. It takes a lot of grass to one rabbit, and many rabbits to one hawk. As a consequence, there are many, many green plants on the Earth, fewer animals that eat plants, and even fewer animals that eat animals; this is known as the energy pyramid. In the bosque, the cotton woods and other plants trap the sunlight energy and provide it in a form usable by the entire collection of other organisms found there. They provide the foundation for life along the river. Although sunlight energy is used up as it is ed along the food chain, fortunately there is an abundant supply of this energy. In contrast, the materials from which all living things are made are limited in supply and must be used over and over. The primary building blocks of all living things include only six materials: carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur. When an organism dies and decomposes, these materials are returned

to the system and are used again. The carbon that was once part of a dinosaur‟s tail may now be in the tomato that you eat for dinner! If these compounds are removed from the cycle in some way, they may become limited in supply. For example, if a tree dies but the wood does not readily decompose, carbon and the nutrients are trapped in the wood and increasing the rate of decomposition, undecomposed wood is now building up and trapping nutrients. This affects the health of the entire ecosystem. One very important cycle is the water cycle. Rain that falls on a hillside percolates down into the ground water, or may flow above ground into a lake or the ocean. Water in the lake or ocean then evaporates, and drops together into clouds, to eventually fall again as rain. Our use of water greatly affects the water cycle. In New Mexico we remove water from the underground aquifer (water present in the bedrock below ground) much faster than it is replenished. Much of this water evaporates directly into the atmosphere while we use it, and may then fall again somewhere else on the planet, thus reducing the amount of water available locally. We also impact the cycling of materials by introducing poisons. As materials are cycled over and over, toxins build up. Concentrations of toxins increase along food chains, since a predator eats many prey with the toxin, a process known as biomagnification. These increasing concentrations of toxins often have devastating effects. Some well-known examples include toppredator species such as bald eagles and peregrine falcons that nearly became extinct due to the effects of DDT or other chemicals. Awareness of these problems may go a long way towards helping to keep our cycles clean. Through the flow of energy and the cycling of materials, all living things are interrelated. A mouse not only gets energy from the seed that it eats, but also gets materials that will help to build more mouse tissue. The mouse breathes out carbon dioxide which is taken in by plants, which in turn give off oxygen used by the mouse. The mouse also depends on plants for finding shelter, and it provides food for a snake or owl. The components of the bosque are interrelated with connections extending to the surrounding uplands as well. Some connections are obvious, such as birds that fly between the bosque and uplands at different times of day or during different seasons, moving materials from one place to another. Others are more subtle, such as water flowing underground. But these connections make our actions even more important. Pesticides applied to our fields may add toxic materials to the river, affecting not only the water itself but also all the organisms that depend on the water. Change is an integral part of the natural world. Changes may occur over geologic time, such as the transition of the Rio Grande from a series of lakes to the river that we know today, or they may occur over much shorter

time periods, such as the transition of a seed to a tree and finally to a fallen log. Change was once an integral part of the natural Rio Grande riparian ecosystem, as the river wandered across the floodplain leaving behind its ever-changing mosaic of vegetation. However, human-induced changes have much different effects on the ecosystem. The rate at which we are causing changes on Earth is much greater than has been known previously, and we do not yet know the ecological consequences of most of our actions. By understanding the ecological systems in which we live, and how we interact with them, we can begin to lessen our impact on Earth. The community of living organism within every ecosystem has a tropic structure, a pattern of feeding relationship, consisting of several levels. The trophic structure determines the route that energy takes in flowing through the ecosystem, as well the pattern for chemical cycling. The sequence of food transfer from trophic level to trophic level is known as food chain (Fig. 1). In Figure compares a terrestrial food chain and an aquatic food chain. Food chains provide an overview of an ecosystem structure and function, but they an oversimplification. A more realistic view of the trophic structure of an ecosystem than a food chain is a food web, a network of interconnecting food chain (Fig. 2). This figure shows a simplified example of a food web in a salt marsh. The orange arrows represent primary consumption while the blue arrows represent secondary consumption. Indicating “who eats whom”, the arrows in the food web diagram the transfer of food from the producers through the trophic levels, moving chemical nutrients and energy through an ecosystem. Figure 1. A terrestrial and an aquatic food chain (Lifted from Campbell et al, 2003) Figure 2/ A food web chain (Lifted from Campbell et al, 2003)

B. Review of Ecological Concepts – Bases of Biological Control

1. Biological control is: 

a manifestation of the natural associations of different kinds of living organisms: parasite and pathogens with their hosts : predators with their prey



dynamic: subject to disturbances by various factors : subject to changes in the environment : subject to the adaptations, properties and limitations of the organisms involved

2. Population of Ecology Populations – are groups of actually or potentially interbreeding individuals at a given locality Characteristics of a Populations 1. Size – changes in the number of individuals due to: a. environmental factors (biotic and abiotic) b. migrations of individuals into or out of the local population 2. Age structure/ population structure - in some species, all the at any time may be approximately the same age or in the same stage of development -

in some insect species however, individuals of all ages occur together, and generations are not synchronized but strongly overlap. This is commonly found in short-lived insects with many generations per year (e.g. aphids, hoppers, mites, etc.)

3. Populations are dynamic with regards to geographic distribution. They tend to spread until some limiting environmental condition is encountered such as geographical barriers like coast, mountain ranges, desert boundary or absence of a required resources like food or habitat.

4. Populations do not exist in isolation. They occur in habitats in association with other species, forming communities.

Importance of Age Structure in Biocon In respect to host populations in which only one or two stages of development are utilizable by a particular natural enemy, a close synchronization between natural enemy and host life cycles must occur if successful control of the host is to be achieved. Importance of Population Studies in Biocon 1. Aids in classifying the role played by natural enemies as well as the other forces 2. In communities, trophic or nutritional association between interacting species can be distinguished: Primary producers – green plants Primary consumers – herbivores Secondary consumers – carnivores, decomposers, scavengers

C. Definition of Pests and Their Characteristics

A pest is: 

Any organism which competes with mankind for a limited resources or is threatening to man‟s health or comfort and possessions.



Ecologically, there are no pests, only consumers. However, when an organism begins to take what mankind wants, that organism becomes a pest (Fig.3)

(Fig.3) Locusts

D. Factors that Determine the Existence of an Organism in an Ecosystem 1. Food Supply – food is the basic need of any organism in an ecosystem. Insufficiency of this factor greatly affects the other vital processes of an organisms like growth and development, mating and reproduction. 

Starvation – blowflies needing flesh may not find necessary corpses or wounds, silkworms hatching before bud opening of mulberry leaves.



Decline in Supply – cabbage aphids at end of seasonal crop or after harvest.



Dependence up food chain e.g. predators of above cannot find prey Potential food exists but is unavailable for consumption

       

Accidental loss of food – lice, fleas fall off host; aphids, grasshoppers blown by wind Interference by other species – humans apply repellent to crops or livestock Insect behavior. Tsetse flies don‟t feed on animals in the open. If host is not close by shade, they will not feed. Cannibalism – If codling moth larvae encounters another in an apple, one is eaten despite plenty of apple for both. Affects of insect feeding on hosts – If buffalo flies are in large numbers, their host will take action to avoid being bitten. Nutrient deficiencies – Mite numbers, egg production and longevity are directly related to N2 content in leaves. Development time in mites is indirectly related to the N2 content in leaves. Lack of food at critical time – often dependent upon weather o Hover flies (syrphid) require spring pollen for maturation of ovaries. If plants not in flower  few eggs. o Sorghum midges diapause over winter as mature larvae, emerging with summer rains as adults for only 2 days and need to find sorghum flowers in that time.

HOW DO INSECTS OVERCOME FOOD PROBLEM?   

Dispersal Polyphagy – eat multiple species of predators or plants Storage of food – social insects – ants, bees.

2. Predator Number The numerical response is ecology is the change in predator density as a function of change in prey density. The term numerical response was coined by M.E. Solomon in 1949. It is associated with the functional response, which is the change in predator‟s rate of prey consumption with change in prey density. As Holling notes, total predation can be expressed as a combination of functional and numerical response. The numerical response has two mechanisms: the demographic response and the aggregational response. The numerical response is not necessarily proportional to the change in prey density, usually resulting in a time lag between prey and predator populations. For example, there is often a scarcity of predators when they prey population is increasing. Demographic Response The demographic response consists of changes in the rates of predator reproduction or survival due to a changes in prey density. The increase in prey availability translates into higher energy intake and reduced energy output. This is different from an increase in energy intake due to increased foraging efficiency, which is considered a functional response. This concept can be articulated in the Lotka-Volterra Predator-Prey Model. dP / dt = acVP – Mp a= conversion efficiency: the fraction of prey energy assimilated by the predator and turned into new predators P = predator density V = prey density m = predator mortality Demographic response consist of a change in dP/dt due to a change in V and/or m. For example, if V increases, then predator growth rate (dP/dt) will increase. Likewise if the energy intake increases (due to greater food availability) and an decrease in energy output (from foraging), then predator mortality (m) will decrease and predator growth rate (dP/dt) will increase. In contrast, the functional response consists of a change in conversion efficiency (a) or capture rate (c).

The relationship between available energy and reproductive efforts can be explained with the life history theory in the trade-off between fecundity and growth/survival. If an organism has more net energy, then the organism will sacrifice less energy dedicated to survival per reproductive effort and will therefore increase its reproductive rate. In parasitism, functional response is measured by the rate if infection of laying of eggs in host, rather than the rate of prey consumption as it is measured in predation. Numerical response in parasitism is still measured by the change in number of adult parasites relative to change in host density. Parasites can demonstrate a more pronounced numerical response to changes in host density since there is often a more direct connection (less time lag) between food and reproduction in that both needs are immediately satisfied by its interaction with the host.

Aggregational Response The aggregational response, as defined by Readshaw in 1973, is a change in predator population due to immigration into an area with increased prey population. In an experiment conducted by Turnbull in 1964, he observed the consistent migration of spiders from boxes without prey to boxes with prey. He proved that hunger impacts prey movement. Riechert and Jaeger studied how predator competition interferes with the direct correlation between prey density and predator immigration. One way this can occur is through exploitation competition: the differential efficiency in use of available resources, for example, an increase in spiders‟ web size (functional response). The other possibility is interference competition where site owners actively prevent other foragers from coming in vicinity.

Ecological Relevance The concept of numerical response becomes practically important when trying to create a strategy for pest control. The study of spiders as a biological mechanism for pest control has driven much of the research on aggregational response. Antisocial predator populations that display territoriality, such as spiders defending their web area, may not display the expected aggregational response to increased prey density

The functional response in ecology is the intake rate of a consumer as a function of food density. It is associated with the numerical response, which is the reproduction rate of a consumer as a function of food density. Following C.S.Holling function responses are generally classified into three types, which are called Holling‟s type 1, II and III. Graphical representation of all three functional responses.

Type I Type I functional response assumes a linear increase in intake rate with food density, either for all food densities, or only for food densities up to a maximum, beyond which the intake rate is constant. The linear increase assumes that the time needed by the consumer to process a food item is negligible, or that consuming food does not interfere with searching for food. A functional response of type I is used in the Lotka-Volterra predator-prey model. It was the first kind of functional response described and is also the simplest of the three functional responses currently detailed.

Type II Type II functional response is characterized by a decelerating intake rate, which follows from the assumption that the consumer is limited by its capacity to process food. Type II functional response is often modeled by a rectangular hyperbola, for instance as by Holling‟s disc equation, which assumes that processing of food and searching for food are mutually exclusive behaviors. The equation is f (R) =

ɑR 1 + ɑhR,

Where f denotes intake rate and R denotes food (or resource) density. The rate at which the consumer encounters food items per unit of food density is called the attack rate, ɑ. The average time spent on processing of food is called the handling time, h. Similar equations are the Monod equation for the growth of microorganism and the Michaelis-Menten equation for the rate of enzymatic reactions. In an example with wolves and caribou, as the number of caribous increases the number of caribou kills per wolf also increases, however, the higher the

density of caribou, the less increasing caribou density increases the number of kills per wolf. At very high caribou densities, wolves need very little time to find prey and spend almost all their time handling prey and very little time searching. Wolves are then saturated and the number of caribou kills per wolf reaches a plateau.

Type III Type III functional response is similar to type II in that at high levels of prey density, saturation occurs. But now, at low prey density levels, the graphical relationship of number of prey consumed and the density of the prey population is more than linearly increasing function of prey consumed by predators. This accelerating function is caused by learning time, prey switching, or a combination of both phenomena. Learning time is defined as the natural improvement of predator‟s searching and attacking efficiency of the natural improvement in their handling efficiency as prey density increases. Imagine a prey density so small that the chance of a predator encountering that prey is extremely low. Because the predator finds prey so infrequently, it has not had enough experience to develop the best ways to capture and subdue that species of prey. Holling identified this mechanism in shrews and deer mice feeding on sawflies. Al low numbers of sawfly cocoons per acre, deer mice especially experienced exponential growth in of the number of cocoons consumed per individual as the density of cocoons increased. The characteristic saturation point of the type III functional response was also observed in the deer mice. At a certain density of cocoons per acre, the consumption rate of the deer mice reached a saturation amount as the cocoon density continued to increase. Prey switching involves two or more prey species and one predator species. When all prey species are at equal prey densities, the predator will indiscriminately select between prey species. However, if the density of one of the prey species decreases, then the predator will start selecting the other, more common prey species with a higher frequency. Murdoch illustrated this effect with guppy preying on fubificids and fruit flies. As fruit fly numbers decreased guppies switched from feeding on the fruit flies on the water‟s surface to feeding on the more abundant tubificids along the bed.

3. Habitat Advantage – the type of soil in a habitat influences an insect‟s distribution and abundance and is easily disturbed by agriculture, e.g.

Irrigation changes moisture and subsequently, the type of pest in a crop. Chemicals in soil affect plant growth and therefore the dependent insects.

Chapter 2

CONCEPTS OF BIOLOGICAL CONTROL OF AGRICULTURAL PESTS

A. Definition and concepts of biological control Biological Control may be defined (in as few words as possible) as: The use of living natural enemies to control pests. In slightly more words, it has been defined as: The active manipulation of antagonistic organisms to reduce pest population densities, either animal or plant, to noneconomically important levels. Both definitions imply that biological control is an action taken by people using what are called ”natural enemies” or “antagonistic organisms.” These may be predators, parasites, parasitoids, pathogens, or competitors of the pest that is to be controlled. There are various ways in which these “natural enemies” or “antagonistic organisms” can be put to work. The four major ways are by what has been called Augmentive Biological Control, Classical Biological Control (otherwise known as Inoculative Biological Control), Inundative Biological Control, and Manipulative Biological Control.

Augmentive Biological Control: Release of large numbers of biological control agent to supplement the small numbers already present, in expectation of a greatly increased effect. Example: A stink bug is causing severe damage to lychee flowers and small fruit in an orchard. A beneficial wasp is naturally present but is killing only 10% of the stink bug eggs. Release of many more of the same wasp species, reared in an insectary, results in many more stink bug eggs being killed. The wasp reproduce and reduce damage to below the economic threshold for the rest of the fruiting season. As an added bonus, use of these wasp, instead of a chemical pesticide, conserves honey bees which are pollinating the lychee flowers.

Classical Biological Control (Inoculative Biological Control):

Importation and release of biological control agents into an area in which they are not already present, with intent to establish a permanent population. Example: USDA researchers import from South America a weevil that attacks an aquatic weed (also from South America). After determining that the weevil attacks only the aquatic weed, they breed the weevil in a laboratory and release a few hundred at many places in the southern USA. The weevil eats the weed and reproduces, and its populations grow and spread to still other places where the weed is causing problems. After a year or two, the weed and the weevil still exist at many places, but both at low numbers, and the weed is no longer a problem. Control is effectively permanent, with no weed for future expenditures. Seasonal Inoculative Biological Control is a subset of this and is defined as: Release of biological control agents in an area in which they cannot survive permanently due to severe climate or other constraints; the expectation is that they will establish a population that will persist for some fraction of a year. Example A: Mexican bean beetle spread decades ago to the northeastern USA where it does severe damage. A wasp was found in India that attacks Mexican bean beetle and its close relatives (but the only close relatives in the USA are also pests). Unfortunately, the wasp cannot survive winters in the northeastern USA. So, small numbers of the wasp are maintained in an insectary and some of them are released at the beginning of each growing season in bean-growing areas. They attack the Mexican bean beetle, reproduce, control the numbers of the beetle throughout the growing season, and die in winter. Example B: Seasonal Inoculative releases of a South American flea beetle are used to control alligatorweed in more temperature areas where the plant has become invasive.

Inundative Biological Control Release of large numbers of biological control agent relative to the numbers of a target species, in expectation of a rapid effect. There is no implication that the released biological control agent will establish a permanent population. Example: A golf course is being damaged severely by white grubs (scarab larvae). The superintendent pays for an application of a biopesticide (in this case an entomopathogenic nematode that happens to be native). The nematode killd 90 of the white grubs within 10 days and then disappears

because white grubs will not reappear until next year. An added bonus is that the nematode is exempt from regulation by the EPA, the golf course does not have to be closed for play during and after the application, and the superintended is seen as a good neighbor by residents of nearby homes (because they don‟t like use of chemical pesticides anywhere near them).

Manipulative Biological Control: The manipulation of elements in the environment to enhance the numbers and/or actions of natural enemies. Example: Channels are dug in a saltmarsh to connect pools of water. This allows naturally-occurring predatory fish to gain access to pools and eat mosquito larvae . Conservation Biological Control is a subset of this because it seeks merely to conserve. Example: Plots of a particular weed are left untreated with herbicide around a sugarcane field; these plants are very important source of nectar for adults of a species of wasp that attacks white grubs (scarab larvae) that damage sugarcane roots. This conserves the wasp population, reduces the pest (scarab) population, and reduces damage to the sugarcane.

B. Unique characteristics of pests as it affect biological control strategies 1. Adaptability Pests particularly insects have great capabilities for adapting to many different environmental conditions. Insects have specific adaptations like defense mechanism, protective coloration and protective mimicry which may hinder or render biological control strategies ineffective. 2. High reproductive potential and abundance – this characteristics can render biological control strategies less effective especially if natural enemies are few in number. 3. Capacity for flight – this characteristics makes it difficult for a parasite or predator to catch up with the host or prey.

4. Protective retreats – insect pests live in burrows, nests, bags, tunnels or galls serving as protective for them not to be reached by either a predator or a parasite.

C. Types of host plant resistance affecting the pests Definition of an Insect-Resistant Plant: Definitions of an insect-resistant plant are many and varied. In the broadest sense, plant resistance is defined as “the consequence of heritable plant qualities that result in a plant being relatively less damaged than a plant without the qualities”. In practical agricultural , an insect-resistant crop cultivar is one that yield more than a susceptible cultivar when confronted with insect pest invasion. Resistance of plants is relative and is based on comparison with plants lacking the resistance characteristics, i.e., susceptible plants. Effect of Insect Pest-Plant Host Relationship: Insect-resistant crop varieties suppress insect pest abundance or elevate the damage tolerance level of the plants. In other words, insect-resistant plants alter the relationship an insect pest has with its plant host. How the relationship between the insect and plant is affected depends on the kind of resistance, e.g. antibiosis, antixenosis (nonpreference), or tolerance. Antibiosis resistance affects the biology of the insect so pest abundance and subsequent damage is reduced compared to that which would have occurred if the insect was on a susceptible crop variety. Antibiosis resistance often results in increased mortality or reduced longevity and reproduction of the insect. Antixenosis resistance affects the behavior of an insect pest and usually is expressed as non-preference of the insect for a resistance plant compared with a susceptible plant. Tolerance is resistance in which a plant is able to withstand or recover from damage caused by insect pest abundance equal to that damaging a plant without resistance characters (susceptible). Tolerance is a plant response to an insect pest. Thus, tolerance resistance differs from antibiosis and antixenosis resistance in how it affects the insect-plant relationship. Antibiosis and antixenosis resistance cause an insect response when the insect attempts to use the resistant plant for food, oviposition, or shelter.

Advantages to the Use of Insect-Resistant Crop Varieties Use of insect-resistant crop varieties is economically, ecologically, and environmentally advantageous. Economic benefits occur because crop yields are saved from loss to insect pests and money is saved by not applying insecticides that would have been applied to susceptible varieties. In most cases, seed of insect-resistant cultivars cost no more, or little more, than for susceptible cultivars. Ecological and environmental benefits arise from increases in species diversity in the agroecosystem, in part because of reduced use of insecticides. Increases in species diversity increase ecosystem stability which promotes a more sustainable system far less polluted and detrimental to natural resources. The IPM concept stresses the need to use multiple tactics to maintain insect pest abundance and damage below levels of economic significance. Thus, a major advantage to the use of insect-resistant crop varieties as a component of IPM arises from the ecological compatibility and compatibility with other direct control tactics. Insect-resistant cultivars synergize the effects of natural, biological, and cultural insect pest-suppression tactics. The “built-in” protection of resistant plants from insect pests functions at very basic level, disrupting the normal association of the insect pest with its host plant. The compatible, complementary role plant resistance to insect pests plays with other direct control tactics is, in theory and practice, in concert with the objectives of IPM. All crop cultivars should contain resistance to insect pests. Plant resistance to insect pest have advantages over other direct control tactics. For example, plant resistance to insects is compatible with insecticides use, while biological control is not. Plant resistance to insects is not density dependent, whereas biological control is. Plant resistance is specific, only affecting the target pest. Often effects of use of insect-resistant cultivars are cumulative over time. Usually the effectiveness of resistant cultivars is long-lasting. The role of plant resistance to insects in IPM has been well defined, at least in theory. However, the specific role a resistant cultivar plays in a particular IPM situation is crucial to successful deployment of the resistant cultivar. The impact of the resistant cultivar on standard cultural, biological, and insecticidal control methods should be well defined. Likewise, the impact of each of these control tactics on the resistant cultivar also must be defined.

Chapter 3

NATURE OF BIOLOGICAL CONTROL AGENTS

A. Parasitoids, Pathogens and predators of Insect and Vertebrate Pests 1. Parasitoids The term parasitoid was coined in 1913 by the German writer O.M. Reuter (and adopted in English by his reviewer, William Morton Wheeler) to describe the strategy in which, during its development, the parasite lives in or on the body of a single host individual, eventually killing that host, the adult parasitoid being free-living. About 10% of described insect species are entomophagous parasitoids. There are four insect orders that are particularly renowned for this type of life history. 1. By far the majority are in the order Hymenoptera. The largest and best-known group comprises the so-called “Parasitica” within the Hymenopteran suborder Apocrita: the largest subgroups of these are the chalcidoid wasps (superfamily Chalcidoidea) and the ichneumon wasps (superfamily Ichneumonoidae), followed by the Proctotrupoidea. Outside of the Parasitica, many other Hymenopteran lineages that include parasitoids, such as most of the Chrysidoidea and Vespoidea, and the rare Symphytan family Orussidae. 2. The flies (order Diptera) include several families of parasitoids, the largest of which is the family Tachnidae, and also smaller families such as Pipunculidae, Conopidae, and others. Other families of flies that are not primarily parasitoids or parasites, or at least not primarily protelean, do nonetheless include protelean species. For example Phoridae have already been mentioned as parasitoidal on ants, and at least some flesh fly species, such as Emblemasoma auditrix, are parasitoidal on cicadas, and have raised great interest because they locate their hosts by sound. The kleptoparaistic flesh fly genus Craticulina has already been mentioned and logically qualifies as a protelean fly genus. Two other orders with parasitoidal are 3. The “twisted-wing parasites” (order Strepsiptera), which is a small group consisting entirely of parasitoids.

4. The beetles (order Coleoptera), which includes at least two families, Ripiphordae and Rhipiceridae, tha are largely parasitoids, and rove beetles (family Staphylinidae) of the genus Aleochara. Occational of others can be parasitoids; one of the remarkable is the moth family Epipyropidae, which are ectoparasitoids of planthoppers and Cicadas. The genus Cyclotorna has even more elaborate habits, beginning its growth period parasitizing plant bugs, and concluding by feding on ant larvae in their colonies. Figure 5. Koinobiont parasitoid on moth larva. Hymenoptera parasitoids often have unique life cycles. In one family, the Trigonalidae, the female wasps deposits eggs into small pockets they cut into the edge of leaves with their ovipositor. A caterpillar chewing these leaves may unknowingly swallow some of the eggs, and when they get into the caterpillar‟s gut, they hatch and burrow through the gut wall and into the body cavity. Later they search the caterpillar‟s body cavity for other parasitoid larvae, and it is these they attack and feed on. Some trigonalids, once in a caterpillar or sawfly larva, need their vehicle to fall prey to a social wasp. The wasp carries the caterpillar back to its nest, and there it is butchered and fed to the wasp‟s young; they will serve as the host for the trigonalid, the eggs of which are in the butchered caterpillar.

2. Predators a. Lady Beetles Often called ladybugs, lady beetles (Figure 6) are the most familiar Insect predator. Most adult lady Beetles are round to oval, brightly colored and often spotted. Lady beetles are further discussed Figure 6. Twospotted lady beetle in fact sheet 5.594, Lady Beetles. laying eggs. The immature or larvae stages, However, look very different and Figure 7. Typical lady beetle larva often are overlooked or misidentified. Lady beetle larvae are elongated, usually dark colored, and flecked with orange or yellow (Figure 7). Adult and larvae feed on large numbers of small, soft-bodied insects such as aphids. One group of small, black lady beetles (Stethorus) is important in

controlling spider mites and others specialize in scale insects. Lady beetles can rapidly control many developing insect problems, particularly if temperature are warm. One species of lady beetle, however, the Mexican bean beetle, is a plant pest. This common Colorado insect is found feeding on bean leaves. It is distinguished from other lady beetles by spotting and color in the adult stage. Larvae of Mexican bean beetle are yellow and spiny. b. Green Lacewings Several green lacewing species (Figure 8) are commonly found in gardens. The adult stage is familiar to most gardeners: a pale green insect with large, clear, highly-veined wings that are held over the body when at rest. Adult green lacewings primarily feed on nectar and other fluids, but some species also consume a few small insects. Figure 8. Green lacewing adult Green lacewings lay a distinctive stalked egg. Lacewing larvae emerge in four to 10 days. These larvae, sometimes called aphid lions, are voracious predators capable of feeding on small caterpillars and beetles, as well as aphids and other insects. In general shape and size, lacewing larvae are superficially similar to lady beetle larvae. However, immature lacewings usually are light brown and have a large pair of hooked jaws sticking out from the front of the head. Figure 9. Green lacewing nymph. Photo courtesy of Harold Larsen c. Syrphid Flies These flies are called by several names, such as flower flies or hover flies./ Most are brightly colored, yellow or orange and black, and may resemble bees or yellowjacket wasps (Fig 10). However, sryphid flies are harmless to people. Usually they can be seen feeding on flowers. Figure 10. Sryphid fly adult It is the larval stage of the sryphid fly (Fig 11) that preys on insects. Variously colored, the tapered maggots crawl over foliage and can eat dozens of small, soft-bodied insects each day. Sryphid flies are particularly important in controlling aphid infestations early in the season, when cooler temperatures may inhibit other predators. Similar in appearance to sryphid fly larvae is a

small, bright orange predatory midge (Aphidoletes). These insects often can be seen feeding within aphid colonies late in the season. Figure 11. Syrphid fly larva d. Predatory Bugs True bugs (Order: Hemiptera) are predators of insects and mites. All feed by piercing the prey with their narrow mouthparts and sucking out body fluids. A red and black species of predatory stink bug, capable of feeding on fairly large insects such as caterpillars and potato beetle larvae, is most conspicuous. More common, but less frequently observed, are the various light brown damsel bugs, also called nabid bugs. Damsel bugs are found on the foliage of all crops, where they seek out aphids, insect eggs and small insect larvae. Figure 12. Hunting wasp (Ammophila) at prey with caterpillar Figure 13. Crab spider Figure 14. Tachinid fly eggs (white) laid near head of hornworm Figure 15. Tachinid fly feeding on nectar Most common of all the predatory bugs are the small (less than 1/8 inch) minute pirate bugs. Minute pirate bugs are most frequently seen in flower or in crevices of a green plant, where they feed on thrips, spider mites and insect eggs. Other predatory bugs common in yards and gardens include ambush bugs and assassin bugs. e. Ground Beetles Various species of ground beetles are found under debris, in soil cracks or moving along the ground. Immature stages are distinctly different from adults and more often are found within the top few inches of soil. Ground beetles are general feeders with powerful jaws./ Almost any garden pest that spends part or all of its life on the soil surface may be prey for these insects. f. Mantids Mantids are uncommon in most of Colorado but are familiar insects to most gardeners. Mantids are general predators that feed on almost any insects of the right size. They have one generations per year with winter spent as eggs within a pod. One species of mantids, the Chinese mantid, is sometimes available for sale. Mantids are discussed in more detail in fact sheet 5.510, Mantis of Colorado. g. Hunting Wasps

A large number of wasps from several families prey on insect pest. Many take their prey, whole or in pieces, back to their mud, soil or paper nests to feed to the immature wasps. These hunting wasps can be important in controlling Garden insect pests. For example, the common Polistes paper wasps, when hunting, may thoroughly search plants and feed on caterpillars, often providing substantial control of these insects. h. Predatory Mites Several mite species are predators of plant-feeding spider mites. Typically, these predatory mites are a little larger than spider mites but are more rounded in shape and faster moving than their prey. Predatory mites often can provide good control of spider mites. Low humidity can restrict their activity. They are also more susceptible to insecticides than are plant-feeding species. i. Spiders

All spiders feed on insects or other small arthropods Most people are familiar with many common web-making species. However, there are many others spiders – wolf spiders, crab spiders, jumping spiders – that do not build webs but instead move about and hunt their prey on soil or plants. These less conspicuous spiders can be important in controlling insect pests such as beetles, caterpillars, leafhoppers and aphids.

3. Pathogens of Insects Pathogens are viruses or microorganisms that cause disease. Like all other organisms, insects are susceptible to a variety of diseases caused by pathogens. Many of these pathogens cause disease that are acute and fatal and therefore are used as model to study processes of infection and pathogenesis as well as to control populations of insects that are pests or vectors of plant and animals diseases. Generally, insect pathogens have a relatively narrow host range and thus are considered to be more environmentally friendly than synthetic chemical insecticides. The pathogens that cause disease in insects fall into four main groups: viruses, bacteria, fungi, and protozoa. This article discusses the primary biological properties of each these pathogen groups, with specific emphasis on how these pathogens have been used to benefit humans. a. Viruses Viruses are obligate intracellular parasites, meaning that they can reproduce only in living cells and are composed in the simplest form of a nucleic acid, either DNA or RNA, and a protein shell referred to as the capsid. More complex viruses also contain a lipoprotein envelop. Insect viruses can be cultured in living hosts (in vivo) or in cultures insect cells (in vitro). In general, insect viruses are divided into two broad nontaxonomic categories, the occluded viruses and the nonoc-cluded viruses. Occluded viruses are so named because after formation in infected cells, the mature virus particles (virions) are occluded within a protein matrix, forming a paracrystalline bodies that are generically referred to as either inclusion or occlusion bodies. In the nonoccluded viruses, the virions occur freely or occasionally form paracrytalline arrays of virioins that are also known as inclusion bodies. These, however, have no occlusion body protein interspersed among the virioins. The five most commonly encountered types of insect viruses are iridoviruses, cytoplasmic polyhedrosis viruses, entomopoxviruses, ascoviruses, and baculoviruses.

Iridoviruses Nonoccluded viruses with a linear double-stranded DNA genome, the iridoviruses (family Iridoviridae) produce large, enveloped, icosahedral virions (125-200 nm) that replicate in the cytoplasm of a wide range of tissues in infected hosts. Virions form paracrytalline arrays in infected tissues, imparting an iridescent hue to infected hosts, from which the name of this virus group is derived. Over 30 types are known, and these have been most commonly reported from larval stages of Diptera larvae, such as mosquito larvae, as well as from larvae of Coleoptera and Lepidoptera. Generally, the irido-viruses occur very broadly, and they are known from other invertebrates, such as isopods, as well as from certain vertebrates including frogs and fish. Observations of natural occurrence in host field populations suggest that one host range of each type is quite narrow, although in the laboratory iridoviruses are readily transmitted from one insect species to another by inoculation. Prevalence and mortality rates in natural populations of host insects are typically less than 1%. Cytoplasmic Polyhedrosis Viruses The cytoplasmic polyhydosis viruses (family Reoviridae) are occluded double-stranded RNA viruses with a genome divided into 9 orn10 segments of RNA. These viruses, commonly referred to as Vs, cause a chronic disease and reproduce only in the stomach of insects, where typically they from large (ca. 0.5-2 im) polyhedral to spherical occlusion bodies in the cytoplasm of midgut epithelial cells. Infection in early instars retards growth and development, extending the larval phase by weeks. The disease is often fatal. In advance stages of disease, the infected midgut is white rather than translucent brown, because of large numbers of accumulated polyhydra. This virus type is relatively common among lepidopterous insects and among dipterous insects of the suborder Nematocera (e.g., mosquitoes, blackflies, midges). Vs are typically easy to transmit by feeding to species that belong to the same family of the host from which they were isolated, and thus the host range of this virus type is quite broad. Entomopoxviruses The entomopoxviruss (family Poxviridae) are occluded double-stranded DNA viruses that produce large, enveloped virions (150 nm X 300 nm) that replicate in the cytoplasm of a wide range of tissues in most hosts, causing an acute, fatal disease. Occlusion bodies vary from being oval to spindle shaped and generally occlude 100 or more virions. These viruses have been most commonly reported from coleopterans, from which there are over 30 isolates, but they are also known from lepidopterous, dipterous (midges), and orthopterous (grasshoppers) insects. This virus type is easily transmitted by feeding, although where the experimental host range of individual isolates has been tested, it has been found to be relatively narrow, generally being restricted to

closely related species. Insect poxvi-ruses are related to vertebrate poxviruses, such as the variola virus, the etiological agent of smallpox, and they may be the evolutionary source of the vertebrate poxviruses. Ascoviruses The ascoviruses (Ascoviridae) are a new family of DNA viruses, at present known only from larvae of species in the lepidopteran family Noctuidae, where they have been reported from several common pest species such as the cabbage looper, cotton budworm, corn earworm, and fall armyworm. Ascoviruses cause a chronic, fatal disease of larvae. The virions of ascoviruses are large (130 nm X 400 nm), enveloped, and reniform to bacilliform in shape; they exhibit complex symmetry and contain a circular, double-stranded DNA genome. During the course of ascoviruses disease, large numbers of virion-containing vesicles accumulate in the blood of infected caterpillars, changing its color from translucent green to milky white. These viroin-containing vesicles are formed by a unique developmental sequence resembling apoptosis (cell death) in which each infected host cell cleaves into a cluster of vesicles as virion assembly proceeds. An interesting ascovirus feature is that transmission from host to host depends on vectoring by female endoparasitic wasps. Ascoviruses are very difficult to transmit by feeding, with typical infection rates averaging less than 15% even when larvae are fed thousands of vesicles in a single dose. In contrast, infection rates for caterpillars injected with as few as 10 virion-containing vesicles are typically greater than 90%, and experiments with parasitic wasps show that these insects can transmit ascoviruses. Baculoviruses Baculoviruses (family Baculoviridae) are large, enveloped, doublestranded, occluded DNA viruses. These viruses are divided into two main types, commonly known as the nuclear polyhedrosis viruses (NPVs) and the granulosis viruses (GVs). Both NPVs and GVs are highly infectious by feeding, and in some insect species periodically cause epizootics, that is, widespread outbreaks of disease, that result in significant (>90%) declines in caterpillar populations. Nuclear Polyhedrosis Virus The NPVs (Fig. 16) are known from a wide range of insect orders but have been most commonly reported by far from lepidopterous insects, from which well over 500 isolates are known. Many of these are different viruses (i.e., viral species). NPVs replicate in the nuclei of cells, generally causing an acute fatal disease. The virions are large (80-200 nm X 280 nm) and consist of one or more rod-shaped nucleocapsids with a double-stranded circular DNA genome enclosed in an envelope. The occlusion bodies of NPVs are referred to commonly as polyhedral because typically their shape is polyhedral.

Figure 16. Nuclear polyhedrosis virus polyhedra. (A) Wet mount preparations viewed with phase microscopy showing refractile poly-hedra in two infected nuclei. (B) Transmission electron micrograph through a single polyhedron showing the enveloped rod-shaped viri-ons, characteristic of NPVs, occluded within the polyhPathogens of Insectsedral matrix. Upon ingestion, this matrix dissolves in the insect midgut, and the virions invade the host through midgut microvilli. Polyhedra are large (ca. 0.5 – 21|im) and form in the nuclei, where each occludes as many as several hundred virions. The NPVs of lepidopterous insects infect a range of host tissue, but those of other orders are rypically restricted to the midgut epithelium. Some NPVs have a very narrow host range and may replicate efficiently only in close related species, whereas others, such as the AcMNPV (i.e., the NPV of the alfalfa looper, Autographa California), have a relatively broad host range and are capable of infecting species in other genera. Granulosis Virus The GVs, of which over 100 isolates are known, ate closely related to the NPVs but differ from the latter in several important respects. The virions of GVs are similar to those of NPVs but contain only one nucleocapsid per envelope. GVs are known only from lepidopterous insects. Like NPVs, they initially replicate in the cell nucleus, but pathogenesis involves early lysis of the nucleus (as virions begin to assemble), which in the NPVs occurs only after most polyhedral have formed. After the nucleus has lysed, GV replication continues throughout the cell, which now consists of a mixture of cytoplasm and nucleoplasm. When completely assembled, the virions are occluded individually in small (200 nm X 600 nm) occlusion bodies referred as granules. Many GVs primarily infect the fat body, whereas others have a broader tissue tropism and replicate throughout the epidermis, tracheal matrix, and fat body. One, the GV of the grapeleaf skeletonizer Harrisina bril-lians, is unusual in that it replicates only in the midgut epithelium.

Use of Viruses as Insect Control Agents The best example of the use of a virus as an insect control agents is the use of the NPV of the European spruce sawfly, Gilpinia hercyniae, as a classical biological control agents. The European spruce sawfly was introduced into eastern Canada from northern Europe around the turn of the last century and had become a severe forest pest by the 1930s. Hymenopteran parasitoids were introduced from Europe in the mid-1930s as

part of a biological control effort, and inadvertently along with these came the NPV, which was first detected in 1936. Natural epizootics caused by the virus began in 1938, by which time the sawfly had spread over 31,000 km2. Most sawfly populations were reduced to below economic threshold levels by 1943 and remain under natural control today, the control being affected by a combination of the NPV, which s for more than 90% of the control, and the wasp parasitoids. Although viruses, particularly NPVs, are frequently associated with rapid declines in the populations of important lepidopterous and hymenopterous (sawfly) pests, G. hercyniae NPV is the only example of a virus that has proven effective as a classical biological control agent. Another rhinoceros, has been a quasi-classical biological control success in that once introduced into populations in certain South Pacific islands, can yield control for several years, but ultimately it dissipates and must be reapplied. Moreover, augmentative seasonal introductions have been effective because of their poor infectivity by feeding. Cytoplasmic polyhedro-sis viruses are not much better because, although highly infectious by feeding, the disease they cause is chronic. Vs have, however, been useful in some situations, such as for suppression of the pine caterpillar, Dendrolimus spectabilis in Japan. Ascoviruses and ento-mopoxviruses have not been developed as control agents for any insect owing to lack of efficacy. For several reasons, the viruses most commonly used or considered as microbial insecticides in industrialized as well as less developed countries are the NPVs. First, NPVs are common in and easily isolated from pest populations. In addition, production in their hosts is cheap and easy, and the technology for formulation and application is simple and adaptable to standard pesticide application methods. Most NPVs, however, are narrow in their host range, infecting only a few closely related species. Furthermore, although several can be grown in vitro in small to moderate volumes (ca. 20 to 300 – 1 cell cultures), no fermentation technology currently exists for their mass production on a scale that would permit repeated applications to hundreds of thousands of acres, which is possible with Bacillus thur-ingiensis (Bt) chemical insecticides. These two key limitations have been major disincentives for the commercial development of NPVs, especially in industrialized countries. Despite theses drawbacks, several NPVs have been ed as microbial insecticides even though the market size for most is small. And ed or not, several are used in many less developed countries, particularly for control of lepidopteran pests of field and vegetable crops. Moreover, over the past decade there has been renewed interest in developing NPVs because recombinant DNA technology offers potential for improving the efficacy of these viruses. In addition to the NPVs, a few GVs have also been successfully developed for pest control. These in the GVs of

the codling moth and potato tuberworm moth, which are, respectively, serious pests of apples and potatoes in many regions of the world. Other Uses In addition to the use of NPVs in insect control, one baculovirus, the AcMNPV noted earlier, has been developed as an expression vector for producing a large number of foreign proteins in vitro. This expression system takes advantage of the strong polyhedron promoter system, which in the wild-type viruses produces large amounts of the polyhedria used to occlude virions. By substituting foreign genes for the polyhedrin gene, it is possible to synthesize in insect cell cultures large quantities of foreign proteins, such as the capsid proteins of viruses that attack the vertebrates used for vaccine development and basic biochemical research.

b. Bacteria Bacteria ate relatively simple unicellular microorganisms that lack internal organelles such as a nucleus and mitochondria, and reproduce by binary fission. With a few exceptions, most of those that cause disease in insect grow readily on a wide variety of inexpensive substrates, a characteristics that greatly facilitates their mass production. A variety of bacteria are capable of causing diseases in insects, but those that have received the most study are spore-forming bacilli (family Bacillaceae), especially B. thuringiensis. Many subspecies of Bt are used as bacterial insecticides and as a source of genes for insecticidal proteins added to make transgenic plant resistant to insect attack, especially attack by caterpillars and beetles. The other bacterial insect pathogens that have received various degrees of study are B. sphaericus, Paenibacillus popilliae, and P. larvae, the latter being the etiological agent of foulbrood, an important disease of honey bee larvae, Serratia entomophila and S. marcescens. Several of these, in order of importance, are discussed here to present the diversity of bacteria that cause disease insects. Bacillus thuringiensis B. thuringiensis is a complex of bacterial subspecies that occur commonly in such habitats as soil, leaf litter, on the surfaces of leaves, in insect feces, and as a part of the flora in the midgets of many insect species. Bts are characterized by the production of a parasporal body during sporulation that contains one or more protein endotoxins in crystalline form (Fig 2). Many of these are highly insecticidal to certain insect species. These

endotoxins are actually protoxins activated by proteolytic cleavage in the insect midgut after ingestion. The activated toxins destroy midgut epithelial cells, killing sensitive insects within a day or two of ingestion. In insects species only moderately sensitive to the toxins, such as Spodoptera species (caterpillars commonly known as armyworms), the spore contributes to pathogenesis. Bt also produces other insecticidal compounds including (3exotoxin, zwittermicin A, and vegetative insecticidal proteins (Vips). The most widely used Bt is the HD1 isolate OF B. thuringiensis subsp. Kurstaki (Btk), an isolate that produces four major endo-toxin proteins packaged into the crystalline parasporal body (Fig. 2B). This isolate is the active ingredient in numerous commercially available bacterial insecticides used to control lepidopterous pests in field and vegetable crops, and in forests. Another successful Bt is the ONR60A isolate of B. thuringiensis subsp. Israelensis (Bti), which is highly toxic to the larvae of many mosquito and blackfly species. This isolate also produces a parasporal body that contains four major endotoxins (Fig. 2B), but these are different from those that occur in Btk. Several commercial products based on Bti are available and are used to control both nuisance and vector mosquitoes and blackflies. Bit has proven to be an important environmentally compatible insecticide, and now replaced the use of synthetic chemical insecticides for control of mosquito larvae in many countries around the world. A third isolate of Bt that has been developed commercially is the DSM2803 isolate of B. thuringiensis subsp. Morrisoni (pathovar tenebrionis). This isolate produces a cuboidal parasporal body toxic to many coleopterous insects and is used commercially to control several beetle pests. All the above-mentioned isolates are essentially used as bacterial insecticides, applied as needed. A variety of commercial formulations are available, including emulsifiable concentrates, wettable powders, and granules, for use against different pests in a variety of habitats. On a worldwide bases, millions of hectares are treated annually with products based on Bt. Estimates indicate the worldwide market is about $ 80-100 million. Although used as a bacterial insecticide, plants have been engineered to produce Bt Cry (crystal) proteins for resistance to insects, and this use has now far sured the use of Bt insecticides worldwide. Crops that produce Bt Cry proteins against lepidopterous and coleopterous pests, such as Bt cotton and Bt maize are now market value in many countries including the United States, China, India, and Argentina, where the total market value now is several billions of dollars per year . This technological development has proven of great environmental benefit in that is has reduced the use of synthetic chemical insecticides by millions of pounds per year. As the technology of Bt crops is improved and public acceptance expands, Bt crops will become commonly used in many other countries during this century.

Figure 17. Sprouting cell of Bacillus thuringiensis and insec-ticidal parasporal bodies. (A) Transmission electron micrograph through a cell of B. thuringiensis subsp. israelensis illustrating a developing spore (Sp) and endotoxincontaining parasporal body (PB) outside the exoporium membrane, E. Bar, 250 nm. (B) Scanning electron micrograph of parasporal bodies (crystal) of B. thuringiensis subsp. kurstaki, a subspecies used widely to control caterpillar pests. The bipyramidal crystals contain three endotoxins (Cry1Aa, Cry1Ab, and Cry1Ac), whereas the smaller cuboidal crystal contains a single endotoxin (Cry1Ac), and the cuboidal crystal has an additional toxin (Cry2A). This toxin complexity s for the broad spectrum of activity of many isolates of B. thuringiensis subsp. kurstaki. (C) Transmission electron micrograph of a parasporal body of B. thuringiensis subsp. israelensis used widely to control the larvae of mosquitoes and blackflies. This parasporal body is also composed of four major endotoxins, a large semispherical inclusion containing Cyt1Aa, a dense spherical body that apparently contains the Cry4Aa and Cry4Ba proteins, and a bar-shaped body that contains Cry11Aa. The endotoxin inclusion of this subspecies are held together by an envelope of unknown composisiton. This parasporal body has the highest specific toxicity of known Bt species, and this is due to synergistic interactions between the Cyt1Aa and Cry proteins as well as synergistic interactions among the Cry proteins. Bt endotoxins act by destroying the insect midgut epithelium (stomach).

Bacillus sphaericus Since the mid-1960s it has been known that many isolates of B. sphaericus (Bs) are toxic to certain mosquito species. Over the past three decades, three isolates have been evaluated for their mosquito control potential, 1593 from Indonesia, 2297 from Sri Lanka, and 2362 from Nigeria. The 1593 and 2297 isolates were obtained from soil and water samples at mosquito breeding sites, whereas 1593 was isolated from a dead adult blackfly. Like Bt, Bs acquires its toxicity as the result of protein endotox-ins that are produced during sporulation and assembled into par-asporal bodies. Bs is unusual in that the main toxin is a binary toxin (i.e., composed of two protein subunits). These are proteolytically activated in the mosquito midgut to release peptides having molecular masses of, respectively, 43 and 39 kDa, that associate to form the binary toxin, with the former protein constituting the binding domain, and the latter the toxin domain. The toxins bind to micro-villi of the midgut epithelium, causing hypertrophy and lysis of cells, destroying the midgut and killing the mosquito larava.

Paenibacillus popilliae P. popilliae is a highly fastidious bacterium that is the primary eti-ologial agent of the so-called milky diseases of scarab larvae. These insects are the immature stages of beetles, such as the Japanese beetle, Popillia japonica, that are important grass and plant pests belonging to the coleopteran family Scarabaeidae. The term “milky disease” is derived from the opaque white color that characterizes diseased larvae and results from the accumulation of sporulating bacteria in larval hemolymph (blood). The disease is initiated when grubs feeding on the roots and vegetative cells invade the midgut epithelium, where they grow and reproduce, changing in form as they progress toward invasion of the homocoel (body cavity). After ing through the basement membrane of the midgut, the bacteria colonize the blood over a period of several weeks and sporulate, reaching populations of 100,000,000 cells ml-1. For larvae that ingest a sufficient number of spores early in development, the disease is fatal. Dead larvae in essence become foci of spores that serve as a source of infection for up to 30 years. Despite decades of reaserch, suitable media for the growth and mass production of P. polliae in vitro have not been developed. Thus, the technical material (i.e., spores) used in commercial formulations is produced in living, field-collected scarab larvae. Nevertheless, a small but steady market remains for P. polliae in the United States because of serious problems due to scarab larvae, such as damage to turf grass by larvae of the Japanese beetle. Serratia entomophila A novel bacterium named. S. entomophila cause amber disease in the grass grub, Costelystra zealandica an important pest of pastures in New Zealand, and has been developed as a biological control agent for this pest. This bacterium adheres to the chitinous intima of the foregut, where it grows extensively, eventually causing the larvae to develop an amber color; the result of infection is death. The bacterium is easily grown and mass-produced in vitro and can now be grown to densities as hogh as 4 X 1010 cells ml_1. Successful mass production of S. entomophila led to its rapid commercialization. It is now used to treat infested pastures in New Zealand at a rate of one liter of product per hectare. Liquid formulations of this living, nonspore-forming bacterium are applied with subsurface application equipment. The rapid development and commercialization of the bacterium, even though the use is rather restricted, shows how microbials can be successful in niche markets, where there are few alternatives, and mass production methods, the most critical factor, are available.

c. FUNGI The fungi constitute a large and diverse group of eukaryotic organisms distinguished from others by the presence of a cell wall, as in plants, but lacking chloroplasts and thus the ability to carry out photosynthesis. Fungi live either as saprophyte or as parasites of plants and animals, and require organic food for growth, obtained by absorption from the substrates on which they live. The vegetative phase, known as a thallus, can be either unicellular, as in yeasts, or multicellular and filamentous, forming a mycelium, the latter being characteristic for most of the fungi that attack insects. During vegetative growth, the mycelium consists primarily of hyphae, which may be septate or nonseptate, and these grow throughout the substrate to acquire nutrients. Reproduction can be sexual or asexual, and during this phase the mycelium produces specialized structures such as motile spores, sporangia, and conidia, typically the agents by which fungi infect insects. Fungi usually grow best under wet or moist conditions, and those that are saprophyte as well as many of the parasitic species are easily cultured on artificial media. The fungi are divided into five major subdivisions, and these reflect the evolution of the biology of fungi from aquatic to terrestrial habitats. For example, species of the genera Coelomomyces and Lagenidium (subdivision Mastigomycotina) are aquatic and produce motile zoospores during reproduction, whereas of the genera Metarhizium and Beauveria (subdivision Deuteromycotina) are terrestrial and reproduce and disseminate via nonmotile conidia. Unlike most other pathogens, fungi usually infect insects by active penetration through the cuticle. The typical life cycle begins when a spore, either a motile spore or a conidium, lands on the cuticle of an insect. Soon after, under suitable conditions, the spore germinates, producing a germ tube that grows and penetrates down through the cuticle into the homocoel. Once in the hemolymph, the fungus colonizes the insect. Hyphal bodies bud off from the penetrate hyphae that grow throughout the insect body. Complete colonization of the body typically requires 7-10 days, after which the insect dies. Some fungi produce peptide toxins during vegetative growth, and in these strains death can occur within 48 h. Subsequently, if conditions are favorable, which generally means an ambient relative humidity of greater than 90% in all immediate vicinity of the dead insect, the mycelium will form reproductive structures and spores, thereby completing the life cycle. Depending on the type of fungus and species, these will be produced either internally or externally as motile spores, resistant spores, sporangia, or conidia. Fungi are one of the most common types of pathogens observed to cause disease in insects in the field. Moreover, outbreaks of fungal disease under favorable conditions often lead to spectacular epizootics that determine populations of specific insects over areas as large as several hundred square kilometers. As a result, there has been interest in using fungi to control insects for well over a century; the first efforts, in Russia in the late 1880s, used Metarhizium anisopliae to control the wheat cockchafer Anisoplia austriaca. Although there

have been numerous attempts since then to develop fungi as commercial microbial insecticides, very few of these efforts have met with success. Thus, at present barely a handful of commercially available fungal insecticides are available for use in industrialized countries, and true commercial success has remained elusive. On the other hand, in developing countries (e.g., Brazil and China), “cottage industry” technology that used to produce viruses has been turned to the production fungi such as M. anisopliae and Beauveria bassiana. A quasi-commerical product Boverin, developed and used in Russia for control of the Colorado potato beetle, proved ineffective in the United States. Current efforts to find alternatives to chemical insecticides have intensified research on fungi, with the aim of identifying new isolates or improving existing strains through molecular genetic manipulation. Over the past decade, several isolates of M. anisopliae, for example, have been used to control orthopteran (locust) pests in Africa and the Middle East, and the use of strains of this species to control the mosquito vectors of malaria are underway in Africa. Reserachers hope to obtain products that will prove more successful as either classical biological control agents or mycoincesticides. The subsections that follow summarize briefly the critical biological features of selected fungi to illustrate the advantages and disadvantages of these as control agents.

Aquatic Fungi Aquatic fungi of two types that attack mosquito larvae have received considerable study: species of Coelomomyces (class Chytridiomucetes: order Blastocladiales) and Lagenidium giganteum (class Oomycetes: order Lagenidium). The genuse Coelomomyces comprises over 80 species of obligately parasitic fungi that have a complex life cycle involving an illustration of sexual (gametophytic) and asexual (sporophytic) generations. The sexual phase parasitizes a microcrustacean host, typically a cope-pod, whereas the asexual generations develops, with rare exception, in mosquito larvae. In the life cycle, a biflagellate zygospore invades the homocoel of a mosquito larva, where it produces a sporophyte that colonizes the body and forms resistant sporangia. The larva dies and subsequently the sporangia undergo meiosis, producing uni-flagellate meiospores that invade the homocoel of a copepod host, where a gametophyte develops. At maturation, the gametophyte cleaves, forming thousands of uniflagellate gametes. Cleavage results in death of the copepod and escape of the gametes, which complete the life cycle by fusing the biflagellate zygospores, which then seek out another mosquito host. The life cycle of these fungi are highly adapted to those of their hosts. Moreover, as obligate parasites these fungi are very fastidious in their nutritional requirements, and as a result no species of Coelomomyces has been cultured in vitro. Coelomomyces, the largest genus of insect-parasitic fungi, has been reported worldwide from numerous mosquito species, many of which are

vectors of important disease such as malaria and filiaria-sis. In some of these species, Anopheles gambiae in Africa, for example, epizootics caused in some areas by Coelomomyces kills greater than 95% of the larval populations. Such epizootics led to efforts to develop several species as biological control agents. For several reasons, however, these efforts were discontinued. One important factor was the discovery that the life cycle requires a second host for completion. Also contributing were the inability to culture these fungi in vitro and the development of Bti as bacterial larvicide for mosquitoes. Although it is unlikely that Coelomomyces fungi will be developed as biological control agents, interest remain in developing L. gigantenum. This oomycete fungus is easily cultured on artificial media and does not require an alternate host. In the life cycle, a motile zoospore invades a mosquito larva through the cuticle. Once within the homocoel, the fungus colonizes the body over a period of 2-3 days, producing an extensive mycelium consisting largely of non-septate hyphae. Toward the end of growth, the hyphae become septate, and out of each segment an exit tube forms which grows back out through the cuticle and forms zoosporangia at the tip. Zoospores quickly differentiate in these, existing through an apical pore to seek out a new substrate. In addition to this asexual cycle, thick walled resistant sexual oospores can be formed in the mosquito cadaver.

Terrestrial Fungi The fungi that have received the most attention for use in biological control are terrestrial fungi, with most emphasis placed on the development of selected species of hyphomycetes such as M. anisopliae and B. bassiana for use as microbial insecticides. In addition, the more specific and nutritionally fastidious entomophthoraceous fungi continue to receive attention, but for their potential use as classical biological control agents rather than as microbial insecticides. Representative examples of these terrestrial fungi are discussed in the subsections that follow.

ENTOMOPHTHORALES These fungi comprise a large order of the class Zygomycetes that contains numerous genera, many species of which are commonly found parasitizing insects and other arthropods. The fungi routinely cause localized and sometimes widespread epizootics in populations of hemipterous and homopterous insects, particularly aphids and leafhoppers, but also in insects of other types such as grasshoppers, flies, beetle larvae, and caterpillars. In addition, a few species of the genus Conidiobolus are able to cause mycoses in some mammals, including humans. Apart from these few species, most of the entomophthoraceous fungi are highly specific, obligate parasites of insects

and therefore their use for biological control poses no threat to nontarget organisms. As with Coelomomyces, however, the complex nutritional requirements, which have thus far prevented mass production in vitro, and high degree of host specificity, make these fungi poor candidates for development as microbial insecticides. Moreover, the conidia are very fragile, providing a challenge to formulation, and the resistant spores, like the oospores of L. gigantenum, are difficult to germinate in a predictable manner. Nevertheless, there is evidence that if cultural practices in crop production are modified, these fungi can provide effective insect control where they occur naturally, and through introduction of foreign strains and species (i.e., a classical biological control approach). The most important genera found attacking insects insects in the field are Conidiobolus (aphids), Erynia (aphids), Entomophthora (aphids), Zoophthora (aphids, caterpillars, beetle) and Entomophaga (grasshoppers, caterpillars). Although many species of these genera cause epizootics and have received considerable study, none really seems to have much potential for development as a commercial microbial insecticides. On the other hand, cultural control, classical biological control, and environmental monitoring methods continue to show promise for using entomophthoraceous fungi for insect control. For example, the introduction of Erynia radicans from Israel into Australia to control the spotted alfalfa aphids, Therioaphis maculate, has proven a classical biological control access. A relatively recent example of apparent classical biological control can be found in the natural outbreaks of Entomophaga maimaiga in larval populations of the gypsy moth, Lymantria dispar, an important pest of deciduous forests throughout several state comprising the middle Atlantic and New England regions of the United States. Outbreaks of E. maimaiga have reduced larval populations to below economic thresholds, and the fungus is spreading westward naturally, and with human assistance, to gypsy moth populations established at that time. Then in the late 1980s, outbreaks of E. maimaiga began to occur in Connecticut and New York, and later in Viginia. In areas where it has established, given sufficient rainfall, the fungus seems to be capable of keeping the gypsy moth population below defoliation levels. It will require another 10 years of evaluation to determine whether this is a valid instances of classical biological control by a fungus.

CLASS HYPHOMYCETES The hyphomycetes fungi belong to the fungal subdivision Deuteromycotina (imperfect fungi), a grouping erected to accommodate fungi for which the sexual phase (perfect state) has been lost or remains unknown. This group contains the fungal species that most workers consider to have the best potential for development as microbial insecticides, B. bassiana and M. anisopliae, the agents of, respectively, the white and green mus-cardine

diseases of insects. Unlike the fungi already discussed, these two species have very broad host ranges and probably are capable of infecting insects of most orders. With respect to the general life cycle of these fungi, the process of invasion, colonization of the insect body, and formation of conidi-ophores and conidia is similar to that described for the other fungi. During invasion and colonization, some fungal species produce peptide toxins that quicken host death. The infectious agent is the conidium (Fig. 3), and the taxonomy for the hyphomycetes is based primarily on the morphology of the reproductive structures, particularly the conidiosphores and the conidia. Most of the hyphomycetes fungi used or under development grow well on a variety of artificial media, and this attribute, along with their ability to infect insects via the cuticle, favors commercial development. In the “cottage industry” commercial operations in Brazil, China, and the former Soviet Union, solid or semisolid substrates are used for production, and the primary ingredients are grain or grain hulls. In general, the development of B. bassiana and M. anisopliae is being targeted for control of insects that live in cooler and moist environments, such as beetle larvae in soil and planthoppers on rice, though the formers species is also being evaluated against whiteflies in glasshouses, as well as grasshoppers, especially locusts, in field crops. In addition to these species, several species with much narrower host ranges are considered to have potential for development, including Paecilomyces fumoso-rosea (for whiteflies), Verticillium lecanii (for aphids and whiteflies in glasshouses), Hirutella thompso-nii (for mites), and Nomurea rileyi (for noctuid caterpillars). With this apparent advantages, it is natural to ask why none of the hyphomycetes fungi have been commercially successful as micro-bial insecticides in developed countries. There are several reasons related to their biological properties. First and foremost is that the production of conidia or mycelia fragments that are used as the active ingredient of formulations is not cost-effective because too much material is required to allow the achievement of an acceptable level of control. In addition to the problem of inefficient yields, the formulations are bulky, and preservation of fungal viability beyond a few months is low because the conidia are fragile. In mosquito and blackfly control, similar constraints apply. In addition, the discovery of cost-effective strains B. thuringiensis and B. sphaericus has generally eliminated imperfect fungi, as well as many other microorganisms, for consideration as biological control agents for these important nuisance and vector insects. In developing countries, B. bassiana and M. anisopliae were used in the past in some crops with considerable success. For example, in China, B. bassiana was used to control the European corn borer and Ostrinia nubilalis in maize. The fungus was produced in large covered pits on maize stalks. In Brazil, a prepation of M. anisopliae known as Metaquino has been used for many years on sugarcane plantations and in pastures to control the spittlebug, Mahanarva posticata. Fungal conodia are produced in sealed plastic bags on rice. Figure indicate that as many as 50, 000 ha are treated annually, and reductions in

Figure 18. Typical reproductive structures of deuteromycete (imperfect) fungi. (A-D) Wet mount preparation of conidia-generating cells and conidia of Verticillium lecanii, which commonly attacks aphids and whiteflies. The conidia visible as free conidia and conidial clusters in (B) and (D) are the principal infective units. When these come in with an insect host, they germinate and penetrate the body, forming a mycelium that colonizes the insect over a period of several days. When conditions are appropriate, typically meaning high relative humidity, hyphae penetrate back out through the cuticle, producing conidiophores, the visible branched structures in these s (A-C), which form reproductive conidia at their tips. spittlebug populations are sufficient to keep populations below damaging levels. In the South Pacific, M. anisopliae has also been used to assist control of the rhinoceros beetle, Oryctes rhinoceros, a serious pest of coconut palms. Application of conidia at a rate of 50 gm~2 of soil yielded 80% larval mortality and improved coconut yields by 25%. Although these are examples of local successes, their applicability to agricultural production in developed countries is questionable. Moreover, the use of Bt crops such as Bt maize, noted above, has proven more effective than using fungi for control of important lepidopterous and coloepterous pests. Thus, in the future, genetic engineering techniques are likely to dominate crop development where insecticidal proteins are available to control the most important pests. In other situations, however, fungi such as M. anisopliae and B. bassiana may prove particularly useful. Examples include the control of sucking insect pests like aphids, whiteflies, and sharpshooters, or insects such as locust and adult mosquitoes, where other effective pathogens are not available. As with bacterial insecticides, these fungi are amenable to genetic engineering techniques. It has already been shown recently in the case of M. anisopliae that its insecticidal efficacy can be improved by adding insecticidal proteins and enzymes to the battery of virulence agents that it produces during the process of infecting insects.

d. PROTOZOA Protozoa is a general term applied to a large and diverse group of eukaryotic unicellular motile microorganisms that belong to what is now known as the kingdom Protista. of this kingdom can be free-living and saprophytic, commential, symbiotic, or parasitic. The cell contains a variety of organelles, but no cell wall, and cells vary greatly in size and shape among different species. Feeding is by ingestion or more typically by adsorption, and vegetative reproduction is by binary or multiple fission. Sexual reproduction, often useful for taxonomy, can be very complex, but asexual reproduction occurs as well. Many protozoa produce a resistant spore stage that is also

used in taxonomy. Divided into a series of phyla is based primarily on mode of locomotion and structure of locomotory organelles, the kingdom includes the Sarcomastigophora (flagellates and amoebae), Apicomplexa (sporozoa), Microspora (microsporidia), Acetospora (haplosporidia, now thought to be a type of parasitic alga), and Ciliophora (ciliates). Protozoa of some types, such as the free-living amoebae and ciliates, are easily cultured in vitro, whereas many of the obligate intracellular parasites have not yet been grown outside cells. As might be expected from such a large and diverse group of organisms, many species of protozoans are associated with insects, and the biology of these associations covers the gamut from being symbiotic to parasitic. Those that are parasitic have the general feature of causing diseases that are chronic. Many of the parasitic types especially the microsporidia, build up slowly in insect populations, eventually causing epizootics that lead to rapid declines in populations of specific species. These epizootics attracted interest in the possibility of using protozoa to control pest insects, and over the past several decades numerous studies have been aimed at evaluating this potential. In general, these studies have shown that protozoa hold little potential for use as fast-acting microbial insecticides because of the chronic nature of the diseases they cause and because commercially suitable methods for mass production are lacking. However, as with the entomophthoraceous fungi, the possibility exists that protozoans, particularly microsporidia, amy be useful as classical biological control agents. Clear examples of the effectiveness of such strategies remain to be demonstrated. The life cycles and biologies that occur among the various protozoa that attack insects are too diverse in relation to their pest control potential for even a few to be covered here. Instead, the group with the most potential – the microsporidia – is described in of general biology and possible use in insect control.

e. General Biology of Microsporidia The microsporidia (phylum Microspora) are the most common and best studied of the protozoans that cause important diseases of insects. Although still referred to as protozoa, recent molecular phylogeny-tic studies have shown that these pathogens evolved from fungi. Well over a 1000 species are known, and most of these have been described from insects. Microsporidia have been most commonly described from insects of the orders Coleoptera, Lepidoptera, Diptera, and Orthoptera, but they are also known from other orders and probably occur in all. The epizootics in insect populations caused by protozoa are usually due to microsporidia. All microsporidia are obligate intracellular parasites and are unusual in that they lack mitochondria. In addition, they produce spores that are distinguished from the spores of organisms of all other known types by the presence of a polar filament (Fig 4),

along coiled tube inside the spore used to infect hosts with the sporoplasm. The typical microsporidian life cycle begin s with the ingestion of the spore by a susceptible insect. Once inside the midgut, the polar filament everts, rapidly injecting the sporoplasm into host tissue. Figure 19. Representative microsporidian spore: transmission electron micrograph through a uninucleate spore of Amblyospora abserrati from a larva of the mosquito Ochlerotatus abserratus. The circular structures on each side of the spore are cross sections through the polar filament that is used to inject the contents of the spore into the mosquito body after ingestion and activation of the spore.

The sporoplasm is unicellular but may be uni- or binucleate. Upon entry into the cytoplasm of a host cell (e.g., the fat body in many species of insects), the sporoplasm forms a plasmodium (meront), which undergoes numerous cycles of vegetative growth (merogony). During these cycles, the cells multiply extensively, dividing by binary or multiple fission and spreading to other cells, and, in many species, to other tissues of the host. After several merogonic cycles, the microsporidian undergoes sporulation. This consists of two major phases, sporogony – a terminal reproductive division committed to sporulation – and spore morphogenesis. In the sexual phase of reproduction, meiosis occurs early during sporogony. The spores, which in general measure several micrometers in diameter and length, have a thick wall and are higly, refractile when viewed by phase microscopy. The disease often lasts for several weeks during which billions of spores may accumulate in the tissues of a single infected host. Microsporidian systematics is based on the size and structure of the spores, life cycles, and host associations. In addition to transmission by ingestion, many microsporidia are transmittedvertically from adult females to larvae via the egg (transovarially). With respect to host range, some species are specific, whereas others occur in many species of the same family or order, and some can be transmitted to insects of different orders.

Microsporidia as Biologucal Control Agents Naturally occurring epizootics caused by microsporidia are periodically very effective in significantly reducing insect pest populations. The problem is that these epizootics cannot be predicted with any degree of accuracy, nor can they be relied upon for adequate control, even though many of the conditions that facilitate their occurrence are known. The epizootics caused by Nosema pyrausta in populations of the European corn borer are often a classic example of this unreliability. These epizootics are useful when they occur, but because this often happens too late to prevent economic damage; reliance on

N. pyrausta alone is insufficient. Thus, efforts have been directed toward developing methods for amplifying spore loads in the field through inundative releases, in essence using microsporidia as a microbial insecticides. Because they are obligate intracellular parasites that lack mitochondria, microsporidia cannot be grown on artificial media. Several species have been grown, however, in established insect cell lines, although this is not practical for field use. For field application, whether for microbial insecticides trials or for introductions into population, spore are grown in living hosts. With such methods the yield can be quite high (109-1010 spores per host). These yields in of the number of larvae that must be grown to treat a hectare and infect most of the target population are comparable to the requirements for nuclear polyhedrosis viruses. Thus, if the microsporidia could cause acute diseases, they would be on an equal footing with many of the NPVs. However, the diseases are chronic, and even if a high percentage of the target pest population is infected, there all too often is little, if any, crop protection. In fact, if advanced instar such as thirds and fourths are treated, the larvae may live longer and cause greater crop damage than if the fields were left untreated. Thus, microsporidia are not useful as microbial insecticides. There is now a general realization for use as microbial insecticides. They may, however, be useful as population management tools.

B. Antagonist of Plant Pathogens Antagonism – The Mechanism of Biocontrol Biological control is principally achieved through antagonism (the inhibitory relationships between microorganisms including plants) which involves: (i) amensalism i.e. antibiosis and lysis, (ii) competition, and (iii) parasitism and predation. Amensalism (Antibiosis and Lysis) Amensalism is a phenomenon where one population adversely affects the growth of another population whilst itself being unaffected by the other population. Generally amensalism is accomplished by secretion of inhibitory substances. Antibiosis is a situation where the metabolites secreted by organism A inhibit organism B, but organism A is not affected. It may be lethal also. Metabolites penetrate the cell wall and inhibit its activity by chemical toxicity. Generally antimicrobial metabolites are produced by underground parts of plants, soil microorganisms, plant residues, etc. Fig 13.1 shows in vitro interaction of colony of Colletotrichum gloeosporioides (a fungal pathogen associated with fruit rot of guava) and Fusarium oxysporum (a saprophyte) and formation of inhibition zone between the colonies.

Substances noxious to certain soil-borne plant pathogens are secreted by roots of maize, clover, lentil (glycine, phenylalanin) and other legumes, flax (hydrocyanin acid), pine (volatile mono-and sesquiterpenes) and by other plant roots. Other plant residues are the source of phenolic and non-volatile compounds. Similarly, antimicrobial substances (antibiotics) produced by microorganisms (soil bacteria, actinomycetes, fungi) are aldehydes, alcohols, acetone, organic acid, nonvolatile and volatile compounds which are toxic to microbes. Changes in microbial structures (cell wall, hyphae, conidia, etc.), may occur when microorganisms lack resistance against the attack by deleterious agents or unfavorable nutritional conditions. A chemical substance (i.e. melanin) is present in their cell walls to resist the lysis. Moreover, cell wall constituents, for example, xylan or xylose containing hetero polysaccharides, may also protect fungal cells from lysis. The potent antagonists e.g. Trichoderma harzianum and T. viride are known to secrete cell wall lysing enzymes, 6-1, 3-glucanase (Chet and Henis, 1975) chitinase, and glucanase (Chester and Bull, 1963). However, production of chitinase and 6-1, 3-glucanase by T. harzianum inside the attacked sclerotia of Sclerotium rolfsii has also been reported by Elad et al. (1984). Siderophores. Siderophores are the other extracellular metabolites which are secreted by bacteria {e.g. Aerobacter aerogenes, Arthrobacter pascens, Bacillus polymyxa, Pseudomonas cepacia, P. aeruginosa, P. fluorescens, Serratia, etc.), actinomycetes (e.g. Streptomyces spp.) yeast (e.g. Rhodotorula spp.) fungi (Penicillum spp.) and dinoflagellates (Prorocentrum minimum) Siderophores are commonly known as microbial iron chelating compounds because they have a very high chelating affinity for Fe 3+ they transport it into the cells. Kloepper et.al (1980) were the first to demonstrate the importance of siderophore production by PGPR in enhancement of plant growth. Siderophores after chelating Fe3+ make the soil Fe3+ deficient for other microorganisms. Consequently growth of other microorganisms is inhibited. When the siderophore producing PGPR is present in rhizosphere, it supplies iron to planys. Therefore, plant growth is stimulated. In recent years, role of siderophores in biocontrol of soil-borne plant pathogens is of much interest. Microbiologist have developed the methods for introduction of siderophore producing bacteria in soil through seed, soil or roots. Fig. 19.1 Colony interaction between Fusarium oxysporum (FO) and) Colletotrichum gloesporioides (CG), and formation of inhibition zone (Courtesy: Dr. R.R. Pandey, Manipur University, Imphal). Gupta, Sharma, Dubey and Maheshwari (1999) have isolated a strain of P. aeruginosa (GRC) from potato rhizosphere which was found to secrete hydroxamate type of siderophore after 48 h of incubation. It is also secreted hydrocyanic acid and indole acetic acid. Moreover, this strain also displayed

antagonistic properties against two plant pathogens, Macrophomina phaseolina and Fusarium oxysporum. Fig. 13.2 shows the inhibition in colony of M. phaseolina (MP) by a fluorescent pseudomonas (FP). Competition Among microorganisms competition exists for nutrients, including oxygen and space but not for water potential, temperature or Ph. Amensalism involves the combined action of certain chemicals such as toxins, antibiotics and lytic enzymes. Success in competition for substrate by any particular fungal species is determined by competitive saprophytic ability (Garrett, 1950) and inoculums potential (Garrett, 1956) of that species. Competitive saprophytic ability is “the summation of the physiological characteristics that make for success in competitive colonization of dead organic substrates” (Garrett, 1956). Garrett (1950) has suggested four characteristics which are likely to contribute to the competitive saprophytic ability (i) rapid germination of fungal propagules and fast growth of young hyphae towards a source of soluble nutrients, (ii) appropriate enzyme equipment for degradation of carbon constituents of plant tissues (iii) excretion of fungistatic and bacteriostatic growth products including antibiotics, and (iv) tolerance of fungistatic substances produced by competitive microorganisms. Inoculum potential is defined under section Inoculum. Possession of any of the four characteristics and inoculums potential‟ is sufficient for a microbe to get success in microbial competition. Biological control of Forties annosus by inoculating the freshy cut stumps of pine with Peniophora gigantea is a result of competition, as is the control of Pseudomonas tolaasii on mushroom by other bacteria (Baker and Cook, 1974). The fate of plant pathogens in competition for food and soace depends on other factors also such as, cellulolysis rate that mediates the speed of saprophytic tissue penetration (Garrett, 1975). Species of Trichoderma and Gliocladium are the two potent antagonists which produce antibiotics to destroy mycelia of other fungi. Some examples of successful competitors competing for nutrients with other microorganism are: bacteria vs. S. scabies (for oxygen), soil amoebae vs. Gaeumannomyces graminis var. tritici (wheat roots), T. viride vs. Fusarium roseum (wheat straw), Chaetomium sp. Vs Cochliobolus sativus (wheat straw), Arthrobacter globiformis vs Fusarium oxysporum f. lini (glucose and nitrate). Fig.19.3. Post-interaction events during mycoparasitism. A, coiling (a, antagonist; h, host hypha); B, penetration; C, barrier formation (by) by host; D, branch formation and sporulation (s) by antagonist; E, chlamydospore (c) formation; F, lysis of host hypha (diagrammatic, after Dubey and Dwived, 1986).

Predation and Parasitism Predation is an apparent mode of antagonism where a living microorganism is mechanically attacked by the other with the consequences of death of the farmer. It is often violent and destructive relationship. Parasitism is a phenomenon where one organism consumes another organism, often in a subtle, non-debilitating relationship. These aspects are dealt with the example of fungi, nematodes and amoebae (Table 13.3). Mycoparasitism When one fungus is parasitized by another one the phenomenon is called as mycoparasitism. The parasitizing fungus is called hyperparasite and the parasitized fungus as hypoparasite (Fig. 13.3). Mycoparasitism commonly occurs in nature. As a result of inter-fungus interaction i.e. fungus-fungus interaction, several events take place which lead to predation viz., coiling, penetration, branching, sporulation, resting body production, barrier formation and lysis (Fig 13.3). In coiling (A) an antagonist, the hyperpasite (a) recognizes its host hyphae i.e. the hypoparasite (h) among the microbes and comes in and coils around the host hyphae. The phenomenon of recognition of a suitable host by the antagonists has been discovered in recent years. Manocha (1985) has given a molecular basis of host specificity and host-recognition by mycoparasites. Cell wall surface of host and non-host contains D-galactose and N-acetyl D-galactosamine residues as lectin binding sites. With the help of lectins present on the cell wall, an antagonist recognizes the suitable sites (residues of lectins) and binds the host hypha. As a result of coiling, the host hypha loses the strength. If the antagonist has capability to secret cell wall, degrading enzymes, it can penetrate the cell wall of host hyphae and enter in lumen of the cells. The event of entering in lumen of host cell is known as penetration (Fig. 13.3 B). Several cell wall degrading enzymes such as cellulose β -1, 3-glucanase, chitinase, etc. have been reported (Elad et al, 1982). Sometimes host develops a resistant barrier (Fig. 13.3B) to prevent the penetration inside the cell. Cytoplasm accumulates to form a spherical, irregular or elongated structure, so that the hypha of antagonist could not towards the adjacent cells of the hypha (Fig 13.2C). Depending upon nutrition, the antagonist forms branches and sporulates (s) inside the host hypha (Fig. 13.2D). Until the host‟s nutrients deplete, the antagonist produces resting bodies, the survival structures, for example, chlamydospores (c) inside the host hypha (Fig.13.2E). Finally post-infection events lead to lysis of the host hypha (Fig.13.2F) due to loss of nutrients and vigor for survival. Example of parasitism and post-infection events are given in Table 13.2 and Fig.13.3 (Dubey and Dwived, 1986).

Examples of predation and parasitism Mode of Plant pathogens Antagonists Organism (hosts) Mycoparasitism Rotrytis alii Gliocladium roseum Cocchliobolus Myrothecium sativus verrucaria and Epicoccum purpurascens Rhizoctonia solani Trichoderma and Fomes viride annosus Sclerotium rolfsii T. harzianum Nematophagy:

Post-infection Penetration of hyphae Antibiosis and penetration

Coiling, cytoplasm coagulation coiling, penetration and lysis Penetration of cysts and egg killing Perforation in conidia

Heterodera Phialospora rostochiensis heteroderae Mycophagy: Cocchliobolus Soil amoebae sativus Gaeumannomyces Soil amoebae Penetration and lysis graminis var. tritici of hypae Source: Various mycological/ microbiological research and review papers. Nematophagy This is the phenomenon of eating upon nematodes by fungi. However, several nematode eating i.e. nematophagous fungi (NF) are known which develop different kinds of trap (T), arrest the pathogenic nematodes (N) and finally kill them (Fig 13.4). Morphological and biochemical aspects of trap formation is discussed by Cook (1977). Examples of nematode trapping fungi are Arthrofyotrys, Dactylaria Dactyleela, etc. Phialospora heteroderae penetrates the cysts and kills the eggs of Heterodera rostochiensis. Besides the fungi eating on nematodes, a spore forming bacterium, Bacillus penetrans kills the nematode and, therefore, is used for the control of Meloidogyne sp. B. penetrans is resistant to nematicides. Being an obligate parasite, this bacterium can not be grown in axenic culture. The bacterium shows host specificity and its spores survive for a long time. These spores adhere to the surface of infectious second-stage female larvae and eat on it. Adherance is followed by infection but it is not apparent until the adult stage comes (Kerr, 1982). Fig. 19.4. Nematophagy : N, a nematode; NTF, a nematode trapping fungus; T, a ring like trap formed by NTF (diagrammatic).

Mycophagy Mycophagy is the phenomenon of feeding on fungi by amoebae. In recent years, mycophagy has become a new field of research as far as biocontrol of soil-borne plant pathogens is concerned. Many soil amoebae are known to feed on pathogenic fungi. For example, take-all disease of wheat caused by Gaeumannomyces graminis var. triticiwas very severe in Australia. Finally it was found that some natural soil exhibited suppressiveness against this disease. Microbiological analysis of soils showed the presence of several amoebae in the soil. This amoebae played a significant role in take-all decline of wheat. The antagonistic soil amoebae e.g. Arachnula, Archelle, Gephyramoeba, Geococcus, Saccamoeba, Vampyrella, etc. (Old, 1977; Chakraborty et al, 1983) make perforation on the hyphal wall of Cochliobolus sativus, graminis var. tritici, Fusarium oxysporum and Phytophthora cinnamomi (Chakraborty and Warcup, 1982; Dwivedi, 1986) and on the conidal wall of C. sativus (Chakraborty and Old, 1983) and F. oxysporum (Pussard et.al. 1979), and develop round cysts on the lysed hyphae (Dwivedi, 1986). Fig. 19.5. Mycophagy (A-C). Feeding of amoebae (a) upon fungal conidia (Ic) ad fungal hypha (II h). A, attachment of amoebae; B, engulfment (I) and hole formation (II); C, digestion of conidium (I) (diagrammatic). Chakraborty et. Al. (1983) have described the following three majors steps of feeding on propagules by soil amoebae (Fig. 13.5A-C). (i) Attachment: Attachment of trophozoites of amoebae (a) to fungal propagules i.e. (c) or hyphae (h) appears to be a matter of chance. It takes place be chemothigmotaxis (Fig. 13.5A). (ii) Engulfment: Fungal propagules (e.g. spores, conidia, fragments of hyphae) engulfed by amoebae (b). The small trophozoites attached the hyphal wall or spore and perforations on it. (iii) Digestion: The completely or partially engulfed propagules/cytoplasm of the host are digested in a large central vacuole formed inside the cysts (Fig. 13.5 C). Based on pot-bioassay study, Dwivedi (1986) has suggested the biological control of wheat by using soil amoebae as a biocontrol agent.

C. Biological Control Agents of Weeds

Biological control of weeds is the deliberate use of natural enemies to reduce the density of a particular weed to a tolerable level. The objective of biological weed control is not eradication but simply the reduction of the weed population to an economically low level. In fact for biological control to be continuously successful, small numbers of the weed host must always be present to assured the survival of the natural enemy. The two most frequently cited examples of successful biological weed control are the destruction of the prickly pear cacti (Opuntia; spp.) in Australia by an imported moth (Cactoblastis cactorum) and the control of St. Johnswort Hypericum perforatum (millepertius perfore) on rangeland in California, Oregon, Washington, and British Columbia. These examples demonstrate that biological control can provide a permanent solution to serious weed problems. The first insects were introduced 50 years ago and the Australian prickly pear is still under control. St. Johnswort is presently controlled at two per cent of its former density in British Columbia. Weeds are plants growing where they are not “wanted” and most of our serious weeds have been introduced from other parts of the world. Of the 107 noxious Canadian weeds, 78 have been introduced from Europe or Asia. One of the reasons why these plants are so noxious in their new habitat is that natural enemies are often absent. Biological control has most frequently been applied against these alien weeds and attempts are made to restore the natural control of these weed pests by introducing one or more host-specific, damaging natural enemies from the native region of the weed. Methods Used in Biocontrol of Weeds The classical approach to biological control of weeds involves the introduction of host-specific natural enemies of alien weeds. Recently the approaches utilizes in biocontrol programs have been expanded to include two other methods: 1. Augmentation of natural enemy populations; 2. Application of “biological herbicides” Augmentation includes the periodic release and/or distribution of natural enemies. Work is presently being conducted in the United States to determine the effect of mass rearing of a native moth Bactra verutana) in the laboratory and releasing it in fields of yellow nutsedge Cyperus esculentus) (souchet comestible).

The application of spore suspensions of plant pathogens as „biological herbicides‟ is discussed in more detail later. Biological Control Agents Insects have been most frequently used as biological control agents of weeds and this will likely continue. The reasons are that there have been major successes using phytophagous insects and almost all of the research has demonstrated the potential of other organisms, including plant pathogens, nematodes, and fish. Procedures in Classical Biological Control The first step in biological weed control program is to determine the suitability of the weed for this approach. Not all weeds are suitable and those with the following characteristics are generally least suited for biological control: 1. Weed species which are valued in other situations are not good candidates for this approach, for example, blue weed Echlum Vulgate) (viperine) is a serious pasture weed, but it is also a desirable honey plant. As the biocontrol agent cannot be limited in area, like chemical or mechanical treatments, „weed‟ that are of values in other situations are not suitable targets for biocontrol. 2. Weeds that are closely related to economic crops are not good candidates for this method. The closer the relationship the less possibility there is that a biotic agent could distinguish between the weed and the crop. For example, no insects have been found that attack wild oats (Avena fatua) (folle avoine) and do not attack cultivated oats and our other cereals. 3. Native weed species are not generally amenable to this approach. However, the native range of an introduced alien weed provides a source from which a parasite can be introduced. 4. Weeds of cropland under intensive cultivation are generally not suited to this approach. Since the biological control agent is specific to only one weed species, little would be gained if one weed, such as lambsquarters, was controlled in the corn crop as numerous other weed species would soon occupy the available space. However, biocontrol is particularly suited to rangeland situations where a single, dominate weed species is troublesome. 5. Minor weed problems are not generally suited to this approach. The target weed should infest large areas. Since there are only four scientists in Canada

working more or less full time in biological weed control, minor weeds usually cannot be considered. 6. If eradication of the weed is desired (e.g., poisonous weeds), the method is generally not applicable. Therefore, in classical biocontrol of weeds, the ideal target weed is an aggressive. Introduced weed which infest areas of marginal land such as rangeland, pastures, and waste areas. The second step in the program is to conduct a survey overseas to determine if there are any parasites available for introduction against the weed. A survey is also conducted in Canada to ensure that a prospective biotic agent is not already present in Canada to determine what parts of the target weed are not attacked in Canadian populations. For example Canada thistle Cirsium arvense; (chardon des champs) is attacked by a number of seed feeders in Canada and, therefore, it would be of little value to introduce other organisms that attack the seed heads. Surveys are being conducted in Europe on the following weeds: English name Bindweed Absinthe Narrow leaved hawk‟s beard White cockle Bladder campion European buckthorn

French name Liseron Armoire Crepis des toits Lychnide blanche Silene enflee Nerpun commun

Scientific name Convolvvulus spp Artemisia absinthium Crepis tectorum Lynchis alba Silence cucubalus Rhamnus cathartica

The third step is to determine the potential effectiveness of the parasite in Canada in an attempt to eliminate ineffective agents before importation and screening tests in Canada. Insects that have been collected in certain parts of Europe may not survive under Canadian conditions. The next and perhaps most important phase of the program is to determine the safety of the selected parasite for release in Canada. Very extensive tests are conducted in the quarantine facilities at the Agriculture. Canada Research Station at Regina, Saskatchewan. These studies involve thorough investigation of the agent‟s biology and its host range to demonstrate that the introduced agent is host-specific and will not become a pest of an economic crop. Some insects that are presently being screened at the Regina laboratory include:

Liothrips spp. a sucking insect which attacks ragweed (Ambrosia artemisiifolia) (petite herbe a poux). Tephritis dilacerate – a seed-head fly attacking perennial sowthisle (Sonchus arvensis) (laiteron des champs). Argyroploce striana – a root moth attacking dandelion (Taraxacum officinale) (pissenlit). If it found during these tests that the candidate biological control agent is not host-specific, the organism is destroyed and the project is terminated. After the screening tests are complete, a report is prepared for t approval by the Canadian and U.S governments. A report on the nematode, Paranguina picridis, which forms galls on Russian knapweed [Acroptilon (Centaurea) repens] (centauree de Russie), was recently submitted and permission for limited field trails has been granted. Once the agent has been determine safe and permission to release it has been given, the fifth step in the program is to establish the biocontrol agent in Canada on infestations of the target weed. This part of the program requires the co-operation of farmers. Infestations of the target weed need to be located and the release site must be maintained for some time with minimal disturbance. This stage is the most critical because they were not adequately cared for during the initial phases of establishment in their new environment. Attempts are being made to establish insect agents on the following weeds in Canada: Nodding thistle Carduus nutans

(chardon

penche) Rhinocyllus beetle)

conicus

(seed-head

Ceutorhynchidis horridis (root weevil) Canada thistle (chardon des champs) Urophora cardui (stem gall fly) (Cirsium arvense) Bull thistle (chardon vulgaire) Russian thistle (soude (Salsola pesttifer)

Urophora stylata (seed-head fly) roulante) Coloephora parthenica (stem mining moth)

Diffuse and Spotted Knapweed Urophora affinis and U.quadrifasciata (centauree diffuse et centauree (seed-head flies) maculae) (Centaurea diffusa, C. Maculosa)

Once the insect agent is established, the step is to determine if the agent is increasing and its effect on the weed. The beetle on nodding thistle and the seed-head flies on the knapweed are well established and are rapidly increasing. Two major projects can be considered complete in Canada. As mentioned earlier the population of St. Johnswort in British Columbia has been reduced to two per cent of its former density by two leaf-feeding Chrysolina beetles. Tansy ragwort Senecio jacobaea) senecon jacobee) has been successfully controlled in Nova Scotia by the defoliating cinnabar moth 9Tyria jacobaeae). From the above examples it can be seen that biological weed control programs have been primarily concerned with introduced insects as biotic agents and the approach has been directed against aggressive, alien weeds in rangeland situations. However, recent research results with other types of biocontrol agents, particularly plant pathogens, has extended the application of biological control to aquatic weeds and those of cultivated land. Plant Pathogens as Biocontrol of Weeds Plants pathogens offer two advantages over insects as biocontrol agents of weeds: 1) they are often more host specific, and 2) they can be applied with conventional spray equipment at a time when the weed is at its most susceptible stage. A. Conventional approach As discussed above, biological control weeds has conventionally been applied against alien weeds by introducing one or more of their natural enemies. Control of a weed with a plant pathogen used in this manner was achieved against skeleton weed (Chondrilla juncea), the major weed of wheat in Australia. A host-specific rust, Puccinia chondrillina, was collected in Italy and introduced into Australia in 1971. The rust spread very rapidly and at present controls the weed over most of its range. Control has been so spectacular that herbicides are no longer being used to control this weed in Australia. Numerous introduced weeds in Canada are known to be attacked by plant pathogens in their native ranges. However, research in this area has not yet been established in Canada.

B. Biological Herbicides A new approach to biocontrol of weeds has recently been pioneered in Arkansa with the application of a spore suspension of an endemic fungus. The fungus controlled 99 per cent of a serious weed (Northern tvetch) in rice, without damage its host because it normally attacked too late in the season. However, when the fungus was applied early (in the seedling stage), it destroyed the weed. The fungus is now being tested on a field scale and there appears to be no biological or technological limitations that would prevent commercialization of the fungus as a „mycoherbicides‟. Possible targets for this approach in Canada are numerous as practically all Canadian weeds have plant pathogens recorded on them. For example, wild oats (Avena fatua) (folle avoine) and other weeds of our cultivated land can be controlled to some extent by cultural and mechanical means. However, they still remain as serious contaminants of our crops. Wild oats is attacked by numerous plant pathogens which normally attack late in the season and do not appreciably damage the weed. If host-specific pathogens of wild oats were applied early in the season, some damage of control could possibly be achieved. An attractive feature of this method is that it does not involve the introduction of any new organism into our environment but merely the use of host-specific pathogens at a time when they are most effective. This method may also prove to be valuable for integrated weed control systems. For example, most weeds are controlled in corn with standard herbicide application, but a few weeds such as quackgrass (Agropyron repens) (chiendent) and yellow nutsedge (Cyperus esculentus) (souchet comestible) are more difficult to control. The application of host-specific pathogens could be integrated into the present weed control system to control these types of weeds. Aquatic Weed Control Biocontrol of water weeds is being investigated in Florida and other parts of the U.S. The possibilities of using insects, plant pathogens, and a fish, the white amur (Ctenopharynogondon idella) are being studied. Water weeds are also a serious problem in many areas of Canada and the developments in biocontrol are being carefully observed. The disadvantages of using herbicides in water systems are numerous and alternative methods should receive high priority. This is considerable controversy concerning the use of the fish for biological control. Research is

continuing and further studies should demonstrate that the advantages will by far outweigh any possible disadvantages. Biological Weed Control Program at Macdonald College The research in biological weed control at Macdonald College is composed of two segments: 1. The release of screened biotic agents on infestations of weeds in Quebec; 2. Investigation of the potential of naïve natural enemies of certain weeds in Quebec. A. Release of Biotic Agents Insects that have been screened and are available for release are shipped to Macdonald College from the Regina laboratory. These insects are then taken to desirable field release sites and liberated on the target weed. This program has involved the co-operation of numerous Quebec farmers and its continued success depends on their . Last year, insects were released on the following weeds in Quebec. Canada thistle (chardon des champs) Urophora cardui (stem gall fly) (Cirsium arvense) Bull thistle (chardon vulgaire) (Circium Urophora stylata (seed-head fly) vulgare) Leafy spurge (euphorbe esule) Hyles euphobiae (defoliating moth) (Euphorbia esula) Additional releases of these insects will be made this year and releases of Chrysolina beetles on St. Johnswort, Tyria. Jacobeae (cinnabar moth) on tansy ragwort and Uronhora spp on spotted knapweed infestations in Quebec are planned for this year. Urophora cardui has previously been released at St.Hyacinthe and Rhynocyllus conicus has previously been established on nodding thistle in the Lac St. Jean region. B. Native Natural Enemies This program involves surveying of weed populations, collection of natural enemies (plant pathogens and insects) and determination of the potential of selected organisms as biocontrol agents. At present, two postgraduate students, Jean-Guy Champagne and Harry Hartmann, are investigating the host-parasite relationships of Canada thistle and its rust pathogen and ragweed and its natural enemies. In addition, endemic plant

pathogens of other weeds, such as quackgrass and yellow nutsedge, are also being collected and studied. Summary Biological weed control has recently received renewed interest because it is an environmentally compatible method of weed control without residue and pollution problems. However, biological weed control has its weaknesses and is not suitable for all weed problems. Indeed, only a few of our enemy noxious weeds have been investigated for biocontrol and so far only two are sufficiently advanced to be considered complete successes. Undoubtedly, with continued interest and research , the general assumption that biological control is used only as a last resort where chemical and other methods have failed will be proven incorrect.

Chapter 4

ASSESSMENT, MASS PRODUCTION AND FIELD RELEASES

A. Assessment of Effectiveness of a Natural Enemy Assessment of effectiveness in augmentative biological control programs is probably as important as releasing the natural enemies. The tools in assessing effectiveness of natural enemies include: 1. Comprehensive life table studies which show the extent of indispensible mortality attributable to a specific natural enemy, and can be used to measure the expected impact on the target pest. 2. An understanding of expected yield gains attributable to natural enemies is also a useful measure that may be used in deciding whether to employ augmentative biological control. This approach will also allow the development of a meaningful measure of effectiveness, viz. to what extent a natural enemy reduces dependence on chemical or other pest management options.

B. Nutrition and Mass Production of Biological Control Agents

1. Nutrition of Natural Enemies The nutrition of entomophagous arthropods was originally discussed in detain bu Doutt (1964) and Hagen (1964). Slansky (1982, 1986) and Thompson & Hagen (1999) illustrates the complex interactions of behavioral, physiological and nutritional factors in arthropod nutrition. Nutrition is thus the action or processes of transforming substances found in foods into body materials and energy to do all the things attributed to life. Nutritional requirements are dependent on the synthetic abilities of the organism, which is controlled genetically. House (1977) stated that “… through nutrition we have a direct and essential connection between an environmental facto, foodstuff and the vital processes of the insect organism. “Most nutrition research with insects has been aimed at improving rearing and not developing a basic understanding of their nutrition. Research has emphasized feeding and the development of artificial diets, which are concerned with dietetics (Beck 1972). Although critical to insect rearing, such research has given only a little understanding of insect nutrition per se.

Qualitative nutritional requirements of all insects are very similar in spite of a great diversity of feeding habits (Beck 1972, Dadd 1973, Hagen 1986b). Although knowledge of dietetics and nutrition das advanced, practical application of principles to insect rearing to biological control is lacking. Rearing each insect species is a unique challenge as there is meager knowledge of nutrition principals that might provide a broad and sound basis for approaching insect species husbandry (house 1777). With entomophaga, foodstuff is in a continuous state of qualitative and quantitative change, and very little is known of the quantitative nutritional requirements for various life stages and physiological functions of these insects. The requirements for many nutrients are often dependent on the presence and concentration of others and correct nutrient balance may be critical for successful nutrition. Those parasitoids and predators for which artificial diets have been may developed may serve as models for in vitro investigation on quantitative requirements for specific nutrients. Thompson (1976, 1082) used a defined artificial medium to examine the quantitative requirements for ing larval growth of Exerister roborator. Parasitoid development is intimately associated with host physiology. Changes in the host‟s physiology following parasitism are adaptive for the parasitoid, which insures successful development (Vinson & Iwansch 1980, Thompson 1986). Changes in composition of the host‟s internal milieu may have significant nutritional consequences for a parasitoid 9Grenier 1986, Thompson 1989). Endocrine interactions seem critical to successful parasitoid development. Synchrony in development between many larval indoparasitoids and their host occurs (Beckage 1985), and this suggests that the host‟s hormones and endocrine physiology influence parasitoid development (Lawrence 1986a). The physiological basis of developmental synchrony is not well understood and knowledge is restricted to investigation of the relationship of Biosteres longicaudatus with its host Anastrepha suspensa (Lawrence 1982, 1986b). Some studies have tested the effects of hormones on the development of parasitoids in vitro with little success. The potential of using insect hormone supplements in artificial media to achieve successful growth and development of parasitoids in vitro deserves research emphasis.

The importance of ecological considerations in the nutrition of insects was discussed by Slansky (1982). It was emphasized that behavior and regulatory physiology of insects are in a state of continuous flux in response to food supply, and that nutrition can be fully understood only by considering the insects “nutritional ecology.” With entomophaga both the ecology of the entomophage as well as that of the host or prey needs to be known.

Dietary and nutritional requirements are genetically based and genetic manipulation holds promise as a way to modify the nutrition of entomophages. Chabora (1970) suggested that nutritional content varies between strains on insects when he demonstrated that yields of two parasitoids, Nasoniau vitripennis (Walker) and M. raptor Girault & Sanders were significantly increased when they were reared on a hybrid of two strains of the host, Musca domestica L. The selection of desired traits for insect rearing was discussed by Collins (1984). The potential for genetic improvement of entomophages was outlined by Rousch (1979) and Hoy (1979, 1986). Most genetic selection has been directed to increase field effectiveness of entomophages, such as improving sex ratio, host finding ability, host preference, pesticides bresistance and improved climatic tolerance. However, genetic improvement must also guarantee the preservation of vigor and vitality of the entomophage. Because these are intimately associated with nutrition, genetic programs may involve selection for nutritionally related traits. Advances in recombinant DNA technology indicate a possibility for genetic manipulation of the nutrition of entomophages (Thompson 1989). The incorporation of foreign or in vitro altered genes for the expression of desirable traits by an organism, is rapidly advancing (Beckendorf & Hoy 1985), but is still not suited for practical application as of 1991. History of Parasitoid Nutrition – Salt (1941) probably was the first to emphasize the complexity of parasitoid nutrition in studies that demonstrated that the host influences growth and survival of the developing parasitoid as well as sex ratio, fecundity, longevity and vigor of the adult wasp (Clausen 1939, Salt (1941). Such complexities were demonstrated in work by Arthur & Wylie (1959), Wylie (1967), Nozato (1969) Sandlan (1979a) and others (Vinson & Iwantsch 1980). It has long been known that there is a relationship between host biomass and size of solitary parasotoids, larger parasitoids developing from larger hosts. This relationship exists for parasitoids which attack every host developmental stage, but applies more generally to parasitoids of host eggs and pupae where host size is fixed (Sandlan 1982).The relationship applies when a parasitoid is reared on different host species of variable size as well as when reared on different sized individuals of a single host species (Salt 1940, Jowyk & Smilowitz 1978, Mellini & Campadelli 1981, Sandlan 1982, Mellini & Beccari 1984). It does not seem to hold with ectophagous parasitoids, however (Legner 1969). The size of adult Trichogramma pretiosum Riley reared on the eggs of five hosts showed a direct correlation between parasitoid size and the volume of the host egg from which it emerged (Bai et al. 1989). A correlation also exists between total parasitoid biomass and/ or numbers with host size in the case of gregarious larval parasitoids (Wylie 1965, Bouletreau 1971, Thurston & Fox 1972). The

means by which gregarious organisms moderate their development relative to host size has been shown (Beckage & Riddiford 1983). The relationship between size oh host and parasitoid is closely associated with food quality and quantity (Arthur & Wylie 1959, Sandlan 1982). Salt (1940) found that adult Trichogramma avanescens Westwood display behavioral dimorphism related to host size. Large females obtained from large hosts failed to oviposit on small hosts, whereas small females accepted hosts of all sizes. Male wing development was influence by host size, and this was also found in Geliscorruptor (Foerster) by Salt (1952). Adult female Coccygomimus (= Pimpla) turionellae L. did not show morphological and behavioral polymorphism, but larger females found it difficult to oviposit in small host. On the other hand small females were more efficient in attacking small hosts. Fecundity was influenced by longevity with the greatest longevity reported for longer individuals reared from large hosts (Sandlan 1982). The success of parasitoids in parasitizing activity is directly related to nutritional factors. Smith (1957) found differences in larval mortality and adult size, sex ratio and reproductive rate of several species when reared on Aonidiellaaurantii (Maskell) and Comperiella bifasciata Howard maintained on different food plants. Habrolepis rouxi Compere displayed limited mortality on A. aurantii when feeding on Acyrthosiphon pisum (Harris) and Rhopalosiphum maidis (Fitch) than on Aphis fabae Scopoli. Coccinellaseptempunctata L. gained more weight when feeding on Lipaphis erysimi (Kaltenbach) than on two other aphid species, and it was demonstrated that L. erysimi had higher protein levels (Atwal & Sethi 1963). Utilization of Food Parasitoids have been thought to show high efficiencies in food utilization. Larvae consume food of high nutritional content and are mostly inactive within the host which offers a limited food supply, which points to selection for high food efficiency (Fisher 1971, 1981; Slansky & Scriber 1985, Wiegert & Petersen 1983). Parasitoids examined for food utilization include Coccygomimum (= Pimpla) instigator (F.), Pteromalus puparum (L.) (Chlodny 1968), Gelis macrurus (Thompson), Hidryta frater (Cresson) (= sordidus) (Edgar 1971), Brachymeria intermedia (Nees) and C. turionellae (Greenblatt et. al. 1982) Diadromusu pulchellus Wesmael (Rojas-Rousse & Kalmes 1978) and Trypatgilum (=Trypoxylon) politum (Say) (Cross et al 1978), Phanerotoma flavitestacea Fischer (Hawlitzky & Mainguet 1976), Ventura (=Nemeritis) canescens (Gravenhorst) (Fisher 1968), Cidaphus alarius glomerata (L.) (Slansky 1978). In these species, the mean net conversion efficiency, (= proportion of assimilated food converted to body mass (Petrusewics 1967, Calow 1977, Hagen et al. 1984) varied broadly (11-62%), with a mean of 37%

that was ≤ than for many groups of insect herbivores and detritivores. Cameron & Redfern (1974) of two studied parasiotids, Eurytoma tibialis Boheman and Habrocytus elevates (Walker), were at the high end of this range. Net conversion efficiencies may not be very high because selection might have been for rapid rather than efficient growth (Slansky 1986). Possibly the well known inverse relationship between growth efficiency and assimilation (Welch 1968) may also be important. In contrast to net conversion efficiency, the above parasitoids had relatively high percentages of assimilation (= percentage of ingested food that is assimilated) ranging from 55-94%, with mean of 67%, compared with means of 40-50% for most herbivores and detritivores. Howell & Fisher (1977) reported the highest nutritional efficiencies for a parasitoid in the ichneumonid V. canescens. Larvae had 65% net conversion efficiently and 95% assimilation when maintained on the host Anagasta (=Ephestia) (Zeller); net conversion efficiency to the adult was 20%. The proportion of food/host available that is consumed by the parasitoid and converted to parasitoid biomass was calculated by Slansky (1986) and Howell & Fisher (1977). Calculated exploitation indices varied among species from 3-80%, and V. canescens larvae consumed 90% of its host‟s biomass and converted 55%, but there was no clear correlation between host size and parasitoid size nor biomass conversion. Food utilization by predators has also been thought to be highly efficient, for reasons similar to that for parasitoids. This is especially true when predators spend much time waiting for their food (Lawton 1971), thus avoiding metabolic expenditure. Studies on food utilization of 11 predators was reviewed by Slansky & Scriber (1985). All had similar net conversion efficiencies (4-64%, mean = 34%), but higher assimilation efficiencies (3798%, mean = 86%) than those of parasitoids. Cohen (1984, 1989) in studies of food utilization by Geocoris punctipes (Say) when reared from 1st instar nymphs to adults on eggs of Heliothis virescens (F.), found an assimilation efficiency of ca. 95%, gross conversion efficiency of 53% and net conversion efficiency of 55%. Nutritional Requirements in Development Qualitative nutritional requirements of insects, determined by use of defined and deficient artificial diets, were presented by several authors (Dadd 1973, 1977, 1985; Friend & Dadd 1982, Hagen et. al. 1984). All insects have similar requirements for ca. 30 chemicals that include protein and/ or 10 essential amino acids 9arginine, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), the B-vitamin complex

(biotin, folic acid, nicotinic acid, panthothenic acid, pyridoxine, riboflavin and thiamin), as well as other water soluble growth factors, including choline and inositol, some fat soluble vitamins , cholesterol or a structurally similar phytosterol, a polyunsaturated fatty acid, minerals and an energy source usually provided by simple or complex carbohydrates and/ or lipids. Nutritional requirements of entomophagous insects are similar, and similar to those of nonentomophagous species. House (1977) referred to this common characteristics of insect nutrition as the “rule of sameness” (House 1966a, 1974). The rule has been confirmed by recent studies with parasitic and predaceous insects. In assessing the need for nutrients, it is important to consider that rearing a single generation on a synthetic or semi-synthetic diet did most studies. Some investigations overlooked the potential contribution of nutrients stored within the egg. Stored nutrients may limited development and, in the case of trace nutrients, supply a sufficient quantity to ensure development of at least one generation. Studies with Itoplectis conquisitor (Say) (Yazgan 1972) and Exeristes roborator (F.) (Thompson 1981a) demonstrated partial larval development on diets lacking various essential amino acids and B-complex vitamins. Other studies have demonstrated that entomophagous insects have no unusual qualitative nutritional requirements. A requirement for asparagine by Eucelatoria bryani Sabrosky (Nettles 1986a) and the absence of a requirement for a polyunsaturated fatty acid by A. housei (House & Barlow 1960) were consistent with finding for nonparasitic Diptera (Dadd 1977). The quantitative balance of different nutrients is a critical and dominating factor determining dietary acceptability and suitability (House 1969, 1974). The predominant foods of both parasitic and predaceous insects are of animal origin and, thus, are generally high in protein content and low in carbohydrate and fat (House 1977). Thompson (1986a) found a high requirement for protein and/ or amino acids in parasitoids. Exeristes roborator at the 6% amino acid level completed larval development without glucose and/or fatty acids (Thompson 1976a). However, glucose markedly improved survival when the amino acid level was reduced to 3% and at 1% amino acid, no development occurred with the carbohydrate. Similar effects of amino cid level on larval development were reported by Yazgan (1972) for I. conquisitor. Adult eclosion was reduced by dietary amino acid levels of < 6% and by deletion of glucose. Fatty acids were only marginally beneficial in enhancing growth and development rates of both species. A polyunsaturated fatty acid, however, was required in small amounts. Adult I. conquisitor (Yazgan 1972) and E. roborator (Thompson 1981a) displayed crumpled wings and/or bent ovipositors without a polyunsaturated fatty acid in the larval diet. Linolenic acid alleviated these deformities in I. conquisitor, and linoleic and linolenic acids were provided together in the case of E. roborator.

Thompson (1983a) described the effect of nutritional balance on larval growth of Brachymeria lasus (Walker). Media containing 0-10% glucose with 2% amino acids, and 1-8% amino acids with or without 2% glucose were tested. All media contained 15% albumin and 2.5% lipids. Weight gain increased on diets containing 2% glucose when the amino acid level was increased from 14%, but was reduced at the higher amino acid levels. Similar effects of varying the amino acid level were obtained with diets lacking glucose, but the overall weight gain was less than observed with the diets containing glucose. On diets containing 2% amino acids, weight gain increased dramatically when glucose was increased from 0.5-4%, but decreased at higher glucose levels. Growth rates on the above diets were generally in the range of 15-200 mg/g/day. The maximal rate, 260 mg/g/day, was obtained on a medium containing 2% glucose and 2% amino acids. The effects of nutrient balance were closely related to the osmolality of the artificial medium (Thompson 1983b). House (1966b) demonstrated similar quantitative requirements to those of hymenopterous parasitoid in the dipteran Agria housie Shewell. Maximal growth and survival were achieved when all nutrients were increased proportionately over the levels in a basal diet that contained 2.25% amino acids, 0.05% salts, 1.16% lipids and 2.25% other ingredients, including glucose, ribonucleic acid, vitamins and agar. When amino acid level alone was increased, survival was reduced. On a diet containing nutrient levels equivalent to pork liver (= 20% amino acids, 4% glucose, 3.5% lipids, 2% salts and 0.75% ribonucleic acid), survival was >80%. House (1967, 1970) showed that the relative balance of amino acids and glucose was critical in determining growth or development and that A. housie larvae selected diets for feeding on the basis of nutrient balance. Quantitative nutritional studies with parasitoids have generally evaluated the effects of nutritional balance by univariate or monofactorial analysis. Grenier et al. (1986) thought that such an approach had servere limitations because it ignored potential interactions between nutrients, including “…additivity, competitivity, antagonism or synergy.” Thus, interpretation of effects of nutrient variation aimed at medium optimization was difficult, and it was suggested that nutritional studies be designed and analyzed in a multidimensional manner that ed for interactions between all nutrients and biological criteria. Canonical correlation analysis, which constructs maximum correlation between all linear combinations of variables within sets, such as between growth and development, and dietary parameters, were reported with Lixophagadiatraeae (Townsend) by Bonnot (1986, 1988). Because biologically meaningless correlations may be generated, accurate interpretation requires knowledge of biological correspondence between variables. Bonnot varied the

concentrations of 30 medium components and determined the effects on 9 developmental criteria. Nine linear correlations were obtained and three had correlation coefficients of >0.95. There is little information about the effects of developmental nutrition on the behavior of parasitoid larvae apart from measurements related to growth and development rate. However, Veerman et al (1985) reported that a photoperiodic response by C. glomerata was influenced by the carotenoid content of its host‟s diet. Vitamin A was essential for photoperiodic induction of diapause and it was suggested that this vitamin or a derivative may function as a photoreceptor pigment. Optimal nutritional balance can be influenced by environmental factors, as was shown by House (1966b) with A. housie that the effects of dietary glucose level on larval survival and development could be modulated by temperature. The nutritive value of a basal medium 9House 1966a) was increased by increasing the temperature from 20 to 25 and 30 ᵒ C at glucose levels between 0-1.5%. At higher glucose levels larval survival and development were reduced with increasing temperature. Two media of different composition were formulated whose superiority for larval growth and development of A. housie was reversed at two different temperatures (15 & 30ᵒ C (House (1972). Such nutritional effects might have ecological significance in affecting insect host range (House 1966b). It was thought that in establishing host range, an insect might be affected differently if the nutrient composition of its food were uniform but the temperature varied within the range rather than if the temperature were uniform but the composition of food was variable. On the other hand, the insect might not be affected if variation in food composition was accompanied by compensatory changes in temperature. Therefore, an insect species that attacks a particular foodstuff in a region with a specific temperature might, if introduced into another area with a different temperature, adapt to a different food source whose nutrient composition is favored at the new temperature. Non-nutritional factors are intimately and intrinsically involved in food acceptance and ingestion. These include physical properties such as form, texture, etc., but also non-nutritive chemicals that elicit specific behavioral and/or physiological responses essential for finding and accepting foodstuff and in some cases for initiating behaviors associated with the feeding process itself (Bernays & Simpson 1982), Bernays 1985). Although such factors have been best shown on phytophagous insects, they also play a role in the biology of entomophaga and will likely be of importance in the development of continuous in vitro culture.

Predator Culture In Vitro The artificial rearing of predators has stressed maintenance of the adult stage for maximizing egg production rather than complete in vitro culture. Predator larvae are the preferred biological control agent, and eggs and larvae produced by adults are placed directly in the field. However, some effort has been aimed at complete artificial culture of predators. Among the first reared artificially from egg to adult was the coccinellid Coleomegilla maculata maculata (DeGeer) by Szumkowski (1952). Adults fed on raw liver or meat being kept for months on these food in the absence of prey. However, survival of larvae was poor on meat products alone and only 38% reached the adult stage. Supplementing vitamins resulted in ca. 86% of the larvae reaching adults. Oviposition and egg viability were increased by addition of vitamin E to the adult diet. The culture methods were refined and a diet of fresh yeast and glucose ed larval development (Szumkowski 1961a, b). Smith (1965, 1966) reared several coccinellid species including C. maculata lengi on dried aphids supplemented with pollen. Success also was achieved on a diet of 40% brewer‟s yeast, 55% sucrose, inorganic salts, cholesterol, RNA, wheat germ oil and vitamins. Adults were fed the same diet supplemented with powdered liver. Attallah & Newsom (1966) reared 8 generations of this coccinellid on a defined diet of casein, sucrose, wheat germ, soybean hydrolysate, glycogen, butter fat, corn oil, a liver factor dextrose, cotton leaf extract (with carotenoids and steroids), brewer‟s yeast, ascorbate, inorganic salts, vitamins and agar. Adults reared in vitro were fecund and mating was stimulated by addition of vitamin E to the diet. The medium failed to growth of Coccinella novemnotata Herbst, Cycloneda spp., Hippodamia convergens Guérin and Olla v-nigrum (=abdominalis) (Mulsant). The last species was cultured in vitro by Bashir (1973). Optimum egg production required inclusion of vitamin E in the larval diet, which was in contrast to the results of Szumkowski (1952) where supplementation of the adult diet alone was insufficient for maximum egg production. Several coccinellid species were reported to be successfully cultured in vitro by Smirnoff (1958). These included Psyllobora (=Thea) virgintiduopunctata (L.), C. septempunctata, Oenopia (=Harmonia) doublieri (Mulsant), O. (=Harmonia) conglobata (L.), Rhizobius lophantae (Blaisedell), R. litura (F.), Rodolia cardinalis (Mulsant), Exochomusanchorifer Allard, E. quadripustulatus (L.) E. nigromaculatus Erhorn, Scymnus suturalis Thunberg, S. pallidivestis Mulsant, S. kiesenwetteri Mulsant, Stethorus punctillum Weise, Chilocorus bipustulatus (L.), Clitostethus arcuatus Rossi, Pharoscymnus numidicus Pie, P. ovoideus Sicard and Mycetaea tafilaletica Smirnoff (Endomychidae). The diet contained sucrose, honey, alfalfa flour, yeast, royal jelly and agar supplemented with dried pulverized prey. Larval rearing in a few species was improved by adding beef jelly. All species developed more rapidly

and lived longer on the artificial diet compared with insects reared under natural conditions, and the adults were very healthy. Harmonia axyridis (Pallas), C. septempunctata and Chilocorus kuwanae Silvestri were reared on Smirnoff‟s (1958) diet and other artificial media by Tanaka & Maeta (1965). Successful culture of all species was obtained but adults failed to lay eggs. Chumakova (1962) reared Crytolaemus montrouzieri Mulsant on similar crude diets supplemented with dried prey. Okada et al (1971a, 1972) and Matsuka et al. (1972) successfully reared H. axyridis on diets containing powdered larvae and pupae of drone honeybees (Aphis mellifera L.). Sixteen generations of H. axyridis and three generations of Menochilus sexmaculatus (F.) were cultured in vitro. Okada & Matsuka (1973) and Matsuka et al. (1982) later improved the rearing method for maintaining adult Rodolia cardinalis. Chilocorus rubidus Hope, Scymnus hilaris Motschulsky, S. otohime Kamiya, Vibidia duodecimguttata Poda and S. hilaris Motschulsky, C. septempunctata, Coccinula crotchi (Lewis), Eocaria muiri, H. axyridis, Harmonia yedoensis Takizawa, H. convergens, Hippodamia tredecimpunctata L., Lemnia beplagiata (Swartz), M. sexmaculatus, Propylea japonica, S. hilaris and S. otohime, Variable results were obtained, but 11, 16 and 25 successive generations of E. muiri, H. axyridis and M. sexmaculatus respectively were cultured from the egg to adult stage. Larval development, adult longevity and fecundity were satisfactory. The fractionation of honeybee powder was described by Matsuka & Okada (1975) who found that the active factor stimulating predator growth was unstable but nonproteinaceous. Expanded attempts to analyze bee powder was described by Niijima et al. (1977). Niijimi et al. (1986) then formulated several chemically defined diets for rearing H. axyridis. Larvae developed from the 1-3rd instar on a diet containing 18 amino acids, sucrose, cholesterol, 10 vitamins and 6 minerals. Kariluoto et al. (1976) described rearing of A. bipunctata. About 60 variations of seven artificial diets were tested. These contained varying amounts of wheat germ, brewer‟s yeast, casein, cotton-leaf extract, egg yolk, sucrose, liver fractions, honey, glycogen, soybean hydrolysate, butter fat, corn oil, amino acids, dextrose, ascorbate, choline, inorganic salts, vitamin E and antibiotics. The best diets yielded 60-80 % of larvae that became adults, but development time was slowed and adult weight lowered. Kariluoto (1978) modified the medium, and Kariluoto (1980) obtained fecund adult of A. bipunctata, C. septempunctata and others reared in vitro. In vitro culture attempts with Chrysopa species did not succeed until Hagen & Tassan (1965) got a complete culture of Chrysoperla carnea (Stephens) on an encapsulated liquid medium (in paraffin droplets). The diet

consisted of enzymatic yeast, protein hydrolysate, ascorbate, fructose, choline and casein hydrolysate. Adults were fecund but development time from the egg stage was ca. 2X that of insects reared on aphids. Vanderzant (1969) then successful cultured C. carnea for 7 generations on pieces of cellulose sponge soaked in enzymatic casein and soy hydrolsates, fructose, inorganic salt, lecithin, cholesterol, choline, ascorbate, vitamins and inositol. Development on this diet was slow, but 50-60% of larvae reached the adult stage compared with 85% when reared on natural insect eggs. Hassan & Hagen (1978) reported obtaining three generations of C. carnea on an artificial diet of honey, yeast flakes, sucrose, casein, yeast enzymatic hydrolysates and egg yolk. Developmental time and pupal weights were similar to those of insects on eggs of Sitotroga cerealella (Olivier). Chrysoperla sinica (Tjeder) was cultured for 10 generations on a diet of egg, brewer‟s yeast, sucrose, honey and ascorbate (Ye et al. 1979). Adults were fed powdered liver, honey and brewer‟s yeast. Cai et al (1983) reared this species on an encapsulated medium of soybean and beef hydrolysates, egg yolk, sucrose, honey, brewer‟s yeast, ascorbate and linoleic acid, with similar success reported by Zhou & Zhang (1983). The hemipteran predator, Geocoris punctipes may be reared on several diets (Dunbar & Bacon 1972). Media were nevertheless supplemented with insects. Cohen (1981) reported in vitro culture of G. punctipes from 1st stage nymph to adult on encapsulated semidefined diets. Six media containing casein hydrolysates, yeast, sucrose, cholesterol, corn oil, lecithin, agar, inorganic salts, phenylalanine and a vitamin mixture were formulated and encapsulated in different forms. The latter included mixtures of polybutene 32, dental impression wax, Vaseline, epoline C-16, candelilla wax, Sunoco, and Paraplast. Best results were with vitamin-enriched medium encapsulated in a mixture of 5% polybutene 32 and 95% dental impression wax. Development of G. punctipes in vitro was better than when reared on Spodoptera exigua (Hubner). The percent of nymphs that reached adults and survival of the in vitro reared predators were significantly greater on the artificial diet. Cohen (1983) then described modifications of media content, preparation and encapsulation and could rear two generations of G. punctipes, Geocoris pallens Stal, H. convergens, H. axyridis and Nabis spp. also successfully fed on the encapsulated medium. In all cases superior results were obtained on medium encapsulated with 30% polybutene 32 and 70% dental wax. A diet composed of equal parts of fresh ground beef and beef liver supplemented with sucrose for continuous rearing of G. punctipes was produced (Cohen 1985). The ingredients were blended into a paste and small aliquots wrapped in stretched Parafilm presented to developing nymphs for feeding. Twelve generations were successfully cultured, and artificially reared predators displayed greater fecundity and adult weight than individuals reared on insect

eggs and coddled larvae (Cohen & Urias 1986). Nevertheless, development was slower on the artificial diet. Parasitoid Cultures In Vitro In vitro culture offers a simple alternative for mass culture (Mellini 1978, Greany et al. 1984) and also enable dietary and nutritional mainioulations for fundamental studies of nutrition and biochemistry. Some benefits of in vitro culture were given by Greany et al. (1984). However, the physiological and metabolic adaptations exhibited by insect parasitoids in relation to their parasitic way of life are of critical importance for successful in vitro culture (Mellini 1975a, Thompson 1981a, Grenier et al. 1986, Campadelli & Dindo 1987). Parasitoid/host relationships are often incorrectly thought to lack the complex physiological interactions typical of the host associations of other Metazoa (Thompson 1985, 1986a, Dindo 1987). The immature stages of many parasitoids are truly parasitic and such parasitoid/host relationships are characterized by extensive physiological and biochemical interaction (Beckage 1985, Thompson 1985, 1986a; Lawrence 1986a). Such interactions are often intimately associated with nutrition and successful development of the parasitoid in the host (Beckage & Riddiford 1983, Thompson 1983a, 1986a). The potential importance of the host endocrine system and of hormonal interaction in in vitro culture was discussed by Mellini (1975b, 1978, 1983) and Grenier et al. hymenopterous larval endoparasitoids. The extent that parasitoid/host physiological interactions need to be considered in the successful development of in vitro culture must still be determined but will undoubtedly vary with the parasitoid species. Diptera. - - A variety of natural foodstuffs, including fish and liver products, were utilized in early rearing attempts with parasitoids. House & Traer (1948) reared the sarcophagid A. housei for many generations on a diet of salmon and liver. Contrasted to 38% pupation among larvae reared on the host, Choritoneura fumiferana (Clemens), 88% pupated when reared on the artificial medium. A related species, Sarcophaga aldrici Parker was reared on the same medium and on liver alone (Arthur & Coppel 1953) and subsequently Coppel et al. (1958) maintained A. housei in the laboratory on fresh pork liver. About 1,000 A. housei larvae were reared on ½ lb. of sliced liver and were not affected by putrefaction of the tissue. Smith (1958) maintained Kellymyia kellyi (Aldrich) for 40 generations on pork liver and was also able to rare larvae on a mixture of powdered milk, powdered egg and brewer‟s yeast moistened with water to form a thick paste. House (1954) developed the first chemically defined medium for rearing a parasitoid, using A. housei. The diet contained 19 amino acids, ribonucleic acid, dextrose, inorganic salts (U.S.P. XII), B vitamins, choline and inositol. It

was prepared aseptically and gelled with agar. About 84% of the larvae reached the 3rd instar, 60% of those pupated and 32% of the pupae emerged as adults. The medium was later refined and many of the developmental nutritional requirements of A. housei were determined (House 1977). Vitamin E was necessary for reproduction (House 1966c). Other dipterous parasitoids have been more difficult to culture outside the host. Many of these species have specialized physiological adaptations associated with parasitism that are lacking in sarcophagids. Tachinids, for example, have relatively high respiratory rates (Ziser & Nettles 1979, Bonnot et al. 1984) and during or immediately following the first stadium form a direct connection to the host‟s tracheal system (Kellen 1944, Fisher 1971). First instar larvae of the parasitoid E. bryani attach to the host‟s tracheal system 12 hrs after hatching, and respiratory considerations were critical for the development of in vitro cultures (Nettles et al. 1980). During initial studies, first instar larvae dissected from the host were placed directly in a liquid artificial diet. They were then transferred to diets gelled with agar, thereby exposing larvae directly to atmospheric oxygen. Improvements in the methods allowed development without transfer. Powdered artificial diet containing 1.5% agar was preconditioned by maintaining it at 5% RH for 24 hrs. The diet was then poured into petri dishes and held at 90% RH. Young larvae dissected from the host 18-24 hrs after larviposition fed on the liquid diet covering the surface of the gelled medium, and this was consistent with the normal feeding habit of first instar larvae that feed on and develop in the host‟s hemolymph. As the liquid was slowly absorbed by the agar gel, the surface of the gelled medium dried and larvae were exposed to the atmosphere. The artificial medium for rearing E. bryani was composed of mixtures of organic acids, amino acids, nucleic acid bases, B and fat soluble vitamins, phospholipids and derivatives as well as ATP, lactalbumin hyydrolysate, bactopeptone, yeastolate, albumin, cholesterol, triolein, glucose and trehalose. When thus reared, larvae developed at an equivalent rate as when reared on the host, H. virescens, and 13% developed into adults with a sex ratio of ca. 66% females. Adults were fecund but produced fewer progeny than host reared insects. The medium was later refined and simplified and some of the basic developmental nutritional requirements of E. bryani were determined (Nettles 1986a). The nutritive values adding albumin or soy flower to the medium was tested, which greatly increased adult yields and fecundity (Nettles 1986b). Other tachinid parasitoids have been successfully reared on artificial media. Larval development of Phryxecaudata Rondani to the 3rd instar was obtained with a liquid artificial diet (Grenier et al. 1974). However, in contrast to the results of Nettles et al. (1980) with E. bryani, development of P. caudata was not improved by rearing larvae on gelled diets (Grenier et al. 1975). It was suggested that this may have resulted from the slower development rate and

respiratory requirements of the latter when reared in vitro (Nettles et al. 1980). Bonnot (1986) discussed the importance of respiratory requirements in the in vitro culture of P. caudate. The first tachinid that was successfully cultured in vitro on artificial media from the first instar larvae to the adult was Lixophaga diatraeae (Townsend) (Grenier et al. 1978). This medium contained organic acids, amino acids, B and fat soluble vitamins, gelatin, enzymatic hydrolysates of casein, soy protein, lactalbumin, ovalbumin, ATP, cholesterol, lecithin and gelled with agarose. Adults were fecund and their progeny developed normally on Galleria mellonella. One critical factor for successful development of both P. caudate and L. diatraeae was osmolality, which could not exceed 450 mOs/Kg (Grenier et al. 1986). Grenier (1979) investigated the embryonic development of P. caudate and L. diatraeae on artificial media. Newly fertilized eggs were removed from adult females and placed on an agarose-gelled medium similar to that for the larvae. Larval yield was equal to that observed in vivo and was mush greater than when reared on a liquid diet. Again, respiratory requirements seemed critical for success. Hymenoptera. - - Simmonds (1944) made the first attempt to rear hymenopterous parasitoids in vitro. There species of ichneumonid ectoparasitoids were maintained as larvae for extended periods on raw beef and gelatin. Although some growth was observed, none could complete their development. Bronskill & House (1957) did succeed in rearing C. turionellae on a slurry of pork liver in 0.8% saline. An autoclaved homogenate of the liver was dispensed into sterile test tubes and surface sterilized eggs were dissected from host pupae and transferred to this medium. Mature larvae were placed in gelatin capsules for pupation and 7% of the eggs developed to adults. When reared naturally on G. mellonella, 50% parasitoid adults were obtained. Culture of ichneumonid I. conquisitor. The diet was a mixture of amino acids, fatty acids, fat soluble vitamins B vitamins and lipogenic growth factors, and glucose, NA and gelled with agar. It was ground into a viscous slurry. Parasitoid eggs dissected from the host were placed directly on this medium, and development from egg to fecund adult was obtained with a development time twice that observed on the natural host, G. mellonella. Exeristes roborator was reared on a diet with a similar nutrient composition (Thompson 1975), but unlike I. conquisitor, larvae of this parasitoid would not tolerate direct with gelled media. Direct exposure to atmospheric oxygen was important for successful in vitro culture of E. roborator and success was achieved by retaining suspensions of the liquid diet in lipipholic Sephadex LH-20 gel filtration medium. Mortality, size and development time of the parasitoid reared in vitro were similar to those of individuals reared on Pectinophora gossypiella (Saunders). Many of the developmental nutritional

requirements of I. conquisitor and E. roborator were determined by Yazgan (1972) and Thompson (1976a, b). Thompson (1989, 1981d) described the various chemically defined diets for rearing various chalcids of the genus Brachymeria. Complete development of B. lasus from egg to adult at rates approximating those observed in G. mellonella were obtained on diets containing heat-denatured albumin, amino acids, glucose, B vitamins, inorganic salts, lipogenic growth factors and Intralipid. The latter, a phospholipid emulsion of soybean oil, was necessary for complete development. Larvae were reared from eggs dissected from host pupae immediately following oviposition and parasitoids were cultured individually in the wells of micro tissue culture plates. Development of larvae was ca. 2X as long on the synthetic medium compared to the insect host, and ca. 80% reached the active adult stage. Interestingly, the yellow coloration of the femur did not develop if vitamin A was lacking. A critical factor in formulating the artificial media for B. lasus was osmotic pressure (Thompson 1983b). The effect of both carbohydrate and amino acid levels was similar and appeared closely related to osmalality. Optimum osmotic pressure in the artificial diets ranged from 550-700 mOs.Kg which was much greater than the 350-450 mOs/Kg of host hemolymph and tissues. Complete development of the pteromalid Pachycrepoideus vendemiae (Rondani) was not obtained on an artificial medium similar to that used successfully for in vitro cultureof Brachymeria (Thompson 1981c). When the amino acid component was replaced with a mixture of the corresponding polymerized amino acids and the osmolality was reduced to ca. 390 Mos/Kg, development from egg to adult was obtained (Thompson et al. 1983c). These studies demonstrate that the importance of osmotic pressure varies with the parasitoid species. Parasitoids such as I. conquisitor and E. roborator are very tolerant of osmotic pressures. Artificial diets that ed in vitro culture of these species had osmolalities of ca. 2,000 mOs/Kg. On the other hand, the tachinids, P. caudata and L. diatraeae (Grenier et al. 1986), and the pteromalid P. vindemiae did not develop at osmolalities of >450 mOs/Kg. Pteromalus puparum was cultured in vitro by Bouleteau (1968, 1972). Complete development on host hemolymph in hanging drop slide mounts was obtained. Similar results were reported by Hoffman et al. (1973). Hoffman & Ignoffo (1974) had limited success with an artificial medium containing yeast hydrolysate, fetal bovine serum and Grace‟s tissue culture medium. Tetrastichus schoenobii Ferriere was reared on modified Gardiner‟s tissue

culture medium supplemented with egg yolk, milk and hemolymph from Anteraea pernyi Guérin (Ding et al. 1980a). About 60% of the parasiotids completed development to the adult stage with no deformities nor abnormal fecundities. Greany (1980, 1981) described studies on the in vitro embryonic development of the barconid Cotesia (= Apanteles) marginiventris (Cresson), maintained in Grace‟s tissue culture medium supplemented with fetal bovine serum, bovine serum albumin and whole egg ultrafiltrate. Insects were reared from the embryonic germ band stage to mature first instar larvae on this diet cocultured with host fat body tissue. Greany (1986) obtained similar results with Microplitis croceipes. Emphasis was placed on the importance of protein nutrition for success and protein secretion by the fat body was considered a factor to explain the need for this tissue for successful embryonic development. Vinson & Iwantsch (1980) found that teratocytes (cells derived from the embryonic membrane of the parasitoid egg) are released into the host homocoel at the time of egg hatching. It was suggested that the teratocytes may play a role in parasitoid nutrition. Sluss (1968) demonstrated that the teratocytes of Perilitus coccinellae (Shrank) increased in volume several times in the coccinellid host and where then subsequently eaten by the developing parasitoid larvae. Graeny (1980) found that teratocytes present in artificial culture medium for C. marginiventris caused dissociation of cocultured fat body and suggested that the teratocytes might facilitate larval growth. Rotundo et al (1988) obtained complete larval development of the braconid Lysiphelebus fabarum (Marshall) on a similar artificial diet that was lacking in fat body and teratocytes. Strand et al. (1988) demonstrated a role for teratocytes in the successful in vitro culture of the egg parasitoid Telenomus heliothidis Ashmead. Embryonic development of T. heliothidis was obtained in Hinks TNH-FH medium containing 30% w/v M. sexta hemolymph. Mature embryos were transferred to a medium containing 40% M. sexta hemolymph, chicken egg yolk, trehalose and milk. Development of the adult stage required one day more than on the host H. virescens and 42% of the larvae became adults. The sex ratio was ca. 50% females. The presence of teratocytes had no effect on larval development to the third instar. However, when teratocytes were removed from the medium during larval development, pupation was greatly reduced and the development time of parasitoids that completed development increased. The authors concluded that the teratocytes aided larval feeding by dispersing the particulate material in the medium and solubilizing nutrients. It was suggested by Strand et al. (1986) that teratocytes of T. heliothidis aided in decomposition and necrosis of host tissue partially due to release of lytic enzymes. Therefore their function in vitro might be the same that occurs

during the normal development of the parasitoid in the host, Heliothis virescens. Culture of Trichogramma pretiosum in vitro was first attained by Hoffman et al. (1975) following unsuccessful attempts by Rajendram (1978) with T. californicum Nagaraja & Nagarkatti. Trichogramma pretiosum completed development on filter paper disc soaked in sterile H. zea hemolymph. In vitro culture to the adult stage required ca. 25% more time than observed on the host, Trichoplusia ni (Hübner). Even though most adults did not fully expand their wings, they mated and laid eggs without difficulty. Progeny from eggs of in vitro cultured parasitoids had a sex ratio of 1.2:1 males/females when reared on host eggs. Hoffman et al. (1975) reported development to the prepupal stage on a semisynthetic artificial diet similar to that described by Hoffman & Ignoffo (1974) for P. puparum,but supplemented with wheat germ oil. Strand & Vinson (1985) obtained complete in vitro culture of T. pretiosum on an artificial medium similar to that used by Thompson (1981d) for B. lasus but supplemented with ca. 40% M. sexta hemolymph, the latter being required to induce pupation. Survival to the adult stage was 70s% and the sex ratio ca. 1:2 males/females. Xie et al. (1986a) also reported that host hemolymph was required for pupation of T. pretiosum and that factors in the host egg influenced adult emergence. Irie et al. (1987) reported that the requirement of host hemolymph for the complete in vitro development was due to the presence of specific factors that could be extracted in 76% ethanol. Purification of the pupation factor by chromatographic methods showed the presence of two active carbohydrate containing factors. Trichogramma dendrolimi Matsumura was cultured in vitro in hanging drop amounts of hemoplymph from A. pernyi Guan et al. (1978). Liu et al (1979) reported success in hanging drop mounts containing media with A. pernyi or Attacuscynthia (Drury) hemoplymh and chicken egg yolk, bovine milk, organic acids and pocine serum. The extent of development of Trichogramma japonicum Ashmead, T. australicum Girault and T. evanescens was not reported, however, Wu et al. (1980, 1982) and Wu & Qin (1982a) obtained successful culture of T. dendrolimi to the adult stage on media without host hemolymph but containing chicken egg yolk, chicken embryo fluid, bovine milk, and amino acid mixture and peptone. However, only 16% of the eggs completed development, and most adults were females of poor vitality. The results did suggest that in contrast to T. pretiosum, the in vitro culture of T. dendrolimi on a medium of yeast hydrolysate, fetal calf serum, Grace‟s tissue culture medium, chicken embryo extract, bovine milk and chicken egg yolk. However, adults were less viable than normal and displayed abnormal wing development. The cooperative Research Group of Hubei Province, China (CRGHP 1979) has carried out extensive studies on the complete in vitro culture of T. dendrolimi in artificial media encapsulated in

artificial eggs into which the adult females oviposited. Gao et al. (1982) reported rearing 35 continuous generations of this species hanging drop mounts of the artificial medium. Some studies have tried to determine the effects of hormone supplementation on parasitoid development in vitro, with generally negative results. The tachinid Gonia cinerascens Rondani depend on its host‟s endocrine system for growth and development, but was not induced to mold from the 1-2nd instar by addition of 20-hydroxy (b) ecdysone to an artificial medium of host tissue homogenate and Grace‟s tissue culture medium. Development from the 2nd instar to adult was reported on artificial medium in the absence of hormones, indicating that some hormones may be necessary for the 1-2nd instar molt in vitro. The 20-Hydorxy ecdysone failed to stimulate development of B. intermedia in vitro (Thompson 1980); however, Greany (1980, 1981) reported that this hormone inhibited egg hatching in C. marginiventris and ecdysone, 20-hydorxy ecdysone and the juvenile hormone analog hydropene had no effect on larval growth or development. The deleterious effect of this hormone could be overcome by simultaneous application of hydroprene. Nenon (1972a,b) demonstrated that hormones greatly increased in vitro survival of developing embryos and larvae of the encyrtid Ageniaspis fuscicollis (Dalman). The parasitoid was maintained on a diet of chicken embryo extract, beef peptone and equine serum. Ecdysteroid or juvenile hormone added in the medium had little effect, but when included together, resulted in nearly 100% survival to the 2nd instar. Further study of the effects of host hormones in vitro culture systems must require careful and detailed experimental design. Hormones act in a complex and often synergistic way, and the timing of their application as well as the method of exposure may prove critical to assessing their potential. There is no doubt as to the importance of hormonal interaction to the successful development of parasitoids in vivo, particularly with regard to synchronizing parasitoid development to the host‟s life cycle. Adult Entomophage Nutrition Adults of many entomophaga must feed, and although adult parasitoids and predators are usually fed in the laboratory, early workers had largely ignored the significance of such feeding in nature. Bierne (1962) considered that many biological control attempts failed as a result. Leius (1967a) gave one of the first field demonstrations of the importance of adult feeding when he reported a relationship between the natural abundance and variety of wild flowers in apple orchards and the incidence of parasitism of Malacosoma americanum (F.) and Laspeyresia (= Carpocapsa) pomonella (L.) by the

parasitoids, I. conquisitor, Apophua (= Glypta) simplicipes (Cresson), Scambus hispae Harris, Telonomus sp., Ooencyrtusclisiocampae (Ashmead), and Eupelmus spongipartus Foerster. Eighteen times as many L. pomonella eggs were parasitized in orchards with an undergrowth of wild flowers when compared with other orchards lacking such flora. The early literature describing how adult parasitoids feed from flowers and other plant parts was reviewed by Leius (1960). Generally insects fed on floral and extrafloral nectars as well as pollens. Although knowledge of the specific nutritional requirements of adult entomophagous insects is limited, much data are available on the chemical and nutritional requirements of adult entomophaga is limited, much is available on the chemical and nutritional composition of these plant products. Floral nectars contain up to 75% by weight of simple sugars, mainly sucrose, fructose and glucose (Baker & Baker 1983), but considerable qualitative and quantitative differences exist between plant species. Free amino acids are also abundant in nectars although most nectars do not contain all 10 essential amino acids. Small amounts of protein, lipids, dextrins and vitamins that are nutritionally beneficial are also found. The composition of extrafloral nectars is also complex (Baker et al. 1978). Pollens have a complex composition of small molecular nutrients and many pollens have high levels of free amino acids (Barbier 1970, Stanley & Linsken 1974). By comparison, pollens generally have higher levels of protein, lipid and polysaccharides. Pollens and nectars together can provide a complete diet sir successful growth, development and reproduction. The predator Coleomegilla maculate lengi Timberlake can complete larval development on pollen alone (Smith 1961); therefore, when prey are scarce, plant products may play a critical role in maintaining predators (Hodek 1973). Hagen (1986a) discussed the complex ecological and evolutionary interactions between plant flowers, nectars and pollens and several insect groups. Leius (1960) examained the plant feeding habits of I. conquisitor, Scambus boulianae (Hartig) and Orgilusobscurator (Nees). The attractiveness of the flowers of wild mustard, white sweetclover, wild parsnip, silky milkweed and annual sowthistle were tested. Except for annual sowthistle, I. conquisitor was attracted to and fed from all flowers tested, but was most attracted to wild parsnip. Similar results were shown with S. boulianae. Orgilus obscurator was attracted to and fed on wild parsnip only, but further tests revealed that this parasitoid also fed on other umbelliferous plant flowers, including those of wild carrot and water hemlock. The nutritive value of various pollens for fecundity and longevity of S. boulianae was reported by Leius (1963). Itoplectis conquisitor and S. boulianae accepted various semi-natural foods also, including honey, sucrose solution with or without plant pollens and raisins. Plant feeding behavior of O. obscurator examined by Syme (1975) showed a broad range of food plants, including species from five families. Adult

parasitoids may emerge prior to the availability of the insect host, and Syme (1977) suggested that a variety of plant species be provided as foods to ensure sufficient longevity of the adult female. Lingren & Lukefar (1977) demonstrated that adult Campoletis sonorensis (Cameron), a parasitoid feeding on the extrafloral nectar of cotton, lives when exposed to extraforal nectaried cotton than nectariless cotton. Parasitism of hosts was higher on the nectaried form. Adejei-Maafo & Wilson (1983) showed that 15 categories of entomophaga, including the predators Deraeocoris signatus (Distant), Geocoris lubra (Kirkaldy), Nabis capsiformis Germar, Chrysopa spp., Laius bellalus Guérin, Coccinella repanda (Thunberg) and Verania frenata Erichson, were present at densities of 2-3 tomes higher on nectaried versus nonnectaried cotton. Although semiochemicals contribute to attraction for plants in these insects, the nutrition provided by nectars and pollens seems to be important. Hemptinne & Desprets (1986) reported that following hibernation Adalia bipunctata (L.) fed on pollens as an alternate food which allows the predators to lay eggs as soon as prey become available. As was discussed in an earlier section, in addition to feeding plants and plant products many parasitoids are host-feeders. Adult female Hymenoptera puncture or damage host larvae or pupae and feed on the hemolymph and/ or internal tissues. Kidd & Jervis (1989) estimated that as much as 1/3 of the world‟s parasitoid fauna (>100,000 species) host feed. Some parasitoids may kill more host individuals by host feeding including ovipositor probing followed by host rejection, than by parasitization (Johnston 1915, DeBach 1943, 1954). Legner (1979) emphasized that consideration of a parasitoid‟s host destructive capacity was important to correctly evaluate the impact of periodic inundative field releases on pest populations, and Greathead (1986) and Yamamura & Yano (1988) suggested that host-feeding behavior was important for assessing the potential of a biological control agent. Kidd & Jervis (1989) recently discussed the significance of host-feeding on parasitoid-host population dynamics. Bartlett (1964) in examining host-feeding in the encyrtid, Microterys flavus Howard, was among the first to correlated host-feeding behavior with nutrition. He hypothesized that host feeding developed coincidentally with depletion of eggs and suggested that host mutilation was a reflection of “frustrated” host feeding when the host failed to bleed readily. Host feeding by M. flavus was usually displayed following egg-laying, and oviposition resumed after host feeding. Reviewing this predatory habit for adults from 20 families of Hymenoptera, Bartlett concluded that the behavior was indicative of the necessity for dietary supplementation of some ubiquitous substances required by many diverse species. He reported that a food supplement of enzymatic

yeast and soy hydrolysate with honey satisfied the nutrient requirements for sustaining reproductive activity in M. flavus, and suggested that a protein nutrient source may be necessary. The difference between proovigenic and synovigenic Hymenoptera was discussed earlier, categories proposed by S. E. Flanders (1950). Females of proovigenic parasitoids complete oogenesis prior to or shortly after emergence and lay eggs over a relatively short period of time principally on larval stages of their host. Host feeding is important for ensuring that the female lives long enough to deposit all eggs. In contrast, females of synovigenic species eclose with a minor fraction to their total egg complement as mature eggs. Synovigenic parasitoids attacks primarily host eggs and pupae, are longer lived than proovigenic species and produce eggs throughout their adult lives. To sustain oogenesis the females of many synovigenic species require additional nutrients. Based on the egg type, Dowell (1978) described two types of synovigenic parasitoids: (1) those producing large anhydropic or yolk-rich eggs that contain sufficient nutrient for completion of embryonic development prior to oviposition. Parasitoids that produce anhydropic eggs obtain nutrition for sustaining egg production by host-feeding; (2) those producing hydropic or yolk-deficient eggs. Embryonic development in hydropic eggs occurs in the host following oviposition, in which case the adult does not require additional nutrient to egg development and has no requirement to host feed. Legner & Gerling (1967) showed the importance of early host feeding and oviposition to pteromalids of the first type, as was previously discussed. Leius (1962, 1967b) demonstrated the importance of feeding habits of fecundity of S. buolianae. Egg production was reduced to 1/3rd and longevity to 2/3rds, when females were permitted to host-feed intermittently or were deprived after 15 days of age. No eggs were laid if females were deprived for 290 days. The effects of feeding host body fluids, in conjunction with honey, pollen and raisins on fecundity and longevity of S. buolianae and I. conquisitoir were examined by Leius (1961a,b). Maximum fecundity and longevity of both species were obtained when host fluids and seminatural foods were provided together. Host feeding was nevertheless essential, and S. buolianae did not lay eggs when deprived of host hemolymph or tissues. The feeding behavior of 140 hymenopterous parasitoids was also reviewed by Jervis & Kidd (1986), who concluded that host feeding was important for maintenance and longevity. Four types of host feeding distinguished were (1) concurrent feeding where the female used different host individuals for feeding and oviposition, (3) the feeding habit may be nondestructive or destructive (the host may survive or may die), and (4) destructive feeding which generally resulted in a host that was most likely when hosts were readily available and that destructive feeding was advantageous when host density was low.

Jervis & Kidd (1986) also gave several models to assess how the energetic demands and constraints on parasitoid affect its host-feeding strategy. One model predicted the feeding strategy for maximizing egg production of a single synovigenic female (see Thompson & Hagen 1999, for formulae). Host feeding also occurs among dipterous parasitoids but is not as common as in Hymenoptera (Clausen 1940). Host feeding by tachinid parasitoids may affect longevity and fecundity (Shahjahan 1968). Nettles (1987b) demonstrated that fecundity was prolonged by feeding E. bryani host hemoplymph compared with feeding a sucrose solution. The effect of host feeding on fecundity could not be simulated by substituting a solution of free amino acids or bovine serum albumin. The excretion of various Homoptera, such as honeydew, may serve as a food for many adult entomophaga. Neuropterans of the genus Chrysoperla and other genera with nonpredaceous adults feed actively on honeydew as well as on nectar and pollen (Principi & Canard 1984). Although honeydew does not contain all the essential amino acids in some nonpredaceous species (Hagen & Tassan 1972). Neuropteran predatory adults also feed on honeydew, but reproductive activity ensues only after prey are eaten (Haggen 1986a). Hagen (1962) found that honeydew alone will not stimulate egg production in coccinellid predators. Dipterous and hymenopterous parasitoids also have been found to feed on honeydew (Clausen 1940, Zoebelein 1956). The importance of honeydew as a supplementary food was suggested by Clausen et al. (1933) in work with Tiphia matura Allen & Jaynes. Female adults traveled long distances from the location of their host to feed on honeydew, which migration occurred annually. Ichneumonids of the genus Rhyssa appeared dependent on honeydew for maintaining the longevity necessary to parasitize and regulate populations of Sirex (Hocking 1967). The nutritional value of honeydew for parasitoids varies with the homopteran source, as Wilbert (1977) showed considerable differences in longevity of several Hymenoptera when fed aphid or coccid honeydew. Nutritional requirements of adult entomophagous insects are obscure. Bracken (1965, 1966, 1969) examined some requirements of the parasitoids Exeristes comstockii (Cresson), finding that adult females fed an artificial medium containing amino acids, sucrose, fatty acids, cholesterol, vitamins and inorganic salts produced eggs at an equivalent rate as individuals fed Galleria mellonella (L.) larvae and sucrose. Egg production was reduced or eliminated when amino acids, sucrose, vitamins or salts were deleted. Sucrose, pantothenic acid, folic acid and thiamine were all essential for egg-laying. Nutritional requirements of adult predators similarly are not well known. Numerous semi-natural diets have been successfully developed for

maintaining chrysopid predators and various adults coccinellids. It seems that predators require a complete and well balanced diet to ensure maximum longevity and reproductive potential. The effects of various diets on fecundity of some chrysopids was summarized by Hagen (1986b), and nutritional data for adults of several other species by Roussett (1984). Continuous Culture on Artificial Media The ultimate goal of studies on in vitro culture of entomophagous insects is continuous artificial culture without the host insect. In order to achieve this goal, careful scrutiny of factors that otherwise would not be considered of direct important to nutrition must be made. Commercial parasitoid culturing requires the direct deposition of eggs or larvae onto an artificial substrate. Artificial food must be acceptable for feeding by all stages of a predator. Behavioral considerations may be critical for the successful continuous culture of many entomophaga. Success with in vitro culture thus far reflects the level of complexity of behavioral interactions between parasitoid and host or predator and prey. The first succeed with parasitoids was achieved with Sarcophagidae, many of which readily oviposit and develop on carrion. Sarcophaga aldrici and K. kellyi were reared for many generations on fish and liver, respectively (Arthur & Coppel 1953, Smith 1958). Agria housei was reared continuously for 756 generations on pork liver. However, the behavioral interaction between many parasitoids and their hosts are complex, involving numerous physical and chemical cues that initiate specific behavior which leads to oviposition. Host selection and successful parasitism is a multistep process which involves host habitat location, host location, host acceptance, host suitability and host regulation, as was discussed in previous sections (Doutt 1959, Vinson 1976, 1984). Factors that influence host acceptance in particular are critical for continuous culture. The different events which lead to successful oviposition, including examination of the host, probing with the ovipositor, insertion and oviposition (Schmidt 1974) may each be stimulated by different chemical as well as physical cues (Arthur 1981, Vinson 1984). These cues may be associated with the host species, the plant or other food source of the host, or may result from interactions, involving both the host and its food (Vinson 1975). Physical factors associated with the host‟s food plant are essential for successful oviposition and parasitism by G. cinerascens (Mellini et. al. 1980). This tachinid deposits microtype eggs on the leaves of certain plants, and host larvae become infected by ingesting the eggs. Leaf color, shape, thickness, size and reflectivity are among the several factors which influence oviposition in this species. Mellini et al (1980) constructed polished, thin, yellow oval-pointed bee‟s wax leaves, 2-7 cm2, on which large numbers of parasitized eggs were laid. This parasitoid readily developed in G. mellonella after host feeding on the artificial leaves. Complex combinations of physical cues, including size, shape, color, texture and movement have been

demonstrated to have influence on oviposition behavior in paarsitoids 9Arthur 1981, Jones 1981, Nordlund et al. 1981). Important roles are played by chemicals in both parasitoid-host and predator-prey interactions (Arthur 1981, Greany and Hagen 1981, Vinson 1984, Hagen 1986a). The involvement of chemicals in host acceptance and oviposition by parasitoids is well documented. During predator-prey relationships, kairomones produced by the prey may serve as attractants, arrestants and/or phagostimulants. Chrysopa carnea adults, e.g.. are attracted to a variety of chemicals such as tryptophan byproducts (Hagen et. al. 1976). Although studies with numerous predaceous insects have demonstrated the role of semiochemicals in prey finding and recognition, their role in feeding is not well established. Deployment of behavior modifying chemicals in continuous artificial culture has involved only a few species. Itoplectis conquisitor accepts a host and oviposits following detection of specific components of host hemolymph during ovipositor probing (Arthur et al. 1969). This parasitoid even oviposited into host hemolymph that was placed on paraffin tubes. The active fraction was colorless, water soluble and gave a strong reaction to ninhydrin and folinphenol reagents. It had a molecular weight of ca. 7,000, was heat stable and nondializable. Arthur et al (1973) concluded that the stimulant was proteinaceous and they were successful in stimulating similar oviposition activity with a variety of amino acid mixture containing trehalose and/or MgCl2. The best results were with a mixture of serine (0.5M), leucine (0.065M), arginine (0.05 M) and MgCl2 (0.025 M). The ovipositional activity observed greatly exceeded that stimulated by the host hemolymph. House 91978) then developed a synthetic artificial host comprised of an artificial diet encapsulated in paraffin. The diet was based on that described by Yazgan (1972) and contained gelatin, casein, inorganic salts, amino acids, glycogen, lipids, trehalose, glucose, water and fat soluble vitamins and agar. Female parasitoids readily accepted and oviposited into the artificial host, and the first successful complete artificial culture of a hymenopterous parasitoid was realized. However, only one single adult male was obtained. There have been considerable studies to determine how chemical and other factors influence adult reproductive capacity in in vitro cultures. Larviposition by E. bryani is stimulated by kairomones which emanate from the host‟s cuticle, and female adults examine artificial hosts coated with cuticular extracts with great care (Burks & Nettles 1978). Tucker & Leonard (1977) extracted a kairomone from the pupae of Lymantria dispar that appeared responsible for ovipositional behavior by Brachymeria intermedia. Tetrastichus schoenobii was stimulated to oviposit in artificial eggs coated with host scales (Ding et al. 1980b).

The parasitoid group, which has received the most attention, is the Trichogrammatidae. There have been more extensive efforts to develop continuous artificial culture with Trichogramma spp. than with other parasitoids. Many aspects of the ovipositional behavior of this genus were described by Salt (1934, 1940) in studies on T. avanescens (Fisher 1986). Recent studies demonstrate the importance of physical (Rajendram & Hagen 1974) and chemical factors, including kairomones (Nordlund et al. 1985) for eliciting oviposition. Rajendram (1978a, b) obtained artificial oviposition by T. californicum into physiological saline or Neisheimer‟s salt solution encapsulated in paraffin. Nettles et al (1982, 1983) reported that a dilute solution of KCl and MgSO4 induced oviposition by T. pretiosum artificial was eggs (Nettles et al. 1984). Leucine, Phenylalanine and/or isoleucine stimulated oviposition by T. dentrolimi in artificial eggs (Wu & Quin 1982b). Adult females laid more eggs than when insect hemolymph was used then employing a complete mixture of all three amino acids, 600, 400 and 320 mg/100ml. A synthetic membrane was developed as an alternative for paraffin through which T. pretiosum would oviposit (Morrison et al. 1983). The siliconpolycarbonate copolymer was clear, highly elastic and adult females oviposited through the surface into an ovipositional stimulants at rates that were comparable to host eggs. The use of polyethylene as an ovipositional stimulants at rates that were comparable to host eggs. The use of polyethylene as an alternative to wax for producing artificial eggs for oviposition by T. dentrolimi was decribed by the Chinese CRGHT (1985). Xie et al. (1986b) demonstrated the potential for large scale continuous artificial culture of T. pretiosum. Three in vitro culture methods were developed as a follow up to earlier work by Xie et al. (1986a) and Nettles et al. 91985). These utilized microtiter tissue culture plates, multiple drop rearing in petri plates and flooded petri plat rearing. The basic diet was 50% heat treated insect hemolymph, 25% egg yolk, 15 g/100 ml dried milk suspension and 0.15% gentamycin. Each method ed large populations of parasitoids larvae. Microbial contamination and subsequent loss of entire petri plats was a major obstacle but several antibiotics were available for reducing losses. Field trials with in vitro reared Trichogramma have been made. Continuous artificial mass culture of T. dentrolimi was described by Li 91982) and Gao et al. (1982), who reported that field release of in vitro reared parasitoids resulted in 93% parasitism of Heliothis armigera (Hubner) eggs in cotton. Artificial mass culture of Chrysopa carnea was described by Yazlovetskij & Nepomnyashchaya (1981) after the development of a suitable artificial medium for ing larval development (Nepomnyashchaya et al. 1979). The medium was microencapsulated and composed of casein hydrolysate, brewer‟s yeast extract, soybean oil, wheat germ extract, sucrose, lecithin, choline, cholesterol and ascorbate. The effectiveness of the artificially

reared larvae against Myzus persicae was equal to that of insects reared on eggs of S. cerealella. A microencapsulated technique for mass producing artificial eggs for C. carnea was also described by Morisson et al. (1975). Contemporary Applications Considerations of how nutrition currently applies in biological control programs, focuses on its purpose as being restricted to use of food and food supplements to enhance the activity and effectiveness of entomophagous insects in the field as suggested earlier (Hagen & Hale 1974, Hagen & Bishop 1979, Greenblatt & Lewis 1983, Hagen 1986a, Gross 1987). Such use is dictated by a lack of synchrony between natural enemies and their hosts and/or isolation of entomophagous insects from the natural environment that normally supplies alternate food sources such as nectars and honeydews (Hagen 1986a). These factors occurring in crop monoculture may intensify following pesticide application. The importance of nutritional supplements for adult parasitoids and predators is well known, and recent studies with Trichogramma demonstrated that fecundity and longevity could be increased be feeding adult insects (Anunciada & Voegele 1982, Bai et al. 1988). The future use of feeding prior to or following release in the field may have a significant effect on biological control successes. Few studies on the effects of feeding parasitoids on the field performance are available, however. Temerak (1976) reported spraying honey solution on sorghum stalks during winter to provide supplementary food to Bracon brevicornisi Wesmael in the absence of pollen, honeydew and nectars. Parasitoid cocoons significantly increased after spraying and the prevalence of hosts decreased. Despite field trials employing kairomones for attracting and stimulating host searching by Trichogramma sp. (Lewis et al. 1979, 1982), no attempt has been to use kairomones in combination with supplemental foods to maintain parasitoid populations when host numbers are low. Supplementary food sprays have been successfully deployed with predaceous insects. Ewert & Chiang (1966) sprayed sucrose solutions on corn to aggregate coccinellid and chrysopid adults. Increased predator density and reproductive activity significantly lowered aphid populations. The numbers of Chysopa sp. And Glischiochilus quadrisignatus (Say) were increased in corn sprayed with sugar or molasses solutions (Carlson & Chiang 1973), with resultant increased predation resulting in significant reductions of Ostrinia nubilalis (Hubner). Hagen et al. (1976) working in sugar sprayed alfalfa plots were able to retain larger numbers of C. carnea and Hippodamia sp. in the field during periods of low host density. Within 24 hrs the population of coccinellid adults increased 20X and that of C. carnea 200X. Populations of Lygus spp. also increased after application of sugar sprays (Lindquist & Sorenson 1970), and Hagen et al. (1971) concluded that sucrose was an

arrestant for adult Lygus were attracted to potato plants sprayed with honey, which suggested a critical role for volatile components (Ben Saad & Bishop 1976a, b). Adding semiochemicals to supplemental foods for C. carnea is useful. The complex interactions of semiochemicals and food in influencing the behavior of C. carnea was described by Hagen & Bishop (1979). The adult responds to a volatile signal, a synomone, from plant habitats in which prey are located and is then attracted to the prey by tryptophan breakdown products from the honeydew (Van Emden & Hagen 1976). Specific behavioral and flight patterns shown by C. carnea in response to these interactions were discussed by Duelli (1980). The habitat synomone affecting the behavior of C. carnea in cotton was shown by Flint et al. (1979) to b e caryophyllene, but several chemicals from other plants also displayed synomone activity for this species (Hagen 1986b). Chrysopa carnea adults were successfully attracted to alfalfa fields by applying artificial honeydews composed of various yeast (Wheast) products and sugar (Hagen et al. 1971). Although the specific synomone of alfalfa is unknown, application of caryphyllene with the kairomone from tryptophan greatly improved attraction of C. carnea during the beginning of flowering (Hagen 1986a). Application of artificial honeydew was also successful for aggregating Hippodamia spp. as well as coccinellids and other predators. Subsequent trials employing the yeast mixture in combination with sucrose and honey or molasses applied to various crops were successful in manipulating C. carnea populations (Hagen & Hale 1974). The sugar was essential for retaining C. carnea adults in the field after attraction. Butler & Ritchie (1971) reported that C. carnea adults were attached to the yeast/sugar mixtures sprayed on cotton, but no increase in egg deposition was noted. Similar studies demonstrated inconsistent oviposition in grape culture (White & Jubb 1980). There was no attraction of chrysopid adults in treated people orchards (Hagley & Simpson 1981) nor in potato fields when only the yeast was applied. Dean & Satasak (1983) gave reasons why food sprays might not be practical in control programs for cereal aphids in England, which included the variable abundance of univoltine C. carnea populations, low plant growth form and the development of sooty mold on plant where food sprays containing sugar were applied. Duelli (1987) did not find an increase in oviposition by chrysopids when artificial honeydews were applied to alfalfa, corn, sunflowers and in prune orchards. It was suggested that the different responses of sibling species of C. carnea in Europe and North America may be related to behavioral differences.

2. Mass Prodcution of Biological Control Agents Mass-production of parasites and predators is often necessary or desirable in connection with many biological control projects, particularly where attempts are made to increase parasitism or predation buy mass releases of entomophages over a wide area at a time in the season when these natural enemies are few or absent. However, the term “massproduction” is vague and individual differ in their concepts of large numbers. Moreover, the actual numbers needed in connection with different biological control problems vary considerably. Some successful parasites introductions have been made with only a dozen or so individuals; others have required very large numbers before establishment has been obtained. There are two main types of biological control, each of which has different requirements with regard to mass-production. As mentioned above, there is that in which large numbers of entomophages are released at critical times in the season in order to increase the destruction by natural enemies. In such cases it is obvious that mass-production methods are essential, the scale depending on the size and duration of the operation. The main difficulties here are to produce the required enormous numbers of entomophages at exactly the correct critical period of the season, particularly when this period is dependent on weather and may vary considerably and somewhat unpredictably from year to year. A thorough survey of the results obtained from this method of control in the number of instances in which it has been used would be very interesting. The second, and far more desirable, procedure is to established a suitable exotic entomophage against a pest which will thrive in the new environment and give perennial reduction in the pest population. This has, of course, been accomplished successfully many times, but where failure has occurred the exact reasons for it are often obscure and it is some Director, Commonwealth Institute of Biological Control, Curepe, Trinidad, West Indies times attributed to the introduction of too few individuals. In such cases it is claimed that massproduction and release might yield better results. Mass-production-weather of parasites or predators, fungi, protozoa, bacteria, viruses, sterile males, or genetically incompatible individuals-has become an integral part of biological control programmes. Large-scale field collection is often the simplest and most economical method of obtaining large numbers of a given species. Often this entails feeding the host or immature predators in the laboratory, where considerable care may be necessary to eliminate hyperparasites and diseases. Mass-production methods usually produce large numbers of entomophages, free from contaminants, which can be released immediately in the field. However, there are virtually no general rules that can be laid down as to optimum conditions of cage-size, environment, feeding and illumination for breeding these. Each problem demands different treatment, depending on the requirements of individual

species and numbers to be produced, and each scheme is usually based on a technique developed in the laboratory and then modified for cheap, largescale production. However, there are a number of general aspects of massproduction of entomophages that should be considered. In many cases it is necessary to provide large numbers of a host species. These may possibly be collected in the field and, if necessary, stored for future use. Such collections may, however, be impossible or impracticable and it may be necessary to propagate host in large numbers, often throughout the year, in heated insectaries. Great care is then necessary to produce normal, healthy adults, rather than small individuals susceptible to disease. In some cases, too, precautions have to be taken to avoid cannibalism. When normal plants are unavailable or unsuitable for mass-production methods, it may be possible to find a more suitable alternate host-plant, or even to provide an artificial, not necessarily synthetic, diet in place of the host. Although the mass production of host-plants does nit actually concern insect vectors of medical importance, it is mentioned here on general grounds. Perhaps the normal host cannot be mass-production. It is then often possible to find an alternative, but unnatural, host on which the entomophage can develop, and which can be produced in large numbers. Sometimes it may be necessary to “condition” such alternative hosts with extracts of normal hosts before oviposition by entomophages can be induced. The logical development from this is research into producing artificial, and even purely synthetic, diets on which entomophages may be massproduced. The use of unnatural or artificial hosts may possibly produce abnormalities in the progeny, and this must be guarded against. The diet of both the preceding larval generation and also of the entomophages themselves may have considerable influence on their longevity, reproductive rate, fertility of eggs, and possibly on the proportion of progeny entering diapause-all of which are of major importance in mass-production schemes. There is the possibility also that the use of unnatural hosts may affect the host preferences of the progeny, a point which should be investigated where necessary. The genetical aspect of breeding and mass-propagation of entomophages is important. In most instances laboratory stocks, or those for mass-production schemes, are derived from a small amount of field material and they are often further inbred during the course of many generations. Genetic variability of the stock will almost certainly be reduced and with it, possibly, the adaptability of the entomophage to different environmental conditions. Hence every effort should be made to maintain or increase genetic variability in mass-propagation programmes.

The determination of optimum mating conditions to provide maximum numbers of progeny with an adequate sex ratio is also essential. In massproduction schemes it is usually easy to eliminate hyperparasites in the case of parasites, or parasites in the case of predators. Disease and mites, however, may often cause losses in crowded mass-production conditions, and if this occurs rearing methods must obviously be suitably altered. Disease transmitted through the eggs are particularly difficult to eradicate. In conclusion it should be stressed that not only does each problem in mass-production of an entomophage present its own difficulties, but also the actual need for, or size of, a mass-production programme should be carefully considered, since once such a programme is initiated there is sometimes a tendency to increase production out of proportion to real needs.

Chapter 5

Methods and Approaches to Biological Control

A. Quarantine and Exclusion The primary function of a biological control quarantine facility is to provide a secure area where the identity of all incoming biological control candidates can be confirmed and undesirable organisms, especially hyperparasitoids, parasitoids of predators, and extraneous host or host plant material, can be eliminated. In fact the quarantine laboratory often represents the last chance to study and evaluate potential biological control agents in the sequence of collection, importation and liberation. The number of quarantine in the United States which are certified to handle incoming shipments of beneficial organisms has increased from four to 26 over the past 40 years. In addition to 24 listed by Coulson & Hagen (1985), new quarantine facilities have been constructed at Montana States University, Bozeman for phytophagous insects and the University of California, Riversides for nematodes. New or expanded quarantine facilities have been constructed in a number of other countries (e.g., Australia, Great Britain, Mexico, and Thailand). The steady increase in quarantine need and capacity is due on part to an increased interest in biological and non-polluting methods of pest control, and the desire to expand on the many successes already achieved through the importation of exotic natural enemies. Also, there is an increase in new pests that are transported throughout the world and which are amenable to biological as well as the stricter prerelease information requirements on behavior and safety of biological control candidates. Lengthy studies on candidates often ties up quarantine areas thereby increasing the need for greater quarantine capacity to avoid limiting the amount of materials that can be handled. For example, in the United States to prove the environmental safety of plant-feeding arthropods for the biological control of weeds can include studies with as many as 10-20 North American native plant species related to the target weed. When such studies are not permitted or feasible in the country of origin of the biological control phytophage, these test must be conducted in a domestic quarantine facility. Similarly, testing parasitoids against indigenous insect species which have been declared legally threatened or endangered and which may be present in areas near or contagious to insect pest infested agricultural crops that are targeted for parasitoid release, may not only require more quarantine space but also delay or prevent the colonization of newly imported organisms. The longer the imported organisms remain in quarantine before these tests can be

conducted, the greater the risk that subtle genetic changes occur, altering the potential fitness of the organism. Increasing concern over the quality of the environment is also causing a proliferation of regulations governing the importation and liberation of beneficial organisms. Explorer collectors, quarantine officers and project scientists must spend increasing time to study and comply with domestics and foreign regulations that cover importation, exportation and liberation of biological control agents. Air travel has reduced the amount of time required to move biological control agents from one continent to another, but the proliferation of international airports has spawned a logistical confusion of unpredictable package routing, delayed agricultural and customs inspection and unscheduled reloading and shipment to the final destination. Frequently material arrives dead or in a weakened state and on occasion may never arrive.

B. Use of Resistant Host Plants Plants have two ways of defense against herbivores. One is direct defense, which affects herbivores directly through physical or chemical means, such as thorns, toxins, digestibility reducers. Then other is indirect defense, which promotes the effectiveness of carnivores (Dicke, 1999). A direct defense is to employ toxins such as alkaloids, phenolics, terpenoids, which are lethal for herbivores in some cases. While another direct defense is to produce substances such as tannins and phenolics as digestibility reducers to delay herbivore development. A plant‟s indirect defense can promote the performance of carnivores in several ways. For example plant morphological character plant substances prolonging herbivore development which results in the herbivore remaining in a stage that is susceptible to carnivores for a longer period of time, or induced plant volatiles that help carnivores to find their herbivores prey (Dicke, 1999; Groot & Dicke, 2001). It has been recognized that many of the plant traits and processes that negatively affect herbivores change following attack. Karban & Baldwin (1997) refer to changes in plants following damage or stress as induced responses. Those induced responses that decrease the negative fitness consequences of herbivore attack on plants are termed induced defenses, including induced direct and indirect defenses. Induced direct defense normally acts through preventing herbivores from converting a plant‟s tissues into their own tissues after damage or stress. In induced indirect defense, two types can be distinguished. One is wound-induced change in the production of extrafloral

nectar. The other acts through the emission of induced volatile compounds in response to herbivory that attracts predators and parasitoids of herbivores. Many induced response to wounding are systemic. In such cases the damage plant tissue may produce a signal that is transmitted systematically throughout undamaged parts of the plant, causing the induction of new morphological or physiological states, the induced response. Several different signal transduction pathways are involved, including chemical and electrical signaling. Signalling compounds include 1) oligosaccharide fragments of plant cell walls; 2) systemin (an oligopeptide); 3) salicylic acid; 4) ethylene, which mediates induced defenses such as induced volatile production; 5) abscisic acid; 6) jasmonic acid and methyl jasmonate; and 7) electrical signals (Karban & Baldwin, 1997; Leon et al., 2001; Lorenzo et al., 2004; Adie et al., 2007). The volatile compounds that plants emit when they are damaged typically are mixtures of C6-alcohols, -aldehydes, and –esters produced by the oxidation of membrane-derived fatty acids, and terpenoids and aromatic compounds such as methyl salicylate and indole. These herbivore \-indiced volatiles released by plants were found to increase the foraging efficiency of carnivores, and carnivores may learn to associate the volatiles with actively feeding herbivores (Vet & Dicke, 1992; Karban & Baldwin, 1997; D cke & van Loon 2000; Turling & Ton, 2006). Plant defenses have been exploited by humans to develop two methods of environmentally-benign pest control: plant resistance based on direct defense and biological control based on indirect defense. Host plant resistance (HPR) can be defined as the inherited property that enables a plant to avoid, tolerate, or recover from injury by insect populations. For crop production, host plant resistance represents the inherent ability of crop plants to restrict, retard or overcome pest infestations and thereby to improve yield or quality of the harvestable product. Three mechanisms of plant resistance to insects are commonly recognized: antixenosis (non-preference), antibiosis, and tolerance. Antixenosis is defined as a relatively low acceptability of a plant as a host to an insect herbivore. Plants that exhibit antixenotic resistance would be expected to have reduced initial infestation or a higher emigration rate of the insect than susceptible plants. The basis of this resistance mechanism can be morphological or chemical. Antibiosis is defined as the mechanism leading to negative effects of a resistant plant on the biology of an insect which has colonized the plant. Both chemical and morphological plant traits can have antibiotic effects. The consequences of antibiotic ristics that can the activities of natural enemy resistance may vary from mild effects that influence fecundity, development times and body size, through to acute direct toxic

effects resulting in increased mortality. Tolerance is the degree to which a plant can an insect population that under similar conditions would severly damage a susceptible plant. That is, when two cultivars are equally infested the less tolerant one has a smaller yield (Thomas & Waage, 1996). Biological control is the suppression of a pest population using predators, parasitoids and pathogens. There are three main forms of biological control: 1) conservation of natural enemies already present in the environment; 2) augmentation and dissemination of natural enemies of pests, such as microorganisms, nematodes, and arthropods. 3) classical biological control-to suppress a pest population permanently through a single introduction of a natural enemy (Thomas & Waage, 1996). A parasitoid is an insect that as a juvenile, lives at the expense of another one (host) and is usually does not kill its host immediately but at the end of juvenile development. Parasitoids may parasitize eggs, larvae, pupae or adult hosts and each species is usually highly specific in this. A predator is an insect that eats more than one other organism during its life and kills its prey immediately. Predators are normally larger than their prey and are generally much more generalistic than parasitoids. In a successful biological control programme the pest kill rate of effective natural enemies should be always higher than the potential maximum rate of population increase of the pest species (Dicke, 1996; Hawkins & Cornell, 1999). The natural enemies of herbivores can be influenced by plant characteristics independently of the herbivore or mediated through herbivore activities. The relevant plant characteristics include the following aspects: i) plant tissues and products that are used as a source of nutrition by natural enemies; ii) plant morphology; iii) visual and vibrational cues; iv) secondary plant chemicals; v) plant volatiles (Thomas & Waage, 1996). Many pest management practitioners have found that host plant resistance is fundamentally compatible with biological control (Verkerk et al, 1998). Positive interactions result where natural enemies use herbivore-induced synomones emitted by plants as cues to find prey (Vet & Dicke, 1992); Dicke et al., 2003; Arimura et al., 2005; D‟Alessandro et al., 2006). Partial plant resistance may also provide benefits for the third trophic level by reducing the growth rate of prey which in turn increases the duration of their availability to natural enemies (Feeny, 1976; Price et al., 1980). However, in some cases, negative interactions between plant resistance and biological control are caused by toxic secondary plant compounds which can be ed on through herbivores to their natural enemies (Hare, 1992; Harvey et al., 2003). They may also be caused by plant factors which can impede natural enemy effectiveness (e.g. leaf toughness, cuticle thickness, trichomes (Price,1986). Therefore it is important to integrate HPR and BC dexterously.

In the future, fundamental and applied pest control research will have be more closely integrated and coordinated to increase efficiency. In this project, the integration of host plant resistance and biological control will be studied.

C. Destruction by Cultural Management or Direct Removal of Infected/Diseased Host Crop termination and alternate host elimination are cultural management methods that increase mortality of insect pests and decrease subsequent damage through destruction of food sources and overwintering habitats. Destroying the crop soon after harvest and eliminating volunteer sorghum plants suppresses insect pest abundance the following year. Destroying alternate host plants eliminates sources of insect pests that infest sorghum. Tillage methods for sorghum vary regionally and have evolved from plowing with a moldboard plow, followed by several secondary tillage and planting into one trip over the field. Whatever tillage is used, sorghum stalks should be destroyed soon after harvest to expose and kill insect pests and eliminate their food supply. It is equally important to destroy volunteer sorghum and alternate host plants where reduced tillage is pests. Herbicides can be used to kill sorghum and alternate host plants where reduced tillage is used. However, failure to destroy overwintering sites mechanically may contribute to an increase in the survival of certain insect pests. Destroying food sources and overwintering habitats reduce abundance of cutworms; sorghum midge; sorghum webworm, Nola sorghiella Riley; sugarcane borer, Diatraea saccharalis (Fabricius); and sugarcane rootstock weevil, Anacentrinus deplanatus (Casey). Johnsongrass, S. halepense (L.) Pers., is a noncultivated host of many sorghum insect pests, including yellow sugarcane aphid, Sipha flava (Forbes); greenbug; and sorghum midge. Destroying this weed is difficult but highly beneficial to efficient sorghum insect pest management and crop production. Crop rotation Crop rotation is a cultural management method that involves alternate use of host and nonhost crops in a field to reduce insect pest abundance and damage. Sorghum benefits most when rotated with a broadleaf or taprooted crop such as cotton, Gossypium hirsutum L., or soybean, Glycine max (L.) Merr. Growing sorghum in a field planted to a different, nonhost crop the previous year significantly reduces the abundance of some insect pests, as well as some diseases and weeds.

Crop rotation is most effective against insect pests with limited host range, long life cycle (one or fewer generations a year), and limited ability to move from one field to another. For example, several species of wireworms, white grubs, and some cutworms have only one generation a year, must have a grass-type crop on which to develop and reproduce, and because they live underground cannot during the damaging larval stage move from one field to another. Thus, growing a nongrass crop such as cotton or soybean the year before growing sorghum reduces abundance of soil-inhibiting insect pest in sorghum fields. Sorghum should be rotated annually with other crops. Variety selection, seedbed preparation, and seed treatment Variety selection, seedbed preparation, and seed treatment are important for managing sorghum insect pests. Only sorghum varieties with seed germination of eighty percent or greater should be used. Poor seed germination results in reduced stands and less competitive plants more susceptible to damage by insects. A sorghum variety adapted to the locale, and preferably one least vulnerable to insect pests and diseases, should be used. Sorghum hybrids selected should be resistant to greenbug. Sorghum midge-resistant hybrids, if available, are useful in more southern regions of the United States. Larvae of corn earworm; fall armyworm, Spodoptera frugiperda (J. E. Smith); and sorghum webworm that consume developing kernels infest sorghum hybrids with open panicles less than those with compact panicles. Also, kernels of open-panicle sorghum varieties are less likely to deteriorate from the combined effects of weather and damage by panicle-infesting bugs and pathogens. Sorghum varieties should be selected that mature as early and uniformly as practical in a locale. These varieties escape infestation by sorghum midge, corn earworm, fall armyworm, sorghum webworm, and sugarcane borer. Sorghum varieties resistant to pathogens and lodging also lessen detrimental effects from insect pests. Insect pests stress sorghum plants, and this stress combined with pathogen infection increases the chance of plant lodging. Greenbug and corn leaf aphid, Rhopalosiphum maidis (Fitch), transmit maize dwarf mosaic virus to sorghum. This problem is best dealt with by using virus-resistant varieties. Iron-tolerant sorghum varieties should be used in areas where iron deficiency is a problem. Well-prepared seedbeds speed seed germination and seedling growth. The trend in planting sorghum is less tillage and fewer trips over the field using appropriate planting equipment. Tillage for seedbed preparation should modify the soil to allow desired control of seed placement, weeds, water infiltration, water evaporation, and erosion. Rapid seed germination is essential to avoiding damage be seedfeeding insects such as wireworms and red imported fire ant, Solenopsis

invicta Buren. Rapidly growing and larger plants better tolerate damage by yellow sugarcane aphid, greenbug, and chinch bug, Blissus leucopterus leucopterus (Say). Insecticide can be used to protect planted seed from insect pests. Recently, sorghum seed treated commercially with a systematic insecticide is a available to protect against seed-feeding insects and some seedling inslect pests. Also, insecticides can be applied to seed in the planter box or in-furrow at planting. Systemic in-furrow insecticide protects seedling sorghum against some insect pests. Insects controlled by systemic seed or in-furrow treatment include wireworms, red imported fire ant, cutworms, southern corn rootworm, Diabrotica undecimpunctata howardi Barber, aphids, and chinch bug. Planting time Planting time of sorghum in most locales should be as early as practical but not when soil is too cool for rapid seed germination and seedling growth. In many areas, early planting takes advantage of seasonal rainfall. Planting early avoids infestation and damage because the sorghum plant is beyond the vulnerable stage when some insect pests are abundant enough to cause damage, or at the very least, the crop is susceptible for a shorter period of time. Early uniform planting of sorghum to avoid a damaging sorghum midge infestation is an excellent example of the benefit of planting early. Planting sorghum early avoids high numbers of corn earworm, fall armyworm, sorghum webworm, stalks borers, and panicle-feeding bugs. Fertilizer and water Fertilizer and water applied to sorghum either can be beneficial or detrimental to insect pests. Using too much fertilizer and irrigation can cause sorghum plants to be especially succulent and attractive to insect pests, amd may extend the time to maturity, increasing the duration of vulnerability. On the other hand, healthy, vigorously growing sorghum plants better tolerate insect pest infestation and other stresses. Chinch bug and Banks grass mite are favored by hot, dry conditions and moisture-stressed plants. Yield of healthy plants is less reduced by most lea-feeding insect pests. In some areas, application of iron is important for production of healthy sorghum plants.

D. Conservation and Augmentation The conservation of natural enemies is probably the most important and readily available biological control practice available to homeowners and gardeners. Natural enemies occur in all areas, from the private garden to the open field. They are adapted to the habitat and to the target pest, and their conservation generally simple and cost-effective. Lacewings, lady beetles, hover fly larvae, and parasitized aphid mummies are almost always present in aphid colonies. Fungus-infected adult flies are often common following periods of high humidity. These natural occurring biological controls are often susceptible to the same pesticides used to target their pest host. Preventing the accidental eradication of natural enemies is termed “simple conservation”. To conserve and encourage pest insect eating birds, including native plants and ornamentals plants that supply berries, acorns, nuts, seeds, nectar, and other vegetative foods, and also bird nest building materials, encourages their presence, health, and new generations. These qualities can also increase the visible population to enjoy in a garden. Using companion planting and the birds‟ insect cuisine habits is a traditional method for biological control agent pest control in an organic garden and any landscape, and in organic farming and sustainable agriculture. Installing specified nest boxes for mosquito-eating bats reduces a pest and increases endangered species conservation. Augmentation This third type of biological control involves the supplemental release of natural enemies. Relatively few natural enemies may be released at a critical time of the season (inoculative release) or literally millions may be released (inundative release). Additionally, the cropping system may be modified to favor or augment the natural enemies. This latter practice is frequently referred to as habitat manipulation. An example of inoculative release occurs in greenhouse production of several crops. Periodic releases of the parasitoid, Encarsia Formosa, are used to control greenhouse whitefly, and the predaceous mite, Phytoseiulus persimilis, is used for control of the two-spotted spider mite. Lady beetle, lacewings, or parasitoids such as those from the genus Trichogramma are frequently released in large numbers (inundative release). Recommended release rates for Trichogramma in vegetable or field crops range from 5,000 to 200,000 per acre (1 to 50 per square metre) per week depending on level of pest infestation. Similarly, entomopathegic nematodes are released at rates of millions and even billions per acre for control of certain soil-dwelling insect pests.

The spraying of octopamine analogues (such as 3-FMC) has been suggested as a way to boost the effectiveness of augmentation. Octopamine, regarded as the invertebrate counterpart of dopamine plays a role in activating the insects‟ flight-or-fight response. The idea behind using octopamine analogues to augment biological control is that natural enemies will be more effective in their eradication of the pest, since the pest will be behaving in an unnatural way because its flight-or-fight mechanism has been activated. Octopamine analogues are purported to have two desirable characteristics for this type of application: (1) they affect insects at very low dosages (2) they do not have a physiological effect in humans (or other vertebrates). Figure 20 A turnaround flowerpot, filled with straw to attract Dermaptera species Habitat or environmental manipulation is another form of augmentation. This tactic involves altering the cropping system to augment or enhance the effectiveness of natural enemy. Many adult parasitoids and predators benefit from sources of nectar and the protection provided by refuges such as hedgerows, cover crops, and weedy borders. Also, the provisioning of natural shelters in the form of wooden caskets, boxes or (turnaround) flowerpots is a form of this. For example, the stimulation of the natural predator Dermaptera is done in gardens by hanging up turnaround flowerpots with straw or wood wool. Mixed planting and the provision of flowering borders can increase the diversity of habitats and provide shelter and alternative food sources. They are easily incorporated into home gardens and even small-scale commercial plantings, but are more difficult to accommodate in large-scale crop production. There may also be some conflict with pest control for the large producer because of the difficulty of targeting the pest species and the use of refuges by the pest insects as well as natural enemies. Examples of habitat manipulation include growing flowering plants (pollen and nectar sources) such as Buckwheat near crops to attract and maintain populations of natural enemies. For example, hover fly adults can be attracted to umbelliferous plants in bloom. Biological control experts in California have demonstrated that planting prune trees in grape vineyards provides an improved overwintering habitat or refuge for a key grape pest parasitoid. The prune trees harbor an alternate host for the parasitoid, which could previously overwinter only at great distances from host vineyards. Caution should be used with this tactics because some plants attractive to natural enemies may also be hosts for certain plant diseases, especially plant viruses that could be vectored by

insect pests to the crop. Although the tactic appears to hold much promise, only a few examples have been adequately researched and developed.

E. Integrated with other Control Tactic for Pest Management IPM system is designed around six basic components: 1. Acceptable pest levels: The emphasis is on control, not eradication. IPM holds that wiping an entire pest population is often impossible, and the attempt can be expensive and environmentally unsafe. IPM programmes first work to establish acceptable pest levels, called action thresholds, and apply controls if those thresholds are crossed. These thresholds are pest and site specific, meaning that it may not be acceptable at one site to have a weed such as white clover, but at another site it may not be acceptable. By allowing a pest population to survive at a reasonable threshold, selection pressure is reduced. This stops the pest gaining resistance to chemicals produced by the plant or applied to the crops. If many of the pests are killed then any that have resistance to the chemical will form the genetic basis of the future, more resistant, population. By not killing all the pests there are some unresistant pests left that will dilute any resistant genes that appear. 2. Preventive cultural practices: Selecting varieties best for local growing conditions, and maintaining healthy crops, is the first line of defense, together with plant quarantine and „cultural techniques‟ such as crop sanitation (e.g. removal of diseased plants to prevent spread of infection). 3. Monitoring: Regular observation is the cornerstone of IPM. Observation is broken into two steps first; inspection and second, identification. Visual inspection, insect and spore traps, and other measurement methods and monitoring tools are used to monitor pest levels. Accurate pest identification is critical to a successful IPM program. Records-keeping is essential, as is a thorough knowledge of the behavior and reproductive cycles of target pests. Since insects are cold-blooded, their physical development cycles modeled in of degree days. Monitor the degree days of environment to determine when is the optimal time for specific insect‟s outbreak. 4. Mechanical controls: Should a pest reach an unacceptable level, mechanical methods are the first options to consider. They include

simple hand-picking, erecting insect barriers, using traps, vacuuming and tillage to disrupt breeding. 5. Biological controls: Natural biological processes and materials can provide control, with minimal environmental impact, and often of low cost. The main focus here is promoting beneficial insects that eat target pests. Biological insecticides, derived from naturally occurring microorganisms (e.g.: nicotine, pyrethrum and insect juvenile hormone analogues), but the toxophore or active component may be altered to provide increased biological activity or stability. Further „biology-based‟ or „ecological‟ techniques are under evaluation. An IPM regime can be quite simple or sophisticated. Historically, the main focus of IPM programmes was on agricultural insect pests. Although originally developed for agricultural pest management, IPM programmes are now developed to encom diseases, weeds, and other pests that interfere with the management objectives of sites such as residential and commercial structures, lawn and turf areas, and home and community gardens.

Process IPM is applicable to all types of agriculture and sites such as residential and commercial structures, lawn and turf areas, and home and community gardens. Reliance on knowledge, experience, observation, and integration of multiple techniques makes IPM a perfect fit for organic farming (sans artificial pesticide application). For large-scale, chemical-based farms, IPM can reduce human and environmental exposure to hazardous chemicals, and potentially lower overall costs of pesticide application material and labor. 1. Proper identification of pest – What is it? Cases of mistaken identity may result in ineffective actions. If plant damage to over-watering are mistaken for fungal infection, spray costs can be incurred, and the plant is no better off. 2. Learn pest and host life cycle and biology. At the time you see a pest, it may be too late to do much about it except maybe spray with a pesticide. Often, there is another stage of the life cycle that is susceptible to preventative actions. For example, weeds producing from last year‟s seed can be prevented with mulches. Also, learning what pest needs to survive allows you to remove these.

3. Monitor or sample environment for pest population – How many are here? Preventative actions must be taken at the correct time if they are to be effective. For this reason, once the pest is correctly identified, monitoring must begin before it becomes a problem. For example, in school cafeterias where roaches may be expected to appear, sticky traps are set out before school starts. Traps are checked at regular intervals so populations can be monitored and controlled before they get out of hand. Some factors to consider and monitor include: Is the pest present/absent? What is the distribution – all over or only in certain spots? Is the pest population increasing or decreasing. 4. Established action threshold (economic, health or aesthetic) – How many are too many? In some cases, a certain number of pests can be tolerated. Soybeans are quite tolerant of defoliation, so if there are few caterpillars in the field and their population is not increasing dramatically, there is not necessarily any action necessary. Conversely, there is a point at which action must be taken to control cost. For the farmer, that point is the one at which the cost of damage by the pest is more than the cost of control. This is an economic threshold. Tolerance of pests varies also by whether or not they are a health hazard (low tolerance) or merely a cosmetic damage (high tolerance in a noncommercial situation). Different sites may also have varying requirements based on specific areas. White clover may be perfectly acceptable on the sides of a tee box on a golf course, but unacceptable in the fairway where it could cause confusion in the field of play. 5. Choose an appropriate combination of management tactics for any pest situation, there will be several options to consider. Options include mechanical or physical control, cultural controls, biological controls and chemicals controls. Mechanical or physical controls include picking pests off plant, or using netting or other material to exclude pests such as birds from grapes or rodents from structures. Cultural controlsinclude keeping an area free of conductive conditions by removing or storing waste properly, removing diseased areas of plants properly. Biological controls can be either through conservation of natural predators or augmentation of natural predators. Augmentative control includes the introduction of naturally occurring predators at either an inundative or inoculative level. An inundative release would be one that seeks to inundative a site with a pest‟s predator to impact the pest population. An inoculative release would be a smaller number of pest predators to supplement the natural

population and provide ongoing control. Chemical controls would include horticultural oils or the application of pesticides such as insecticides and herbicides. A Green Pest Management IPM program would use pesticides derived from plants, such as botanicals, or other naturally occurring materials. 6. Evaluate results – How did it work? Evaluation is often one of the most important steps. This is the process to review an IPM program and the results it generated. Asking the following questions is useful: Did actions have the desired effect? Was the pest prevented or managed to farmer satisfaction? Was the method itself satisfactory? Were there any unintended side effects? What can be done in the future for this pest situation? Understanding the effectiveness of the IPM program allows the site manager to make modifications to the IPM plan prior to pests reaching the action threshold and requiring action again.

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