A STUDY OF THE PARASITISM EFFICIENCY OF VESTALIS

(HALIDAY) ON , PLUTELLA XYLOSTELLA (L.),

(LEPIDOPTERA: PLUTELLIDAE)

BY

GILSON CHIPABIKA

UNIVERSITY OF ZAMBIA

LUSAKA

2014

A STUDY OF THE PARASITISM EFFICIENCY OF

(HALIDAY) ON DIAMONDBACK MOTH, PLUTELLA XYLOSTELLA (L.),

(LEPIDOPTERA: PLUTELLIDAE)

BY

GILSON CHIPABIKA

DIP., UZ, CAC, Zimbabwe, BSc., UNZA, Zambia.

A DISSERTATION SUBMITTED TO UNIVERSITY OF ZAMBIA IN

PARTIAL FULFILMENT OF THE REQUIREMENT OF THE DEGREE OF

MASTER OF SCIENCE IN AGRONOMY (PLANT SCIENCE –

ENTOMOLOGY)

ii

UNIVERSITY OF ZAMBIA

LUSAKA - ZAMBIA

2014

DECLARATION

I, GILSON CHIPABIKA, wish to declare that this work is of my own origin of my studies and has never been submitted for a degree anywhere else.

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Signature

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Date

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APPROVAL

The dissertation of GILSON CHIPABIKA presented is approved as fulfilling part of the requirements for the award of the degree of Masters of Science in Agronomy

(Plant Science - Entomology) of the University of Zambia.

Name and signature of Examiners Date

1. Name: ------

Signature: ------

2. Name: ------

Signature: ------

3. Name: ------

Signature: ------

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DEDICATION

I dedicate this dissertation to the Chipabika family for their splendid support during my studies and to the memory of my late sisters, Mary and Loveness.

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ABSTRACT

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) is an pest of cruciferous crops in the world including Zambia. It causes 52 – 92% loss in marketable produce in India. Control of P. xylostella has mainly depended on use of . However, the pest has developed resistance to pesticides used against it. One option is biological control. In Zambia, an attempt to control the pest using biological control agents was done between 1977 and 1984 when Cotesia vestalis and Diadromus collaris from Pakistan were released together with indigenous Oomyzus sokolowskii. The results were not successful probably because Cotesia vestalis was not efficient hence the motivation to investigate the parasitism efficiency of C. vestalis on P. xylostella under laboratory conditions.

Colonies of P. xylostella and C. vestalis were established from larvae of P. xylostella collected from cabbage fields in Chilanga, Kafue and Lusaka districts. Completely randomized design experiment with three replications was set up in the laboratory.

Plutella xylostella were exposed to C. vestalis at 24, 48, 72, 96 and 120 hours after emergence of C. vestalis at ratio levels of 2:1 (P2) and 4:1 (P4). In this study, the field parasitism percentage was 34.85 and contributed 73% to total larval mortality.

In the laboratory, parasitism rates of 55% and 50% were observed in the first 24 hrs of exposure at P2 and P4 levels respectively from this study. There was significant difference in Searching efficiency of (p < 0.01) with exposure time in treatment ratio P2. Significant difference in damage score were observed to cabbage plants treatment levels P2 (p < 0.001) and P4 (p < 0.003). There was negative correlation between % parasitism of P. xylostella and damage score of cabbage. As parasitism increased from 0 in the control to 55% and 50% in treatment ratios P2 and

P4 respectively, damage score decreased from 6 in the control to 1 and 2.5 in

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Treatments ratios P2 and P4 in that order. These results therefore, show that C. vestalis has the potential to be used as a biological control agent against P. xylostella in cruciferous crops in Zambia. Parasitism efficiency needs to be investigated under field conditions and where insecticides were not applied.

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TABLE OF CONTENTS

Title Page

DECLARATION ...... iii APPROVAL ...... iv DEDICATION ...... v ABSTRACT ...... vi LIST OF TABLES ...... x LIST OF FIGURES ...... xi LIST OF APPENDICES ...... xii CHAPTER 1. INTRODUCTION ...... 1 1.1 Background ...... 1

1.2 Significance of study...... 4

1.3.1 Specific objectives ...... 6 1.3.2 Hypotheses tested in the study ...... 6 CHAPTER 2. LITERATURE REVIEW ...... 7 2.1 of Plutella xylostella ...... 7 2.2 Origin and distribution of Plutella xylostella ...... 8 2.4 Economic importance of Plutella xylostella ...... 9 2.5 Management of Plutella xylostella...... 10 2.5.1 Use of insecticides ...... 10 2.5.3 Biology of Cotesia vestalis ...... 12 2.5.4 Searching efficiency of Cotesia vestalis ...... 13 2.5.6 Biology of Diadromus collaris ...... 15 2.5.7 Parasitism percentage of Cotesia vestalis ...... 16 2.5.8 Sex ratio of parasitoids ...... 17 2.5.9 Leaf damage by P. xylostella ...... 18 CHAPTER 3. MATERIALS AND METHODS ...... 20 3.1 Experimental site ...... 20 3.3 Collection of Plutella xylostella and Cotesia vestalis ...... 22 3.4 Rearing of Plutella xylostella and Cotesia vestalis ...... 24 3.5 Treatments and Experimental design ...... 25

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3.6 Parasitism efficiency of Cotesia vestalis...... 26 3.7 Measurements of Parasitism efficiency ...... 27 3.7.1 Parasitism percentage ...... 27 3.7.2 Searching efficiency ...... 28 3.7.3 Killing Power ...... 29 3.7.4 Sex ratio ...... 29 3.8 Leaf damage ...... 29 3.9 Data analysis ...... 30 CHAPTER 4. RESULTS ...... 32 4.1 Collection of Plutella xylostella larvae from farms ...... 32 4.2 Emergence of Cotesia vestalis from Plutella xylostella collected in farms ...... 32 4.3 Parasitism percentage ...... 34 4.4 Searching Efficiency ...... 35 4.5 Sex ratio of Cotesia vestalis ...... 36 4.6 Leaf damage ...... 37 CHAPTER 5. DISCUSSION ...... 39 5.1 Collection of Plutella xylostella and Cotesia vestalis ...... 39 5.2 Parasitism percentages of Plutella xylostella exposed to Cotesia vestalis at different time intervals ...... 41 5.3 Searching Efficiency ...... 43 5.5 Sex ratio of parasitoids ...... 44 6.1 Conclusion ...... 49 6.1.1 Determination of Parasitism percentage, Searching efficiency, Killing power and Sex ratio of parasitoids at two levels of parasitoid/ host densities.. 49 REFERENCES ...... 50 Appendices ...... 69

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LIST OF TABLES

Table 1. Types of natural enemies for Plutella xylostella...... 4

Table 2. Insecticides farmers use to control Plutella xylostella in Chilanga, Lusaka

and Kafue districts...... 22

Table 3. Treatments and Experiment layout in the laboratory...... 26

Table 4. Level of parasitism of Plutella xylostella collected from ten farms of

Chilanga, Lusaka and Kafue districts ...... 33

Table 5. The effect of time on two levels of female parasitoid densities on the

Searching efficiency and Killing power of Cotesia vestalis ...... 36

Table 6. Sex ratio of Cotesia vestalis at five constant exposure period of time in the

laboratory...... 37

Table 7. Mean damage score per head of cabbage under screen house conditions....38

Table 8. Effect of parasitism on mean leaf damage score per head of cabbage ...... 38

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LIST OF FIGURES

Figure 1: Forewing venation of Cotesia vestalis…………………………………....12

Figure 2: Hind leg of Cotesia vestalis ………………………………………………12

Figure 3: Rearing cage side view……………………………………………………20

Figure 4: Rearing cage rear view...... 20

Figure 5: Cabbage seedlings in screen house...... 21

Figure 6: Map of Chilanga, Lusaka and Kafue districts showing sites where Plutella

xylostella and Cotesia vestalis were collected...... 23

Figure 7: Exposure cages...... 27

Figure 8: Cabbage damaged by Plutella xylostella...... 31

Figure 9: Emergence of Cotesia vestlis from field collected Plutella xylostella...... 32

Figure 10: Emergence of Cotesia vestalis from Plutella xylostella collected at CQ

and Miyoba farms...... 34

Figure 11: Effect of age on Cotesia vestalis to Parasitism percentages of Plutella

xylostella...... 35

Figure 12: Left, damage by parasitized Plutella xylostella larvae in the cage...... 47

Right, damage by un parasitized Plutella xylostella larvae in the cage…48

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LIST OF APPENDICES

Appendix 1.0 Parasitism percentage ...... 71

1.1 Analysis of variance ratio1:2 (P2) ...... 71

1.2 Tables of contrasts ...... 71

1.3 Parasitism% ratio 1:4 (P4) ...... 72

1.3.1 Tables of contrasts ...... 72

Appendix 2.0 Searching efficiency ...... 73

2.1 Analysis of variance ratio 1:2 (P2) ...... 73 3.0 Regression analysis for % parasitism in relation to damage searching efficiency

and killing power ...... 74

4.0 Searching efficiency ...... 74

4.1Analysis of variance ratio 1: 4 (P4) ...... 74

5.0 Variate: Killing power Ratio 1:2 (P2) ...... 75

5.1 Analysis of variance ...... 75

5.2 Variate: Killing power ratio 1:4 (P4) ...... 75

5.2.1 Analysis of variance ...... 75

6.0 Damage score Ratio 1:2 (P2) ...... 76

6.1 Analysis of variance ...... 76

7.0 Regression analysis Ratio 1:2 (P2) ...... 77

7.1 Response variate: Damage...... 77

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ACKNOWLEDGEMENT

I am profoundly grateful to my supervisors Dr. Philemon H. Sohati and Dr. Felix S.

Mwangala for their guidance not only during my research work but when writing the dissertation. I am also indebted to Mt. Makulu Research Management especially the

Entomology Team for allowing me to use some the facilities during my Research. I am thankful to the Department of Plant Science for availing me an opportunity to undertake this programme at University of Zambia. Special thanks go to Organic

Agriculture Development in Sub – Saharan Africa (ODISSA) for sponsoring this

Research.

My heartfelt thanks go to all my relatives and friends who assisted me in one way or another for their dedicated and tireless physical support and moral encouragement during my studies.

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CHAPTER 1. INTRODUCTION

1.1 Background

The diamondback moth, Plutella xylostella L. (Lepidoptera: Plutellidae), also known as cabbage moth, is one of the most important cosmopolitan insect pests of Cruciferous crops in the world (Talekar and Shelton, 1993) including Zambia (Mingochi et al. 1995).

Host plants include both cultivated and wild plants of the family Brassicaceae, as well as several ornamentals such as wallflower, candytuft, stocks, and alyssum (Fleischer, 2003;

Mau and Kessing, 2007). Cultivated crops that are attacked include Broccoli, Brussels

Sprouts, Cauliflower, Chinese cabbage and Common cabbage. Weed hosts, such as mustard and radish, are important reservoir hosts for the . Plutella xylostella is a cosmopolitan pest that is thought to have originated in the Mediterranean region (Mau and Kessing, 2007). This pest is widely distributed and is found in North America, the southern portion of South America, southern Africa, Europe, India, South East Asia, New

Zealand, and parts of Australia (Hardy, 1938). Since P. xylostella is a well distributed cosmopolitan pest (Hill, 1975), it is able to survive in diverse climatic conditions that are found in different parts of the World. Plutella xylostella attacks the crop from the nursery stage until maturity. In certain parts of India, it causes as high losses as 52 to 90% of marketable yield (Kumar et al. 1986; Verkerk and Wright, 1996; Oke, 2008). The last stage is a voracious feeder; it causes more injury than the first three larval instars

(Mau and Kessing, 2007).

Application of insecticides to control P. xylostella on crucifers is the most common method used among small scale farmers. Application to plant foliage may be as frequently as twice per week.

However, the use of insecticides to control P. xylostella is a serious problem in terms of pest resistance. This is not only in sub – Saharan Africa (Kibata, 1997; Sereda et al.

1997) but also in the temperate climate (Talekar and Yang, 1993).

Insecticides have adverse effects on the environment and none targeted organisms. The application of insecticides in most cases is not quite precise and none target organisms get exposed in the area where they are applied and even kill beneficial organisms such as bees. Humans, livestock and indeed other organisms suffer the harmful effects of insecticides (Lah, 2011). Insecticides effects on humans include damage to the nervous system, reproductive system and other organs, development of abnormalities, disruption of hormone functions as well as immune dysfunction (Purezing, 2007). Research has found that thirty out of thirty seven crop chemicals, mostly fungicides such as dimethomorph and imazalil as well as like pirimiphos-methyl and λ- cyhalothrin interfere with the action of testosterone (Orton et al. 2011; Macrae, 2013).

Natural enemies respond differently to different classes of insecticides. Mortality of C. vestalis is observed when exposed to different insecticides. Miyata et al. (2001) reported that mortality of C. vestalis exposed to different insecticides was 58 to 100% within 48 hours after application. However, parasitism by surviving adults was also seriously affected. Over use of insecticides cause contamination of soil, water and vegetables

(Rowell, 2000).

However, there are other control alternatives like cultural method that are being practiced by farmers to control P. xylostella. Intercropping with other crops such as onion (Allium cepa L.) that have repellent properties reduce damage on cabbage crop (Beyene, 2007).

Farmers also use trap crops such as the Indian mustard which is highly preferred by P. xylostella. Indian mustard is grown as a trap crop in the cabbage field as it is more 2 attractive to P. xylostella than Cabbage (Charleston and Kfir, 2000; Hasheela et al. 2010).

This type of protection is cheaper than the use of insecticides. Some farmers use irrigation to reduce the effect of P. xylostella on cabbage. Over head irrigation of cabbage reduces the P. xylostella infestation by 37.5 to 63.9% and best results are achieved by intermittent daily application between 20.00 hours and 23.30 hours (McHugh Jr and

Foster, 1995). This type of irrigation disturbs moths from laying eggs on the leaves of cabbage and when a lot of water is applied in the field, first to second instars of P. xylostella larvae drown and die (Diez, 2007). Farmers also use time of planting as a control strategy. Cabbage planted in the rain season is usually less damaged by P. xylostella because a large proportion of young larvae are killed by rainfall and consequently fewer larvae will develop to adults that will deposit eggs (Capinera, 2012).

Equally the cabbage that is planted in the cold season is not heavily damaged because the rate of development is reduced.

Other farmers use botanical pesticides that have insecticidal properties. Extracts from trees such as the neem, Azadirachta indica and syringa, Melia azadirachta have been found to give control to P. xylostella (Charleston, 2013). Water extracts of both plants have proved to be most effective even at low concentrations of 12.5 g of seed kernels per liter of water (Schmutterer, 1992). Another alternative control method of P. xylostella is by use of biological control agents and this is through introductions and augmentation or inundations. Governments through national and international institutions conduct this activity. Natural enemies of different types have been released in different countries to control P. xylostella at different growth stages and some of them are shown in (Table 1).

Best and Beegle (1977) and Clark et al. (1994) pointed out that predaceous ability in a laboratory does not exactly reflect suppressive effect on pests in the fields and therefore predators have not been found to be effective in the field. 3

Table 1. Types of natural enemies for Plutella xylostella.

Parasitoid species host stage attacked life style

Diadegma larval solitary

Microplitis larval solitary

Cotesia larval solitary

Oomyzus larval gregarious

Diadromus larval solitary

Entomopathogens

Bacteria ( (Bt)) larval

Fungi (Isaria fumosorosea) larval

Viruses (Ascovirus HvAV-3e) larval

Predators

Carabid beetles (Coleoptera: Carabidae) eggs and larval

Ladybird beetles (Coleoptera: Coccinellidae) eggs and larval

Syrphid flies larva (Diptera: Syrphidae) eggs and larval

Lacewings (Neuroptera: Chrysopidae) larval

Rove beetles (Coleoptera: Staphylinidae) larval

Spiders (Araneae) Larval

(Larson, 2007; Canola Council of Canada, 2010; Dosdall et al. 2011)

1.2 Significance of study. Plutella xylostella is considered a major pest of cruciferous crops in all countries of eastern and southern African region (Nyambo and Pekke, 1995), Zambia inclusive

(Mingochi et al. 1995). Management of the pest had dependent heavily on insecticide 4 applications that have since proved futile as the pest has developed resistance to most of the insecticides (Miyata et al. 2001; Shelton and Wyman, 1992). The alternative control method was by introduction of different species of parasitoids of P. xylostella that have been released in countries such as Kenya, Uganda, South Africa, Benin and others.

Introduction of semiclausum from Asian Vegetable Research and

Development Center, Taiwan in 2001 to East Africa led to successful establishment, spread and 50% reduction in cabbage damage in Kenya, Uganda and parts of Tanzania

(Gichini et al. 2008). Consequently 50% of farmers also stopped using insecticides (Löhr et al. 2007). In Jamaica, the reintroduction of Cotesia vestalis in 1989 resulted into an increase in parasitism from 5.4 to 88.7% and plant damage also reduced from 75% before introduction to 38% after one year (Alam, 1992). In South Africa, C. vestalis released in

1999 at St Helena, up to 80% P. xylostella larval parasitism was recorded and infestations remained so low that no chemical control was needed (Kfir and Thomas, 2001) and is now the most efficient parasitoid often accounting for more than 90% of total parasitism

(Kfir, 2003).

In Zambia, despite the introduction of biological control agents C. vestalis and D. collaris from Pakistan that were introduced in Chilanga, Kafue and Lusaka districts between 1977 and 1984 to reduce the pest problem (Mingochi et al. 1995), the density of P. xylostella and damage levels are still high (Sohati, 2012). From the above stated evidence therefore, the study was important to understand why the two biological control agents have not been effective in Zambia.

1.3 Aim and objective of the study

The aim of the study was to establish the parasitism efficiency of C. vestalis on P. xylostella under laboratory conditions. 5

1.3.1 Specific objectives

This study addressed the following specific objectives:

 To determine Parasitism percentage, Searching efficiency, Killing power

and Sex ratio of parasitoids at two levels of parasitoid/ host densities.

 To evaluate the impact of C. vestalis on P. xylostella in reducing cabbage

damage.

1.3.2 Hypotheses tested in the study

 Cotesia vestalis has high Parasitism percentage, Searching efficiency,

Killing power at two levels of parasitoid/ host densities.

 Cotesia vestalis has an impact on P. xylostella and consequently reduction

in cabbage damage.

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CHAPTER 2. LITERATURE REVIEW

2.1 Taxonomy of Plutella xylostella

Powell and Opler (2009) and Shaw, (2003) classified P. xylostella as:

Kingdom - Animalia

Phylum - Arthropoda

Class - Insecta

Order - Lepidoptera

Super family - Yponomeutoidea

Family - Plutellidae

Genus - Plutella

Species - xylostella

Binomial name - Plutella xylostella (L.)

Common name - Diamondback Moth

The adult diamondback moth is a small gray or brown moth about 12 to 15 mm in length.

At rest, wings are folded rooflike over its body. Light tan marks can be seen on the margin of the forewing. Male moths have three diamond-shaped markings on the back of the forewing when the wings are folded together, hence the name diamondback moth.

Wing tips are fringed with long hairs (Knodel and Ganehiarachchi, 2008). Wingspan of adult P. xylostella is from 12-15 mm (Covell, 1984). Forewing length, range from 5.5-7.5 mm (Powell and Opler, 2009).

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2.2 Origin and distribution of Plutella xylostella

Plutella xylostella is a destructive pest of Brassicaceous crops in the world. It is a cosmopolitan species that probably originated in the Mediterranean region (Talekar and

Shelton, 1993). It is found over much of North America, the southern portion of South

America, southern Africa, Europe, India, South East Asia, New Zealand, and parts of

Australia (Hardy, 1938; Diez, 2007).

2.3 Biology of Plutella xylostella

The life cycle of the pest takes about 32 days to develop from egg to adult under optimum or natural condition of 20 to 22oC (Henry and Baker, 2008; Oke, 2008). Eggs are laid singly or in groups of 2 to 8 on the upper or undersides of the leaves. They hatch in 2 to 8 days and there are 4 larval stages (instars). The larval period varies from 6 to 30 days depending on temperature (Oke, 2008) and the pupa period averages 8 days.

Females live from 7 to 47 days, averaging 16.2; and males live from 3 to 58 days, averaging 12.1. The number of eggs produced by a single female is influenced by photoperiod, temperature, age and quality of food taken as larvae (Harcourt, 1957).

At 27oC, there could be as many as 30 generations per year (Ho, 1965). However, time to complete a generation may vary from 21 – 51 days depending on weather and food conditions (FAQ, 2012) and there are several generations per growing season depending on temperature, being quicker in warm weather and slower in cool weather (Henry and

Baker, 2008). The number of eggs laid per female may range from 18 to 356, the mean being 159. Oviposition normally begins on the day of emergence lasting about 10 days, the peak occurring on the first night of oviposition except when temperature is below

19oC (Harcourt, 1957) at sunset. The number of eggs produced by a single female is influenced by photoperiod, temperature, and age or condition of larval food (Harcourt,

1957). The P. xylostella populations from different areas may produce different number 8 of eggs, those of Cotonou, Parakou, Malanville, Natitingou and Songhai in Benin produced on average 210 eggs/female. The least productive were the populations from

Agoué with 115 eggs/female and Lokossa with 170 eggs/female (Arvanitakis et al. 2002).

2.4 Economic importance of Plutella xylostella

Plutella xylostella greatly reduces the yield and quality of the crop and accounts for the majority of insecticide use in crucifer production (Diaz-Gomez et al. 2000). If no sprays were applied for control of P. xylostella, it would be reasonable to conclude that all plants would be unmarketable. Losses of cabbage and cauliflower due to the moth can reach

90% without the use of insecticides (Uijtewaal, 2006). In China, when no sprays were applied to control P. xylostella, the losses of summer cabbage in Shanghai were 99% in

1992 and 80% in 1994. In Texas, it has been suggested that 100% of cabbage and at least

20% of broccoli would be unmarketable if it were not treated with insecticide (Shelton,

2001). In Africa, Ghana’s cabbage fields many times were lost due to P. xylostella damage and farmers abandoned their farms (Cobblah, unpublished). In Ethiopia, losses have been reported to reach 91.2 percent (Ayalew, 2006) and 70 percent (Webb, 2012) in the absence of control measure respectively. In Zambia, P. xylostella may cause losses of up to 70 percent in the absence of control (D. Mingochi, personal communication, 2013).

Most of the crop damage by P. xylostella is caused during the hot dry months from July to the onset of rains, generally in October or November (Ahmed, 1977).

The cost of chemical control is one billion US dollars per year worldwide (Javier, 1992;

Talekar and Shelton, 1993). In India, costing up to 168 US$ per year in damage and control have been recorded (Sander, 2004). In the United States and Canada, estimated cost of Canadian $50 million was spent to control P. xylostella (Dosdall et al. 2004). In

California a severe infestation by P. xylostella in 1997 resulted in crop losses estimated to

9 be greater than $6 million (Shelton et al. 2000). In Australia, crop loss due to P. xylostella damage in an average year was estimated to be ca. $A 8 million and control costs $A 12 million (Shelton, 2001). As a consequence, farmers tend to apply insecticides frequently and indiscriminately to minimize the damage and the loss (Orr et al. 1999).

2.5 Management of Plutella xylostella

2.5.1 Use of insecticides

Management of P. xylostella by farmers in most cases is by use of insecticides. However, over use of broad spectrum insecticides for P. xylostella control has obscured the contribution of indigenous parasitoids (Rowell et al. 2005) and some populations of P. xylostella have developed resistance to almost all the insecticides used against them. The development of resistance is a serious problem not only in the tropics but also in temperate climates (Talekar and Yang, 1993), including sub-Saharan Africa (Kibata,

1997; Sereda et al. 1997; Mota- Sanchez et al. 2002; Sarfraz and Keddie, 2005).

2.5.2 Biological control of Plutella xylostella

Successful integrated pest management programmes are more dependent on investigation and knowledge about the biology of the pest, natural enemies and their interactions with host plants (Sequeira and Dixon, 1996; Awmack and Leather, 2002).

Among the control methods of P. xylostella is the use of biological control agents such as

C. vestalis, O. sokolowskii, D. collaris and D. semiclausum. Cotesia vestalis, a solitary endoparasitoid of P. xylostella larvae takes 10 – 15 days to develop from egg to adult at temperature of 25 – 35oC. Cartwright et al. (1987) and Delahaut (2004) found the level of

0.30 P. xylostella larvae per plant at cupping as useful in pest control in commercial cabbage field as this number is a threshold that triggers control measure to be implemented. In the presence of effective parasitoids, parasitized larvae do not cause

10 much damage to cabbage. Choh et al. (2008) observed that leaf area consumed by parasitized larvae was significantly lower than that by non-parasitized larvae

2.5.2.1 Origin and distribution of Cotesia vestalis

Cotesia vestalis is one of the most important biological control agents of P. xylostella in the world (Rowell et al. 2005) and is probably the most widely distributed solitary larval endoparasitoid of P. xylostella. It is believed to have originated from Europe and later spread throughout the world with P. xylostella (Lim, 1992; Waterhouse and Norris, 1987;

Talekar and Shelton, 1993). It has been recorded in the Philippines and former U.S.S.R.

(Telenga, 1964), China (Liu et al. 2000), Malaysia (Ooi, 1980), Taiwan (Talekar and

Yang, 1991), Thailand and United States (Waterhouse and Norris, 1987; Rattanayat,

1998; Talekar, 1997), Pakistan (Mustaque and Mohyuddin, 1987; Shi et al. 2002), India

(Simmonds, 1971; Waterhouse and Norris, 1987) and South Africa (Kfir, 1997).

2.5.2.2 Taxonomy of Cotesia vestalis

Shaw (2003) and Myers et al. (2013) classified C. vestalis as:

Kingdom - Animalia

Phylum - Arthropoda

Class - Insecta

Order -

Super family - Braconidea

Family -

Genus - Cotesia

Species - vestalis

Binomial name - Cotesia vestalis (Haliday)

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The length of the adult body of C. vestalis is less than 3 mm. The antennae of the female is long reaching apex of metasoma. The forewing of C. vestalis (Figure 1) is not smoky over most of the area.

The leg (Figure 2) has a hind femur that is mostly yellowish to orangish sometimes darker base. Hind coxa (Figure 2) is usually dark brown to black and slightly paler at the apical (Shaw, 2003).

nbaii.res.in nbaii.res.in

Figure 1: Fore wing venation of Cotesia Vestalis Figure 2: Hind leg of Cotesia vestalis

2.5.3 Biology of Cotesia vestalis

Life stages of the C. vestalis include egg, three larval instars, pupa and the adult. In long- term oviposition trials, of C vestalis females laid eggs on P. xylostella larvae for up to 10 days (Alizadeh et al. 2011). Larval development of the parasitoid in host only required

6.47 days: the first instar larva required 3.25 ± 0.047 days; the second instar larva needed

2.78 ± 0.1 days and the third instar larvae exited the host and pupated in, 0.4 ± 0.07 days

(Alizadeh et al. 2011). Pre-pupa and pupa period of were 1.9 ± 0.06 and 2.13 ± 0.09 days respectively.

Unmated female and male longevity of wasp were 16.83 ± 0.37 and 16.25 ± 0.17 respectively and sex ratio is male-biased. When a mixed group of instars were presented to parasitoids, the 2nd and 3rd instars larvae were preferred and the 4th instar was less attractive for selection (Alizadeh et al. 2011).

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2.5.4 Searching efficiency of Cotesia vestalis

Searching efficiency of a parasitoid is the ability of a female parasitoid to find a host and parasitize it. This is the phenomena of nature for them to perpetuate their survival and reproduce the next generation (Powell and Poppy, 2001). It is through searching that the parasitoid will be judged for its efficiency to regulate the pest population. Hassell and

Rogers (1972) stated that the searching efficiency of parasitoid is affected by its response to pest density, its response to other parasitoids and distribution of searching females in relation to host distribution. According to other studies, Alphen and Jervis (1996) stated that oviposition rate is reduced per female as a result of competition that emanates from low density of the host. The high parasitoid densities have influences on sex ratio, brood, increased rate of dispersal of females and decision on super parasitism on the host

(Hassell, 1978; Visser et al. 1999).

In searching efficiency study of C. vestalis, the required number of females is kept together with test number of P. xylostella in an experimental cage. Twenty four hours later, both the parasitoids and hosts are removed but the later (if larvae) is further reared to the pupa stage on host plant. Pupae are kept until the emergence of the adult parasitoids. Experiments in the laboratory are conducted at room temperature of 25 -

27°C (Alizadeh et al. 2011).

The area of discovery, which is a measure of searching efficiency, is calculated using the formula by Hassell (1971): a = 1/ p log Ce Ni/ Nr

Where:

a = Area of discovery,

Ni = Initial host density,

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Nr = final (un parasitized host densities),

p = parasitoid density, and

Ce = exponential.

The relationship between a and the density of parasitoids searching for hosts can be described by the formula (Hassell and Varley, 1969):

a = QP - m

Where

a = Area of discovery

Q = is the quest constant or the level of efficiency of one parasitoid

- m = the interference constant between searching parasitoids

when expressed in logarithms, the equation becomes linear:

log a = log Q - m log P

The killing power of the parasitoid, K, is calculated by using formula:

K= log Ni/Nr

Where:

Ni = initial host density, and

Nr = final unparasitized host density

2.5.5 Taxonomy of Diadromus collaris

Myers, P. et al. (2014) classified D. collaris as:

Kingdom - Animalia

Phylum - Arthropoda

Class - Insecta

Order - Hymenoptera

14

Super family - Inchneumonoidea

Family - Inchneumonidea

Genus - Diadromus

Species - collaris

Binomial name - Diadromus collaris (N)

It is an endo parasitoid of P. xylostella. It is a small slender wasp with body length of 6 mm. The head is black with large eyes and the antenna is 4 mm long. The thorax is robust and black, both wings clear with solid triangular sector mid-anterior edge and brown veins. The abdomen is anterior ascending, short, black and posterior long, cylindrical, light brown with some black rings or segments. The legs are yellowish to yellow-brown.

It differs from other similar species in having rather cylindrical abdomen (McCormack,

2007).

2.5.6 Biology of Diadromus collaris

Diadromus collaris deposits its eggs only in P. xylostella cocoons. Its life cycle is 15 days from egg to adult emergence (Lui et al. 2000). The development rate of the parasitoid increases linearly with temperature from 15 to 30oC (Lui et al. 2000). Rates of survival from larva to adulthood are about 90% between 20 and 28oC and decreases as temperature decreases or increases. No immature survive to adulthood at 35oC. When provided with honey solution, the females live on average 7.0 and 11.5 days (Lui et al.

2000). Unmated females produce male progeny only whilst mated females produce progeny of both sexes (Lui et al. 2001; Rowell et al. 2005). When the temperature is as low as 20oC the parasitism percentage is 26 percent but as the temperature rises the parasitism also increases. At 30oC the parasitism percentage increases up to 46% (Lui et

15 al. 2000) and beyond that the parasitism percentage declines. Successful development and function parasitoids occur at temperatures that stray into regions of intermediate temperature conditions. Thus temperature is an important driving force of insect population metabolism (Bommarco, 2001). For each insect species, there are threshold temperatures where no development occurs if temperatures are above or below. The are inactive at cold temperature, active at median temperature and inactive at hot temperature (Jervis and Copland, 1996). Within this temperature range, activity and developmental rate increases with temperature and levels off at the optimum. Thus the relationship between temperature and development is linear at intermediate temperatures

(Jervis and Copland, 1996; Roy et al. 2002).

2.5.7 Parasitism percentage of Cotesia vestalis

Parasitism is a symbiotic relationship in which one organism lives in or on another organism (the host) and consequently harms the hosts while it benefits (de la Torre-

Bueno, 1985). Successful parasitism is an important parameter as it gives an estimate of the number of individual parasitoid entering the subsequent generation (Jervis and

Copland, 1996). Alizadeh et al. (2011) indicated that C. vestalis females oviposited on the third instar larvae of P. xylostella for up to 10 days, but high proportion of eggs were laid on the first three days in choice experiment, the percentage parasitism of 2nd, 3rd and

4th instars was 78.58, 69.94 and 4.36%, respectively. This rapid oviposition rate increases the potential of the parasitoid to biologically suppress P. xylostella populations because the likelihood of mortality for the parasitoids due to exposure to detrimental environmental factors or generalist predators increases with time (Muniappan et al. 2004;

Alizadeh et al. 2011).

16

Some attempts at classical biological control of P. xylostella in Africa are as follows:

South Africa in 1936 with the introduction of D. semiclausum from England (Evans,

1939), Zambia in 1977 with introductions of C. vestalis and D. collaris from Pakistan

(Yaseen, 1978), St. Helena island in 1999-2000 with C. vestalis and D. collaris from

South Africa (Kfir and Thomas, 2001) and in Kenya in 2001 with D. semiclausum from

Taiwan (Gichini et al. 2008). The African parasitoids D. mollipla, O. sokolowskii and four minor species were exported to Hawaii in 1953 (Löhr and Kfir, 2004). Highest parasitoid diversity was recorded in South Africa where parasitism rates often reached greater than 90% (Kfir, 2001). It was suggested that in view of parasitoid diversity, P. xylostella may have originated in South Africa, where parasitoids appear to be able to maintain the pest below damage thresholds (Löhr and Kfir, 2004). Parasitoid diversity and effectiveness is much lower in other parts of Africa. In tropical West Africa, the most abundant parasitoid was C. vestalis while O. sokolowskii was predominant in Sahelian countries. In large surveys in East Africa, parasitism rates were below 15% and the most frequent parasitoids were D. mollipla and O. sokolowskii (Löhr and Kfir, 2004).

2.5.8 Sex ratio of parasitoids

Sex ratio is the proportion of adults in a population that are male or female. The sex ratio of parasitoid indicates the power of increase of parasitoid. High C. vestalis densities are known to result in male-biased sex ratios in progeny (Nofemela, 2004). Alizadeh et al.

(2011) found that sex ratio in C. vestails is male-biased. Male-biased sex ratios in progeny at high female densities of some parasitoids are explained through Local Mate

Competition (LMC) theory. The LMC models predict what offspring sex ratio a mother should produce given the numbers of other mothers present (Hamilton, 1967; 1979; King,

1993). If encounters between searching females are more frequent than encounters with males, this situation results in sons being more valuable than daughters and thus there is a 17 strong selection on the mothers to bias their progeny sex ratios towards males (King,

1993).

A mother's ability to pass on her genes to future generations is significantly affected by her offspring sex ratio (proportion of male offspring), and so offspring sex ratio is expected to be under strong selection (Fisher, 1930; Hamilton, 1967; Werren and

Charnov, 1978; Charnov, 1982). The particular sex ratio favored may vary according to environmental conditions; in which case, selection will favor mothers with the ability to manipulate sex ratio in response to current environmental conditions. Two environmental conditions that may affect what sex ratio a mother should produce are:

Amount of resources that will be available to her offspring (Trivers and Willard, 1973;

Charnov, 1979; Charnov et al. 1981) and number of mothers present (Hamilton, 1967).

Haplodiploid female parasitoid are particularly good organisms for testing the theory. Females store sperms in a spermathecal capsule. When an egg is passing through the oviduct and sperms are not released, the egg will pass through unfertilized and develop into a son. This is the same phenomena with Apaonagyrus lopezi (De Santis) where unmated females of arrhenotokous lay only male eggs in host Phenacoccus manihoti (Matile-Ferrero) (Umeh, 1988). In contrast, if a female releases the sperm when an egg is passing through, the egg will be fertilized and a daughter will develop

(Flanders, 1956). Equally, eggs laid by mated A. lopezi females produce males and females in the ratio of 1:3 in favor of females. Thus by controlling egg fertilization, mothers can control their offspring sex ratios.

2.5.9 Leaf damage by P. xylostella

The P. xylostella lays singly or cluster small eggs of two to eight on the underside of leaves of cabbage. When they hatch, the first instars larvae burrow inside the leaves

18

(Talekar and Shelton, 1993) and sometimes feeds in the spongy plant tissue beneath the leaf surface forming shallow mines that appear as numerous white marks. These mines are usually not longer than the length of the body. The larvae are surface feeders in all subsequent stages. These larvae feed on the lower leaf surface 62-78% of the time, chewing irregular patches in the leaves eventually all the leaf tissues are consumed except the veins (Harcourt, 1957). On some leaves, the larvae feed on the upper epidermis creating a "windowing" effect. The last stage larva is a voracious feeder; it causes more injury than the first three larval instars (Mau and Kessing, 2007). Plutella xylostella attacks the crop from the nursery stage onwards and causes 52% - 90% loss in marketable yield in cabbage in parts of India (Kumar et al.1986; Oke, 2008). Sometimes damage to cabbage plants reaches 90% during outbreak of P. xylostella in Southeast Asia, (Verkerk and Wright, 1996. In Zambia, the mere presence of insect damage on vegetables at harvest results in considerable loss of crop marketability. though no monetary loss has been quantified

19

CHAPTER 3. MATERIALS AND METHODS

3.1 Experimental site

This study was conducted at Mount Makulu Research Station in Chilanga situated

15o33’S and 28o15’E at an elevation of 1168.9 m above sea level. The experiment was conducted both in the laboratory and screen house.

The culture of C. vestalis was established from individuals that emerged from parasitized

P. xylostella larvae collected from cabbage fields in Chilanga, Kafue and Lusaka districts. The collections in one litre jars covered with micro nylon mesh were used for transportation of P. xylostella larvae from the field to the laboratory. The cages of dimension 60 x 45 x 45 cm covered with micro mesh on two sides and a glass on the opposite side of the door were used as mating and rearing cages (Figures 3 and 4). On the front part of the cage there was an opening of 25 cm diameter which was covered by sleeve attached to the nylon. This served as an opening for putting and removing plants, insects as well as for irrigating plants.

Figure 3:Rearing cage side view Figure 4: Rearing cage rear view Picture by G. Chipabika Picture by G. Chipabika 20

The other cages of size 30 x 30 x 30 cm and made up of perplex glass and an opening attached with a black sleeve were used for exposing second instars P. xylostella larvae to

C. vestalis in the laboratory. Petri dishes of size 3 (28 cm2) were used to put P. xylostella larvae for exposure to parasitoids. Another one of the same size was topped with cotton wool that contained 10% honey solution that served as source of food for C. vestalis.

3.2 Cabbage plant production

Composted livestock manure was manually admixed with soil at the rate of 2kg/m before seeding following the recommended practice in the Bio-farm at Kasisi (personnal communication, 2012). Watering was done every other day until germination. Four week old seedlings of Riana variety were transplanted into the pots of 20 cm with soil admixed with composted manure at the rate of 200 kg/ha (Figure 5). They were taken for experimental use when they were eight weeks. During experiment, watering and aeration was done every other a day and fortnightly respectively.

Figure 5: Cabbage seedlings in screen house, by G. Chipabika

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3.3 Collection of Plutella xylostella and Cotesia vestalis

Plutella xylostella larvae were collected from farms growing cabbage in Chilanga, Kafue and Lusaka districts (Figure 6) from September to December 2012. The intensive survey was conducted every month with the view to recover parasitoids that were released between 1977 and 1984 in the respective districts. During collections, farmers indicated that P. xylostella was a problem and were using insecticides shown in Table 2.

Table 2. Insecticides farmers use to control Plutella xylostella in Chilanga, Lusaka and

Kafue districts.

Product Chemical Group Chemical group Active Pre harvest

name number Ingredient interval

Malathion organophosphate 1B Malathion 7days

Dimethoate organophosphate 2 Dimethoate 14 days

Decis 5 EC pyrethroids 3A Deltamethrin 7 days

Silencer pyrethroids 3A Lamba- cyhalothrin 7 days

Pyrinex organophosphate 1B Chlorpyrifos 21 days

Cartap organophosphate 4C Cartap Hydrochloride 10 days

22

CHILANGA

DISTRICT LUSAKA 23 DISTRICT

KAFUE DISTRICT

Figure 6: Map of Chilanga, Lusaka and Kafue districts showing sites where Plutella xylostella and Cotesia vestalis were collected

In each field visited, second to fourth instars larvae of P. xylostella were identified and collected using camel hair brush and put into one litre glass jar loaded with a leaf of cabbage. At each collection point the Geographical position system (GPS) reading was recorded and the specimen numbered. The collections were taken to Mt. Makulu Station where the larvae were transferred into rearing cages on 8 – week old potted cabbage plants in the green house where there was enough light for plants to carry out photosynthesis. The larvae were reared until pupation and emergence of adults in the cages. The C. vestalis emerged from parasitized P. xylostella larva whilst un-parasitized larva pupated and emerged as P. xylostella moths. The emerged C. vestalis from P. xylostella larvae from each site were identified by using insect identification keys and references of C. vestalis samples from School of Agricultural Sciences, Department of

Plant Science collections at University of Zambia. The parasitoids were recorded and transferred to rearing cages that had second instars larvae of P. xylostella in the green house and consequently established the colonies for both the host and parasitoids. The parasitism percentages of emerged parasitoid species from larvae of P. xylostella were determined using formula by Russell (1987):

Percent of parasitized parasitoids = ((Number Parasitized)/ (Number Non-Parasitized +

Number Parasitized)) x 100

3.4 Rearing of Plutella xylostella and Cotesia vestalis

In order to obtain the same age group of P. xylostella larvae, 8 week old potted cabbage plants were placed inside oviposition cages of 60 x 45 x 45 cm. Five pairs of newly emerged P. xylostella (1 male: 1 female) were introduced and left for 24 hours. There after all the plants were removed and placed in another cage of the same dimension. This operation continued until the end of the experiment. The eggs that were laid on the leaves were monitored until they hatched. The larvae emerged after 8 days in the screen

24 house under natural environmental conditions. The larvae fed on cabbage leaves until they were second to third instars.

Following the establishment of second to third instars larvae of P. xylostella, pairs of C. vestalis were put into the mating cage for 24 hours and 10% honey solution as source of food was provided through soaked cotton wool that was placed on the Petri dish. The sugar solution provided the insect with the food it required. Twenty four hours later, second/ third instars P. xylostella larvae were introduced and withdrawn after 48hrs. The environmental conditions in the laboratory were maintained at 26 ± 2oC with relative humidity of 65 ± 5%. The photo to dark period was kept at 10: 14 hour this was as described by Talekar and Lin (1998). The removed larvae were reared on new eight week old cabbage plants in the rearing cage of 60 x 45 x 45 cm in the screen house. In the screen house, C. vestalis was reared according to Talekar et al. (1997) with some modifications in that standard constant environment (25±1°C, 65±5% relative humidity and a photoperiod of light 16: dark 8 hours were not used. During rearing of parasitized

P. xylostella larvae, there was no change of food until parasitoid emergence.

3.5 Treatments and Experimental design

From the established colonies of P. xylostella and C. vestalis the experiment was carried out in the laboratory and screen house in the exposure and rearing cages of dimensions 30 x 30 x 30 cm and 60 x 45 x45 cm respectively. Fifty pupae of C. vestalis were collected and put on tissue in two Petri dishes. Each Petri dish had twenty five pupae and was placed in each perplexes cage. Five days later parasitoids emerged and were sexed, removed and put in another similar cage. Ten percent honey solution topped with cotton wool to keep it moist was served as food. Two days later, Cotesia vestalis had mated and ready to be used in the experiment.

25

The treatments were set in a completely randomized design with three replications as shown in Table 3 and were:

(1) Ten Cotesia vestalis with Twenty second instars Plutella xylostella larvae

(1C. vestalis : 2P. xylostella larvae)

(2) Two Cotesia vestalis with eight second instars Plutella xylostella larvae

(1C. vestalis : 4P. xylostella).

(3) The control (8P. xylostella larvae without C. vestalis).

Table 3. Treatments and Experiment layout in the laboratory.

R 1 R II RIII

1C.vestalis: 2P. xylostella Control (P. xylostella) 1C. vestalis: 4P. xylostella

Control (P. xylostella) 1C. vestalis: 4P. xylostella 1C.vestalis: 2P. xylostella

1C. vestalis: 4P. xylostella 1C.vestalis: 2P. xylostella Control (P. xylostella)

3.6 Parasitism efficiency of Cotesia vestalis

To determine the parasitism efficiency of C. vestalis on P. xylostella, two sets of 24 hours old mated C. vestalis females were confined with second instars P. xylostella larvae in exposure cages.

The P. xylostella larvae were exposed to C. vestalis for twenty four hours after which another set of P. xylostella larvae was introduced and exposed to the same parasitoids.

This process was repeated until the death of C. vestalis.

Consequently the exposure interval was at 24, 48, 72, 96 and 120 hours. In the exposure cage (Figure 7), the parasitoids were fed on 10% honey solution and the food source was changed every day.

26

Figure 7: Exposure cages . Picture by G. Chipabika

3.7 Measurements of Parasitism efficiency

3.7.1 Parasitism percentage

The rearing of exposed larvae from each treatment was conducted in individual rearing cages in the screen house until the emergence of P. xylostella/ C. vistalis. The emergence of C. vestalis took 18 to 21 days and the number of parasitoids that emerged were counted and recorded. The number of adult P. xylostella both in the treatments and control were recorded as well as the dead ones.

To standardize with natural mortality of parasitoids in the experiment Sun-Shepard's formula (Finney, 1971) was used:

Correction % = ((Mortality % in treated plot + Change % in control plot population)/

(100 + Change % in control plot population)) x 100.

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After getting the correction percentage, the number of larvae that died due to parasitism was calculated by deducting correction percentage from (natural mortality + mortality due to parasitism). Thereafter parasitism percentage was also calculated using (Hassell and Varley, 1969; Russell, 1987) formula:

Parasitism % = ((C. vestalis)/ (Moths emerged + C. vestalis)) x 100

3.7.2 Searching efficiency

In this study, ten and two C. vestalis females were kept together with twenty and eight P. xylostella larvae in experimental cages for 24 hours respectively as shown in Figure 7.

After this only the hosts (P. xylostella) were removed before another set of P. xylostella was introduced for another 24 hours. It was during this period that C. vestalis was able to discover hosts (P. xylostella larvae) and parasitized some of them. Through the exposure of P. xylostella larvae to C. vestalis in each experimental unit (exposure cage) and after getting Parasitism percentage, the area of discovery and killing power was calculated using Hassell’s 1971 formula (Chua and Ooi, 1986). a = 1/ p log e Ni/ Nf

Where:

a = searching efficiency

Ni = initial P. xylostella larvae density,

Nr = final un parasitized P. xylostella larvae density,

e = exponential, and

p = the parasitoid density.

The relationship between a and p using Hassell and Varley’s (1969) formula:

a = QP – m expressed in logarithms the equation become linear, and m is the

slope of regression of log a and log p

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a = QP - m

Where:

Q = is the quest constant or the level of efficiency of one parasitoid

- m = the interference constant between searching parasites

P = the parasitoid density

When expressed in logarithms, the equation becomes linear

Log a = Log Q – m log P

3.7.3 Killing Power

Killing power of parasitoids (K) was calculated using the formula given in Ooi

(1980).

K = log 10 Ni/Nr

3.7.4 Sex ratio

The sex ratio was determined from the progeny of parasitoids that emerged from exposed

P. xylostella.

The parasitoids were sexed using identification key in order to recognize the male from the female. Female antennae are longer than male, reaching apex of metasoma or nearly so. Entire mesosoma in lateral view is not flattened dorso ventrally (Shaw, 2003; URL: http:www.sharkeylab.org/cotesia/cotesia.cgi#.)

3.8 Leaf damage

Damage by P. xylostella was assessed on cabbage used in the experiment on parasitism

(Figure 8). The exposed P. xylostella larvae to C. vestalis and non exposed (control) were reared on fresh 8 - week old cabbage plants in cages according to their respective

29 treatments until emergence of adults. After collecting data of parasitoids that had emerged and adult P. xylostella from respective rearing cages, cabbage plants were accessed for damage. The rating system developed by Chalfant and Brett (1965) was used. Damage per leaf was scored in six classes:

Class 1 = without damage, class 2 = damage between 0 and 1 %, class 3 = damage between 1 and 5 %, class 4 = damage between 6 and 10 %, class 5 = damage between 11 and 30 %, class 6 = damage more than 30%. The classification of damage was based on portion of damage to entire leaf.

Threshold of leaf damage was based on the market acceptability of head of cabbage and is usually from 0 – 10% and > 30% is un marketable (Ortiz, 2011).

Figure 8: Cabbage damaged by Plutella xylostella Picture by G. Chipabika

3.9 Data analysis

The data on Parasitism percentage, Searching efficiency, Killing power by C. vestalis on

P. xylostella and total leaf damage by P. xylostella was subjected to an Analysis of variance (ANOVA) using statistical program GenStat Release 14.2 ( PC/Windows 7

30 copyright, 2011). To separate the means, least significant difference (LSD) at 5 % was used. In order to determine the time when parasitism was highest, orthogonal test was conducted. Further, regression and correlation analysis was conducted to find relationships between parameters – parasitism percentage, damage score, searching efficiency, and killing power were conducted.

31

CHAPTER 4. RESULTS

4.1 Collection of Plutella xylostella larvae from farms

During the survey a total number of 505 P. xylostella larvae were collected from 10 farms that were visited and 176 C. vestalis emerged from the samples in the laboratory (Figure

9). Highest recovery of C. vestalis was in crops of above 24 weeks old followed by 16 weeks old. There was no recovery of O. sokolowskii in all the fields sampled.

Figure 9: Emergence of Cotesia vestlis from field collected Plutella xylostella

4.2 Emergence of Cotesia vestalis from Plutella xylostella collected in farms

From 505 P. xylostella larvae collected, 34.85% larvae died due to parasitism from C. vestalis whilst 12.87% died out of natural death Unparasitized larvae that developed to adult P. xylostella were 52.27%. The total larvae mortality was 47.72%. The contribution of parasitism to larval mortality was 73.03% (Table 4).

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Table 4. Level of parasitism of Plutella xylostella collected from ten farms of Chilanga,

Lusaka and Kafue districts

Parameter Total No. Percent

Larva collected 505

Non parasitized (other causes of larvae mortality) 65 12.87

Emerged P. xylostella adults 264 52.27

Larvae mortality due to parasitism 176 34.85

Total larva mortality 241 47.72

Contribution of parasitism to larval mortality 73.03

33

Figure 10: Emergence of Cotesia vestalis from Plutella xylostella collected at CQ and Miyoba farms 4.3 Parasitism percentage

More larvae were parasitized when they were exposed to treatment ratio 1:2 (p < 0.04) and treatment ratio 1:4 (p < 0.02) in 48 hours than at 72, 96 and 120 hours exposure periods (p > 0.99) respectively (Appendices 1). Parasitism percentages ranged between

54.9 and 22.9% and 50.1 to 6.7% in the treatment ratios 1:2 (P2) and 1:4 (P4) respectively (Figure 11).

34

Figure 11: Effect of age on Cotesia vestalis to parasitism percentages of Plutella xylostella 4.4 Searching Efficiency

There was significant difference p < 0.001 in searching efficiency with time in the ratio

1:2 (P2). It increased with time at 96 and 120 hours. Significant differences in both treatments (P2) p < 0.002 and (P4) p < 0.001) were observed in the killing power of parasitoids with exposure time. The significance was detected at 24 and 48 hours for treatment (P2) and up to 72 hours for treatment (P4). An increase in exposure time resulted in the decrease in killing power (Table 5).

35

Table 5. The effect of time on two levels of female parasitoid densities on the Searching efficiency and Killing power of Cotesia vestalis.

Area of discovery Killing power

Exposure time (hours) P2 P4 P2 P4

24 0.11 0.34 0.48 0.54

48 0.11 0.35 0.34 0.43

72 0.11 0.34 0.18 0.24

96 0.28 0.55 0.20 0.10

120 0.77 0.36 0.24 0.08

LSD 0.15 0.29 0.12 0.10

CV 17. 4% 19.4% 23.9% 20.7%

4.5 Sex ratio of Cotesia vestalis

There were 38 females and 54 males progenies in treatment ratio 1:2 (P2) whilst treatment ratio 1:2 (P4) treatment produced 20 females and 15 males. The progeny ratios are shown in (Table 6).

36

Table 6. Sex ratio of Cotesia vestalis at five constant exposure period of time in the laboratory.

Treatments

(Sex ratio; Female: Male)

Time (hours) Treatment ratio Treatment ratio 1:2 (P2) 1:4 (P4) 24 1:1.1 (36) 1:1 (14)

48 1:1.7 (27) 1:1 (10)

72 1:2 (18) 5:1 (6)

96 1: 1.6 (8) 2:1 (3)

120 2:1 (3) 1:1 (2)

Note. Numbers in brackets are total number of Costalis vestalis at each time interval.

4.6 Leaf damage

There was a significance difference (p < 0.01) of cabbage damage between parasitized larvae and unparasitized larvae (control) (Table 7). In the control, damage was higher than in (P2) and (P4). However, there was no difference at 72, 96 hrs exposure periods for both treatment ratios 1:2 (P2), 1:4 (P4) and the control.

37

Table 7. Mean damage score per head of cabbage under screen house condition

Treatments

Time (hours) 24 48 72 96 120 LSD

Ratio1:2 (P2) 1.00 1.33 2.33 4.00 5.33 1.24

Ratio 1:4 (P4) 2.67 3.33 4.67 5.00 5.33 1.5

Control 6.00 6.00 6.00 6.00 6.00

In regression analysis there were highly significant difference p < 0.001 in damage with respect to parasitism and time in P2 and P4 treatments (Appendices 2). Correlation analysis showed a significant negative correlation (p < 0.05) between parasitism percentage of P. xylostella larva and mean leaf damages (Table 8).

Table 8. Effect of parasitism on mean leaf damage score per head of cabbage

Treatment Treatment ratio 1:2 P2 Treatment ratio 1:4 P4

Time (hours) Parasitism% Damage Parasitism% damage

24 54.9 1.00 50.1 2.67

48 36.7 1.33 39.3 3.33

72 23.3 2.33 24.9 4.67

96 23.0 4.00 10.3 5.00

120 22.9 5.33 6.7 5.33

38

CHAPTER 5. DISCUSSION

5.1 Collection of Plutella xylostella and Cotesia vestalis

In all ten vegetable farms visited for collections of P. xylostella larvae, farmers use a wide range of insecticides to control the pest (Table 2). This is a common trend practiced by farmers who apply insecticides frequently and indiscriminately to minimize the damage (Orr et al. 1999). In Cameron highland, Chau and Ooi (1986) reported that 23.3 % of all farmers practice chemical control to combat P. xylostella.

During collections, the higher recovery of C. vestalis was in old crop, abandoned and ratoon cabbage fields (Figure 9) where the use of insecticides was minimized or abandoned at York and CQ farms. This is contrary to low recoveries made from

Liempe, Mandondo, Miyoba, Mwanza and Sianjele farms (Figure 9) where cabbage production was still active and bi - weekly application of insecticides such as cypermethrin done. The application of insecticides may have killed the natural enemies. This is in conformity with findings of Rowell et al. (2005) who reported that over use of broad-spectrum insecticides for P. xylostella control killed natural enemies and obscured the contributions of parasitoids to control P. xylostella in Thailand.

At CQ farm in Chilanga district, highest recoveries of C. vestalis were made on mature and old crop that was ready for harvest and as compared to the young one. This was due to the fact that the farmer had lessened the frequency or ceased insecticide spray

(Figure 10) as the thresh hold of P. xylostella to cabbage had dropped to 5% from the time heads started forming. This is similar to what is recommended in the Commercial

Vegetable Production Guide that suggests a threshold of 20% infestation to cabbage plants prior to heading, then drops the threshold to 5% when heads begin to form

(Fleischer, 2003). Low recoveries of C. vestalis at Miyoba farm (Figure 10) agrees to

39 findings of Ortiz (2011) where parasitism percentage in the field in Nicaragua were estimated between 32 and 49% on farms that used insecticides and between 52 and

63% on farms that did not use insecticides. Parasitism of P. xylostella by D. insulare reached 95% in plots without the pyrethroid treatment. Kfir (2003) and Muckenfuss et al. (1990) reported similar results in which incidences of parasitism levels were as high as 95 % in plots that were not treated with insecticides. These results therefore indicate that application of insecticide to control P. xylostella in cruciferous vegetables reduced the population and parasitism percentage of parasitoids. The other problem caused by insecticide use is development of resistance by the host. Lim (1992), for example, showed that insecticides (carbaryl + malathion) are toxic to parasitoids but not to P. xylostella. It is also assumed that non recovery of O. sokolowskii from P. xylostella larvae could also be attributed to over use of insecticides that may have reduced its population to levels that are difficult to recover. The non recovery of O. sokolowskii is in agreement with the findings of Shi et al. (2004) who reported that some insecticides used by farmers were extremely harmful to O. sokolowskii but slightly toxic to C. vestalis. Another reason is that O. sokolowskii is a parasitoid of low land where high parasitism is achieved at temperatures of 30oC and 35oC (Talekar,

1996) contrary to temperatures that obtain in the area of study which range from 18 to

29oC (Meteorological Dept, 2012). In this study, the field parasitism percentage was

34.85 and contributed 73% to total larval mortality (Table 4).

Other causes of mortality contributed 12.87% and among them is predation by other (Table 1) that are present in Zambia. Predators (hoverflies - Syrphidae; spiders - Lycosidae; rove beetle – Staphylinidae) accounted for up to 72% of larval mortality.

40

5.2 Parasitism percentages of Plutella xylostella exposed to Cotesia vestalis at different time intervals The total mean of parasitoids emergence for treatment ratios 1:2 (P2) and 1:4 (P4) per female in their oviposition period of five days were 32.2 and 26.2 respectively.

Parasitism percentage was higher at all levels at the peak (P2 = 54.9 and P4 = 50)

(Figure 9) than the one obtained from the field at 34.8 (Table 4). It was observed that the higher population level of parasitoids in (P2) had higher leverage in parasitism percentage than in P4 whose ratio was higher (1:4). However, the parasitism percentage was higher in the first (24 hours) in both treatments, (P2) (54.9 %.) and

(P4) (50.1%) than any of the other days that followed (Appendices 1.3). The reduction in the number of eggs produced by female as time increased is due to advancement in age (Harcourt, 1957). This is similar to findings of other researchers as it is evident that egg laying decreased with increased age of parasitoids (Figure 11).

Although the females oviposited up to five days, there were no significant differences between treatment ratios 1:2 (P2) (p > 0.3; Mean =36.7) and 1:4 (P4) mean = 24.9) in the mean number of parasitoids’ oviposition from day three to fifth day in respective treatments. Fifty percent of parasitism was on the first day and 25 % on the third day in both treatments. This confirms the findings of Alizadeh et al. (2011) where parasitoids laid more eggs on the first day than any other when C. vestalis oviposition experiment was conducted. This was also reported by Gauld and Hanson (1995) and

Luo et al. (2007) that many endoparasitoids are pro-ovigenic where bicoloratus’ oviposition was heavily skewed towards the first few days with approximately one third of the eggs laid on day one and over 50% laid by day three on larvae of the cotton leafworm, Spodoptera litura. Muniappan et al. (2004) observed similar phenomena where the gregarious larval parasitoid, Euplectrus maternus, laid eggs on the second instars larvae of fruit piercing moth, Eudocima fullonia for thirty

41 days but larger number of eggs were laid during the first week of exposure. The endo parasitoids have been reported to be pro- ovigenic meaning that eggs are fully developed upon adult emergence and most of them are ready to be oviposited upon coming across a suitable host in which they hatch within a short period of time. This is one of the conditions that lead to the oviposition of eggs into the host in the early days of its lay period. Equally P. xylostella lays 60 – 70% eggs in the first 4 days of oviposition and it takes 2 to 8 days to hatch depending on temperature (Arvanitakis et al. 2002). The 2nd and 3rd instars larvae take 2.06 ± 0.28 and 2.14 ± 0.14 days to develop respectively (Alizadeh et al. 2011). The two stages are the most suitable for

C. vestalis oviposition of eggs and development of larvae. Therefore, in this study it was observed that C. vestalis oviposited 50% of its eggs within 72 hrs so that the off springs will have enough food for development and emerge before the P. xylostella larvae could pupate and survivability took less than six days. However, in this study the short period of C. vestalis survivability was not clearly understood. Other studies have achieved survivability up to ten days which is contrary to what was observed in this study. This could have been due to inadequate nutrition of the host plant that may have equally affected the quality of P. xylostella larvae. Parasitoid larval growth provides a direct measure of host quality because it reflects the nutritional status that the host plant provides to the pest to which the parasitoids benefit and perform throughout the course of parasitism. Because nutrient reserves accumulated by larvae feeding on different quality plants may not be the same, these differences could have consequences on adult life-history parameters, including fecundity, longevity and body size of the parasitoids. Similar observations were made by (Boggs, 1981;

Sequeira and Mackauer, 1992). Body size is an important fitness index because it often affects reproductive success through variations in fecundity, longevity, dispersal,

42 searching efficiency, and host handling strategies (Charnov and Skinner, 1984; Visser,

1994; Kazmer and Luck, 1995).

Studies on reproduction of C. vestalis tended to assume that the fecundity schedule was shaped mainly by survivorship which Dixon and Agarwala (2002) alluded to.

Naturally very few individuals live long enough to reproduce in the second half of their lives than in the first. Thus natural selection would be expected to favour individuals that invest more in early reproduction even if it has an adverse effect on their potential longevity. It has been observed in this experiment that the number of progenies produced by the two sets of parasitoid per day represented higher fecundity in the first two days conforming earlier studies done by other people.

Using data of several parasitoid species that have been used in classical biological control programs against a variety of insect pests, Lane et al. (1999) tested the hypothesis that a parasitoid species with higher fecundity was a better biological control agent because of its ability to kill a greater number of hosts over the course of its lifetime. Plutella xylostella lays its eggs at night and the greatest number of eggs are laid in the first night and first 3 days after emergence and there after continue for ten days. Cotesia vestalis also laid many eggs in the first 72 hours so that there was synchronization in and survivorship for C. vestalis. This agrees to findings of

(Harcourt, 1957; Mau and Kessing, 2007; Henry and Baker, 2008).

5.3 Searching Efficiency

The searching efficiency was lower in ratios 1:2 (P2) than ratios 1:4 (P4) (Table 5.) because parasitoids spent more time encountering each other while searching for P. xylostella larvae. This is similar to other studies that have shown that female parasitoids react to competition in various forms when the host population is low.

Some fight or delay searching for host upon encountering a competitor (Aphen and

43

Jervis, 1996). For the increased searching efficiency at 96 and 120 hours exposure periods, it is assumed that due to time lapse and quest to survive, intensification for search of hosts increased in order to leave as many offsprings as possible in the next generation. This is in agreement with observations by Powell and Poppy (2001). It has been found that there is a strong association between the rate of host encounter and lifetime offspring production (Vet, 2001). Encounters with parasitized hosts lead to a greater incidence of leaving the patch in some parasitoid species (Outreman et al.

2001). At the ratio 1:4 (P4), the parasitoids were spending less time in search for host as it was readily available.

According to Tenhumberg et al. (2001), a long patch residence time will reduce the percentage of hosts escaping parasitism as was the situation in the ratio 1:4 (P4) especially in the first 24 hours of exposure period.

5.4 Killing Power

The significant differences in Killing power for both treatments at 24, 48 and 78 hours exposure periods (Table 5) could have been due to high parasitism that was observed in parasitism percentage (Figure 9). The parasitoids were initially full of energy and eager to lay eggs in order to maintain the reproduction and survivability. There is negative correlation between time of exposure and killing power. In this study, as period to exposure of parasitoids to P. xylostella increased the killing power decreased

(Table 5). The oviposition reduced with age of parasitoids hence there is positive correlation between age of parasitoids and killing power. As the parasitoid grew old, the oviposition reduced so was killing power.

5.5 Sex ratio of parasitoids

Sex ratio is the proportion of adults in a population that are male or female. The sex ratio of parasitoid indicates the power of increase of parasitoid. In this study, however,

44 the increase in parasitoid density appeared to influence progeny sex ratio towards males in the ratio 1:2 (P2). In contrast, a decrease in parasitoid density resulted in more female-biased in the ratio 1:4 (P4) (Table 6).

Sex ratio is influenced by two factors:

The amount of resources that will be available to her offspring (Trivers and Willard,

1973; Charnov, 1979; Charnov et al. 1981) and the number of mothers present

(Hamilton, 1967). Alizadeh et al. (2011) found that sex ratio in C. vestalis is male- biased. These findings are similar to what has been observed in this experiment. This observation was more exhibited in the ratio 1:2 (P2) where parasitoid density was higher. The reason for the male-biased sex ratios in progeny at high female densities is explained through Local Mate Competition (LMC) theory. The LMC models predict what offspring sex ratio a mother should produce given the numbers of other mothers present (Hamilton, 1967; 1979; King, 1987; 1993). If encounters between searching females were more frequent than encounters with males, this situation resulted in sons being more valuable than daughters and thus there was a strong selection on the mothers to bias their progeny sex ratios towards males. This agrees to finding of King,

1993 and this study that I undertook.

Another reason is that the behavior of C. vestalis is similar to that of Apaonagyrus lopezi (De Santis) of being arrhenotokous. Unmated females of A. lopezi lay only male eggs in its host Phenacoccus manihoti (Matile-Ferrero), eggs laid by mated females produce males and females in the ratio of 1:3 in favor of females (Umeh,

1988). When C. vestalis mate, the females store sperms in spermathecal capsules and when the eggs passed through the oviduct, the females did not release the sperms, the eggs passed through unfertilized and developed into males. This phenomenon happens

45 when the host is of poor quality and also when the mother wants more males due to unfavorable environmental conditions and this could have been the case with P. xylostella that was used in the study and could be a contributing factor in most cases where sex ratio was biased towards males.

Further studies are required on effects of size of the host in the wide environment.

5. 6 Leaf damage

Less damage was observed from parasitized than unparasitized P. xylostella larvae treatments (Table 7). In treatments ratios 1:2 (P2) and 1:4 (P4) there was no significant difference in damage at exposure periods 24, 48 and 72 hours (Table 7). However, lesser damage was observed in the first 24 hours of exposure period than the rest of the time though for ratios 1:2 (P2) it was highly significant even at 48 and 72 hour

(Appendices 2). This was as a result of high parasitism. Parasitized larvae had no energy to feed ferociously hence the less extensive damage (Figure 12).

Figure 12: Left, damage by parasitized Plutella xylostella larvae in the cage,

Right, damage caused by un parasitized Plutella xylostella larvae in the cage.

According to Chalfant and Brett (1965) ratings, there was no significant difference in damage between the two treatments at 96 and 120 hours of exposure and the control as

46 damage was over 35 % per plant meaning the produce was not marketable. In the control cages there was total failure of the crop due to heavy infestation of the pest

(Figure 12). This is similar to the findings of Ayalew and Ogoal (2006) who reported similar crop loss in central rift valley areas of Ethiopia due to lack of control measure of any sort. As observed in (Table 8) at 96 and 120 hrs exposure periods, lower parasitism percentage in P4 and absence of parasitoids in the control resulted in the higher number of healthy larvae that caused extensive damage. Marketing of this crop from these cages could have led to rejection due to presence of larvae in the florets/head and bigger holes on the lamina. This is in agreement to situation described by Capinera (2001) who stated that damage caused by larvae feeding on leaves or by the presence of larvae, contaminate and lower the quality of the product.

The damage increased with reduced parasitism percentage (Table 8) because fecundity of parasitoids diminished with time and age.

In treatment (ratio 1:4) P4, because C. vestalis to P. xylostella ratio was higher the parasitism percentage was lower hence some P. xylostella larvae escaped parasitism and were feeding with minimum interruption from the parasitoids and consequently resulted in the damage being higher than in treatment ratio 1:2 (P2) (Tables 8). This concurs with findings of Okine et al. (1996) and Sarfraz and Keddie (2005) where P. xylostella larvae parasitized by D. insulare consumed significantly less foliage than non-parasitized counterparts on the eight Brassicaceae that were evaluated. Monnerat et al. (2002) also observed similar behavior in which P. xylostella larvae parasitized by an unknown species of Diadegma consumed 35% less leaf surface of cabbage compared with non-parasitized larvae. Similarly, Yang et al. (1994) reported that

P. xylostella larvae parasitized by D. semiclausum consumed 1.07 to 1.24-fold less cabbage foliage than their non-parasitized counterparts. Additionally, findings of

47 reduced food consumption by parasitized larvae as a result from solitary parasitoid has been reported on Diatraea saccharalis (F.) parasitized by Lixophaga diatraeae

(Townsend) (Brewer and King, 1978). Similarly, reduced food consumption by P. xylostella resulted from parasitization by C. vestalis. This behavior has previously been reported for larvae of Ostrinia nubilalis (Hubner) parasitized by L. diatraeae

(Hubner and Chiang, 1982) and for larvae of Spodoptera frugiperda (Smith) parasitized by Ophion flavidus (Rohlfs and Mack, 1983). Studies of solitary

Kionobiont have indicated that many parasitoids are known to regulate host growth by reducing total food consumption, with parasitized hosts attaining only a fraction of the size of non-parasitized hosts. Consequently it has been suggested that these parasitoids are of direct importance to the plant. This is the same scenario where parasitized larvae of C. vestalis consumed a fraction that of un parasitized larvae.

Correlation analysis showed that where there was high parasitism there was reduced leaf damage (Table 8).

48

CHAPTER 6. CONCLUSION AND RECOMMENDATIONS

6.1 Conclusion

6.1.1 Determination of Parasitism percentage, Searching efficiency, Killing power and Sex ratio of parasitoids at two levels of parasitoid/ host densities.

The investigation in the laboratory revealed that C. vestalis has the potential to contribute to management of P. xylostella because of its high parasitism percentage of over 50, good host Searching efficiency that range from 0.11 to 0.77 and Killing power with the range from 0.10 to 0.54. There was no much difference in sex ratio as there were a total of 58 female against 69 male progenies though the treatment ratio

1:2 produced more males than treatment ratio 1:4.

6.1.2 Evaluation of the impact of C. vestalis on P. xylostella with regard to cabbage leaf damage. The damage to cabbage by P. xylostella reduced as parasitism percentage increased.

The negative correlation between P. xylostella parasitization and reduced leaf damage that has been observed demonstrates that C. vestalis has the capacity to be used as a biological control agent in cruciferous crops in Zambia.

6.2 Recommendations.

Deducing from this study the following recommendation would be good to consider:

6.2.1 For biological control to be more effective intervention through augmentation

of new females that would continue parasitizing P. xylostella is required.

6.2.2 Parasitism efficiency needs to be investigated under field conditions and where

insecticides are not applied.

6.2.3 More studies are required to test insecticides that would be compatible with

biological control agent in integrated pest management.

49

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Appendices

Appendix 1.0

1.1 Parasitism percentage

Analysis of variance ratio1:2 (P2)

Source of variation d.f. s.s. m.s. v.r. F pr.

Time 4 3147.8 786.9 1.39 0.284

Residual 15 8474.7 565.0

Total 19 11622.5

Tables of means

Grand mean 32.2

Time 24 48 72 96 120

54.9 36.7 23.3 23.0 22.9

LSD .(5%) 35.82

Cv% 73.9

1.2 Tables of contrasts

Source of variation d.f. s.s. m.s. v.r. F pr.

Time 4 3147.8 786.9 1.39 0.284

24 Vs 48, 72, 96 and 120 1 2592.6 2592.6 4.59 0.049

48 Vs 72, 96 and 120 1 554.8 554.8 0.98 0.337

72 Vs 96 and 120 1 0.3 0.3 0.00 0.982

96 Vs 120 1 0.0 0.0 0.00 0.997

Residual 15 8474.7 565.0

Total 19 11622.5

69

1.3 Parasitism% ratio 1:4 (P4)

Analysis of variance Ratio 1:4 (P4)

Source of variation d.f. s.s. m.s. v.r. F pr.

Time_hrs 4 5523.2 1380.8 3.10 0.048

Residual 15 6672.0 444.8

Total 19 12195.2

Tables of means

Variate: Parasitism%

Grand mean 26.2

Time_hrs 24 48 72 96 120

50.1 39.3 24.9 10.3 6.7

L.SD (5%). 31.79

Cv% 80.30

1.3.1 Tables of contrasts

Source of variation d.f. s.s. m.s. v.r. F pr.

Time_hrs 4 5523.2 1380.8 3.10 0.048*

24 Vs 48, 72, 96 and 120 1 2855.8 2855.8 6.42 0.023*

48 Vs 72, 96 and 120 1 1927.4 1927.4 4.33 0.055*

72 Vs 96 and 120 1 714.5 714.5 1.61 0.224

96 Vs 120 1 25.5 25.5 0.06 0.814

Residual 15 6672.0 444.8

Total 19 12195.2

70

Appendix 2.0

2.1 Searching efficiency

Analysis of variance ratio 1:2 (P2)

Source of variation d.f. s.s. m.s. v.r. F pr.

Rep stratum 2 0.023128 0.011564 1.89

TIME_Hrs 4 0.987740 0.246935 40.36 <.001

Residual 8 0.048941 0.006118

Total 14 1.059810

Variate: searching efficiency

Tables of means

Grand mean 0.276

24 48 72 96 120

0.110 0.108 0.110 0.281 0.772

L.S.D 0.1473

Cv% 17.4

2.3 Regression analysis for % parasitism in relation to damage, searching efficiency and killing power.

Correlation analysis

%PAR -

Damage - 0.8433 -

Searching efficiency 0.1696 0.1657

Killing power 0.9057 -0.6312 0.4210 -

Time -0.4558 0.5908 0.5360 -0.3246

% Parasitism Damage Searching efficiency Killing power Time

71

2.4 Searching efficiency

2.4.1Analysis of variance ratio 1: 4 (P4)

Source of variation d.f s.s. m.s. v.r. F pr.

Rep stratum 2 0.05691 0.02846 1.19

TIME_Hrs 4 0.10142 0.02536 1.06 0.435

Residual 8 0.19138 0.02392

Total 14 0.34972

Tables of means

Variate: Searching efficiency

Grand mean 0.388

Means

TIME_Hrs 24 48 72 96 120

0.338 0.354 0.338 0.551 0.358

L.S.D. 0.2912

Cv % 39.90

2.5 Variate: Killing power Ratio 1:2 (P2)

2. 5.1 Analysis of variance

Source of variation d.f. s.s. m.s. v.r. F pr.

TIME_Hrs 4 0.186093 0.046523 9.89 0.002

Residual 10 0.047043 0.004704

Total 14 0.233136

Tables of means

Grand mean 0.287

72

TIME_Hrs 24 48 72 96 120

0.481 0.335 0.177 0.198 0.242

L.S.D 0.1248

Cv% 23.90

2.5.2 Variate: Killing power ratio 1:4 (P4)

2.5.3 Analysis of variance

Source of variation d.f. s.s. m.s. v.r. F pr.

TIME_Hrs 4 0.491640 0.122910 37.35 <.001

Residual 10 0.032909 0.003291

Total 14 0.524548

Tables of means

Grand mean 0.278

TIME_Hrs 24 48 72 96 120

0.543 0.426 0.236 0.103 0.080

L.S.D. 0.1044

Cv% 20.7

2.6 Damage score Ratio 1:2 (P2)

2. 6.1 Analysis of variance

Source of variation d.f. s.s. m.s. v.r. F pr.

Time_hrs 4 40.4000 10.1000 25.25 <.001

73

Residual 10 4.0000 0.4000

Total 14 44.4000

Tables of means

Grand mean 2.80

Time_hrs 24 48 72 96 120

1.00 1.33 2.33 4.00 5.33

LSD (5%) 1.151

Cv% 6

2.7 Regression analysis Ratio 1:2 (P2)

2.7.1 Response variate: Damage

Fitted terms: Constant + Time + %PAR.Time

Summary of analysis

Source d.f. s.s. m.s. v.r. F pr.

Regression 9 76.633 8.5147 13.81 <.001

Residual 10 6.167 0.6167

Total 19 82.800 4.3579

Estimates of parameters

Parameterestimate s.e. t(10) t pr.

Time 5.826 0.772 7.55 <.001

%PAR.Time 24 -0.0651 0.0121 -5.38 <.001*

%PAR.Time 48 -0.0920 0.0179 -5.13 <.001*

%PAR.Time 72 -0.1155 0.0281 -4.11 0.002

%PAR.Time 96 -0.0390 0.0275 -1.42 0.186

%PAR.Time 120 -0.0279 0.0287 -0.97 0.354

74

Parameters for factors are differences compared with the reference level:

Factor Reference level

Time 24

Correlations between parameter estimates

Parameter ref correlations

Constant 1 1.000

Time 48 2 -0.710 1.000

Time 72 3 -0.711 0.504 1.000

Time 96 4 -0.720 0.511 0.512 1.000

Time 120 5 -0.709 0.503 0.504 0.510 1.000

%PAR.Time 24 6 -0.861 0.611 0.612 0.620 0.611 1.000

%PAR.Time 48 7 0.000 -0.605 0.000 0.000 0.000 0.000 1.000

%PAR.Time 72 8 0.000 0.000 -0.603 0.000 0.000 0.000 0.000

%PAR.Time 96 9 0.000 0.000 0.000 -0.590 0.000 0.000 0.000

%PAR.Time 120 10 0.000 0.000 0.000 0.000 -0.606 0.000 0.000

1 2 3 4 5 6 7

%PAR.Time 72 8 1.000

%PAR.Time 96 9 0.000 1.000

%PAR.Time 120 10 0.000 0.000 1.000

8 9 10

75

Accumulated analysis of variance

Change d.f. s.s. m.s. v.r. F pr.

+ Time 4 30.3000 7.5750 12.28 <.001

+ %PAR.Time 5 46.3327 9.2665 15.03 <.001

Residual 10 6.1673 0.6167

Total 19 82.8000 4.3579

2.8 Variate: Damage score ratio 1:4 (P4)

Analysis of variance

Source of variation d.f. s.s. m.s. v.r. F pr.

Time_hrs 4 15.7333 3.9333 8.43 0.003

Residual 10 4.6667 0.4667

Total 14 20.4000

Variate: Damage4

Tables of means

Grand mean 4.20

Time_hrs 24 48 72 96 120

2.67 3.33 4.67 5.00 5.33

L.S.D. 1.243

Cv% 16.3

76

2.9 Regression analysis Ratio 1:4(P4)

Response variate: Damage

Fitted terms: Constant + Parasitism% + Parasitism%.Time_hrs

Summary of analysis

Source d.f. s.s. m.s. v.r. F pr.

Regression 5 29.100 5.8201 23.62 <.001

Residual 14 3.450 0.2464

Total 19 32.550 1.7132

Estimates of parameters

Parameterestimate s.e. t(14) t pr.

Constant 6.020 0.202 29.79 <.001

Parasitism% -0.04807 0.00502 -9.58 <.001

Parasitism%.Time_hrs 48 0.00481 0.00617 0.78 0.449

Parasitism%.Time_hrs 72 0.02212 0.00680 3.25 0.006

Parasitism%.Time_hrs 96 0.0175 0.0100 1.74 0.104

Parasitism%.Time_hrs 120 0.0175 0.0117 1.49 0.159

2.10 Correlations between parameter estimates

Parameter ref correlations

Constant 1 1.000

Parasitism% 2 -0.576 1.000

Parasitism%.Time_hrs 48 3 -0.057 -0.511 1.000

Parasitism%.Time_hrs 72 4 -0.128 -0.419 0.408 1.000

77

Parasitism%.Time_hrs 96 5 -0.316 -0.152 0.289 0.287 1.000

Parasitism%.Time_hrs 120 6 -0.270 -0.130 0.247 0.245 0.228 1.000

% Parasitism constant 24 48 72 96 120

78