Seasonal Abundance and Distribution of Leafminer, Liriomyza Trifolii (Diptera: Agromyzidae) and Its Parasitoids on Bean Crop in South Florida

SEASONAL ABUNDANCE AND DISTRIBUTION OF LEAFMINER, LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOIDS ON BEAN CROP IN SOUTH FLORIDA

JIAN LI

A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2011

To my parents and my friends

ACKNOWLEDGMENTS

I sincerely thank my advisor Dr. Dakshina Seal for his academic guidance and support during my graduate study and research. I also extend my gratitude to my co- chair advisor Dr. Gary Leibee and my graduate committee Dr. Oscar Liburd. I thank them for their unending instruction and assistance in promoting my research project. I thank all the technicians, C. Sabines, C. Carter, E. Arias and J. Betancourt in Vegetable

Insect Pest Management Laboratory for their hard work in planting and maintaining the bean crops for my research. I would like to extend my appreciation to Dr. Gary Steck

(Division of Plant Industry, Gainesville, Florida) for the leafminer identification and Dr.

Sonja Scheffer (Systematic Entomology Laboratory, US Department of Agriculture,

Maryland) for the assistance of parasitoid identification. I appreciate the opportunity studying at University of Florida. I really enjoyed my graduate study in the Entomology and Nematology Department, and I thank all of the faculties who ever instructed me in the courses etc.

I thank my great parents Pizeng Li and Yulan Feng for their tremendous love and support overseas. I thank Charles Stuhl for his help in my study and my life. I also thank all of my friends both in China and USA for their help and company.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... 4

LIST OF TABLES ...... 7

LIST OF FIGURES ...... 8

ABSTRACT ...... 10

CHAPTER

1 LITERATURE REVIEW OF THE LEAFMINER LIRIOMYZA TRIFOLII (BURGESS) ...... 12

Biology and Life Cycle ...... 12 Economic Importance ...... 13 Management ...... 14 Chemical Control and Insecticide Resistance ...... 14 Physical Control ...... 15 Biological Control ...... 16 Ecological Study ...... 18 Research Objectives ...... 19

2 SEASONAL ABUNDANCE AND SPATIAL DISTRIBUTION OF LEAFMINER LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOID OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) IN SOUTH FLORIDA ...... 21

Materials and Methods...... 23 Study Sites and Bean Production ...... 23 Seasonal Density of Leafminer and Parasitoid ...... 24 Spatial Distribution of Leafminer and Parasitoid ...... 25 Parasitism and Host Density ...... 26 Distribution Pattern and Insects’ Density ...... 27 Results ...... 27 Seasonal Density of Leafminer and Parasitoid ...... 27 Spatial Distribution of Leafminer ...... 28 Spatial Distribution of Parasitoid ...... 30 Parasitism and Host Density ...... 31 Distribution Pattern and Insects’ Density ...... 31 Discussion ...... 31

3 DIEL DENSITY PATTERN OF LEAFMINER, LIRIOMYZA TRIFOLII AND THE PARASITOIDS, OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) AND DIGLYPHUS SPP. (HYMENOPTERA: EULOPHIDAE) ...... 45

Materials and Methods...... 46 Study Site and Beans Preparation ...... 46 Plot Design and Diel Activity ...... 47 Statistical Analysis ...... 48 Results ...... 48 Discussion ...... 51

4 THE COMPOSITION AND SEASONAL ABUNDANCE OF HYMENOPTERAN PARASITOIDS OF LIRIOMYZA TRIFOLII ON BEANS IN SOUTH FLORIDA ...... 62

Materials and Methods...... 63 Study Site ...... 63 Leaf Sampling and Insect Rearing ...... 63 Results and Discussion...... 64

5 CONCLUSION ...... 79

LIST OF REFERENCES ...... 82

BIOGRAPHICAL SKETCH ...... 88

LIST OF TABLES

Table page

2-1 Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in September 2010 ...... 40

2-2 Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in November 2010 ...... 40

2-3 Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in December 2010 ...... 41

2-4 Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in January 2011 ...... 41

2-5 Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in February 2011 ...... 42

4-1 Number of leafminer L. trifolii and its parasitoids (%) reared from the bean foliages (300 leaves / month) from September 2010 to February 2011 in south Florida ...... 78

LIST OF FIGURES

Figure page

2-1 Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 1, from September to October 2010...... 35

2-2 Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 2, from October to December 2010...... 36

2-3 Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 3, from December 2010 to February 2011...... 37

2-4 Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus in each month, from September 2010 to February 2011...... 38

2-5 Density (mean ± SE / yellow sticky card / 24 h) of L. trifolii and O. dissitus adults in each month, from September 2010 to February 2011...... 39

2-6 Bean crop field (50 m  30 m) was divided into 15 equal plots (10 m  10 m).... 43

2-7 Bean foliages were sampled and placed in the laboratory...... 43

2-8 Leafminer and the parasitoids were reared in the laboratory...... 44

3-1 Bean site 1, Nov-09-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h interval...... 54

3-2 Bean site 1, Nov-16-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h...... 55

3-3 Bean site 1, Nov-23-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h...... 56

3-4 Bean site 1, Dec-01-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h...... 57

3-5 Bean site 1. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 EST...... 58

3-6 Bean site 2. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 EST...... 59

3-7 Seasonal density: mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. per 15 yellow sticky traps / 10 h at bean site 1, 2010 and site 2, 2011...... 60

3-8 Fifteen yellow sticky traps were set in bean field from 8:00-18:00 EST...... 61

3-9 The caught insects during different 2 h interval were marked on the yellow sticky card...... 61

4-1 Liriomyza trifolii (Agromyzidae)...... 70

4-2 Opius dissitus (Braconidae)...... 70

4-3 Euopius sp. (Braconidae)...... 71

4-4 Diaulinopsis callichroma (Eulophidae)...... 71

4-5 Diglyphus begini (Eulophidae)...... 72

4-6 D. intermedius (Eulophidae)...... 72

4-7 D. isaea (Eulophidae)...... 73

4-8 Neochrysocharis sp. (Eulophidae) ...... 73

4-9 Closterocerus sp. (Eulophidae)...... 74

4-10 Zagrammosoma lineaticeps (Eulophidae)...... 74

4-11 Z. muitilineatum (Eulophidae)...... 75

4-12 Pnigalio sp. (Eulophidae)...... 75

4-13 Chrysocharis sp. (Eulophidae)...... 76

4-14 Halticoptera sp. (Pteromalidae)...... 76

4-15 Seasonal abundance of parasitoids, O. dissitus, D. callichroma and Diglyphus spp. on snap bean crop from September 2010 to February 2011...... 77

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

SEASONAL ABUNDANCE AND DISTRIBUTION OF LEAFMINER, LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOIDS ON BEAN CROP IN SOUTH FLORIDA

Jian Li

August 2011

Chair: Dakshina R. Seal Major: Entomology and Nematology

The leafminer, Liriomyza trifolii (Burgess) is a phytophagous fly infesting a wide range of vegetable and ornamental plants. Knowledge of the biology of this pest is essential to develop an effective management program. Various aspects of its biology and parasitoids were studied in snap bean (Phaseolus vulgaris) fields in Miami-Dade

County from 2010 to 2011. L. trifolii showed a seasonal preference having high population density during December 2010 (17.9 ± 1.5 adults / 5 leaves) and January

2011 (30.3 ± 2.7 adults / 5 leaves) when the temperature was relatively low. Opius dissitus, the major parasitoid of leafminer, showed a pattern of population density similar to L. trifolii, abundant during December 2010 (4.5 ± 0.45 adults / 5 leaves) and

January 2011 (5.4 ± 0.73 adults / 5 leaves). Both L. trifolii and O. dissitus tended to show an aggregated pattern of distribution when their densities were higher during

December 2010 and January 2011, but a regular pattern when densities were lower in

September 2010 and February 2011.

Diel density patterns of L. trifolii and its parasitoids, O. dissitus and Diglyphus spp. were evaluated by using yellow sticky traps in bean fields. L. trifolii density was found to

be more abundant from 8:00 to 10: 00 EST than any other time throughout the day during fall 2010, but did not show any clear pattern of diel abundance in spring 2011.

There was no significant difference in diel density pattern of O. dissitus in this study.

Diglyphus spp. was the most abundant from 10:01 to 12: 00 EST in fall 2010 and from

12:01 to 14: 00 in spring 2011.

The composition and seasonal abundance of hymenopteran parasitoids of L. trifolii was surveyed on snap beans in south Florida. Thirteen species or genera of parasitoids were collected from the bean foliages. O. dissitus was the most abundant larval-pupal endoparasitoid during all the bean seasons, from September 2010 to February 2011.

Diaulinopsis callichroma (Crawford) was the most abundant larval ectoparasitoid, and it was prevalent during fall 2010. Other parasitoids reared from bean foliages include

Euopius sp., Diglyphus begini (Ashmead), D. intermedius (Girault), D. isaea (Walker),

Neochrysocharis sp., Closterocerus sp., Chrysocharis sp., Zagrammosoma lineaticeps

(Girault), Z. muitilineatum (Ashmead), Pnigalio sp., and Halticoptera sp. Among these parasitoids, the D. isaea reared from Liriomyza leafminer is the first record in Florida.

The morphological characteristics of the leafminer parasitoids were described, and the photos containing key characters were presented for identification.

CHAPTER 1 LITERATURE REVIEW OF THE LEAFMINER LIRIOMYZA TRIFOLII (BURGESS)

Biology and Life Cycle

The leafminer, Liriomyza trifolii (Burgess) is a worldwide pest feeding on vegetable and ornamental plants. The adult female causes leaf puncturing by its ovipositor and feeds on the exudates from the injuries (Musgrave et al.1975). The Liriomyza adult females can live 15 - 20 days and males can live 10 - 15 days depending on temperature and food supply (Parrella and Bethke 1984). Adults mate soon after the emergence, and their mating activity can be affected by temperature (Dimetry 1971).

Adult L. trifolii is about 2 mm long, and the wing is 1.25 to 1.9 mm long. L. trifolii head is yellow, eyes are red, thorax and abdomen are mostly gray and black, ventral surface and legs are yellow. L. trifolii has a grayish black mesonotum, which differs from the closely related species, L. sativae (Blanchard) with shiny black mesonotum. The hind margins of L. trifolii eyes are yellow, while those of L. sativae are black. L. trifolii differs from L. huidobrensis (Blanchard) in having yellow femora while L. huidobrensis has dark femora (Capinera 2001).

Liriomyza trifolii has a relatively short life cycle. It requires about 19 days from egg deposition to adult emergence at 25°C. Development rate increases with the increase of temperature up to 30°C (Leibee 1984). Adult females deposit eggs in the feeding punctures. The total fecundity of a L. trifolii female can be 200 to 400 eggs on celery

(Leibee 1984). The egg is white in color and deposited in the plant tissue below the epidermis through the adaxial or abaxial leaf surface. The egg is oval shaped, about 0.1 mm long and 0.2 mm wide (Capinera 2001).

The larva of L. trifolii is yellow in color and cylindrical in shape. Larva goes through four instars. Capinera (2001) described different instars of L. trifolii. The body length and mouth size of the first instar are ~ 0.39 mm and ~ 0.10 mm, the second instar are ~ 1.00 mm and ~ 0.17 mm, and the third instar are ~ 1.99 mm and ~ 0.25 mm, respectively.

The fourth instar occurs between the pupariation and pupation.

The period of pupariation is about 2 - 4 hours depending on temperature. Leibee

(1984) indicated that mature third instar larva of L. trifolii exits the leaf mine in the morning and pupates on the ground. He also found the pupariation of L. trifolii larva could be delayed for a short time by continuous lighting condition (Leibee 1986). The pupae show a golden brown color at the early stage and turn to be a dark brown color during the late stage. Pupal development time is about 8 - 11 days under greenhouse or field conditions (Parrella 1987). The L. trifolii pupa has been reported to exhibit a diapause at 16°C (Suss 1984).

Economic Importance

Twenty-three species of Liriomyza leafminer are economically important pest of agricultural and ornamental plants (Spencer 1973). Among these, L. trifolii is a worldwide pest with a broad range of host plants. There were 55 hosts reported from

Florida including bean, pepper, potato, squash, beet, carrot, celery, cucumber, eggplant, lettuce, melon, onion, pea and tomato (Stegmaier 1966). Besides the crop hosts, several genera of weeds were found as the alternative host of Liriomyza leafminers in tomato field (Schuster 1991). Liriomyza leafminers can cause serious economic damage to its host crops. In the instance of severe infestation, it can cause total defoliation. Schuster (1978) reported that 90% of the tomato foliages might be lost in absence of an effective control method. In Miami Dade Co., leafminer is the

predominant pest infesting foliages of snap beans, and causes serious economic loss annually.

Both larvae and adults cause damage to the host plants. The larva begins feeding on the host immediately after the eclosion. The larvae live inside the foliage and form mines in the mesophyll layer. The rate of mine diameter and formation increases as larval development progresses, and Fagoonee (1984) found that leaf material consumption and feeding rate by the L. trifolii third instar is 643 and 50 times greater than the first instar, respectively. Female adults injure mesophyll cells by its ovipositor and feeds on the punctures. Bethke (1985) indicated that all punctures should be considered as injuries because females feed at all of these sites. The stipples and mines caused by L. trifolii activity significantly reduce the photosynthesis parameters on plant hosts. Parrella et al. (1985) reported that the leaf stipples reduced the photosynthesis parameters up to 75%. It was reported that the injuries caused by L. trifolii activities allow the entry of plant pathogens, such as bacteria of Pseudomonas chichorii (Broadbent 1990).

Management

Chemical Control and Insecticide Resistance

Liriomyza trifolii has been a pest of vegetable crops in Florida since 1945

(Wolfenbarger 1947). The most commonly used method of leafminer control is insecticide application. Application of nicotine sulphate was first used to control leafminer in Florida when leafminer was present (Leibee and Capinera 1995).

Chlordane was recommended for controlling leafminer on potato crops in south Florida

(Wolfenbarger 1947). Genung et at. (1979) indicated that the use of diazinon, naled, and azinphosmethyl reduced both vegetable seedlings mortality and yield reductions by

leafminer before 1974, but they were not effective after 1974. In Florida, methamidophos and permethrin were used to control leafminer on celery, but permethrin became ineffective in less than two years (Leibee and Capinera 1995). Poe and Strandberg (1979) reported that oxamyl was effective for controlling leafminer.

Cyromazine, an insect growth regulator (IGR), was used to control leafminer in celery industry in 1982. Leibee and Capinera (1995) confirmed the presence of a strain that was highly resistant to cyromazine. They found that the resistant strain survived 300 ppm of cyromazine, which was the highest label of concentration in the field. Abamectin, a GABA agonist, was applied to control leafminer on celery in early 1990, and cyromazine was used as the rotation. Spinosad and abamectin were highly effective for controlling leafminer on vegetable crops (Seal and Betancourt 2002). Ferguson (2004) found that all strains of L. trifolii, resistant to cyromazine, abamectin and spinosad, reverted to susceptible after five generations in the absence of insecticide selection pressure. Webb (2002) reported that spinosad and emamectin benzoate could be applied to control L. trifolii, and they are relatively compatible with the natural parasites.

It was reported that Coragen® (rynaxypyr) and Venom® (dinotefuran) were used to control leafminer in recent years (Webb and Stansly 2008). In Turkey, Civelek and

Weintraub (2003) demonstrated bensultap was effective in controlling L. trifolii larvae under the high larval density. They found all insecticide treatments (1.5, 2.0 2.5 and 3.0 kg / ha) had significantly fewer alive larvae than control (P < 0.01) within one day of application and the number of live larvae decreased after 10 days of treatment.

Physical Control

The yellow sticky cards can be used to monitor leafminer adult abundance, adult movement and field dispersion. Yellow sticky traps used for monitoring L. trifolii on

chrysanthemum in greenhouse situation provided consistent information (Parrella and

Jones 1985). Aluminum foil mulch was also found to effectively repel Liriomyza adults on tomato and squash and reduce the crop infestation (Wolfenbarger 1968). In addition, various cultural practices can be helpful in reducing L. trifolii on various hosts.

Destruction of weed hosts and soil deep ploughing can effectively reduce leafminer alternative hosts and the abundance of pupae in the soil.

Biological Control

Biological control plays an important role in the integrated pest management (IPM) of a pest. Several parasitoid wasps are used in the biological control of Liriomyza leafminers. Valladares and Salvo (2001) indicated that species richness and density of leafminer and the parasitoid community were positively correlated in the forest of central

Argentina. They found that parasitism was greater when parasitoid community has a higher species number and lower dominance. Capinera (2001) reported that at least 14 species of L. trifolii parasitoids occur in Florida. Among all the parasitoid families,

Eulophidae, Braconidae and Pteromalidae are the most studied for controlling leafminer.

The eulophids, Diglyphus isaea (Walker), D. begini (Ashmead), D. intermedius

(Girault) and D. carlylei (Girault) are solitary ectoparasitoids (larval parasites) of dipteran leafminers occurring in North American (Lasalle and Parrella 1991). The female

Diglyphus adult lays one or more eggs attached to the leafminer late instar larvae

(Minkenberg 1987). The parasitoid larvae hatch out of eggs and feed on the leafminer larva externally, eventually killing the leafminer larvae. The parasitoid larva develops through three instars and pupates in the mine before emerging as an adult.

Development time is temperature dependent. D. isaea is one of the most effective

biological control agents of Liriomyza leafminer in greenhouse (Minkenberg 1987; Boot et al. 1992). D. isaea takes about 10 days to complete egg to adult development on L. trifolii and L. huidobrensis at 25°C (Bazzocchi et al. 2003).

Endosymbionts, such as Wolbachia, have been studied for improving the effectiveness of leafminer biological control. Tagami et al. (2006 a) reported that

Wolbachia showed strong cytoplasmic incompatibility (IC) and perfect vertical transmission in L. trifolii, and it may be applied in leafminer control. In another study,

Tagami et al. (2006 b) indicated that endosymbiont, Rickettsia might cause thelytokous reproduction in the leafminer parasitoids, Neochrysocharis formosa (Westwood)

(Hymenoptera: Eulophidae), and it may increase the effectiveness of leafminer biological control.

Nematodes have also been studied as a biological control agent for controlling leafminer L. trifolii. LeBeck et al (1993) found that all L. trifolii larval stages were susceptible to the entomopathogenic nematode, Steinernema carpocapsae, and the second instar was the most susceptible to S. carpocapsae. They indicated the mines produced by L. trifolii larvae were suitable for S. carpocapsae to survive. The nematodes enter the mines through the oviposition punctures on the leaf and penetrate the leafminer larva via the anus, mouth or spiracle. It was found using nematodes and parasitoid wasps D. begini together resulted in a higher effective result in controlling leafminer L. trifolii than using either biological control agent alone (Sher et al. 2000).

The importance of biological control and insecticide application in leafminer control promotes further studies on compatibility between the biological control agents and common insecticides. Weintraub (2001) found that cyromazine and abamectin

significantly reduced the population of parasitoid D. isaea compared to non-treated control. Parasitoid population from abamectin treatment recovered sooner than in cyromazine treatment. Kaspi (2005) found that the percentage emergence of D. isaea from the mine and longevity of the emerged adult were not affected by the abamectin treatment when applying to chrysanthemum plants. The use of reduced risk insecticides decreases the impact on the biological agents in an IPM program.

Ecological Study

Knowledge of seasonal population dynamics and spatial distribution of pests and natural enemies are essential for developing IPM strategies. Few studies on seasonal abundance of leafminer and parasitoids have been conducted on bean crops in south

Florida. Valladares (2001) reported that the parasitism of leafminer was higher in summer but lower in winter in Central Argentina. Shepard et al. (1998) determined that leafminer L. huidobrensis has a seasonal incidence in various crops in Indonesia. They found that the abundance of L. huidobrensis was very low on potato during the dry season. The abundance of parasitoid Hemiptarsenus varicornis (Hymenoptera:

Eulophidae) was always high when L. huidobrensis number was low. Temperature has a direct effect on its abundance. Abou-Fakhr Hammad (2000) reported that the population density of L. huidobrensis reduced in September and October 1994 in

Lebanon due to the high daily average temperature of 31.2°C and 29.7°C respectively.

Various studies indicated that parasitoid’s foraging efficiency increased when their host had an aggregative distribution, and it lead to a direct density dependent parasitism

(Hassell and May 1974; Head and Lawton 1983). The leafminer, Ophiomyia maura showed a Poisson distribution in the early season but later became weakly clumped

(Ayabe and Shibata 2008).

Both biotic and abiotic factors affect the distribution of leafminer and the parasitoids. The distribution of leafminer O. maura showed a Poisson distribution in the early growth season but later became weakly clumped in Okazaki City, Japan (Ayabe and Shibata 2008). Saito et al. (2008) found leafminer Chromatomyia horticola (Diptera:

Agromyzidae) and parasitoids of D. isaea and D. minoeus were abundant in the cool season from December to May, in Japan. However, the parasitoid Chrysocharis pentheus was abundant in the warm season from May to June. The natural enemy should coincide with their host distribution and have a similar thermal requirement

(Kang Le at el., 2009). Tantowijoyo (2010) determined that altitude could affect the distribution of leafminer L. huidobrensis and L. sativae. They found L. huidobrensis was more abundant at 700 m above sea level and L. sativae was more abundance below

600 m. Temperature was the overriding influence on their altitudinal distribution.

In addition, both plant leaf age and life history can affect female leafminer adult’s oviposition preference. Facknath (2005) demonstrated that the infestation by L. trifolii starts from the lower leaves and proceeds to middle and upper leaves on both bean and potato. They found the larval survival was significantly lower in the smaller upper leaves than the lower and older leaves. Parrella (1983) determined that intraspecific competition of L. trifolii larvae leads to small larvae, fewer pupae and less vigorous adults. The older and thick leaves have more mesophyll and can supply sufficient space and food. In another study, it was reported that some insects could increase their nitrogen utilization in the old plant parts (Williams et al. 1998).

Research Objectives

This research study was conducted to develop an effective IPM strategy for controlling L. trifolii. Information about pest density levels at different times of the year is

essential to monitor, initiate management programs and select appropriate management tools. The spatial distribution of L. trifolii and its parasitoids indicates their ecological behavior and interactions with host plants. This information is essential for developing a successful leafminer management programs. In addition, studying the composition and seasonal abundance of leafminer hymenopteran parasitoids leads to exploration of potentially effective biocontrol agents. My specific research objectives are:

1) Determine seasonal abundance and spatial distributions of leafminer, L. trifolii and its parasitoid, O. dissitus on bean crops in south Florida.

2) Determine diel density pattern of leafminer, L. trifolii and its two parasitoids of O. dissitus and Diglyphus spp. on beans.

3) Determine the composition and seasonal abundance of hymenopteran parasitoids of L. trifolii on beans in south Florida.

CHAPTER 2 SEASONAL ABUNDANCE AND SPATIAL DISTRIBUTION OF LEAFMINER LIRIOMYZA TRIFOLII (DIPTERA: AGROMYZIDAE) AND ITS PARASITOID OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) IN SOUTH FLORIDA

The leafminer, Liriomyza trifolii (Burgess), is a phytophagous fly feeding on a wide range of ornamental and vegetable plants. The species is distributed in the temperate and tropical regions worldwide. The adult female injures the plant tissues with its ovipositor, and adult female feeds on the punctures. The eggs are deposited in the punctures and hatch into larvae. The mining activity of larvae causes damage to the mesophyll layer of the leaf. Leaf consumption and feeding rate increases rapidly as the larvae developed (Fagoonee 1984). L. trifolii is one of the main pests of vegetable crops in south Florida (Seal and Betancourt 2002). The mature larvae exit the mine, drop to the ground and pupate.

The Opius dissitus wasp is one solitary larva-pupal endoparasitoid of L. trifolii. O. dissitus female deposits its egg directly inside the leafminer larva, and the host larva continues to develop until pupation (Bordat et al. 1995 a). O. dissitus develops inside the host pupa and finally emerges out of the pupa. Generally, only one parasitoid emerges from one pupa. Several studies reported that O. dissitus was reared from L. trifolii from infested celery leaves, tomato leaves and weeds in Florida (Stegmaier 1972;

Schuster and Wharton 1993; Schuster and Gilreath 1991). O. dissitus was the most abundant parasitoid of L. trifolii on the snap bean crop in our preliminary study.

Effective monitoring of leafminer and parasitoid density is essential for making management decisions in biological control. Recent studies showed that rapid increase in leafminer population is primarily due to intensive insecticide applications leading to the development of resistance (Saito 2004) and a reduction in natural enemy densities

(Minckenberg and van Lenteren 1986). Some other abiotic factors also contribute to leafminer seasonal abundance. Temperature is an important factor affecting leafminer and parasitoid densities (Shepard et al. 1998; Saito et al. 2008). High daily temperatures (≥ 30°C) reduced leafminer adult’s density (Abou-Fakhr-Hammad 2000).

For parasitoid O. dissitus, Bordat et al. (1995 b) reported that 20°C was optimal for both adults male and female, and the optimum temperature for female reproduction was at

25°C. Light condition could also affect leafminer density level, and the pupariation of emergent L. trifolii larva could be delayed for a short time by continuous lighting condition (Leibee 1986). In addition, Shepard et al. (1998) reported that moisture level might be another factor affecting leafminer density. They found that the infestation on potato by L. huidobrensis was more severe during the wet season possibly indicating that parasitoid’s density in different season is affected by various abiotic factors.

Insect spatial distribution allows us to understand insect ecological behavior in the field and their interactions with parasitoids. Some authors believe that parasitoid’s foraging efficiency increases when their host is in an aggregative distribution pattern, which leads to a direct density dependent parasitism and better biological control effectiveness (Hassell and May 1974; Head and Lawton 1983). In general, insect spatial distribution pattern might be affected by several factors. The leafminer adult female prefers the high quality foliages of host plants for oviposition (Faeth 1991), so that the larvae can have better quality food and a better performance. In addition, leafminer larval density can affect parasitoid female’s oviposition behaviors (Nelson and Roitberg

1995).

The objectives of this study were (1) to determine the seasonal abundance of the leafminer, L. trifolii and its parasitoid, O. dissitus, (2) to characterize their spatial distribution patterns on bean growth season (September 2010 to February 2011), (3) to determine the relationship between parasitism and host density, and (4) to assess the relationship between insect spatial distribution pattern and density.

Materials and Methods

Study Sites and Bean Production

The study was conducted in Homestead, Dade County, FL. In this area, bean crops are grown commercially in open field conditions. There are two growing seasons per year, extending from October to February. In this study, three bean sites (each 50 m

 30 m, as shown Figure 3-6), were prepared at the Tropical Research and Education

Center (TREC), University of Florida. Site 1 was planted with bean from September to

October 2010, site 2 from October to December 2010, and site 3 from December 2010 to February 2011. The soil type of the study area is Krome gravelly loam soil, which consists of about 33% soil and 67% pebbles. The snap bean (Phaseolus vulgaris) seeds were supplied from Harris Moran seed Company, Modesta, California. Pre-plant herbicide, halosulfuron methyl (51.9 g / ha, Sandea®, Gowan Company LLC., Yuma,

Arizona), was applied to control nutsedge and broad-leaf weeds. At the time of bean planting, granular fertilizer 6: 12: 12 (N: P: K) was applied at 1345 kg / ha. Liquid fertilizer (4: 0: 8) was applied at the rate of 0.56 kg N / ha / day in furrow by the side of the seed row, 3, 4 and 5 weeks after planting. Plants were irrigated once a day delivering one inch (2.54 cm) / each time to maintain optional soil moisture. The fungicide chlorothalonil (2.81 L / ha, Bravo®, Syngenta Crop Protection, Inc.,

Greensboro, North Carolina) was used during early plant growth to prevent fungal diseases. No insecticide was used at these study sites.

Seasonal Density of Leafminer and Parasitoid

For the purpose of sampling, each bean site was divided into 15 equal plots (10 m

 10 m). Sampling was initiated when the bean plants had two primary leaves fully unfolded, and continued once a week until the beans were harvested. Five bean leaves, one leaf / plant, were randomly collected in each plot (total 75 leaves) per week. When the bean plants produced new leaves and the primary leaves dropped off, the older bottom leaves from the plants were always collected as samples because of L. trifolii feeding preference to the older mature leaf (Facknath 2005).

The sampled leaves from each plot were placed separately into a Petri dish (10 cm diameter). Each Petri dish was labeled with plot number and sampling date (Figure

2-7). All samples were transported to the IPM laboratory, TREC and were placed in growth chamber at 25°C, 70% RH and 14: 10 (L: D) h for further development of L. trifolii and O. dissitus. The samples were checked every day for leafminer pupae in each

Petri dish (10 cm diameter). Pupae were carefully separated from the leaves, and transferred into one new Petri dish and marked with the same information to detect their origin. The number of pupae from each plot was recorded. The pupae were placed in the same growth chamber for further development into adults. The numbers of emerged adults of L. trifolii and parasitoid O. dissitus from the pupae were recorded (Figure 2-8).

As another monitoring method, 15 yellow sticky traps (7.6 cm  12.7 cm), as another monitoring method, were set in the center of each experimental plot (one trap / plot) for 24 hours. At the time of collection, each trap was wrapped with transparent

polyethylene sheets to avoid inclusion of dusts. This method also facilitated transportation and storing of the traps in the laboratory for further study. The traps were checked in the laboratory using a binocular microscope at 10-30  to identify and record number of leafminer and various species of parasitoids. The traps were placed once per week until the beans were harvested.

The density of L. trifolii was calculated based on the number of emerged pupae and adults from the leaf samples, and the number of adults caught on sticky traps. The density of O. dissitus was calculated as the number of emerged adults from leafminer pupae and the adults caught by yellow sticky traps. The density of L. trifolii and O. dissitus was analyzed by analysis of variance (ANOVA, PROC GLM, SAS institute Inc.

2003) to determine the difference by growing month and by bean sites, and the means were separated by Least Significance Difference (LSD) (P < 0.05).

Spatial Distribution of Leafminer and Parasitoid

This study was conducted in the same field in the seasonal abundance study. Plot design, sample collection and sample preparation were as described in the previous study. The spatial distribution was determined based on data collected from two different sized plots: a) 10 m2 of 15 plots; and b) 30 m2 of 5 plots. Spatial distribution patterns of L. trifolii and O. dissitus were evaluated by using Taylor’s power law (Taylor

1961) and Iwao’s patchiness regression (Iwao 1968). The Taylor’s power law linear regression model is:

log s2 =b log +log a

Where the slope b is the index of aggregation, and a is the factor related to sample size. Iwao’s patchiness regression was also used to access the variance to mean relation and to evaluate the distribution patterns. The linear regression model is:

x* =  + 

Where x* is the mean crowding (Lloyd 1967) expressed as x* = + s2 / -1, slope  is the index of regression, and  is the sampling factor. In both of the models, when the slope (b and ) value is not significantly different from 1, it indicates a random distribution pattern; slope significantly > 1 indicates an aggregated distribution pattern; and slope significantly < 1 indicates a regular distribution pattern (P < 0.05). The fitness of each data set to the linear regression model was evaluated by r2 value. The student t test was used to determine the significance of the slopes in both of the models (P <

0.05).

Parasitism and Host Density

The relationship between O. dissitus parasitism proportion and leafminer density was analyzed by transformed log linear regression analysis (PROC GLM, SAS

Institute Inc. 2003).

y = b log + a

The fit of data set to the linear regression was evaluated by r2 value. The parasitism efficiency was expressed as parasitism proportion in each sampling plot calculated as: y = number of emerged O. dissitus / number of the leafminer pupae. means the density of leafminer pupae. The slope b values were used to determine the relationship between the parasitism proportion and host density (P < 0.05). When slope b is significantly > 0, it indicates a direct density dependence; significantly < 0 indicates

a reverse density dependence (P < 0.05). The regression analyses were carried out on all three sites’ data combined.

Distribution Pattern and Insects’ Density

The L. trifolii and O. dissitus distribution patterns on snap beans were determined in each month. The densities of L. trifolii and O. dissitus were assessed by two methods discussed before. The relationship between the insects’ distribution pattern and their population density of was evaluated.

Results

Seasonal Density of Leafminer and Parasitoid

The density of L. trifolii (0.4 ~ 4.9 pupae / 5 leaves) and O. dissitus (0.0 ~ 1.9 adults / 5 leaves) was low in the bean field at site 1, from September to October 2010

(Figure 2-1). At this site, density of L. trifolii (4.9 ± 0.7 pupae / 5 leaves) and O. dissitus

(1.9 ± 0.3 adults / 5 leaves) were significantly higher (F = 12.88, df = 7, 112, P < 0.0001)

(F = 5.38, df = 7, 112, P < 0.0001) in the middle of the bean growth period than the beginning and end of the bean growth periods (less than 1.0 / 5 leaves).

At the bean site 2, the density of leafminer (2.2 ~ 3.3 pupae / 5 leaves) and the parasitoid (0.0 ~ 2.1 adults / 5 leaves) was low in early growth period during October

2010, but it increased rapidly at the end of November 2010 (16.2 ± 2.3 pupae / 5 leaves;

3.1 ± 0.5 parasitoids / 5 leaves). The density of L. trifolii (17.9 ± 1.5 pupae / 5 leaves) and O. dissitus (4.5 ± 0.45 adults / 5 leaves) were both significantly higher (F = 51.99, df

= 9, 140, P < 0.0001) (F =21.7, df = 9, 140, P < 0.0001) during the late growth period during December 2010 than earlier growth period at this site (Figure 2-2).

At the bean site 3, the density of L. trifolii (53.1 ± 5.8 pupae / 5 leaves) and O. dissitus (14.4 ± 1.8 adults / 5 leaves) reached the highest density level in early January

2011 and significantly higher (F = 61.1, df = 8, 126, P < 0.0001) (F = 35.6, df = 8, 126, P

< 0.0001) than the rest growth period (Figure 2-3). The density level of L. trifolii and O. dissitus decreased at the end of January 2011, and it was in a low level (0.87 ~ 4.1 pupae / 5 leaves; 0.2 ~ 0.67 parasitoids / 5 leaves) in February 2011.

Overall, the seasonal abundance of L. trifolii was low in September and October

2010 and February 2011. The leafminer density level started to increase in the middle of

November 2010 and reached the highest average density level (30.3 ± 2.7 pupae / 5 leaves) (F = 61.5, df = 5, 354, P < 0.0001) in January 2011 (Figure 2-4). The parasitoid density level was the highest (5.4 ± 0.73 adults / 5 leaves) (F = 30.95, df = 5, 354, P <

0.0001) during January 2011, and O. dissitus density was always high when leafminer population was abundant.

The yellow sticky traps data showed a similar pattern of seasonal density level of

L. trifolii with the foliage sampling method (Figure 2-5). The abundance of leafminer was low in the September 2010 (1.6 ± 0.4 adults / trap), October 2010 (1.4 ± 0.5 adults / trap) and February 2011 (1.7 ± 0.9 adults / trap), and it was high in November 2010

(10.6 ± 0.98 adults / trap), December 2010 (25.7 ± 2.7 adults / traps) and January 2011

(4.6 ± 0.9 adults / trap). The number of caught parasitoid O. dissitus adults was relatively low when compared with the result of emerged parasitoid from the samples.

The average temperature was relatively lower (mostly less than 20°C) during November

2010 to January 2011 than other months during the bean growth season, shown from

Figure 2-4 and Figure 2-5.

Spatial Distribution of Leafminer

In September 2010, the distribution pattern of L. trifolii (Table 2-1), when plot size was 10 m2, was aggregated based on Taylor’s power law (b = 1.22, P = 0.006, r2 =

0.49), but a regular distribution pattern ( =0.60, P = 0.164, r2 = 0.23) based on Iwao’s patchiness regression. When plot size was 30 m2, L. trifolii showed a regular distribution pattern based on both Taylor’s power law (b = 0.98 P < 0.0001, r2 = 0.72) and Iwao’s patchiness regression (b = 0.74, P =0.035, r2 = 0.44).

In November 2010, L. trifolii showed an aggregated distribution pattern in both sizes of plots, 10 m2 plot (b = 1.20, P = 0.0038, r2 = 0.38) ( =1.20, P < 0.0001, r2 = 0.8) and 30 m2 plot (b = 1.58, P = 0.008, r2 = 0.52) ( = 1.45, P = 0.001, r2 = 0.67) plots

(Table 2-2) based on Taylor’s power law and Iwao’s patchiness. In December 2010, L. trifolii had an aggregated distribution pattern (b = 1.63, P = 0.0045, r2 = 0.47) ( = 1.10,

P < 0.0001, r2 = 0.95) when plot size was 10 m2 based on both analyzing methods.

When plot was 30 m2, it showed a regular distribution pattern (b = 0.98, P = 0.0352, r2 =

0.13) based on Taylor’s power law, but an aggregated distribution ( = 1.05, P = 0.0004, r2 = 0.86) based on Iwao’ patchiness regression (Table 2-3). In January 2011, L. trifolii presented an aggregated distribution pattern (Table 2-4) based on two analyzing methods in both plot sizes of 10 m2 (b = 1.74, P = 0.0019, r2 = 0.42) ( = 1.12, P <

0.0001, r2 = 0.92) and 30 m2 (b = 1.41, P = 0.0009, r2 = 0.69) ( = 1.05, P < 0.0001, r2 =

0.99).

The results in February 2011 (Table 2-5) indicated L. trifolii, in both plot sizes of 10 m2 and 30 m2, showed a regular distribution pattern (b = 0.81, P = 0.038, r2 = 0.23) (b =

0.98, P = 0.054, r2 = 0.32) based on Taylor’s power law, but an aggregated distribution pattern ( = 1.23, P = 0.0002, r2 = 0.54) ( = 1.20, P = 0.0012, r2 = 0.67) based on

Iwao’s patchiness.

Spatial Distribution of Parasitoid

In September 2010, based on Taylor’s power law and Iwao’s patchiness regression, parasitoid O. dissitus had an aggregated distribution pattern (b = 1.37, P =

0.033, r2 = 0.56) ( = 2.74, P = 0.069, r2 = 0.45) when plot size was 10 m2, and a regular distribution pattern (b = -0.32, P = 0.77, r2 = 0.023) ( = -0.38, P = 0.88, r2 = 0.0066) when plot size was 30 m2 (Table 2-1).

In November 2010 (Table 2-2), when plot size was 10 m2, O. dissitus had a regular distribution pattern (b = 0.97, P = 0.0004, r2 = 0.53) based on Taylor’s power law, but an aggregated distribution pattern ( = 1.11, P < 0.0001, r2 = 0.70) based on

Iwao’s patchiness regression. O. dissitus showed an aggregated distribution pattern when the plot size was 30 m2 based on both methods (b = 1.10, P = 0.0013, r2 = 0.66)

( = 1.15, P = 0.0004, r2 = 0.73). In December 2010, O. dissitus presented an aggregated distribution pattern in both of plot sizes, 10 m2 (b = 1.90, P = 0.0034, r2 =

0.50) ( = 1.42, P < 0.0001, r2 = 0.81) and 30 m2 (b = 1.51, P = 0.082, r2 = 0.37) ( =

1.19, P = 0.0013, r2 = 0.79), based on both analyzing methods (Table 2-3). In January

2011, O. dissitus showed an aggregated distribution (b = 1.13, P = 0.005, r2 = 0.38) ( =

1.09, P < 0.0001, r2 = 0.88) when plot size was 10 m2, based on both methods. When plot size was 30 m2, O. dissitus presented an aggregated distribution (b = 1.10, P =

0.024, r2 = 0.41) based on Taylor’s power law, but a regular distribution ( = 0.99, P <

0.0001, r2 = 0.89) based on Iwao’s patchiness regression (Table 2-4).

In February 2011 (Table 2-5), based on Taylor’s power law, parasitoid O. dissitus showed a random distribution pattern (b = 1.00, P < 0.0001, r2 = 0.72) when plot size was 10 m2 and a regular distribution pattern (b = 0.87, P = 0.001, r2 = 0.86) when plot

size was 30 m2. Based on Iwao’s patchiness regression, O. dissitus presented a regular distribution pattern ( = 0.63, P = 0.11, r2 = 0.17) ( = 0.47, P = 0.189, r2 = 0.23) in both of 10 m2 and 30 m2 size plots.

Parasitism and Host Density

The results of emerged leafminer pupae and emerged O. dissitus adults from each week sampling were used to analyze the relationship between O. dissitus parasitism and L. trifolii density. A direct density dependent relationship between O. dissitus parasitism proportion and L. trifolii density was observed based on the combined data of three bean sites (b = 0.095, df = 1, 21, r2 = 0.17, P = 0.049).

Distribution Pattern and Insects’ Density

In comparing the results on density and distribution of L. trifolii and O. dissitus, it is distinct that distribution pattern of O. dissitus followed the distribution pattern of L. trifolii.

The spatial distribution of L. trifolii tended to be an aggregated pattern when their density was high (4.2 ~ 30.3 pupae / 5 leaves), from November 2010 to January 2011.

L. trifolii tended to have a relatively regular distribution pattern when their abundance was relatively low (1.7 ~ 2.6 pupae / 5 leaves) in September 2010 and February 2011.

O. dissitus showed a similar pattern of abundance and distribution as its host during the above mentioned period of study.

Discussion

Based on the results from 3 different bean sites, the present study demonstrated that density of L. trifolii and O. dissitus did not have any preference during a single bean site season. No specific pattern of population density of L. trifolii and O. dissitus was found at any specific growth period of bean crop (Figure 2-1, Figure 2-2, Figure 2-3).

The density of L. trifolii and O. dissitus was high from late November 2010 to January

2011, but low during other growing season. Therefore, it indicated the abundance of leafminer and the parasitoid had a seasonal preference on bean crop.

Temperature is an important factor affecting the leafminer and the parasitoid abundance during different growing seasons. Abou-fakhr-Hammad (2000) reported that population density of L. huidobrensis was reduced by the high daily average temperature. Saito et al. (2008) found leafminer Chromatomyia horticola (Diptera:

Agromyzidae) were abundant when it was cool season in Japan. Homestead is located within a tropical area, and the leafminer L. trifolii was more abundant when the average monthly temperature was relatively low (< 20°C). The seasonal density level of the parasitoid, O. dissitus had a similar trend to the leafminer. O. dissitus was much more abundant when leafminer density was high, and less abundant when leafminer density was low. The present findings on the density level of L. trifolii and its parasitoid O. dissitus agreed with Valladares and Salvo (2001) who stated that abundance of the parasitoid, O. dissitus was positively correlated with leafminer host density.

The distribution of L. trifolii and O. dissitus showed an aggregated pattern when their abundance was high, but regular pattern when their abundance was low. It was observed that L. trifolii distribution pattern was affected by the plant leaf size, and smaller size of the leaf caused an aggregated distribution pattern in L. trifolii (Ayabe and

Shibata 2008). In this study, the number of total leaves and total leaf area / bean plant increased with the progression of the plant growing period, which did not increase in population density and aggregation level. Therefore, I conclude that leafminer and the parasitoid’s spatial distribution pattern might be affected by the density, but not by the plant host foliage size. In addition, both L. trifolii and O. dissitus tended to have an

aggregated distribution pattern during the cool season, but a regular pattern during the warm season. Therefore, temperature might be another factor affecting L. trifolii and O. dissitus distribution pattern. The plot size also affected their distribution pattern.

Population of L. trifolii and O. dissitus tended to be aggregated distribution in the plot size of 10 m2, and regular distribution when the plot size was 30 m2.

The regression slope of transformed equation showed a density dependent parasitism by O. dissitus. However, the density dependent relationship between the host density and parasitism proportion was weak in the present study. Nelson and

Roitberg (1995) reported that leafminer parasitoid, O. dimidiatus tended to have a density-dependent behavior when host mine density increased. For spatial distribution, some authors indicated that host aggregated distribution pattern would increase the parasitoid foraging efficiency, and this may lead to a density dependent parasitism

(Hassell and May 1974; Heads and Lawton 1983). However, our study did not show this aggregate distribution pattern for L. trifolii and O. dissitus. There was no significant higher parasitism rate found when leafminer had an aggregated distribution pattern.

This result supported that spatial aggregation pattern of parasitism was not the condition for increasing biological control efficiency (Reeve and Murdoch 1985).

The density of leafminer, L. trifolii and parasitoid, O. dissitus had a strong seasonal preference. In our study, yellow sticky traps showed an effective monitoring result for L. trifolii but not for O. dissitus. There was not a significant density level of O. dissitus adult caught by yellow sticky trap through the whole bean growth period (September 2010 to

February 2011), and the open field environment might impact the parasitoid survival and activity. The parasitoid, O. dissitus was found to be the most abundant larval-pupal

endoparasitoid of leafminer during the whole bean growth season, and it is a high potential leafminer biological control agent in south Florida.

Figure 2-1. Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 1, from September to October 2010.

Figure 2-2. Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 2, from October to December 2010.

Figure 2-3. Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus at bean site 3, from December 2010 to February 2011.

Figure 2-4. Density (mean ± SE / 5 leaves) of leafminer pupae, emerged L. trifolii and O. dissitus in each month, from September 2010 to February 2011.

Figure 2-5. Density (mean ± SE / yellow sticky card / 24 h) of L. trifolii and O. dissitus adults in each month, from September 2010 to February 2011.

Table 2-1. Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in September 2010

Plot size (m2) Taylor’s power law Iwao’s patchiness regression r2 a b r2  

L. trifolii 10 0.49 0.013 1.22AGG 0.72 0.64 0.98REG

30 0.23 0.46 0.60REG 0.44 2.06 0.74REG

O. dissitus 10 0.56 0.027 1.37AGG 0.45 -2.27 2.74AGG

30 0.023 0.40 -0.32REG 0.0066 3.77 -0.38REG

AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly < 1 (P ≤ 0.05). The division number of 15 and 5 for plot sized at 10 m2 and 30 m2, respectively.

Table 2-2. Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in November 2010

Plot size (m2) Taylor’s power law Iwao’s patchiness regression r2 a b r2  

L. trifolii 10 0.38 -0.052 1.20AGG 0.8 -0.24 1.20AGG

30 0.52 - 0.22 1.58AGG 0.67 - 1.02 1.45AGG

O. dissitus 10 0.53 -0.084 0.97REG 0.7 -0.19 1.11AGG

30 0.66 - 0.0065 1.10AGG 0.73 - 0.114 1.15AGG

AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 (P ≤ 0.05). The division number of 15 and 5 for plot sized at 10 m2 and 30 m2, respectively.

Table 2-3. Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in December 2010

Plot size (m2) Taylor’s power law Iwao’s patchiness regression r2 a b r2  

L. trifolii 10 0.47 -0.63 1.63AGG 0.95 -0.76 1.10AGG

30 0.13 0.16 0.98REG 0.86 0.76 1.05AGG

O. dissitus 10 0.5 -0.73 1.90AGG 0.81 -0.16 1.42AGG

30 0.37 -0.25 1.51AGG 0.79 - 0.33 1.19AGG

AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 (P ≤ 0.05). The division number of 15 and 5 for plot sized at 10 m2 and 30 m2, respectively.

Table 2-4. Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in January 2011

Plot size (m2) Taylor’s power law Iwao’s patchiness regression r2 a b r2  

L. trifolii 10 0.42 -0.80 1.74AGG 0.54 -0.31 1.23AGG

30 0.69 - 0.06 1.41AGG 0.67 -0.33 1.20AGG

O. dissitus 10 0.38 -0.013 1.13AGG 0.88 0.69 1.09AGG

30 0.41 0.06 1.10AGG 0.89 1.27 0.99REG

AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 (P ≤ 0.05). The division number of 15 and 5 for plot sized at 10 m2 and 30 m2, respectively.

Table 2-5. Taylor’s power law and Iwao’s patchiness regression parameters pertaining to the distribution of L. trifolii and O. dissitus on beans in February 2011

Plot size (m2) Taylor’s power law Iwao’s patchiness regression r2 a b r2  

L. trifolii 10 0.23 -0.11 0.81REG 0.54 -0.31 1.23AGG

30 0.32 -0.11 0.98REG 0.67 -0.33 1.20AGG

O. dissitus 10 0.72 0.00 1.00RAD 0.17 0.16 0.63REG

30 0.86 -0.13 0.87REG 0.23 0.05 0.47REG

RAN, random distribution, b is not significantly different from 1 (P ≤ 0.05). AGG, aggregated distribution, slop b is significantly >1. REG, regular distribution, b is significantly <1 (P ≤ 0.05). The division number of 15 and 5 for plot sized at 10 m2 and 30 m2, respectively.

Figure 2-6. Bean crop field (50 m  30 m) was divided into 15 equal plots (10 m  10 m).

Figure 2-7. Bean foliages were sampled and placed in the laboratory.

Figure 2-8. Leafminer and the parasitoids were reared in the laboratory.

CHAPTER 3 DIEL DENSITY PATTERN OF LEAFMINER, LIRIOMYZA TRIFOLII AND THE PARASITOIDS, OPIUS DISSITUS (HYMENOPTERA: BRACONIDAE) AND DIGLYPHUS SPP. (HYMENOPTERA: EULOPHIDAE)

Agromyzid leafminer is an important phytophagous fly feeding on a wide range of ornamental and vegetable plants. The American serpentine leafminer, Liriomyza trifolii

(Burgess) is a serious pest of snap bean crop in south Florida. Both adults and larvae can cause economic damage to the plants. The females puncture the leaves with their ovipositors and insert their mouthparts at the punctures to feed on cell contents. The larvae consume mesophyll tissues of the leaf (Musgrave et al. 1975; Fagoonee 1984).

The feeding of larvae and egg laying by adults can significantly impact plant physiology

(Parrella 1985) and create a proper environment for pathogens (Broadbent 1990).

Chemical control is the main method to manage leafminers on various commercial crops. Frequent use of these insecticides enhances development of resistance in leafminers, resurgence of secondary pests and elimination of natural enemies. Thus, insecticide resistance development limits the control of leafminers (Leibee and Capinera

1995).

To reduce sole dependence on insecticides, various studies have been conducted to manage insect pests using biocontrol agents alone or in combination or alteration with insecticides and other control techniques. Several hymenopteran parasitoids were found to be effective for controlling agromyzid leafminers. There are at least 14 species of L. trifolii parasitoids in Florida (Capinera 2001). The parasitoid wasps O. dissitus and

Diglyphus spp. were found abundant on bean crops in south Florida (unpublished report, Li. 2011). It was reported that Opius sp. and Diglyphus spp. were reared from L.

trifolii from celery and tomato leaves in Florida (Stegmaier 1972; Schuster and Wharton

1993).

In developing an integrated pest management program, knowledge of the biology of the pest and its natural enemies is essential. Negative response of natural enemies to the commonly used insecticides can’t be avoided. Considering the importance, Kaspi

(2005) and Weintraub (2001) found that parasitoid D. isaea was more compatible with abamectin than cyromazine.

Various studies are available on the biology of L. trifolii. The activity of L. trifolii and its parasitoids is affected by various factors. The leafminer L. trifolii activity is affected by temperature, light, moisture and host plants type. Temperature is an important factor affecting the activity of Agromyzidae leafminer and their parasitoids in the natural environment (Abou-Fakhr Hammad 2000; Bordat et al. 1995). Temperature tends to vary throughout the day. We assumed the activity or density levels of L. trifolii and its parasitoids will be different throughout various periods of the daytime. Few studies ever assessed the diel density pattern of L. trifolii and its parasitoids.

Understanding the diel density pattern of L. trifolii parasitoids is crucial in reducing the insecticide impact on these natural enemies in integrated pest management program.

To create more effective IPM decisions for controlling L. trifolii, the objective of this study was to determine the diel density pattern of leafminer, L. trifolii, and its parasitoids, O. dissitus and Diglyphus spp.

Materials and Methods

Study Site and Beans Preparation

The study was carried out in the TREC research plots, Homestead, FL. Two bean fields (site 1 and site 2), each 0.4 ha, were selected for the present study (Figure 3-8).

The soil type of the study sites was Krome gravelly loam soil, which consists of about

33% soil and 67% pebbles. Both fields (site 1 and site 2) were planted with snap bean

(Phaseolus vulgaris, supplied from Harris Moran seed Company, Modesta, CA, USA) on 3, Oct. and 10, Dec. 2010, respectively. Pre-plant herbicide halosulfuron methyl (51.9 g / ha, Sandea®, Gowan Company LLC., Yuma, Arizona), was applied 3 weeks before planting for controlling nutsedge and broad-leaf weeds the seeds. The beans were seeded directly in rows at the rate of three seeds / foot. The bean seeds were spaced 3 feet between two adjacent rows. Granular fertilizer 8: 16: 16 at the rate of 1345 kg / ha was used at planting in a band 1 m apart from the seed rows. Additionally, liquid fertilizer (4: 0: 8) at the rate of 0.56 kg / ha / day was used five weeks after planting at each site. The plants were irrigated through drip tubes twice every day delivering 1.0 inch (2.54 cm) each time. During the early bean growth period, fungicide chlorothalonil

(2.81 L / ha, Bravo®, Syngenta Crop Protection, Inc., Greensboro, NC) was used to prevent fungal disease. Both sites were managed for conducting present study until 15,

Dec. 2010 and 22 Feb. 2011, respectively. No insecticide was used during the study period.

Plot Design and Diel Activity

Each study site was divided into 15 equal plots, each (10 m  10 m). One yellow sticky trap (7.6 cm  12.7 cm) was set in the center of each plot from 08:00 to 18:00

EST within a day. For the purpose of studying diel activity pattern of leafminer and its parasitoids, sticky taps were checked at 2h intervals (08:00 – 10:00, 10:01 – 12:00,

12:01 – 14:00, 14:01 – 16:00 and 16:01 – 18:00 EST) once in every week at the 4th, 5th,

6th and 7th week after the bean planting in each site. The cards were checked 20 min

before each interval. At the time of checking sticky cards, each insect on the card deemed to be leafminer and its parasitoids was marked with colored pen (Figure 3-9), different marks for different intervals. At the end of the day, all traps were collected and wrapped individually with transparent polyethylene sheet to avoid any trapping of unwanted insects. Each trap was marked with plot number, date, and period. All traps were transported to the IPM laboratory, TREC, Homestead, FL. The numbers of leafminer and various parasitoids were recorded using a binocular microscope. The leafminers were sent to Division of Plant Industry (DPI) for identification. The parasitoids were sent to Systematic Entomology Laboratory (USDA, MD) for confirmation of identification to genus and species levels. Each week, a new set of sticky traps was used on the day of study.

Statistical Analysis

The diel densities of insects were calculated as the trapped ones during each 2h interval. The mean number of trapped L. trifolii, O. dissitus and Diglyphus spp. from each different period was compared by analysis of variance (ANOVA, PROC GLM, SAS

Institute Inc. 2003). The means were separated by the Least Significance Difference

(LSD) (P < 0.05).

Results

Site 1. The population abundance of L. trifolii, O. dissitus and Diglyphus spp. in each two hours interval within the day was assessed on November 09, 2010 (Figure 3-

1). The abundance of leafminer was significantly higher (F = 10.58; df = 4,74; P <

0.0001) in the first (8:00 – 10:00 EST) and second 2h interval (10:01 – 12:00 EST) than other intervals. Population abundance of Leafminer decreased as day light hours increased. Lowest activity of leafminer was observed during the fifth interval.

Population abundance of Diglyphus spp. was significantly higher (F = 3.33; df =

4,74; P = 0.0148) in the second, third and fourth intervals than in the first and fifth intervals. Diglyphus spp. was almost absent during the first 2h interval (8:00 – 10:00

EST). Abundance of O. dissitus did not show any significant difference (F = 2.04; df =

4,74; P = 0.0979) among the intervals within the day (Figure 3-1). Overall, O. dissitus poplulation was low during this study.

On November 16, 2010, the density of L. trifolii was significantly higher (F = 4.24; df = 4,74; P = 0.004) in the first 2h interval than in the second and fifth intervals (Figure

3-2). However, population abundance of leafminers in the first two hours did not differ from the third and fourth intervals within the day. The highest peak of leafminers populations was observed at 8:00 – 10:00 followed by 14:01 – 16:00; 12:01 – 14:00;

16:01 – 18:00 and 10:01 – 12:00 EST.

Diglyphus spp. was significantly more abundant (F = 5.06; df = 4,74; P = 0.0012) in the second two hours interval than in the other intervals (Figure 3-2). Diglyphus spp. was almost absent in the first two hours interval. Like Diglyphus spp., O. dissitus was absent in the first interval (08:00 – 10:00 EST); and increased insignificantly in the rest of the intervals (F = 0.88; df = 4,74; P = 0.4778).

On November 23, 2010, the L. trifolii density was significantly higher (F = 17.8; df

= 4,74; P < 0.0001) in the first 2h interval (08:00 – 10:00 EST) than the rest of the intervals within a day (Figure 3-3). Diel density of leafminer decreased as the day time progressed. The lowest activity of leafminer was observed at the end of the day (16:01

– 18:00 EST). The peak increase in diel activity of Diglyphus population was observed during the second interval (10:01 – 12:00) (F = 4.28; df = 4,74; P = 0.0037) (Figure 3-3).

Decrease in activity of Diglyphus sp. was observed before and after the second interval.

Population abundance of O. dissitus was low in all intervals (F = 1.5; df = 4,74; P =

0.2128) (Figure 3-3). No clear peak in the diel pattern of O. dissitus was observed during this study (F = 1.5; df = 4,74; P = 0.2128).

On December 01, 2010, the L. trifolii population abundance was still significantly higher in the first 2h interval (F = 12.77; df = 4,74; P = 0.0002) (Figure 3-4) followed by the fourth, third, second and fifth intervals. Population activity of leafminers was minimum at the end of the day. Population of Diglyphus spp. peaked at the second two hours interval (F = 6.46; df = 4,74; P < 0.0001) (Figure 3-4). Activity of Diglyphus decreased thereafter with the increase or decrease of day light hours. However,

Diglyphus populations were present all across the day. Population of O. dissitus was significantly lower during this study (Figure 3-4). No significant difference (F = 0.2; df =

4,74; P = 0.9394) of the O. dissitus density was found among all the intervals.

When data across all intervals of a day were combined, a distinct pattern in the diel activity of leafminers was observed (Figure 3-5). Peak activity of leafminers was observed during the first 2h interval, which was significantly different (F = 7.22; df =

4,19; P = 0.0019) from all other intervals. The abundance of leafminers did not differ among the rest of intervals. Unlike leafminer, the density of Diglyphus spp. was highest

(F = 7.6; df = 4,19; P = 0.0015) during the second two hours interval than other intervals

(Figure 3-5). The lowest density level of Diglyphus was observed during the first and last 2h intervals. No significant difference (F = 1.44; df = 4,19; P = 0.2696) was found in

O. dissitus density level among all the intervals within the day.

Site 2. The diel activity pattern of L. trifolii was studied based on the combined four days’ data from 15 yellow sticky traps. The leafminer density did not show any significant difference (F = 0.7; df = 4,19; P = 0.6049) (Figure 3-6) within the daytime.

Unlike site 1, the Diglyphus spp. had a significantly higher density level (F = 6.43; df =

4,19; P = 0.0032) (Figure 3-6) in the third two hours interval (12:01 – 14:00 EST). The

O. dissitus density presented a significant difference (F = 3.8; df = 4,19; P = 0.0251) during the daytime, and its density was highest in the second two hours (10:01 – 12:00

EST) among all the intervals.

Discussion

Temperature was a major factor for L. trifolii and its parasitoid abundance. High daily temperatures (≥ 30°C) could reduce the adult’s density (Abou-Fakhr Hammad

2000). At the bean site 1 (November to December 2010), the average temperature within the daytime changed greatly (22 to 27°C), and it was relatively low (< 25°C) in the morning and high (> 25°C) during the rest of the day. The diel density of leafminer, L. trifolii was significantly higher in the first two hours interval when the temperature was lower (< 25°C) than other periods of the day. The leafminer population started to decrease when the temperature increased above 25°C after the second two hours interval. Therefore, the low temperature in the first 2h (8:00 – 10:00 EST) might be the main reason for higher density of leafminer in the morning than other periods of a day.

The parasitoid, O. dissitus did not show any significant density difference among the periods within the day. It was reported that the optimum temperature for both of male and female adults was 20°C, and female had a higher reproduction at 25°C

(Bordat et al. 1995b). The Diglyphus spp. density was always higher in the second two

hours interval than any other periods when the temperature was above 25°C within the day.

There were three different species in Diglyphus spp. found in our study, indicating

D. begini (Ashmead), D. intermedius (Girault) and D. isaea (Walker). The Diglyphus spp., is a larval ectoparasitoid, which has been used in leafminer biological control program in greenhouse (Minkenberg 1987). The optimum temperature for rearing D. isaea from L. trifolii ranged between 32.3°C and 32.6°C (Bazzocchi et al. 2003). The relatively low temperature (< 25°C) in the first two hours interval in the daytime might reduce the Diglyphus spp. density level. The density of Diglyphus increased to a higher level at the second two hours interval when the temperature increased (> 25°C).

The 4 days’ combined data in site 2 in spring 2011, shown in the figure 3-6, did not provide a similar diel density pattern as in bean site 1. The average daily temperature was relatively high during late February and early March. The temperature in the morning was low but not significantly different from the temperature at other periods within the day, and it may not affect the diel density level of L. trifolii. O. dissitus density is relatively higher in the second two hours interval. The highest density level of

Diglyphus spp. was in the third two hours (12:01 – 14:00 EST) interval. These results agreed with the study of temperature influence on O. dissitus and Diglyphus spp.

(Bordat et al. 1995b; Bazzocchi et al. 2003).

Insects’ abundance might be another factor affecting the diel density pattern. The abundance of L. trifolii was higher in the bean site 1 than in bean site 2 (Figure 7), which might be a reason that the leafminer showed a significant density level in the first two hours interval (8:00 – 12:00 EST) within a day in the site 1. However, there was not a

significant diel density level of leafminer during daytime at the site 2 where the leafminer density was very low.

Yellow sticky traps were used for monitoring leafminer and its parasitoids diel density pattern in the study, and it showed an effective monitoring result. The yellow sticky traps had more chance to catch leafminer when the density was high, but less chance when leafminer density was low. Parrella and Jones (1985) revealed yellow sticky trap was a useful and rapid tool to estimate L. trifolii density on Chrysanthemum in greenhouse. In our study, the results indicated that the yellow sticky trap was also an effective tool for monitoring the seasonal abundance of Diglyphus parasitoids density.

Therefore, yellow sticky traps can be used as an effective tool in the research on leafminer and some parasitoid ecology.

The parasitoids of O. dissitus and Diglyphus spp. were found to be the most abundant parasitoid wasps of leafminer L. trifolii on bean crops. O. dissitus is a larval- pupa endoparasitoid of Liriomyza leafminer and adults emerge from the leafminer pupae. Diglyphus parasitoids were ectoparasitoid of leafminer larvae and adults emerge from the mines on foliages. Understanding the diel biological behavior of leafminer and parasitoid could improve IPM strategies in the open field crops. Releasing the leafminer parasitoids during the appropriate time and season could enhance the biological control effectiveness in the open field environment. In addition, insecticides application during the selective time period could relatively reduce the impact on the parasitoids.

Figure 3-1. Bean site 1, Nov-09-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h interval after 8:00 EST. Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-2. Bean site 1, Nov-16-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h interval after 8:00 EST. Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-3. Bean site 1, Nov-23-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h interval after 8:00 EST. Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-4. Bean site 1, Dec-01-2010. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / yellow sticky trap during each 2 h interval after 8:00 EST. Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-5. Bean site 1. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 EST (based on combined data of Nov-09, Nov-16, Nov-23, Dec-01, 2010). Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-6. Bean site 2. Mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. / 15 yellow sticky traps during each 2 h interval after 8:00 EST (based on combined of Jan-31, Feb-17, Feb-24, Mar-01, 2011). Means with the same letter are not significantly different (P < 0.05, LSD test).

Figure 3-7. Seasonal density: mean ( SE) number of the L. trifolii, O. dissitus and Diglyphus spp. per 15 yellow sticky traps / 10 h at bean site 1, 2010 and site 2, 2011. The mean temperature was in November 2010 to February 2011.

Figure 3-8. Fifteen yellow sticky traps were set in bean field from 8:00-18:00 EST.

Figure 3-9. The caught insects during different 2 h interval were marked on the yellow sticky card.

CHAPTER 4 THE COMPOSITION AND SEASONAL ABUNDANCE OF HYMENOPTERAN PARASITOIDS OF LIRIOMYZA TRIFOLII ON BEANS IN SOUTH FLORIDA

The phytophagous leafminer (agromyzidae: Liriomyza spp.) has a significant economic impact on vegetable and ornamental production worldwide. The leafminer

Liriomyza trifolii (Burgess), L. sativae (Riley) and L. huidobrensis (Blanchard) are the important species threatening the agriculture in the USA (Parrella 1987; Capinera 2001;

Schuster and Wharton 1993). The traditional control of leafminer is using broad- spectrum insecticides. This has a high potential for developing leafminer resistance

(Leibee and Capinera 1995), and it also impacted the natural enemies density (Johnson et al. 1980).

Biological control is an important component in integrated pest management (IPM) of leafminer. Research has been conducted to evaluate the biological control effectiveness by using parasitoid wasps in leafminer management (Johnson et al. 1980;

Patel et al. 2003). For minimizing the impact of insecticides on parasitoids, studies have been performed to evaluate the compatibility of traditional and new insecticides with leafminer parasitoids (Ferguson 2004; Weintraub 2001; Kaspi 2005).

Several studies have been conducted to identify leafminer parasitoids in various regions and on different plant hosts in North America (Lasalle and Parrella 1991;

Stegmaier 1966; Stegmaier 1972; Schuster and Wharton 1993). Schuster and Wharton

(1993) reported 4 families and 15 species of leafminer parasitoid on tomato in Florida.

Snap bean is an important agricultural crop in Florida, and it valued $138.4 million during 2000 - 2001 (Mossler and Nesheim 1999). Leafminer L. trifolii is the most significant pest on beans. The leafminer hymenopteran parasitoids were found in the bean crop during various growth seasons. Understanding the parasitoids’ complex and

status is the primary step for investigating new potential biological control agents. It is also important to reveal the leafminer and its parasitoid interactions, and community structure in the crop field in management. For identifying the leafminer parasitoids group on snap bean crops and exploring potential effective biological control agents, the objective of this study research was to study the complex and seasonal abundance of leafminer parasitoids on beans in south Florida.

Materials and Methods

Study Site

The leafminer parasitoids complex was investigated in the same bean crop fields mentioned in chapter 2 in the Tropical Research and Education Center (TREC),

University of Florida. The site 1 was planted to bean on September and maintained until

November 2010, site 2 was from October to December 2010, and site 3 was from

December 2010 to February 2011. The snapbean (Phaseolus vulgaris) seeds were supplied from Harris Moran seed Company, Modesta, CA, USA. The bean fields were prepared with cultural practices as the same as discussed in chapter 2 and chapter 3.

No insecticide was used during the study.

Leaf Sampling and Insect Rearing

The parasitoids were obtained from the bean foliages. The sampling began when the bean plant had two primary leaves fully unfolded. To address all variability in the sampling, the field was divided into 15 equal plots. Five leaves, one leaf / plant, were randomly collected from each plot weekly (75 leaves / week). When the bean plants became mature and their primary leaves dropped off, the older bottom leaves were always collected from the plants because of L. trifolii feeding preference to the older mature leaf (Facknath 2005; Williams et al 1998). The sampled leaves from each plot

were placed into a 15 cm diameter Petri dishes (5 leaves per Petri dish) and were marked with date and plot number. The samples were then transported to the laboratory and placed into a growth at 25°C, 70% RH and 14:10 (L: D) h for the development of leafminer and its parasitoids. The bean leaves were checked every day to collect leafminer pupae. The pupae in each Petri dish were separated from the leaves and transferred into one new Petri dish labeled with the sampling date. The pupae were checked every day to record larval-pupal endoparasitoid emergence. The leaves were also checked every day to collect larval ectoparasitoid from leaf mines.

All the emerged parasitoids were collected and preserved in the 75% alcohol for identification. The leafminers were identified following morphological characters described by Capinera (2001). For further confirmation, the leafminers were sent for identification to the Division of Plant Industry (DPI), Gainesville, FL. The parasitoids were identified based on the external characters used in previous studies (Lasalle and

Parrella 1991; Gordh and Hendrickson 1979; Asadi et al 2006) and further verified by

Systematic Entomology Laboratory, USDA, MD.

Results and Discussion

In the present study, about 99% of the leafminer collected from the beans was identified as L. trifolii (Figure 4-1). The L. trifolii adult is about 2 mm long and the wing length is 1.25 to 1.9 mm (Capinera 2001). The red eyes, grayish black mesonotum and yellow hind margins of the eyes are the key characters of L. trifolii. The L. trifolii abundance was high in December 2010 and January 2011 (Table 4-1). The average temperature of these two months was lower (15 to 19°C) than other planting months (20 to 26°C). Saito et al. (2008) reported that leafminer Chromatomyia horticola (Diptera:

Agromyzidae) was abundant in the cool season on the pea crop. Abou-fakhr-Hammad

(2000) reported that population density of L. huidobrensis was reduced by the high daily average temperatures.

Opius dissitus (Figure 4-2). Two different Braconidae parasitoids, O. dissitus and

Euopius sp. were observed on beans. O. dissitus is a solitary larval-pupal endoparasitoid of L. trifolii (Nelson and Roiterg 1995; Bordat et al. 1995 a; Bordat et al.

1995 b). O. dissitus adult is small in size (about 1.50 mm). It is black in color with long antennae, which are thin and black (Figure 4-2). O. dissitus was found to be the most abundant leafminer parasitoid on snap beans in the Miami-Dade Co. (Table 4-1). The parasitoid females deposit their eggs inside the leafminer larvae. The parasitized host larvae continue to develop until pupae stage. This observation is supported by Bordat et al. (1995 a) reported that the parasitoid females lay their eggs directly inside their host larvae. The development of O. dissitus takes place inside the host pupae. At the end of the development, adult emerged out from the pupae. One O. dissitus adult was found to emerge from a single leafminer pupa in our study. The density of O. dissitus was positively correlated with leafminer density within the whole growth season. The density of O. dissitus was high when the temperature was cool (15 ~ 19°C) in December 2010 and January 2011 (Figure 4-15). The optimum temperature for both of O. dissitus male and female was 20°C, and female had a higher reproduction at 25°C (Bordat et al. 1995 b). The O. dissitus was ever reported from celery and infested tomato leaves in Florida

(Stegmaier 1972; Schuster and Wharton 1993).

Euopius sp. (Figure 4-3). Euopius sp. is also a larval-pupal endoparasitoid. One

Euopius sp. adult emerged from a single host pupa. Euopius sp. has almost the same body size and antennae as O. dissitus. It has an overall yellow color body, which is

clearly different from O. dissitus being black in color. The abundance of Euopius sp.

(Table 4-1) was low on the bean foliage in the study during fall 2010, and no Euopius sp. was found after December 2010. The Euopius sp. was reared from Liriomyza leafminer infested weeds on Bidens alba (Schuster and Gilreath 1991).

Diaulinopsis callichroma (Figure 4-4). There were ten different species of parasitoids found in the family of Eulophidae. The D. callichroma was found to be the second largest group of leafminer parasitoid in this study (Table 4-1). Its abundance was high in October and November 2010, however, was low in January and February

2011 (Figure 4-15). The adult hind femora are basal dusky, fore and middle femora are pale (Gordon and Hendrickson 1979). The male adult’s basal antennal segments are black and enlarged. The body size of adults is about 1.10 -1.30 mm. This parasitoid is a larval ectoparasitoid of leafminer. The D. callichroma larvae feed on the host larva directly and kill the host eventually. The parasitoid larvae pupate inside the mine and emerged from the mine. The abundance of D. callichroma might be affected by both of the host density and temperature condition.

Diglyphus spp. (Figure 4-5; Figure 4-6; Figure 4-7). Three different species of

Diglyphus parasitoids (D. begini, D. intermedius and D. isaea) were reared from the bean foliages. They were not present until November 2010 and became abundant in

February 2011 (Figure 4-15). The D. begini abundance was relatively higher than D. intermedius and D. isaea (Table 4-1). The D. intermedius and D. isaea had a relatively the similar density to each other. Diglyphus spp. parasitoids were the third largest group in the study. Diglyphus spp. is a larval ectoparasitoid, and female adult generally lays more than one egg beside of the host larva (Minkenberg 1987). Diglyphus sp. is

characterized by their antennae with two funicular segments. The forewings of these three species are dense setose. The basal cell is uniformly dense setose. These three species can be differentiated from each other based on the dark area on their hind tibia:

D. begini basal hind tibia has a short and less than 25% metallic dark area (Figure 4-5).

D. intermedius basal tibia has a relatively larger area of 25 - 35% dark color and with extended dusky color (Figure 4-6); D. isaea basal hind tibia with over 75% metallic dark color proportion (Figure 4-7) (Lasalle and Parrella 1991; Gordh and Hendrickson 1979).

Schuster and Wharton (1993) reported D. intermedius and D. begini were reared from tomato crop in Florida. The Diglyphus parasitoids were observed the most abundant larval ectoparasitoid on tomato crop (Schuster and Wharton 1993). However, in our study, the most abundance larval ectoparasitoid on bean crop was D. callichroma. The

D. isaea was used as an effective biological control agent in greenhouse for controlling

Liriomyza leafminer (Minkenberg 1987; Boot et al. 1992). Several studies were conducted on Diglyphus parasitoid and its compatibility with common insecticides

(Bazzocchi et al. 2003; Kaspi and Parrela 2005; Weintraub 2001).

Neochrysocharis sp. (Figure 4-8) was reported as an endoparasitoid of leafminer larvae, and the adults emerge from the mines on the leaves. The adult body is overall metallic green and eyes are red. The fore and middle legs, and hind tibia are pale. The species of N. punctiventris was ever reported as the fourth abundant leafminer parasitoid on tomato crops in Florida (Schuster and Wharton 1993).

Closterocerus sp. (Figure 4-9). The population abundance of Closterocerus sp. was low on bean foliage during this study. It is an endoparasitoid of leafminer young stage larvae (Asadi et al. 2006). The adult body length is about 1.0 mm. The forewings

of Closterocerus are with characteristic dark bands. Stegmaier (1966) reported C. cinctipennis was a parasitoid of leafminer L. trifolii in Florida.

Zagrammosoma spp. (Figure 4-10; Figure 4-11). Zagrammosoma lineaticeps and

Z. muitilineatum are the larval ectoparasitoid of Liriomyza leafminer. Both of Z. lineaticeps and Z. muitilineatum were found in Florida (Stegmaier 1972; Stegmaier

1972; Schuster and Wharton 1993). The Z. lineaticeps is black or very dark color and the forewing has a dark line along the apical margin (Figure 4-10), while Z. muitilineatum is predominately yellow and mesoscutum with dark stripes (Figure 4-11).

The abundance of both species was high in October and November 2010. In this study,

Z. muitilineatum was relatively more abundant (20 adults) than Z. lineaticeps (10 adults)

(Table 4-1).

Pnigalio sp. (Figure 4-12) is a larval ectoparasitoid of Liriomyza leafminer and adult emerges from the mine on the bean foliage. This genus of parasitoid was also reported to parasitize on Diptera, Lepidoptera, Hymenoptera and Coleoptera (Asadi et al. 2006). The body length of adult females is 1.7 ~ 1.9 mm and male is 1.5 ~ 1.7 mm.

Its antennae are with 4 funicular segments, and male has a laterally branched structure antenna (Asadi et al. 2006). Lasalle and Parrella (1991) indicated that only one Nearctic species of P. flavipes attacks Liriomyza. However, Schuster and Wharton (1993) reported species of P. maculipes was reared from the tomato foliage in Florida. The species of Pnigalio in this study was unknown.

Chrysocharis sp. (Figure 4-13) was the larval-pupal endoparasitoid of leafminer, and it was the second most abundant larval-pupal endoparasitoid (Table 4-1). In our study, Chrysocharis adults emerged from leafminer pupae. It only appeared in spring

season in January and February 2011. Chrysocharis sp. post-marginal vein is always longer than the stigma vein, and petiole is present and distinct.

Halticoptera sp. (Figure 4-14) was found in the family of Pteromalidae.

Halticoptera sp. petiole is present and relatively long, and antenna has 6 funicular segments. This species is another larval-pupal endoparasitoid of leafminer in beans.

The abundance of this species was very low and only present in October and November

2010. Schuster and Wharton (1993) reported a high abundance of Halticoptera sp. on tomato. The H. circulus was reported to be the only one Nearitic species of Halticoptera known to parasite Liriomyza leafminer (Lasalle and Parrella1991).

Many biotic and abiotic factors, as temperature, rainfall, host crops, alternative plant hosts, and intra- or inter-competition, may affect the leafminer parasitoids population. In our study, 13 species in three families of leafminer parasitoid were reared from the bean foliages in south Florida. Four species were leafminer larval-pupal endoparasitoid, two were larval endoparasitoid, and seven were larval ectoparasitoid.

The O. dissitus was the most abundant larval-pupal endoparasitoid and D. callichroma was the most abundant larval ectoparasitoid on snap beans. Our study is the first report on D. isaea in Florida. We assumed the nursery industry in Miami Dade Co. might bring and release this species of parasitoid for controlling leafminer on the ornamental plants.

The recent study on parasitoid complex on bean crop will provide significant information of potential biological agents of leafminer for IPM. In addition, the pictures in this study will benefit leafminer hymenopteran parasitoids identification in the future’s work.

Figure 4-1. Liriomyza trifolii (Agromyzidae).

Figure 4-2. Opius dissitus (Braconidae).

Figure 4-3. Euopius sp. (Braconidae).

Figure 4-4. Diaulinopsis callichroma (Eulophidae).

Figure 4-5. Diglyphus begini (Eulophidae).

Figure 4-6. D. intermedius (Eulophidae).

Figure 4-7. D. isaea (Eulophidae).

Figure 4-8. Neochrysocharis sp. (Eulophidae)

Figure 4-9. Closterocerus sp. (Eulophidae).

Figure 4-10. Zagrammosoma lineaticeps (Eulophidae).

Figure 4-11. Z. muitilineatum (Eulophidae).

Figure 4-12. Pnigalio sp. (Eulophidae).

Figure 4-13. Chrysocharis sp. (Eulophidae).

Figure 4-14. Halticoptera sp. (Pteromalidae).

Figure 4-15. Seasonal abundance of parasitoids, O. dissitus, D. callichroma and Diglyphus spp. on snap bean crop from September 2010 to February 2011.

Table 4-1. Number of leafminer L. trifolii and its parasitoids (%) reared from the bean foliages (300 leaves / month) from September 2010 to February 2011 in south Florida Family and species Sep Oct Nov Dec Jan Feb Total Agromyzidae L. trifolii (Burgess) 101 18 132 583 1805 66 2805 Braconidae Opius dissitus (Muesebeck) 41 (73%) 12 (11%) 91 (44%) 202 (79%) 401(83%) 31 (26%) 778 (63%) Euopius sp. 3 (5.0%) 3 (3.0%) 2 (1.0%) 2 (0.7%) 0 0 10 (0.8%) Eulophidae Diaulinopsis callichroma (Crawford) 8 (14%) 73 (69%) 78 (38%) 18 (7.2%) 27 (5.6%) 9 (7.0%) 213 (18%) Diglyphus begini (Ashmead) 0 0 4 (2.0%) 10 (4.0%) 17 (3.5%) 21 (17%) 52 (4.2%) D. intermedius (Girault) 0 0 1 (0.5%) 4 (1.6%) 5 (1.0%) 20 (16%) 30 (2.4%) D. isaea (Walker) 0 0 1 (0.5%) 2 (0.7%) 9 (1.9%) 14 (12%) 26 (2.1%) Neochrysocharis sp. 2 (4.0%) 7 (7.0%) 7 (3.5%) 8 (3.2%) 2 (0.4%) 14 (12%) 40 (3.3%) Closterocerus sp. 2 (4.0%) 1 (1.0%) 2 (1.0%) 4 (1.6%) 1 (0.2%) 0 10 (0.8%) Zagrammosoma lineaticeps (Girault) 0 3 (3.0%) 2 (1.0%) 0 0 0 5 (0.4%) Z. muitilineatum (Ashmead) 0 6 (5.0%) 13 (6.0%) 0 1 (0.2%) 1 (0.8%) 20 (1.6%) Pnigalio sp. 0 0 4 (2.0%) 5 (2.0%) 1 (0.2%) 0 10 (0.8%) Chrysocharis sp. 0 0 0 0 19 (4.0%) 11 (9.2%) 30 (2.4%) Pteromalidae Halticoptera sp. 0 1 (1.0%) 1 (0.5%) 0 0 0 2 (0.2%)

CHAPTER 5 CONCLUSION

Leafminer, Liriomyza trifolii is an important pest infesting vegetable and ornamental plants all over the world. It became a problem on celery and other vegetables since 1945 in Florida (Wolfenbarger 1947). Managements have been established to control this pest on economic crops, including chemical control, biological control and cultural practice. The importance of integrated pest management (IPM) of leafminer has been realized due to the problems caused by chemical controls. The development of insecticides resistance and impact on the natural enemies in the traditional chemical control reduce the effectiveness of leafminer management. L. trifolii is one of the serious pests on bean production in south Florida. For enhancing the IPM control of leafminer, several studies have been conducted for assessing leafminer L. trifolii seasonal abundance, spatial distribution and its hymenopteran parasitoids complex on bean crops in south Florida.

Liriomyza trifolii abundance presented a seasonal preference, and its density was high during the cool season, but low in the warm season. The parasitoid, O. dissitus had a similar seasonal density trend as L. trifolii density level. Both L. trifolii and

O. dissitus showed an aggregated distribution in the bean field when the abundance was high and a regular pattern when their abundance was low. This information will be helpful for monitoring leafminer density and determining leafminer economic threshold in bean production.

The diel density of L. trifolii and its two parasitoids was studied in snap bean field based on five divided 2h intervals within a day. L. trifolii presented a higher density level during the first 2h (8:00 – 10:00 EST) than any other time within a day in cool season

during November and October 2010. There was no significant difference in parasitoid O. dissitus density throughout the day. Diglyphus spp. presented a peak density level in the second 2h (10:01 – 12:00) within a day in the cool season and the third 2h (12:01 -

14:00) in the warm season. Information of their diel activity can guide a chemical control strategy, which reduces the direct impact on these parasitoids. Chemical insecticides can be applied when leafminer density is high and parasitoids density is low. This information also benefits the biological control application, and releasing leafminer parasitoids during its preferred period within a day can enhance the control effectiveness.

Hymenopteran parasitoids have been used to control Liriomyza leafminers. In my study, thirteen different genera or species of parasitoids belonging to three families were collected from beans. The Braconidae parasitoids include O. dissitus, Euopius sp.

The Eulophidae parasitoids include Diaulinopsis callichroma, Diglyphus begini, D. intermedius, D. isaea., Neochrysocharis sp., Closterocerus sp., Zagrammosoma lineaticeps, Z. muitilineatum, Pnigalio sp. and Chrysocharis sp. One genus of

Halticoptera sp. was found in family Pteromalidae.

Opius dissitus was found the most abundant leafminer parasitoid, and it is a larval-pupal endoparasitoid. D. callichroma was the second abundant parasitoid, and it is a larval ectoparasitoid. Diglyphus spp. was the third abundant parasitoid group, and they are also larval ectoparasitoid. The importance of surveying the composition and seasonal abundance of leafminer parasitoids is to provide information for developing management program incorporating effective biocontrol agents. It will also guide

growers to properly apply insecticides and to reduce their harmful impact on the natural enemies.

This research was established to assess leafminer L. trifolii biology characters, including its seasonal abundance and field spatial distribution. This study also investigated the composition and seasonal abundance of its natural hymenopteran parasitoids. O. dissitus was found to be the most abundant parasitoid of L. trifolii on snap bean crop. This study will help make pest management strategies and enhance the effectiveness of IPM in leafminer control.

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BIOGRAPHICAL SKETCH

Jian Li was born in Shenyang, Liaoning Province, China, in 1986. He graduated from Shenyang No. 27 high school in 2005, and began his undergraduate study at

Shenyang Agricultural University. He received a bachelor’s degree in plant protection in

2009. His undergraduate education included plant pathology, entomology, and plant chemical control. His undergraduate research was focused on integrated pest management of root knot nematode, Meloidogyne incognita under the supervision of

Professor Duan Yuxi. His undergraduate thesis was ranked first place in the Plant

Pathology program. He was awarded “Outstanding Undergraduate” in 2009.

Jian enrolled at University of Florida in 2009 and began his master’s study in entomology under the supervision of Dr. Dakshina Seal. Jian’s research involved

Liriomyza leafminer seasonal abundance, spatial distribution, and leafminer hymenopteran parasitoids complex. He received a GPA of 3.77 in his master’s courses.

He participated in extension activities to help growers and crop management advisors.

He also shared his research information with the local growers and industry researchers. He presented his research study of “Spatial distribution of leafminer,

Liriomyza trifolii and its parasitoid Opius sp. on beans in south Florida” in the annual meeting of Entomology Society of America, Southeastern branch, Puerto Rico, 2011.

He won the 3rd place in the Student Oral Presentation Competition in Florida State

Horticulture Society Annual Meeting, St. Petersburg, 2011.

Jian will start his PhD study in Horticultural Science at University of Florida in fall

2011. His prospective research will focus on the tomato breeding with molecular biology techniques and enhance the tomato resistance to pathogens. His career goal is to earn a position as a professor with a university or researcher with a corporation.