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Life History of Triaspis eugenii Wharton and Lopez-Martinez (Hymenoptera: Braconidae) and Evaluation of its Potential fo...

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PAGE 1

LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE) AND EVALUATION OF ITS POTENTIAL FOR BIOLOGICAL CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA: CURCULIONIDAE) By ESTEBAN RODRIGUEZ-LEYVA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Esteban Rodrguez-Leyva

PAGE 3

This work is dedicated to my parents, Benito Rodrguez and Pilar Leyva, and brothers, Martn, Vernica, and Vctor, who have b een teaching me the importance of having resolutions in life, social gatheri ng and education included, within an admirable familial strength. Without their support it would be harder to complete this study, to my nieces and nephew. Este trabajo lo dedico a mis padres, Benito Rodrguez y Pilar Leyva, y a mis hermanos, Martn, Vernica, y Vctor, quienes me han mostrado la impo rtancia de tener metas en la vida, incluido el convivir y la educacin, en el seno de una extraordinaria fuerza familiar. Sin su apoyo completar este trabajo hubiese sido ms difcil. A mis sobrinas y sobrino.

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iv ACKNOWLEDGMENTS Many people and institutions contributed to the success of this dissertation. I am extremely grateful to Consejo Nacional de Ciencia y Tecnologa (National Council of Science and Technology) and Colegio de Po stgraduados from Mxico which supported tuitions, living expenses, and laborer situa tions through all the process for getting the goals of this study. My gratitude and appreciati on go to my advisor, Dr. Philip A. Stansly, he found resources for conducting and assi sting my dissertation and gave me independence to express ideas and objectives. Additionally, for the many hours that he spent reading, correcting, and re -reading drafts of this dissert ation. I am grateful to Dr. David J. Schuster who offered encouragemen t, support, and always friendship. I would like to recognize the rest of my committee, Drs. Norman C. Leppla, Ru Nguyen, and Steven A. Sargent, for being available and offering experiences to improve my academic development. Special thanks go to Dr. Susa n Webb for allowing me to use resources in her laboratory in Gainesville, and Mike Miller for being such an amiable person. Special recognition goes to Debbie Hall, Myrna Litchfield, and Nancy Sanders for providing constant support, always with professionalism and amiability. I want to thank also the personnel in the methods and quarantine facilities at the Division of Plant Industry, Gainesville, FL, and many people of the South West Florida Research and Education Center (SWFREC) at Immokalee, which supported me in the implementation of a bunch of activities. I th ank James M. Conner, Carmen Gmez, Jaime Martnez, Alexandra Delgado, and Ronald D. French. At the same time, I thank those

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v who helped me have a nice time in the center, C. Vavrina, F. Jaber, R. Pluke, M. Ozores, P. Roberts, R. Muchovej, S. Sukla, K. Cush man, J. Knowles, K. Jackson, C. Pandey, M. Triana, J. Castillo, F. Roka, J. & K. Hill, K. & R. Systma, B. & B. Hyman, A. Garcia, D. Spencer, and L. Rigby. I also thank Emily Va squez and Steven Davis of the entomology group at Gulf Coast Research and Edu cation Center (GCREC) in Bradenton. I thank Eugenio Mariscal for his help duri ng the field trips to Nayarit, Mxico. I thank my friends in Gainesville, I will always remember academic, cultural, and gastronomical moments, excursions and of c ourse “intoxicating mo ments of life” that I spent with all of them.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES...........................................................................................................xi ABSTRACT......................................................................................................................x ii CHAPTER 1 INTRODUCTION........................................................................................................1 Literature Review.........................................................................................................4 Origin of Capsicum spp. and Distribution of Pepper Weevil................................4 Economic Importance............................................................................................5 Description............................................................................................................6 Biology and Life History.......................................................................................7 Host Plant Association..........................................................................................9 Capsicum spp.................................................................................................9 Solanum spp.................................................................................................11 Management Strategies.......................................................................................12 Host plant resistance.....................................................................................12 Cultural and chemical control......................................................................13 Biological control.........................................................................................14 Objectives............................................................................................................19 2 REARING METHODOLOGY AND IN FLUENCE OF FOOD QUALITY ON BIOLOGY OF PEPPER WEEVIL.............................................................................20 Introduction.................................................................................................................20 Materials and Methods...............................................................................................22 Laboratory Weevil Colony..................................................................................22 Crowding Effects on Oviposition........................................................................24 Fruit Quality........................................................................................................25 Adult Feeding and Oviposition...........................................................................26 Life Table............................................................................................................27 Nitrogen Content of Fruits and Buds...................................................................28 Results........................................................................................................................ .29

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vii Crowding Effects and Oviposition......................................................................29 Fruit Quality........................................................................................................30 Feeding and Oviposition in Peppe rs versus Floral Buds (7d).............................30 Oviposition with or without Floral Buds (7d).....................................................31 Life Table............................................................................................................31 Nitrogen Content of Fruits and Buds...................................................................32 Discussion...................................................................................................................33 Crowding Effects and Oviposition......................................................................33 Fruit Quality........................................................................................................34 Adult Feeding and Oviposition...........................................................................35 Life Table............................................................................................................37 3 LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE).......................................................................40 Introduction.................................................................................................................40 Materials and Methods...............................................................................................43 Surveys................................................................................................................43 Parasitoid Rearing...............................................................................................44 Host-Age Range..................................................................................................46 Role of the Oviposition Plug in Host Location...................................................47 Effect of Weevil: Fr uit Ratio on Parasitoid Production......................................49 Life Cycle............................................................................................................49 Fecundity.............................................................................................................51 Adult Longevity...................................................................................................52 Results........................................................................................................................ .52 Surveys................................................................................................................52 Host-Age Range..................................................................................................54 Role of the Oviposition Plug in Host Location...................................................54 Effect of Weevil: Fr uit Ratio on Parasitoid Production......................................55 Life Cycle............................................................................................................56 Fecundity.............................................................................................................61 Adult Longevity...................................................................................................62 Discussion...................................................................................................................63 Surveys................................................................................................................63 Host-Age Range..................................................................................................64 Role of the Oviposition Plug in Host Location...................................................66 Life Cycle............................................................................................................67 Potential of T. eugenii as a Biological Agent of the Pepper Weevil...................69 4 RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE) IN FIELD CAGES AND IN THE FIELD...72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................74 Insects..................................................................................................................74 Field Cages..........................................................................................................74

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viii Effect of Honey Supplement...............................................................................76 Survival................................................................................................................78 Effect of Host-Damaged Plants...........................................................................78 Field Release and Establishment.........................................................................80 Results........................................................................................................................ .81 Effect of Honey Supplement...............................................................................81 Survival................................................................................................................82 Effect of Host-Damaged Plants...........................................................................82 Field Release and Establishment.........................................................................84 Discussion...................................................................................................................86 Effect of Honey Supplement...............................................................................86 Effect of Host-Damaged Plants...........................................................................87 Field Release and Establishment.........................................................................89 5 CONCLUSIONS........................................................................................................92 Rearing Methodology and Influence of Food Quality on Biology of Pepper Weevil..............................................................................................................92 Life History of Triaspis eugenii ..........................................................................93 Field Cage and Field Release..............................................................................94 LIST OF REFERENCES...................................................................................................98 BIOGRAPHICAL SKETCH...........................................................................................110

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ix LIST OF TABLES Table page 1 Biological data of pepper weevil, selected references until 2005..............................9 2 Non Capsicum Solanaceae used by pepper weevil*................................................12 3 Natural enemies of the pepper weevil, references until 2005..................................17 4 Characteristics of ‘Jalap eo Mitla’ used for nitrog en content determinations.........28 5 Numbers of eggs per female per day fo r different numbers of females and males per single ‘Jalapeo’ pepper fruit.............................................................................30 6 Feeding and oviposition of pepper w eevil adults when given a choice of ‘Jalapeo’ pepper fruit or floral buds (7d)...............................................................30 7 Oviposition of pepper weevil females fed w ith ‘Jalapeo’ pepper fruits only or with pepper fruits plus floral buds............................................................................31 8 Life table parameters for A. eugenii females (n =14) reared on ‘Jalapeo’ peppers at 27 + 2C, and 14:10 (L:D) photoperiod..................................................32 9 Nitrogen content (Mean + SD) of anthers in floral buds and ‘Jalapeo’ fruits at three maturation stages.............................................................................................33 10 Life table parameters for the pepper weevil.............................................................38 11 A. eugenii and parasitoids emerged from approximately 50 kg of infested hot peppers collected in th e state of Nayarit, Me xico, April and May, 2003.................53 12 Parasitism of pepper weevil eggs by T. eugenii when given the choice of two ages of eggs..............................................................................................................54 13 Parasitism of pepper weevil eggs by T. eugenii when given no choice of egg age.54 14 Parasitism of pepper weevil eggs by T. eugenii when given the choice of eggs covered with an oviposition plug and eggs with no plug.........................................55 15 Parasitism of pepper weevil eggs by T. eugenii when given no choice of eggs covered with an oviposition plug and eggs with no plug.........................................55

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x 16 T. eugenii produced with two pe pper weevil densities............................................56 17 Developmental times of T. eugenii in days (Mean SD) at 27 1C.....................57 18 Demographic parameters (+ 95% CL) for T. eugenii reared on pepper weevil eggs in ‘Jalapeo’ fruits for di fferent exposure intervals at 27 + 2C......................63 19 Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and 14 pepper weevil adults at 27 + 2C........................................................................71 20 Releases of T. eugenii during 2005 at the SWFREC in Immokalee, FL..................80 21 Number of T. eugenii adults released in field ca ges and subsequently observed on pepper fruits (n=5) hung on pepper plants at 28.2 + 4.7C, 55 + 9% RH...........83 22 Adults per 50 terminals, and the number of weevil lifestages per 10 fruits, 1 or 2 d before field releases of T. eugenii at SWFREC, Immokalee, FL, 2005................85 23 Parasitoid and pepper weevil adults r ecovered from ‘Jalapeo’ pepper following field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005........................85

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xi LIST OF FIGURES Figure page 1 Device for exposing different densities of weevil adults to ‘J alapeo’ peppers......24 2 Mean fecundity of adult pepper weevil females (n = 14) reared on ‘Jalapeo’ peppers at 27 + 2 C and 14:10 (L:D) photoperiod..................................................32 3 Longitudinal section of an infested ‘J alapeo’ fruit showing a pepper weevil egg and oviposition plug (ov-p)......................................................................................48 4 Immature stages of T. eugenii : (A) Egg 22 h after oviposit ion, (B) First instar, 30-33 h after oviposition, with well developed mandibles......................................58 5 Larva of T. eugenii (A) Emerging from the poste rior end of the host, (B) Beginning to feed on the host even before emerging completely............................59 6 T. eugenii adults. (A) Female, (B) Male..................................................................60 7 Fecundity of T. eugenii females at 27 + 2C either exposed to hosts changed every 1.5 h (n=5) or exposed to hosts per 9 h (n=5)................................................62 8 Field cages used at the SWFREC. (A) General view of a unit containing two cages. (B) Organdy partition and zipper between two experimental units..............75 9 Honey-wasp-refuge. (A) General view of the set up. (B) Using a unit as T. eugenii release point.................................................................................................77 10 Survival of T. eugenii adults in plastic cups wi th and without honey, when the cups were placed inside the canopy of plants, 25.4 + 4.9C, 67 + 19% RH............82 11 Daily temperatures and humidity during T. eugenii field releases on ‘Jalapeo’ pepper at the SWFREC in Immokalee, FL, 2005....................................................84

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xii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE) AND EVALUATION OF ITS POTENTIAL FOR BIOLOGICAL CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA: CURCULIONIDAE) By Esteban Rodrguez-Leyva May 2006 Chair: Philip A. Stansly Cochair: David J. Schuster Major Department: Entomology and Nematology The pepper weevil, Anthonomus eugenii Cano, is considered the most important pest of peppers ( Capsicum spp.) in Tropical and Subtropi cal America. Juvenile stages develop inside buds and immature fruits, so that only adults are suscep tible to insecticidal control. No viable biologi cal tactic has been develope d to combat this pest but Triaspis eugenii a braconid recently collected in Mexico that attacks pepper weevil eggs, could offer an important addition to its management. The parasitoid was collected from Nayarit, Mexico, during 2003, and a rearing methodol ogy was developed using pepper weevil reared on immature ‘Jalapeo’ peppers. Low le vels of parasitism and high levels of superparasitism hampered the rearing and reli able estimation of pa rasitoid demography until the weevil rearing system was improved. Confining individual mated females to a single immature ‘Jalapeo’ fruit maximized weevil fecundity, and also the number of oviposition punctures that were plugged by female. Weevils fed with pepper floral buds,

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xiii a high nitrogen source, and im mature ‘Jalapeos’ laid 5.5 eggs per female per day over a life span of 64.5 d at 27 2C. The net repr oductive rate (Ro=158.1) and intrinsic rate of increase (rm = 0.14) obtained in a life table study were higher than any previous report. T. eugenii is a solitary egg-prepupa l parasitoid of the peppe r weevil. At 27 1C, T. eugenii eggs hatched at 23 1 h. Females deve loped in 16.6 0.9 d and males in 16.4 0.9 d. Females (n=10) laid 402 199 eggs of which less than 50% reached the adult stage because of high levels of superparasit ism in the laboratory. Ovipositing females lived 16.5 3.02 d, and parasitoids died in fewer than 48 h without carbohydrates (honey). Net reproductive rate (Ro) was estim ated at 106 and 167, and intrinsic rate of increase (rm) at 0.24 and 0.26 with and without superparasitism respectively. The oviposition plug deposited by the pepper we evil played a decisive role in the ability of T. eugenii to find and to parasitize weevil eggs. Crowded rearing conditions inhibited deposition of oviposition plugs and consequently efficiency of parasitism. Wasps were able to find hosts in field cages and semiochemicals from weevil-damaged plants appeared to play a role in host patch location. However, T. eugenii did not stay more than 120 min in the pa tch regardless of the presence of weevil damaged plants. Superparasitism was less frequent in the fi eld cages (28%) compared to the laboratory (55-64%). The relatively short duration of fo raging on large patch size compared to the rearing environment might explain why. T. eugenii was recovered in low numbers from field releases in spring 2005 at Immokalee, FL, and at least one generation was completed in the field. No wasps have been recovered after that although sa mpling has been limited. Even if T. eugenii does not establish in Florida, its host specificity, its ability to parasitize weevil eggs, and its almost

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xiv double intrinsic rate of increase, compared to the pest, make this parasitoid an excellent prospect for biological control by au gmentation against the pepper weevil.

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1 CHAPTER 1 INTRODUCTION The pepper weevil Anthonomus eugenii Cano (Coleoptera: Curculionidae), is considered one of the most important insect pests of all cultivated varieties of chile = pepper ( Capsicum spp.) in the New World. Pepper weevil is considered a key pest of this crop in the southern United States, Hawaii, Mexico, Central America, and Puerto Rico (Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz 1985, Riley and King 1994, Coto 1996, Arcos et al. 1998). Pepper weevil biology, control tactics, di stribution, and sampling methods have been studied (Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992, Riley et al. 1992a,b, and Toapanta 2001). Nevertheless, ins ecticidal control is necessary once the pest is present in pepper crops (Riley a nd King 1994, Riley and Sparks 1995, Arcos et al. 1998, Mariscal et al. 1998). Unfortunately, the us e of insecticides as the principal method of control can provoke many adverse effects su ch as marketing rest rictions, exposure of non-target organisms, environmental contamin ation, pesticide resistance, and secondary pest outbreaks within the same crop (Dou tt and Smith 1971, Van Driesche and Bellows 1996). Biological control could be an alternative strategy to incorporate into the integrated pest management (IPM) of pepper weev il (Riley and King 1994, Mariscal et al.1998, Schuster et al.1999). However, little is known of the natural enemies of this pest. Pepper weevil arrived or was incident ally introduced from Mexico to the United States at the beginning of the last century (Walker 1905, Pratt 1907). Early reports indicated two

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2 species of ants, three pteromalids, one braconid, and one mite that att ack this pest in the United States (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut 1971). However, the effect of those en emies on pepper weevil populations was not considered important (Pratt 1907, Elmore et al. 1934, Elmore and Campbell 1954). A few decades ago, Wilson (1986) reported on the ectoparasitoid Catolaccus hunteri Crawford (Hymenoptera: Pteromalidae) attacking pepper weevil in Florida. C. hunteri is the dominant parasitoid attacking this pest in Florida and Puerto Rico (Wilson 1986, Schuster et al. 1999), and one of the mo st abundant in some states of Mexico (Mariscal et al. 1998, Schuster et al. 1999, Rodriguez et al. 2000). C. hunteri has been collected from a closely related pest of co tton, the cotton boll weevil Anthonomus grandis Boheman (Coleoptera: Curculi onidae), in several countries in Central and South America, Mexico, and the United States (Pierce et al. 1912, Towsend 1912, Cross and Mitchell 1969). Bi ological studies showed that C. hunteri has a higher fecundity than that of the pepper w eevil (Rodriguez et al. 2000, Seal et al. 2002). Those findings stimulated and supported the evaluation of C. hunteri for controlling this pest (Schuster et al. 1999, Rodriguez et al. 2000). Releasing C. hunteri in field plots in Mexico a nd Florida produced equivocal results; in Mexico this parasitoid was not effective for combating pepper weevil on bell pepper (Corrales 2002). However, some augmenta tive releases of this parasitoid prior to and during the pepper season suggested that this parasitoid might have potential to reduce weevil damage (D. Schuster, UFL, personal communication). These contradictory results may be due, in part, to the fact that C. hunteri attacks the 3rd instar host that is usually inaccessible deep within the pepper fruit. Therefore, augmentative releases of C. hunteri

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3 prior to the crop cycle could reduce pepper weevil populations in alternative host plants like nightshade, while releases in early infest ations of pepper weevil, or in small fruited varieties, might be effective because floral buds and small fruits allow C. hunteri to reach its host. Once the pepper fruits increase in si ze, there is no way to expect parasitism. In fact, Riley and Schuster (1992) indicated that C. hunteri was not detected in fallen fruits larger than 2.5 cm in diameter. According to this information, an effective biological control agent for pepper weevil might be one th at attacks earlier and more accessible life stages. The search for biological control agents in the native region of the target pest is one of the first steps in a successful biolog ical control program (DeBach 1964, Hagen and Franz 1973, Huffaker and Messenger 1976). Mexi co and Central America have been indicated as a center of or igin and domestication of Capsicum annuum L., the most important cultivated species of chile = pe pper (Vavilov 1951, MacNeish 1964, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, 1980, Loaiza-Figueroa et al. 1989). Furthermore, it was in Mexico that th e pepper weevil was first reported as a pepper pest (Cano and Alcacio1894). Mariscal et al. (1998) collect ed nine different genera of hymenopteran parasitoids from pepper weevil in Nayarit, Mexico, in cluding a subsequently described species Triaspis eugenii Wharton & Lopez-Martinez 2000 (H ymenoptera: Braconidae). Only Mexico appears to have this diversity of parasitoids of pepper weevil, and additional search would likely yield more potential species. T. eugenii was the most abundant parasitoid emerging on pepper weevil in Nayarit. Incidence of parasitism ranged from 18 to 40%, making it the most important parasitoid

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4 of this pest in the field (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This level of parasitism in unspr ayed fields suggests that T. eugenii could be a good candidate for inclusion in an IPM program against pe pper weevil in the United States, Mexico, and Central America. Nevertheless, informati on on the biology and ecology of this braconid is needed to evaluate its potential as a biological control agent. Literature Review Origin of Capsicum spp. and Distribution of Pepper Weevil Central and southern Mexico and part of Central America, a region called Mesoamerica according to anthropologists, has been indicated as a center of origin and domestication of the most important cultivated species of chile = pepper, Capsicum annuum L. (Vavilov 1951, MacNeish 1964, Pickersgill 1969, 1971, Eshbaugh 1976, Loaiza-Figueroa et al. 1989, C onciella et al. 1990). The othe r four important species of cultivated chiles (or peppers) are Capsicum frutescens “Tabasco”; C. chinense Jacquin “Habanero or Scotch Bonnet” ; C. pubescens Ruiz and Pavon “Manzano or Pera”; and C. baccatum L. All are endemic to South America (Heiser and Smith 1951, Smith and Heiser 1957, Pickersgill 1969, 1971, Pickersgil l and Heiser 1971, Eshbaugh 1976, McLeod et al. 1982). A Mesoamerican origin of the pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is suggested by th e following facts: a) the origin of the most important species of cultivated pepper ( C. annuum ) is probably Mesoamerica; b) the pepper weevil has not been re ported from South America (Clark and Burke 1996); and c) this insect was found for the first time damaging peppers in Central Mexico in Guanajuato (Cano and Alcacio 1894).

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5 A few years after the report from Guan ajuato, Mexico, the pepper weevil was reported from Texas (Walker 1905). According to Pratt (1907) this insect was introduced from Mexico on infested fruit shipments. Afte r affecting pepper crops in Texas, the insect was reported from California in 1923, New Me xico and Arizona in 1927, and Hawaii in 1933 (Elmore et al. 1934). It was reported fr om the west coast of Florida in 1935 (Goff and Wilson 1937), and the east coast in 1972 (Genung and Ozaki 1972). During 19351972, when high levels of organochlorine insectic ides were used on the east coast, there were no records of pepper weevil in the z one, but in 1972 it was a widespread pest (Burke and Woodruff 1980). Presently, the pepper weevil is a pest in all pepper growing areas of the United States including North Caro lina and New Jersey (Burke and Woodruff 1980). It was once reported in greenhouses in Canada, probabl y introduced on seedling plants from California (Riley and King 1994). Mexico, Guatemala, El Salvador, Honduras, Nicaragua, and more recently Puerto Rico, Costa Rica, Jamaica, and Panama have been added to the list of countries where this inse ct is considered a se rious problem on peppers (Cano and Alcacio 1894, Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz 1985, Andrews et al. 1986, Riley and Ki ng 1994, Clark and Burke 1996, Coto 1996). Economic Importance The pepper weevil lays eggs, feeds, and develops completely inside the fruits, which contribute to the difficulty of controlli ng the insect. The damage to flowers and/or young pods causes abscission and diminishes yield up to 30 to 90% if treatment is not implemented (Campbell 1924, Elmore et al. 1934, Goff and Wilson 1937, Velasco 1969, Genung and Ozaki 1972, Riley and Sparks 1995). In 1990, losses in the United States due to the pepper weevil were estimated at 23 million dollars on the 31,000 ha grown in

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6 California, New Mexico, Flor ida and Texas (Riley and King 1994). By 2003, the growing area of the crop in the United States had incr eased to close to 32,000 ha and the value of production reached 550 million dollars (Nationa l Agriculture Statistics Service 2004). Using the same proportion of damage (10 %) estimated by Riley and King (1994), the economic damage in 2003 could have reached more than 50 million dollars. In Mexico, Central America, and the Cari bbean islands, serious economic impact is indicated but not quantified (Riley and King 1994). Additional co sts include the many adverse affects of pesticides such as exposure of non-target organisms, environmental contamination, pesticide resistance, and secondary pest out breaks within the same crop (Doutt and Smith 1971, Van Driesche and Bellows 1996). The pepper weevil shares many similari ties with its congener the cotton boll weevil: Mesoamerica as the likely origin, the damage to their hosts by feeding and developing inside the fruits, and the history of dispersal in th e United States (Burke et al. 1986). However, the difference in acreage a nd, consequently, economic importance of cotton and pepper in the United States, expl ains why pepper weevil has never received the same attention as the boll weevil, and why even a hundred years after the original detection, there is relative ly little information about biogeography, evolution, and biological control of the pepper weevil (Riley and King 1994). Description The pepper weevil was described from sp ecimens collected from Guanajuato, Central Mexico, by Cano and Alcacio (1894). The author mentioned as a motive the importance of the insect as a pest of pepper crops in that region. This weevil belongs to the subfamily Anthonominae and shares many characteristics with the other 330 described species of the genus Anthonomus (Burke 1976, Anderson 1992, Clark and

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7 Burke 1996): basically robust and convex body, elonga tion of anterior part of the head to form a rostrum longer than broad, reduced mand ibles on the tip of this rostrum, and flat scales like hair covering elyt ra, scutellum, and to some extent other parts of the body (Burke 1976). Detailed descriptions of immature lifes tages and adults have been written by Elmore et al. (1934), Burke (1968), and Clark and Burke (1996). Pepper weevil adults are usually not longer that 3 mm, and males possess larger metatibial mucrones than females, a characteristic useful to determine sex (E ller 1995). As with boll weevils, pepper weevil males have a notch visible on the 8th tergum of the abdomen, which females do not have (Agee 1964, Sappington and Spurgeon 2000). However, the small size of the pepper weevil makes the last characteristic difficult to see (Eller 1995). Biology and Life History The biology of the pepper weevil was fi rst described by Cano and Alcacio (1894), Walker (1905), and Pratt (1907); however, Elmo re et al. (1934) made the first complete biological study. The pepper weevil feeds and de velops on several species of Solanaceae, but it is only a pest on peppers, Capsicum spp. (Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992). Adults feed on buds, flower s, fruits, and even leaves. Larvae feed and develop completely inside floral buds and immature fru its. Premature abscission is a consequence of feeding and developing inside buds and fruits re sulting in loss of production (Cano and Alcacio 1894, Walker 190 5, Pratt 1907, Elmore et al. 1934). Even if there were no abscission of fruits, there could be loss, as the fruits have larvae inside and are often spoiled. Actually, abscission of fruits c ould be considered an advantage because fallen fruits can be ga thered to avoid reinfestation of the crop (Berdegue et al. 1994) and because fewer damaged fruits make it to the market.

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8 There are three instars and the number of generations per year depends only of temperature and availability of food res ources (Elmore et al. 1934). Pepper weevil develops within a wide range of temperature (10-30C), but the ideal range is from 27 to 30C (Toapanta et al. 2005). Other reports that included informati on on generation time, oviposition, fecundity, fertility, and host plant associations have advanced our understanding of the biology of this species (Genung and Ozaki 1972, Bu rke and Woodruff 1980, Wilson 1986, Gordon and Armstrong 1990, Patrock and Schuster 1992, Arcos et al. 1998). In addition, a life table study by Toapanta et al. (2005) provide d additional demographic parameters such as net reproductive rate, intrinsic rate of increas e, and finite rate of increase at different temperatures. With this information the bi ology of the pepper weevil is reasonably well known (Table 1), but is not complete. Various f actors that were not included in fecundity experiments could affect fitness of this spec ies, including type and quality of food. It is well known that the boll weevil needs to f eed on floral buds, because of their high nitrogen content, for ovarian development and oviposition (Hunter and Hinds 1905, Isley 1928). Other studies demonstrated that fecund ity was optimal in boll weevil fed with a diet containing high nitrogen content (10% cottonseed flour as th e amino acid nitrogen source) but drastic nitrogen reduction (2.5% cottonseed fl our) reduced egg production (Hilliard 1983). Oogenesis and oviposition in pepper weevil proceeds only on peppers (almost any reference in Table 1). However, adu lts prefer to feed on floral buds instead of fruits (Patrock and Schuster 1992), so the importa nce of this type of food and its effect on fecundity should be considered for future studies.

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9 Table 1. Biological data of pepper weevil, selected references until 2005 Parameter (average in da ys) Conditions Reference Generation time 25 Walker 1905 20.9 Summer 32.1 Fall Elmore et al. 1934 17-18 25.7-27.7C; 70% RH; on an artificial diet Toba et al. 1969 17.5 23-27C Genung & Ozaki 1972 14.2 25.7-27.7C; 40-100% RH Wilson 1986 22.7, 13.9, 12.9 21.0, 27.0, and 30C, 60% RH; 14:10 (L:D), respectively Toapanta et al. 2005 Longevity of adults 78.7 Laboratory reared Elmore et al. 1934 90 Insectary Goff & Wilson 1937 31.7 22-27C; 60-70% RH Gordon & Armstrong 1990 Oviposition period 72 Laboratory reared Elmore et al. 1934 30 Insectary Goff & Wilson 1937 76, 50, 52 21.0, 27.0, and 30C, 60% RH; 14:10 (L:D), respectively Toapanta et al. 2005 Oviposition rate (eggs/ female/ day) 4.7 Laboratory reared Elmore et al. 1934 6.6 Insectary Goff & Wilson 1937 6.0 23-27 C Genung & Ozaki 1972 7.1 25.7-27.7C; 40-100% RH Wilson 1986 8 22-27 C; 60-70% RH Gordon & Armstrong 1990 1.9, 1.7, 3.1 21.0, 27.0, and 30C, 60% RH; 14:10 (L:D), respectively Toapanta et al. 2005 Fecundity (eggs/female) 341 Elmore et al. 1934 198 Insectary Goff & Wilson 1937 253 22-27C; 60-70% RH Gordon & Armstrong 1990 144, 85, 161 21.0, 27.0, and 30C, 60% RH; 14:10 (L:D), respectively Toapanta et al. 2005 Population values a Ro = 25.15, 11.76, 33.57 b rm = 0.06, 0.07, 0.11 c T = 52.51, 35.79, 32.39 21.0, 27.0, and 30C, 60% RH; 14:10 (L:D), respectively Toapanta et al. 2005 a Net reproductive rate (Ro); b Intrinsic rate of increase (rm); c Generation time (T) Host Plant Association Capsicum spp. Host plants utilized by pepper weevil for reproduction are confined to the genera Capsicum and Solanum both in the family Solanaceae. All of the five species of pepper

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10 grown as crops ( C. annuum C. frutescens, C. chinense C. pubescens or C. baccatum) are suitable for oviposition and development of the pepper weevil (Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992). The ma jority of studies describing feeding, oviposition, survival and reproduction have been developed in these hosts. Pepper weevil adults feed on floral buds, fl owers, fruits, and sometimes leaves of pepper plants; they lay eggs inside fl oral buds and young pepper pods. For feeding, females and males bore a small hole with the mandibles, which are located at the end of the rostrum, to reach internal tissu e (Walker 1905, Elmore et al. 1934). For oviposition, females spend a short time in selecting a site on the fruits, after which they bore a small hole with the mandibl es, turn completely around and deposit an individual egg into the hole. Once the egg is in the cavity, the female deposits a yellowish or brownish substance that turns black on drying and seals the hol e (=oviposition plug) (Elmore et al. 1934, Goff and Wilson 1937, Ge nung and Ozaki 1972). After hatching, the larva bores into the interior of the fruit until reaching the placenta and seeds. Once development is completed, the insect pupates inside the fruit (Pratt 1907, Elmore et al. 1934, Goff and Wilson 1937, Burke and Woodr uff 1980, Riley and Sparks 1995). The particular feeding and reproduction behavior of this insect make it inaccessible to many natural enemies, and also to most insectic ides (Pratt 1907, Elmore et al. 1934, Elmore and Campbell 1954). The size, quality, and availability of buds, fl owers, and fruits could be factors that affect the selection of ovi position sites for pepper weevil (Walker 1905, Elmore et al. 1934, Riley and King 1994). More eggs were laid in fruits than in flowers (Patrock and Schuster 1992). For feeding and oviposition, adults prefer small fresh fruits rather than

PAGE 25

11 mature fruits (Walker 1905, Wilson 1986). Acco rding to Bruton et al. (1989), the pepper weevil preferred developing bell pepper fruits from 1.3 to 5.0 cm in diameter rather than smaller (less than 1.3 cm in diameter) or la rger mature fruits (b igger than 5 cm in diameter) for oviposition. Solanum spp. The suitability for feeding, oviposition a nd development of the pepper weevil has been confirmed for at leas t 10 species of the genus Solanum (Table 2). Six additional species of the family Solanaceae served as food, but were not suitable for oviposition in non-choice tests (Wilson 1986, Patrock and Schuster 1992). Nevert heless, laboratory results do not necessary apply to the field. Fo r example, the pepper weevil was able to develop in eggplant ( S. melongena ) in the laboratory, but rarely is observed to attack this crop in the field. The developmental time of pepper weevil re ared on bell peppers was not different from those reared in American black nightshade ( S. americanum ) or eastern black nightshade ( S. ptycanthum ), but the dry weight of adults from pepper was greater than those from the other hosts (Patrock and Schuster 1992). A hypothesis of restriction of resources, where bigger fruits could offer more food for the developing larva, could help to explain why pepper weevil females prefer fr uits instead of flowers for oviposition. The same hypothesis could explain the greater dry weight of pepper weevil adults reared from bell pepper fruit than those from nightsha de fruit (Patrock and Schuster 1992). The qualitative differences in suitability am ong hosts of pepper weevil have not been evaluated.

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12 Table 2. Non Capsicum Solanaceae used by pepper weevil* Plant species Feeding Oviposition Development Solanum americanum Mill L, F, Fr1 Fr S4 S. carolinense L L, F, Fr F, Fr S S. dimidiatum Fav. L, F, Fr F, Fr S S. eleganifolium Cav L, F, Fr F, Fr S S. melongena L L, F, Fr F, Fr S S. pseudocapsicum L L, F, Fr Fr S S. pseudogracile Heiser L, F, Fr Fr S S. ptycanthum Dun L, F, Fr Fr S S. rostratum Dunal L, F F S S. triquetrum Cav L, F2 F2 S S. tuberosum L. L, F2 N3 Datura stramonium L. L, F N Lycopersicon esculentum Mill. L, F, Fr N Nicotiana alata Link and Otto L, F N Petunia parviflora Vilm F N Physalis pubescens L. L, F, Fr N *Source: Wilson (1986), Patrock and Schuster (1992) 1L= Leaves, F = Flowers, Fr = Fruits. 2Fruit unavailable. 3No oviposition observed on flowers or fruits. 4Successful development. Management Strategies Host plant resistance Host plant resistance is an important component of IPM (Painter 1951, NAS 1969). Berdegue et al. (1994) indicated that some types of pepper exhibited less damage from pepper weevil because of an escape mechan ism –concentrated production before the presence of the pest – instead of antibiosi s. Quiones and Lujan (2002) indicated that some ‘Jalapeo’ lines might have some tole rance mechanism against damage from this pest. Seal and Bondari (1999) indicated that only two commercial varieties of peppers, one of them Habanero, had lower rates of infe station by pepper weevil than the remaining

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13 nine varieties tested in field evaluations. Un fortunately, there is not a single cultivated variety known to provide any important resist ance characteristic against this pest. Cultural and chemical control Current management practices against pe pper weevil consist of a combination of cultural and chemical control. According to Riley and King (1994), control practices against pepper weevil focused on cultural control during almost the first 40 years following introduction into the United States. Th e lack of other tactics was explained by the difficulty of reaching the insect insi de the fruit (Walker 1905). The inorganic insecticides, available during those years, esp ecially calcium arsenate could not be used because of human toxicity (Elmore et al. 1934, Elmore and Campbell 1954) and were observed to provoke secondary pest problem s in the crop (Folsom 1927, Elmore et al. 1934). Insecticides have improved in selectiv ity for other pests, but they have not improved in efficacy against pre-imagin al stages of the pepper weevil. Recommendations for avoiding damage incl uded establishing a pepper free period —some months if possible— to redu ce populations by food deprivation. Other recommended practices included 1) using weevil-free seedlings to establish new crops, 2) removing alternative hosts inside and around the fields, 3) collecting and destroying fallen fruits to reduce reinfe station, and 4) destroying cr op residues immediately after harvest (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore at al. 1934, Goff and Wilson 1937, Watson and Lobdell 1939). From the mid 1940’s to 1980’s, chemical control gained favor due to the availability of synthetic insecticides (Elmore and Campbell 1954, Riley and King 1994). Increasing knowledge of dispersal and sa mpling techniques (Andr ews et al. 1986, Riley et al. 1992a b, Riley and Schus ter 1994) aided in managing this pest without calendar

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14 spraying of pesticides (Rile y et al. 1992a b, Riley and King 1994). When 5% of the terminals were damaged (Cartwright at al. 1990) or when one adult pepper weevil was present per 400 terminal buds (Riley and Sc huster 1992 b), significant economic loss in highly productive crops resulted. These cr iteria could serve as action thresholds. Azadirachtin, bifenthrin, cy fluthrin, cryolite, esfenvale rate, oxamyl, permethrin, acetamiprid, and thiamethoxam are some of the insecticides labeled for combating the pepper weevil in Florida (Olson et al. 2005, P. Stansly, personal communication). Biological control Known natural enemies of the pepper w eevil are summarized in Table 3. Although biological control is usually considered an in tegral part of the IPM of any pest, natural enemies of pepper weevil in Florida have not been shown to play an important role in suppression of this pest (Elmore and Ca mpbell 1954, Wilson 1986, Schuster et al. 1999). For this reason, although efforts to introduce na tural enemies against pepper weevil have been considered, no specific parasitoid had b een introduced and releas ed for this pest in the United States until the present study (Elmore et al. 1934, Elmore and Campbell 1954, Clausen 1978, Schuster et al. 1999). Entomopathogenic nematodes ( Heterorhabditis sp. and Steinernema sp.) have proved effective against certain insect pests that develop some part of their life cycle in the soil (Glaser 1931, Bell at al. 2000). Studies in Texas have shown that S. riobravis can kill boll weevil larvae inside abscised squares and bolls of cotton if applied to the soil under ideal conditions of moisture (Cabanill as 2003). In a similar way, entomophagous nematodes might attack the pe pper weevil inside buds or pods that are on the ground, and could thus diminish pest populat ions; however, there is little information in regard to entomopathogenic fungi, as well as viruses or ba cteria that might att ack this pest. In a

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15 recent work in Florida, a commercial formulation of Beauveria bassiana (Balsamo) was used against pepper weevil adults, but proved to be ineffective in the field (Schuster et al. 1999). Three pteromalids, one braconid, two species of ants, and one mite were identified attacking the pepper weevil in the United States (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut 1971). Neverthele ss, their impact on the pest was not considered important (Elmore and Campbell 1954, Genung and Ozaki 1972). The cited natural enemies that are endemic to the United States would be unlikely to act effectively against an exotic pest. Before 1950, two attempts of classical biological c ontrol of this pest were made in the United States. Eupelmus cushmani Crawford and Catolaccus (Heterolaccus) hunteri Crawford were collected from Guatemala and released in Hawaii during 1934-37, where they established, but did not provide enc ouraging results (Clausen 1978). The other attempt during 1942-1943 occurred in California with the release of Triaspis vestiticida Viereck, a parasitoid of Anthonomus vestitus Boheman, the Peruvian cotton boll weevil in Peru (Berry 1947, Clausen 1978). However, th e parasitoid was never recovered from peppers (Clausen 1978). These three natural enemies were originally obtained for biological control programs against the boll weevil ( Anthonomus grandis Boheman) and evaluated against pepper weevil in the hope that similarities between these two pests, might make possible the use of the same natural enemies (Clausen 1978). Nevertheless, there are no reports of E. cushmani or T. vestiticida ever parasitizing pepper weevil. C. hunteri has the widest distribution of any parasitoid of boll weevil or pepper weevil in the United States, Mexico, and Cent ral America (Pierce et al. 1912, Cross and

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16 Mitchell 1969, Cross & Chesnut 1971, Mari scal et al. 1998, Schuster et al. 1999, Rodriguez et al. 2000). It is the most impor tant parasitoid atta cking pepper weevil in Florida (Wilson 1986, Schuster et al. 1999), and one of the most widely distributed in Mexico (Mariscal et al. 1998, Schuster et al 1999, Aguilar and Servn 2000, Rodriguez et al. 2000). It is also present in Honduras (R iley and Schuster 1992) Costa Rica (Schuster et al. 1999), and it is the only one known from Puerto Rico (Schuster et al. 1999). C. hunteri is a generalist that is know n to attack at least 17 species of Curculionidae and 2 of Bruchidae (Cross and Mitche ll 1969, Cross and Chesnut 1971); it usually develops on the last instar of its host (Wilson 1986, Cate et al. 1990, Rodriguez et al. 2000). The particular biology of this species was useful for mass rearing at a moderate scale on the cowpea weevil, Callosobruchus maculatus F. (Rodriguez et al. 2002, Vasquez et al. 2005). Those findings, and so me biology studies that showed that C. hunteri has greater fecundity and in trinsic rate of increase than pepper weevil (Rodriguez et al. 2000, Seal et al. 2002), stimulated and supported the plan of evaluating C. hunteri as a biological control agent. Releases of 1050 C. hunteri per hectare in Sinaloa, Mexico, were not effective in combating pepper weevil on bell pepper (Corrales 2002). Nevertheless, in Florida results of week ly releases of the equivalent of 7900 C. hunteri per hectare suggested that this parasitoid has potential to reduce damage of pepper weevil on peppers (D. Schuster, personal communi cation). One of the most important disadvantages of using C. hunteri to combat pepper weevil is the fact that it attacks the 3rd instar host, which is usually inaccessible deep within the fruit of commercial pepper varieties.

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17 Table 3. Natural enemies of the pepper weevil, references until 2005 Natural enemies Reference Predators Hymenoptera: Formicidae Solenopsis geminata Fab. Pratt 1907, Hinds 1907 Tetramonium guinense Fab. Wilson 1986 Passeriformes: Icteridae Sturnella magna (Easter meadowlark bird) Genung & Ozaki 1972 Parasitoids Pteromalidae Zatropis incertus Ashmead (= Catolaccus incertus Ashmead) Pierce 1907, Cross & Chesnut 1971 Catolaccus hunteri Crawford Pierce et al. 1912, Cross & Chesnut 1971, Mariscal et al. 1998 Habrocytus piercei Crawford Cross & Chesnut 1971 Braconidae Bracon mellitor Say Pratt 1907, Pierce et al. 1912, Cross & Chesnut 1971 Triaspis eugenii Wharton & Lopez-Martinez Mariscal et al. 1998, Wharton & Lopez-Martinez 2000, Toapanta 2001 Urosigalphus sp. Bracon sp. Mariscal et al. 1998, Toapanta 2001 Aliolus sp. Eulophidae Euderus sp. Syempiesis sp. Eupelmidae Mariscal et al. 1998 Eupelmus sp. Mariscal et al. 1998, Toapanta 2001 Eurytomidae Eurytoma sp. Mariscal et al. 1998 Mites Acarina: Pyemotidae Pyemotes ventricosus Newport Pierce et al. 1912, Cross & Chesnut 1971 Even though Mexico and Central America ha ve been indicated as the center of origin and domestication of the most important cultivated species of peppers (Vavilov 1951, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, Loaiza-

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18 Figueroa et al. 1989, Conciella et al. 1990), and the pepper weevil was first described as a pest of that crop in Mexico (Cano and Al cacio 1894), no significant surveys for natural enemies were undertaken until 1997 (Mariscal et al. 1998). A survey by these authors in the west central coastal state of Nayarit, Mexi co, detected four speci es of Braconidae, one of Pteromalidae, two of Eulophidae, and one each from Eupelmidae and Eurytomidae (Table 3). Of these, the most abundant was a new species, later described as Triaspis eugenii Wharton and Lopez-Martinez (Hymenop tera: Braconidae) (Wharton and LopezMartinez 2000), which was reported to reach in cidences of 18-40% parasitism under field conditions (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This is a solitary parasitoid which was reported to parasitize th e egg, and to complete its life cycle in less than three weeks with a life span of less than four (Mariscal et al. 1998, Toapanta 2001, Rodriguez et al. 2004). The genus Triaspis Haliday (Hymenoptera: Braconid ae) is placed in the tribe Brachistini of the subfamily Helconinae (M artin 1956, Sharkey 1997). This genus could be represented by 100 species in the New Worl d, but just a few of them have been described from this region (Sharkey 1997). In general, biology and life history of the genus Triaspis is poorly known. Some species of Brach istini are egg-larval parasitoids of weevils, bruchids, and anthribids, and the last larval instar has an ectoparasitic phase (Clausen 1940, Berry 1947, Saw and Huddlest on 1991, Sharkey 1997). In spite of the fact that the biology of the genus Triaspis is not completely know n, some species of this genus have been used in biological control programs in North America. T. thoracicus was imported to Canada and the United States to combat species of Bruchidae, especially the pea weevil, Bruchus pisorum L. Apparently this species failed

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19 to establish because it has a preference for layi ng eggs in plant tissue instead of dry seeds (Martin 1956, Clausen 1978). Efforts to comb at the cotton boll weevil in USA with Triaspis vestiticida Viereck, were previously mentioned. T vestiticida is a solitary parasitoid which attacks the e gg of the host (peruvian cotton boll weevil) and develops as an endoparasitoid in early instars. At the end of the last instar, the larva emerges from the host to feed as an ectopara sitoid, leaving only the host cephalic capsule (Berry 1947). Although these habits may prevail throughout the tribe, more detailed studies of T. eugenii are necessary to understand its habits and potential use. Objectives The high incidences of parasitism by T. eugenii under natural conditions in Nayarit, Mexico and the recent informati on that confirms that this species attacks the weevil egg make it a good candidate for biological control of pepper weevil. Attempts to use this species for biological contro l will require more specific information on host/parasitoid relationships. Therefore, th e objectives of the presen t study were the following: 1. Clarify key aspects of the biology of the pepper weevil, in cluding demographic parameters, to improve rearing methodologies 2. Evaluate oviposition behavior and population dynamics of T. eugenii to optimize rearing procedures and to assess control potential 3. Test the ability of T. eugenii to control pepper weevil

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20 CHAPTER 2 REARING METHODOLOGY AND INFL UENCE OF FOOD QUALITY ON BIOLOGY OF PEPPER WEEVIL Introduction Adult pepper weevils, Anthonomus eugenii Cano, feed on floral buds, flowers, fruits, and even pepper leaves, but the larvae feed and develop completely inside floral buds and immature fruits (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al. 1934). When feeding in floral buds, weevil larvae damaged especially the anthers (Walker 1905, Elmore et al. 1934, Patrock a nd Schuster 1992). During the oviposition process on fruit, females spend a short time in selecting a site on th e pod, usually close to the calyx. They then bore into the pod, turn completely, and deposit an individual egg in the hole. Once the egg is in the cavity, the female deposit s a brownish substance that turns black on drying and seals the hole (o viposition plug) (Walker 1905, Elmore et al. 1934, Goff and Wilson 1937). The pepper weevil laid more eggs in pepper fruits than in flowers (Patrock and Schuster 1992), and adults preferred small and immature fruits to mature fruits for feeding and oviposition (Elmore et al. 1934, Wils on 1986, Bruton et al. 1989). Although the pepper weevil pepp er weevil has been consid ered the major pest of peppers for more than eight decades in Mexi co and the United Stat es (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al. 1934, Riley and King 1994), no artificial rearing system has been developed to facili tate biological studies. Consequently, most studies have depended on weevils emerging of infested peppers collected from the field

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21 (Elmore et al. 1934, Wilson 1986, Patrock and Sc huster 1992). An attempt to rear pepper weevil on artificial diet can be excluded fr om this generalization (Toba et al. 1969). These authors were able to rear 6 generati ons of weevils using diet. However, females would not lay eggs in the diet, so the proce sses of collection and ma nipulation of eggs are laborious and ineffective (Toapanta 2001). Toapanta et al. (2005) described a met hodology to produce weevils in peppers, but these authors did not define the quality of fruits offered. A similar methodology with a few variations is practiced at the Golf Co ast Research and Education Center (GCREC), Wimauma, FL. Both methodologies consist ba sically of exposing peppers for 48-72 h to weevil adults in cages 50x40x40 cm. Fruits are subsequently removed and held in ventilated plastic containers until the emergen ce of weevils. Toapanta (2001) used mainly ‘Serrano’ peppers, whereas ‘Jalapeo’ peppe rs from the supermarket were used at GCREC. Toapanta (2001) also indicated that the largest numbe r of weevils, 1.7 adults per fruit, was obtained using 5 weevils per fruit. At GCREC a ratio of 15 adults per fruit is used, although an evaluation of efficiency ha s not been conducted (D. Schuster, personal communication). These methodologies have been useful to maintain dozens of generations in the laboratory, but efficiency could be im proved by considering factors such as competition within fruits, fruit d ecay, and effect of food quality. Therefore, modifying fruit quality and decreasing competition could improve the laboratory rearing of the weevil. Even though there is a basic understanding of the biology of pepper weevil, there are no studies that refer to food quality and its effect on f itness of this species. For example, there are no reports that explain why weevils prefer feeding on floral buds

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22 instead of fruits. This might be related to food quality (nitrogen concentration or protein content). That is suggested because the ova rian development and oviposition of the boll weevil, Anthonomus grandis Boheman, are biological processes that depend upon the presence of cotton squares, a high nitroge n content food (Hunter and Hinds 1905, Hunter and Pierce 1912, Isley 1928). The pepper weev il does not need floral buds for ovarian development as has been proved by authors w ho reared the weevil in the laboratory with only peppers available for feeding and ovipos ition (Toapanta et al. 2005, D. Schuster, personal communication). However, studies that include feeding on floral buds are necessary to understand nutritiona l factors in this species. The biology of the pepper weevil has b een studied by several authors (as summarized in Riley and King 1994), but mo st, including Toapanta et al. (2005), reported a fecundity of no more than 150 or 160 eggs per female. Only Elmore et al. (1934) reported a fecundity as high as 340 eggs per female. Possibly a better understanding of the effects of food quality and the availabili ty of immature fruits and floral buds could allow a further realization of the reproductive potenti al of this species and thereby improve the rearing system. The obj ectives of this chapter were to determine the effects of crowding on oviposition and the effects of fruit quali ty on rearing of the pepper weevil, and to provide demographic parameters when immature fruits and floral buds are offered to adults. Materials and Methods Laboratory Weevil Colony A colony of the pepper weev il was established in a la boratory at the Entomology and Nematology Department in Gainesville, UFL, with 200-250 insects that emerged

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23 from infested fruits collected during th e Spring of 2003 at the Southwest Florida Research and Educational Center (SWFREC) Immokalee, FL. Genetic enrichment was provided by addition of insects collected peri odically from the same site, particularly during Spring 2004 and 2005. The insects were reared continuously on fresh peppers, ‘Jalapeo M’ (Harris Seeds Rochester, NY), and ‘Jalapeo Mitla’ (Otis S. Twilley Seed Co. Hodges SC), but all the experiments were conducted with ‘Mitla’ fruits. The colony was moved in April 2004 to a re aring room at the SWFREC maintained at 27 + 2C 60-70% RH, and 14:10 (L:D) h photoperi od, and the experiments with the pepper weevil were conducted at those conditions, unless otherwise indicated. Pepper fruits were exposed to unsexed adult weevils using a ratio of 10:1 insects per fruit. Plastic jars 3.8 L (14x14x25 cm Rubbermaid Home Produc ts Wooster, OH), with four lateral holes 3 cm in diameter covered with polyethyl ene screen, were used as oviposition cages. The cages had a knit sleeve (Kimberly-Clark Co. Roswell, GA) 50 cm long taped to the opening of the jar to prevent escape of ins ects during manipulations. Each cage held ca. 100 unsexed weevil adults. Water was provided in a cotton wick placed in 28-mL plastic cups, and lines of honey were dispensed ever y day on the upper side of the cage. Every 24 h, 10 small fresh ‘Jalapeos’ (ca. 5 cm l ong) were placed in the cages with the weevils, and 10 fruits placed 24 h previously were removed. The fruits were then held in plastic containers, 60-70 fruits each, until the emergence of adults. These containers (33x21x11 cm Rubbermaid Home Products Wooster, OH) had six lateral holes 3 cm in diameter covered with polyethylene screen to ensure ventilation. Weevils that emerged within the same week were co llected and confined in a new oviposition cage. Each cage

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24 was used for four or five weeks, because f ecundity was assumed to decline thereafter and because many weevils died during daily manipulations. Crowding Effects on Oviposition Production of eggs and presence or absen ce of oviposition plugs was evaluated for different numbers of mated females alone and for different numbers of males and females together. Weevils were held in clear plas tic cups 250 mL, each provided with a single fresh ‘Jalapeo’ pepper (4.95 + 0.61 cm long, 1.86 + 0.24 cm wide, and 8.57 + 2.02 g weight, n=49). Individual peppers were vert ically oriented using adhesive poster putty (Henkel Consumer Adhesives, Inc. OH) on th e tip of the fruits. Water was provided by daily saturating a cotton wick placed in the bottom of each cup (Fig. 1). Figure 1. Device for exposing different densitie s of weevil adults to ‘Jalapeo’ peppers. The treatments were 1 female, 1 female/ 1 male, 2 females, 2 females/ 2 males, 5 females, 5 females/ 5 males, and 10 females. The large metatibial mucrones of males were used to sex weevils (Eller 1995). To f acilitate sexing, groups of 10-12 weevil adults were placed in Petri dishes over a wh ite background and were observed through a dissection microscope at 16X magnification. An average of 22 females and 16 males

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25 could be sexed in 10 min using this method (n =4). These seven treatments were arranged in a randomized complete block design with th ree replications. Because it is assumed that fecundity of pepper weevil is higher in the fi rst weeks of its life (Patrock and Schuster 1992) and because of the availability of ins ects, each block was se t up at different time using insects 7-10 days old. ‘Jalapeo’ fruits were replaced daily for 7 d and the number of eggs per day, and number of eggs with or without an oviposition plug, was evaluated. To compare the number of eggs per female per day, data were analyzed using an analysis of variance (PROC ANOVA, SAS In stitute 2000) and means were compared by Least Significant Difference (LSD) (P < 0.05). Data were normalized prior to the analysis using the square root transformation (P ROC CAPABILITY, SAS Institute 2000), but data are presented in the original scale. The presence or absence of oviposition plugs by treatment was analyzed using a chi-square test (df = 1, P < 0.05) to determine differences between observed and expected (1:1) valu es (PROC FREQ, SAS Institute 2000). When one of the cells had expected c ounts less than 5, the Fisher’s exact test to contrast the null hypothesis was used (PROC FREQ, SAS Institute 2000). Fruit Quality The number of adult weevil progeny obtai nable using the ratio of 10 unsexed weevils per fruit was compared for two fruit qualities: immature green and green mature (marketable fruits). Prior to evaluation oviposition cages (3.8 L) containing 100 + 10 unsexed weevils 1-5 d old were provided with 10 immature fruits and 5 or 10, depending on availability, floral buds. The fruits and buds were replaced daily for 3 days. After that, one cage was assigned to one of two treatme nts: 10 immature green fruits (4.75 + 0.61 cm long, 1.77 + 0.25 cm wide, and 7.66 + 2.01 g weight, n=37), harvested from potted plants grown outdoors, or 10 mature green ‘Jalapeo’ fruits (6.67 + 0.77 cm long, 2.06 +

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26 0.41 cm wide, and 14.96 + 3.58 g weight, n=37), bought at the supermarket. Both qualities of fruit were washed with liquid di sh detergent and rinsed up with tap water before offering to the weevils. Fruits were replaced every 24 h (5:00 PM to 5:00 PM) and exposed fruits were held until adult emergence in the ventilated plastic containers used in colony maintenance. Each cage of weevils wa s considered an experimental unit and was evaluated for 22 days. Because of the availa bility of peppers three replicates were conduced at different times during July and September of 2003. After checking the data for normality (PROC CAPABILITY, SAS Institute 2000), statistical differences were determined using the student’s t-distri bution (PROC TTEST, SAS Institute 2000). Adult Feeding and Oviposition One pepper weevil female and one male both 18 h old or younger were placed in individual 250 mL plastic c ups. A lid closed each cup and a 3 cm diameter organdysealed hole provided ventil ation (Fig 1). Each weevil pair was considered an experimental unit and assigned to one of two treatments: a) peppers only or b) peppers plus floral buds. Every day dur ing the first 7 days of life, each weevil pair was provided a cotton wick saturated in water, as well as either a single youn g pepper (length 3.23 + 0.40, width 1.07 + 0.15, n = 15) or a single young pepper plus three floral buds before anthesis. The peppers were vertically oriented in the cups, as indi cated above, and floral buds were placed in the bottom of the cups. Each treatment had 8 replicates. Because there were not enough floral buds for doing all re plicates at the same time, the replicates were performed in groups at different times (3, 3, and 2). After 24 h of exposure, food items were replaced and observed under a ster eoscopic microscope to evaluate number of feeding and/or oviposition punctu res. Punctures were dissected to determine the presence or absence of eggs. Before the analysis, th e data were normalized by the arcsine square

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27 root transformation (PROC CAPABILITY, SAS Institute 2000), but means are presented in the original scale. Statistical differences were determined using the student’s t -test (PROC TTEST, SAS Institute 2000). Results from weevils that received both floral buds and fruits in the same cup were further analy zed to compare the preference of feeding and ovipositing in floral buds or fruits using student’s t-test (PROC TTEST, SAS Institute 2000). Life Table Fourteen weevil pairs 18 h old or younger we re caged individually in plastic cups as previously described. Males were removed after 7 d to avoi d possible interference with oviposition behavior (Elmore et al. 1934). A male was subse quently introduced into each cage for 2 d every 4 wk for mating. The females, or pairs when the males were present, received one fresh ‘Jalape o’ pepper daily (length 3.23 + 0.40, width 1.07 + 0.15, n = 25). The pepper was removed and dissected to count the number of eggs. Two floral buds were offered every day in addition to the fr uits for the lifetime of the female, except during week 2-4 due to short supply. The procedures were followed until death of the females. The sex ratio of the species was estimated from field and laboratory samples, and survivorship was evaluated from a sample of 102 peppers with only one weevil egg per fruit from the laboratory colony. In addition, fe rtility was estimated from a sample of 171 eggs laid in immature peppers from the same colony. Previous observations indicated that an oviposition plug ensured the presence of an egg in 91% of all cases (SE = 0.018, n = 216). Therefore, removing oviposition plugs to check for the presence of eggs was not necessary. A permanent marker was used to mark the oviposition plugs and the fruits were dissected 4 d later to look for larvae.

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28 Using LIFETABLE.SAS (Maia et al. 2000) the whole data set was used to estimate net reproductive rate (Ro), defined as the number of females produced by one adult female during its mean life span; gene ration time (T), the period between the birth of parents and the birth of the offspring; doubling time (Dt), the time necessary to double the initial population; intrinsic rate of increase (rm), the potential growth of a population under given conditions; and fi nite rate of increase ( ), the daily rate of increase of each cohort (Birch 1948, Maia et al. 2000, Sout hwood and Henderson 2000, Toapanta et al. 2005). Data reported by Elmore et al. (1934) and Toapanta et al. (2005) were subjected to the same analysis and compared to results obtained in the present study. Nitrogen Content of Fruits and Buds The nitrogen content of anthers, pericarp and a mix of placenta and seeds of three maturation stages of ‘Jalapeos Mitla’ wa s evaluated. These maturation stages were classified according to size and appearance as: a) immature green, b) mature green, c) mature red (Table 4). Four samples were collected from plants 2-4 months old which were grown in a greenhouse. During each sampling, 70-90 floral buds just prior dehiscence and 6-7 peppers of each matura tion stage were collected. Anthers were removed with forceps, and fruits were dissect ed to separate perica rp from placenta and seed. Because of possible variation among ha rvests (June and July 2005), each sampling was considered as a block. Table 4. Characteristics of ‘Jalapeo Mitla’ used for nitrogen content determinations Pepper quality (n=10) Length, cm Width, cm Wall thickness Immature Green 4.24 + 0.24 1.12 + 0.14 0.18 + 0.02 Mature Green1 6.76 + 0.60 2.42 + 0.24 0.39 + 0.03 Mature Red 7.72 + 0.67 2.57 + 0.20 0.41 + 0.03 1 Similar to fresh market quality

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29 The samples of each botanical structure were placed in individual paper bags, dried at 50 C for three days, and ground to a powde r. Nitrogen content was determined by the soil laboratory at the SWFREC using the alternative dry Micro-Dumas combustion analysis for total nitrogen in solid phase samples. This method is based on transformation to the gas phase by extremely rapid and complete flash combustion of the sample material (Matejovic 1993, 1995, Anonymous 1997). There were four replications (samples) in a randomized block design. The data were normalized by square root transformation (Sokal and Rohlf 1969, O tt and Longnecker 2001) prior to ANOVA (PROC ANOVA, SAS Institute 200 0); however, the means and standard deviations are presented in the original scale. Means we re compared by the LSD test (LSD) (P < 0.05) when the F value was significant. Results Crowding Effects and Oviposition The number of weevils per fruit had a signifi cant influence on the numbers of eggs per female per day (F = 12.47; df = 8,138; P < 0.0001). The largest number of eggs per female per day was obtained with a single female per fruit followed by one female and one male (Table 5). There were no differences among the remaining treatments (LSD test P < 0.05). Increasing the number of weevils from 1 female to 2 females/2 males or even 5 females had no effect on the number of plugge d eggs (Fisher’s exact test P = 0.266 and 0.139, respectively). However, the percentage of unplugged eggs increased 5 or 6 fold at densities of either 5 females/5 males or 10 females (Table 5). There was no differences between these treatments (X2 = 2.61, P = 0.1064).

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30 Table 5. Numbers of eggs per female per day for different numbers of females and males per single ‘Jalapeo’ pepper fruit Weevil density Eggs/female/day (Mean + SD)1 Unplugged eggs (%) 1 5 (4.76) 1 1 2 (2.82) 2 1 (1.16) 2 2 3 (3.61) 5 4 (2.26) 5 5 25 (13.66) 10 5.0 + 2.43 a 3.38 + 1.99 b 2.05 + 1.37 c 1.97 + 0.8 c 1.68 + 0.54 c 1.74 + 0.82 c 1.57 + 0.48 c 30 (9.10) 1Means with the same letter are not significantly different (LSD P < 0.05) Fruit Quality Immature green and green marketable fru its were both able to support the rearing of pepper weevil, although there were signifi cant differences. Using the 10 weevils per fruit density 2.65 + 1.16 weevil adults emerged per immatu re fruit, compared to only 1.11 + 0.81 weevils per marketable fruit (t = 8.81, df = 65, P < 0.0001). Feeding and Oviposition in Peppers versus Floral Buds (7d) When floral buds and peppers were offered at the same time, females fed and laid eggs in both (Table 6). Nevertheless, weevils preferred feeding on fl oral buds instead of fruits (t = 13.39, df = 55, P < 0.0001). In contrast, females pref erred to lay eggs in pepper fruits rather than floral buds (t = 9.59, df = 55, P < 0.0001). Table 6. Feeding and oviposition of pepper w eevil adults when given a choice of ‘Jalapeo’ pepper fruit or floral buds (7d) Parameter Pepper fruits Floral buds t-test (P < 0.05) Feeding punctures per day 3.27 + 4.99 18.75 + 8.64 Eggs per day 12.73 + 10.46 0.16 + 0.49

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31 Oviposition with or without Floral Buds (7d) Differences in oviposition were observed be tween females that were provided with pepper fruits only, compared to those that were provided with both flor al buds and fruits (Table 7). Females provided with fruits and buds laid many more eggs the first day of oviposition (t = 2.81, df = 14, P = 0.0261), and over the entire 7 d test period (t = 5.08, df = 14, P = 0.0002) than females provided only with fruits. Pepper weevil females laid only 1.25% of eggs in the floral buds when both fl oral buds and fruits were provided at the same time. Table 7. Oviposition of pepper weevil females fe d with ‘Jalapeo’ pepper fruits only or with pepper fruits plus floral buds Parameter Peppers Pepper plus floral buds t-test (P < 0.05) Days to begin oviposition 2.87 + 0.99 2.62 + 0.52 ns Eggs at first day of oviposition 3.50 + 1.41 11.87 + 8.30 Total number of eggs (7 d) 23.75 + 11.16 89.12 + 34.65 Life Table The 14 females survived 28 to 103 d, with an average of 64.5 + 25.8 d. During this time 8 of 14 females (57%) began oviposit ion on the second day, and 13 of 14 females were depositing eggs by the third day (Fig. 2) The remaining female took seven days to begin oviposition. Fecundity ranged from 0 to 32 eggs per female per day with an average of 5.51 + 5.31 eggs per female per day, and d eclined to near zero by 86 d. The fertility of eggs deposited in immature fruits was 97.66% (n = 171, SD = 0.15) and the survivorship of all preimaginal stages wa s 89% (n = 102, SD = 0.31). These data and the sex ratio of the species, that was not significantly different from 1:1 e ither in the field or

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32 in the laboratory (X2 =0.95, P < 0.05, X2 =0.35, P < 0.05, respectively), were used to estimate the demographic paramete rs of pepper weevil (Table 8). Figure 2. Mean fecundity of adult pepper weevil females (n = 14) reared on ‘Jalapeo’ peppers at 27 + 2 C and 14:10 (L:D) photoperiod. Table 8. Life table parameters for A. eugenii females (n =14) reared on ‘Jalapeo’ peppers at 27 + 2C, and 14:10 (L:D) photoperiod Parameter Value (+ 95% confidence limits) Net reproductive rate (Ro)a 158.1 (115.1 201.1) Intrinsic rate of increase (rm) b 0.14 (0.12 0.15) Generation time (T)c 36.04 (31.26 41.22) Doubling time (Dt)c 4.93 (4.42 5.49) Finite rate of increase ( ) d 1.15 (1.13 1.17) a Female/female, b loge (Ro)/T, c Day, d Female/female/day. Nitrogen Content of Fruits and Buds Nitrogen content was different among botanical structures and maturation stages of peppers (F = 41.61; df = 6, 27; P < 0.0001). Highest nitrogen c ontent occurred in the anthers of floral buds. An intermediate con centration occurred in pl acentas and seeds at 0 2 4 6 8 10 12 14 16 18 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 Age in daysEggs/ female/ day

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33 any maturation stage, and in pericarp of th e immature green fruits. The lowest nitrogen concentration was detected in pericarp of mature green and mature red fruits (Table 9). Table 9. Nitrogen content (Mean + SD) of anthers in floral buds and ‘Jalapeo’ fruits at three maturation stages Maturation stage Botanical struct ure N content (%) dry weight Floral buds Anthers 5.23 + 0.09 a1 Immature green Placenta and seeds 3.39 + 0.04 b Mature red Placenta and seeds 3.21 + 0.25 bc Immature green Pericarp 3.19 + 0.38 bc Mature green Placenta and seeds 2.96 + 0.09 c Mature green Pericarp 2.25 + 0.42 d Mature red Pericarp 2.01 + 0.35 d 1Means followed by the same letter are not significantly different (LSD, P < 0.05) Discussion Crowding Effects and Oviposition Increasing density of female pepper w eevils per fruit redu ced oviposition and increased the number of eggs lacking th eir normal oviposition plugs. An individual female produced more eggs than a female and a male together, and both treatments had higher number of eggs per female than two females together. The first comparison confirmed a previous study by Elmore et al. (1934) that suggested that the presence of males could inhibit oviposition behavior of females. However, the second comparison indicated that inhibition of oviposition also could occur with the presence of more females. Similar results with unsexed w eevils were reported by Toapanta (2001), who indicated that a ratio of five unsexed weevils per fruit were more efficient for producing adults than larger ratios (10:1, 15:1, 20:1).

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34 Crowding by either males or females interf ered with the ovipos ition process. Males may interfere by seeking to mate with females, and females may interfere by contending with each other for oviposition resources. The final phase of oviposit ion is deposition of the plug, so the same factors might have influenced the presence of eggs lacking oviposition plugs. Feeding could also contribute to the reduction of ovipositi on plugs by crowding. Pepper weevil prefers both to oviposit a nd to feed close to the calyx (Walker 1905, Elmore et al. 1934, Wilson 1986, Toapanta et al. 2005). More weevils per fruit would mean more feeding punctures and, consequentl y, more mechanical damage to tissue close to the calyx, including the plugs. These results demonstrated why rearing the pepper w eevil by using large numbers of weevils per fruit is inefficient. A small nu mber of insects per fresh ‘Jalapeo’, even a single mated female, may be the most efficient combination. Additionally, this system maximizes the number of oviposition plugs which may prevent dehydration of eggs as well as exposure to opportunistic pr edators, such as mites or ants. Fruit Quality Quality of the host can exert an importan t influence on fecundity and survival of insects (Awmack and Leather 2002). Exposing pe pper weevil females to immature green peppers resulted in more offspring than exposi ng females to marketable fruits (= mature green). The nitrogen content in the pericarp of immature fruits was higher than in mature green fruits (Table 9). Partitioning of nitroge n by fruit age and tissue could affect the capacity of both larvae and a dults to acquire nitrogen. Eggs are laid generally in the pericarp of peppers (Walker 1905, Elmore et al. 1934, Goff and Wilson 1937) due to the small size of the pepper weevil proboscis (Clark and

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35 Burke 1996). Eggs laid in older fruits will produce larvae that have to bore through a thicker pericarp, which is lower in nitrogen compared to an immature fruit (Tables 4, 9). Additionally, the maturation process in peppers includes substituting soft tissues with fiber, the clearest example being the seed coat on mature fruits (Bosland and Votava 1999). The high nitrogen content in mature s eeds may not be accessible to the larva. Thus, thickening of the pericarp and redistri bution of nitrogen to s eeds during maturation of pepper fruits could explain why the pepper weevil prefers to feed and to oviposit on immature fruits, and why the rearing process of weevils could be improved if immature fruits are used. Adult Feeding and Oviposition Early observations of pepper weevil adu lts in the field indicated a feeding preference for floral buds compared to fruits (Walker 1905, Elmore et al. 1934). These observations were later corroborated with laboratory tests (Wilson 1986, Patrock and Schuster 1992). The data presente d here corroborate this beha vior in ‘Jalapeo’ floral buds and fruits, and demonstrate the likel y cause. Many more feeding punctures were made per floral bud compared to ‘Jalapeo’ fruit. Patrock and Sc huster (1992) reported a mean of 0.74 and 0.75 feeding punctures for fl oral bud and fruit, respectively, produced by a single female. This contrasted with 3.27 feeding punctures in fruits against 18.75 feeding punctures on floral buds using a pair of weevils in this study. Patrock and Schuster (1992) used 10-20 d old females in bell pepper fruits (‘Early Calwonder’) at 22 + 4C, while 1-7 d old weevils in ‘Jalapeo’ fruits at 27 + 2C were used in the present study. More information related to the quality of the varieties of peppers and the vigor of the insects used could help to better understand th ose differences.

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36 Patrock and Schuster (1992) indicated that a female laid more eggs on immature fruits than floral buds (0.96 and 0.36 eggs, respectively), because fruits offered more resources to support larvae. Data collected here (0.16 eggs per floral bud and 12.73 eggs per fruit) followed the same trend, although the number of eggs laid per fruit was 10 times greater. The seven day oviposition experiment provide d information on the ability of floral buds to increase fecundity. The preoviposition period was not affected by access to floral buds, and a similar value (2 or 3 days) was reported by Elmore et al. (1934), Wilson (1986), Gordon and Armstrong (1990), and Toap anta et al. (2005). Nevertheless, the number of eggs at the first day of oviposition (3.5 vs 11.9) and the total number of eggs during the first seven days after emergence ( 23.7 vs 89.1) was always higher for females that received anthers. The tota l number of eggs deposited in th e first seven days of life of females provided with immature ‘Jalapeo’ fr uits and floral buds was greater than any number reported before for this species in previous works (Elmo re et al. 1934, Wilson 1986, Gordon and Armstrong 1990, Patrock and Sc huster 1992, Toapanta et al. 2005), probably because of feeding on anthers. An e ffect from feeding on floral buds (= cotton squares) has been reported for the boll weevil which needs to feed on floral buds for the ovaries to develop (Hunter and Hinds, 1905, Hunter and Pierce 1912, Isley 1928, 1932). The pepper weevil is able to obtain suffi cient nutrients for egg production from pepper fruits alone, as the majo rity of references in Table 1 indicate, and it is common practice to rear the weevils on a strict di et of fruit. The present comparison was conducted for only 7 days, so it is not possible to say that total fec undity would be lower on a strict fruit diet. Possibly, the resulting ni trogen deficiency c ould be compensated by

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37 increasing consumption, as has been observed for other species including Celerio euphorbiae L. (Lepidoptera: Sphingidae), and Pieris rapae L. (Lepidoptera: Pieridae) (House 1965, Slansky and Feeny 1977, Awmack and Leather 2002). In addition to nitrogen content, a proper balance of amino acids is critical for growth and reproduction in insects (Li nding 1984, Weis and Berenbaum 1989, Nation 2002). Therefore, the increased fecundity obser ved for the pepper weevil could also be due to the balance of amino acids that might be present in floral buds. Suitable diets for the boll weevil were developed only after de veloping a basic knowledge of the proteins and free amino acids in anthers of young cott on flowers (Earle et al. 1966, Hilliard 1983). Further studies that identify the proteins a nd amino acids of anthers of peppers could be useful in the development of a better ar tificial diet for pepper weevil adults. Life Table Pepper weevils laid a mean of 341 eggs per female with an average of 4.7 eggs per day over an average life span of 78.7 d (E lmore et al. 1934). No temperature was recorded in that study. Goff and Wilson (1937) reported a fecundity of 198 eggs with an average of 6 eggs per day. Gordon and Arms trong (1990) reported 253.6 eggs over a life span of 31.7 d, with an average of 8 eggs per day at 22 to 27C. Recently, Toapanta et al. (2005) reported a wide range of fecundity fo r this species according to the number of fruits offered to the weevils and temper ature. Using eight females per group, these authors reported 3.1 eggs per day at 30C and 158 eggs per fe male in single peppers over 51 days. Offered 3 peppers per day at 27C, fe males were able to lay 3.9 eggs per day or 281 eggs over 72 d. The fecundity of pepper w eevil females fed with floral buds observed in this study was 355 eggs per female, with an average of 5.5 eggs pe r day and a life span of 64.5 d.

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38 During the adult stage of insects, the repr oductive potential is fully realized only if the females have an adequate supply of en ergy for surviving and of nutrients for egg production (Hilliard 1983, Awmack and Leathe r 2002). The biology of the pepper weevil has been studied for more than a hundred year s, but just three studi es (Elmore et al. 1934, Gordon and Armstrong 1990, and the present) prov ided floral buds along with fruits to females when estimating fecundity. Not surp risingly, these studies reported the highest fecundity for this species. Fecundity is the major component determining population dynamics of a species and, along with surv ivorship, influences all demographic parameters (Birch 1948, Southwood a nd Henderson 2000). Consequently, the demographic parameters based on fecundity we re higher for data reported by Elmore et al. (1934) and the present study (Table 10). Table 10. Life table parameters for the pepper weevil Parameter Elmore et al. (1934)1 Toapanta et al. (2005) 30C This study 27 + 2C Net reproductive rate (Ro) 153.45 33.57 158.10 Intrinsic rate of increase (rm) 0.09 0.11 0.14 Generation time (T) 53.22 32.39 36.04 Doubling time (Dt) 7.33 6.35 4.93 Finite rate of increase ( ) 1.10 1.11 1.15 1The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively. No temperature data provided. The Ro value for pepper weevil females fed with immature ‘Jalapeo Mitla’ and floral buds was 4.6 times greater than the highest value reported by Toapanta et al. (2005). These authors fed the insects only with ‘Serrano’ or ‘Jalapeo’ peppers, but no floral buds. Furthermore, the condition or quality of fruits was not indicated. The

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39 Generation time (T), and Doubling time (Dt) reported by Elmore et al. (1934) were larger than those observed in this study. This coul d be a consequence of distributing the eggs evenly over the entire oviposit ion period. It was not possible to know the true distribution of eggs over time to make a better estima tion. Therefore, both parameters might be overestimated. If the generation time of th is study is used with the approximation indicated by Birch (1948) to calculate the intrinsi c rate of increase (rm = loge Ro / T), the rm for Elmore et al. (1934) data set is similar to this study (rm = 153.45/36.04 = 0.14). The difference in results in this study ve rsus Elmore’s data are due to generation time, with fecundity (as indi cated by Ro) being the same. Th ese values represented the potential of this species when feeding on floral buds and immature fruits, a situation that would occur in the field. It might explain in part the success of this species in infesting a young pepper crop, when floral buds and im mature fruits are abundant. These demographic parameters could be useful fo r: a) comparisons of different rearing conditions, b) comparisons of population dyna mics with natural enemies, and c) predictions of the potential of biological control agen ts to control this pest.

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40 CHAPTER 3 LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE) Introduction The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is considered one of the key pests of peppe rs in the United States, Mexico, Central America, and some Caribbean Islands (Elmore et al. 1934, Riley and King 1994, Mariscal et al.1998, Schu ster et al.1999). Adults lay eggs in floral buds and immature peppers, and larvae develop inside beyond th e reach of insecticides. Consequently, chemical control targets only adults. An acti on threshold of a singl e adult weevil per 400 pepper terminals has been proposed (Riley et al. 1992 a,b). This low action threshold, and the use of insecticides as th e principal method of control, can result in adverse effects including marketing restric tions, exposure of non-target organisms, environmental contamination, pesticide resistance, and s econdary pest outbreaks (NAS 1969, Luckmann and Metcalf 1982, Doutt and Smith 1971). Unfort unately, no other viable control strategy for the pepper weevil has been developed dur ing the one hundred years that have elapsed since the pest was first reported in Me xico (Cano and Alcacio 1894) and the United States (Walker 1905, Pratt 1907). Biological control could be an alternative tactic to incor porate into integrated pest management (IPM) of the pepper weevil; howev er, sufficiently effective natural enemies have yet to be discovered. One of the natu ral enemies of pepper weevil which has received attention as a potential biolog ical control agent is the generalist and

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41 cosmopolitan parasitoid Catolaccus hunteri Crawford (Hymenoptera: Pteromalidae). This ectoparasitoid of the last inst ar of the pepper weevil has a gr eater fecundity than its host (Rodriguez et al. 2000, Seal et al 2002). It has been recorded as one of the most common parasitoids of pepper weevil in some states of Mexico (Maris cal et al. 1998, Rodriguez et al. 2000), Central America, and Florida (Cross and Mitchell 1969, Wilson 1986, Riley and Schuster 1992, Schuster et al. 1999). Howe ver, releasing the equivalent of 1150 C. hunteri adults per hectare per week in field pl ots at Sinaloa, Mexi co, was not effective against this pest (Corrales 2002). On the other hand, results of releasing 7900 C. hunteri per hectare, prior to and duri ng the early pepper season, indi cated that this parasitoid could reduce damage by the pepper weev il in Florida (D. Schuster, personal communication). Divergent results may be due, in part, to the fact that C. hunteri attacks the 3rd instar host that is inaccessible deep w ithin the pepper fruit. Augmentative releases of this parasitoid prior to the crop cycle on the alternative host pl ant nightshade, which has small fruits, could reduce pepper weevil pop ulations. Releases early in the crop cycle when fruits are small, or in small fruited va rieties, might be effective because the host is more accessible to C. hunteri Unfortunately, once the fruits increase in size the effect of the parasitoid is limited. In fact, R iley and Schuster (1992) indicated that C. hunteri was not detected in fallen fruits larger than 2.5 cm in diameter. Therefore, it is desirable to find species that may be effective as biological control agents of this pest, preferably those that attack earlier and more accessible life stages. A candidate parasitoid was recently re ported in Mexico, where pepper, pepper weevil, and parasitoids likely evolved. This species is considered the most important parasitoid of the pepper weevil in Nayarit, Mexico, where it causes the highest incidence

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42 of parasitism on pepper weevil (from 18 to 40 %) in field samples (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This soli tary endoparasitic wasp was described in 2000 as Triaspis eugenii Wharton & Lopez-Martinez (Hymenoptera: Braconidae). However, there is still little information av ailable on its biology and behavior (Sharkey 1997, Mariscal et al. 1998, Toapanta 2001). This Chapter focuses on the biology and life history of T. eugenii Initial efforts to maintain the parasitoid colony were hampered by low levels of parasi tism and high levels of superparasitism. These f actors limited reproductive pote ntial and, thus, reliable estimates of demographic parameters. In addi tion, inefficient rearing demanded excessive resources (peppers and hosts). Preliminary observations in the la boratory suggested two possible causes for low levels of parasitism: 1) a limited acceptable range of host-age for parasitizing, and 2) the inabil ity of females to find or recognize the host. One hypothesis to explain this latter difficulty was that exposing pepper fruit to too many weevil adults in the colony resulted in fewer eggs sealed with an oviposition plug (Cha pter 2). The lack of acceptable hosts could then result in eith er retention of eggs or in excessive superparasitism. These questions required a solution before demographic parameters could be reliably estimated and before the potential of the paras itoid as a biological control agent could be evaluated. The objectives of this study then were to determine the host lifestage suitable for parasitization by T. eugenii to determine the parasitoid’s ability to recognize and to parasitize plugged and unplu gged host eggs, to describe the life cycle of the parasitoid, and to estimate dem ographic parameters for this species.

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43 Materials and Methods Surveys Based on results of Mariscal et al. (19 88) and Toapanta (2001), two surveys for T. eugenii were conducted in pepper growing regions at Nayarit, Mexico. In April and May of 2003, sampling was conducted in Puerta de Mangos, Caada del Tabaco, Villa Jurez, Los Corchos, Los Medina, and Palma Grande, all located in San tiago Ixcuintla county, Nayarit, between 21 36' and 22 17' north latitude, and between 104 53' and 105 40' west longitude (INEGI 1999). Hurricane Kenna made land fall in San Blas on October 25, 2002, just 30 km from Santiago Ixcuintla and many pepper fields were destroyed. Late replanting resulted in delayed harvest, with the result that fields included in the first survey in April 2003 were still being managed for weevils with insecticides. During th e second survey in May 2003, the majority of peppers were collected fr om abandoned pepper crops. Pepper fruits with signs of damage of the weevil (includi ng presence of feeding and/or oviposition punctures) were collected from the plants and/or from the ground. A random sample (n=57) of these fruits was dissected to c onfirm weevil presence. Around 25 kg of fruit or about 3,300 peppers were collected in each survey. All were Capsicum annuum L. The dominant variety was ‘Serrano’ but local cultiv ars such as ‘Serranillo’, ‘Caloro’, and a few ‘Jalapeo’ were also collected. Fruits were held in plastic nets of 2 kg in the field and later at room temperature to avoid water condensation. Within 3 d the plastic nets containing the peppers were pl aced in individual Ziploc plastic bags (S.C. Johnson & Son, Inc. WI) provided with a paper towel to absorb moisture, and transported by air to the United States in two insulated coolers provided with ice substitute (Rubbermaid Inc.,

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44 Wooster, OH). The material was allowed to enter in the United States under the permit 46799 of the Animal and Plant Health Inspection Service (APHIS), USDA. Upon arrival in Miami, the material wa s brought immediately to the quarantine facilities at the Florida Depa rtment of Agriculture and Consumer Services (FDACS), Division of Plant Industry (DPI), in Gainesville, Florida. In the presence of the quarantine officer, the peppers were distributed into lidded plastic containe rs (30x23x10 cm). Each container had 4 lateral holes, 5 cm in diam eter, covered with fine mesh screen for ventilation. A paper towel was placed at the bottom of each contai ner to absorb excess moisture. These emergence containers were held in the maximum security room in quarantine at 27 + 3C 50 to 70% RH, and 12:12 (L:D) h photoperiod. All the parasitoids except T. eugenii were preserved in 70% ethano l and labeled and identified with the aid of available literat ure. Identifications of braconids and chalcids were verified by R. Wharton and R. Lomel, respectivel y, both of Texas A&M University, College Station. Voucher specimens were deposited at the Florida State Colle ction of Arthropods at DPI. Parasitoid Rearing Wasps were held in 3.8 L plastic cages (Cha pter 2) with the la teral holes sealed with organdy. Parasitoids had free access to water in a cotton wick placed in 28-mL plastic cups, and to lines of honey dispensed every day on the upper side of the cage. In addition, a 7-8 cm long pepper flush (of leaves and floral buds) was inserted in a 28 mL plastic cup filled with water and placed in each cage to simulate natural host finding conditions. The flush had been exposed fo r 48 h to three pepper weevil adults. Pepper weevil hosts were obtained from a colony held in Florida Reach-In Incubation chambers (Walker et al. 1993) maintained at 27 + 1C and 70-80% RH, and

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45 14:10 (L:D) h photoperiod. Immature ‘Jalapeo’ fruits (4.7 + 0.77 cm length, 1.56 + 0.27 cm width, and 5.57 + 1.07 g weight, n = 25) were exposed to weevils for 24 h at a ratio of 10 weevils per fruit. Under these conditions, pepper weevil eggs take 2.9 + 0.1 d to hatch, and an additional 1.7 + 0.1 d to reach the second in star (Toapanta et al. 2005). Accordingly, two age ranges of eggs (2-24 h and 26-48 h after weev il exposure) and first or early second instars (4 to 5 d after infestation), were o ffered to the wasps. Each cage contained one or two T. eugenii females. At least two pepper fruits of each age category were offered daily to the wasps beginni ng the second day after emergence. Following exposure, fruits were held in ventilated containers until emergence of insects. When it became apparent that parasitoids were only recovered from peppers that had weevil eggs, the exposure period of fru its to weevils in the colony was limited to 24 h. Fruits were replaced between 4:00 to 6:00 PM, held at 22 + 2C until 10:00 AM the next day, exposed to parasitoids fo r 24 h, and then held as above. After the second generation of parasitoids, 10-15 females and 10-12 males were included per cage to maintain a ratio of one pa rasitoid female per infested fruit per day. Each cage was used 10-12 d. Females emerging from one cage were used to mate with males emerging from a different cage to prev ent inbreeding within the colony. Using this rearing system, 80-100 females and 100 males were held each gene ration until the colony could be moved from quarantine during October and November, 2003. The colony was then held at the Entomology and Nematol ogy Department, University of Florida, Gainesville, at 27 + 1C and 70-80% RH, and 14:10 (L:D) h photoperiod for five months. The colony was moved in April 2004 to the S outh West Florida Research and Education Center (SWFREC) at Immokalee FL, and wa s maintained in a rearing room at 27 + 2C,

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46 60-70% RH, and 14:10 (L:D) h photoperiod. Ex periments were conducted under these environmental conditions, unless otherwise in dicated. ‘Jalapeo Mitl a’ was the variety used most in rearing the colony and in conduc ting experiments, but when this cultivar was not available, ‘Jal apeo Ixtapa’ was used. Host-Age Range Choice tests were conducted during Decem ber 2003 to compare the acceptance of eggs either 24-40 h or 48-64 h old. ‘Jalapeo’ fruits 5.25 + 0.53 cm length, 1.68 + 0.15 cm width, and 7.41 + 0.70 g weight, (n = 15), were exposed the required time to 10 weevil females per fruit. Fruits with eggs were offered to the parasitoids for 8 h. A no-choice test was conducted during J une 2005 and included eggs either 3.5, 27.5, 51.5 or 75.5 + 3.5 h old. ‘Jalapeo’ fruits (4.73 + 0.68 cm length, 1.48 + 0.26 cm width, and 5.26 + 0.67 g weight, n = 10) were exposed seven hours to female weevils at a density of one weevil per fruit. Fruits with e ggs of the first age class were used without storage while fruits in the othe r age classes were held at 22 + 2C and 50-60% RH for 2472 h before being offered to T. eugenii Infested fruits were exposed to parasitoids for 2 h. Three randomly selected weevil eggs we re removed under a dissecting microscope from peppers in each age class to observe development. Heat units for each age, including the time exposed to the wasp, were proportionally estimated using the average temperature for each period. Heat units = (maximum temperature + minimum temperature/2) – threshold temperature. The threshold temperature wa s considered to be 11C. For the choice test, an experimental un it was considered a group of 10 infested peppers, five for each egg age class, offered at the same time to 20 T. eugenii females.

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47 Five replicates were run. For the no-choice te st, the experimental un it was five infested peppers of each egg age class, which were offered simultaneously to 10 T. eugenii females, there were three replicates. Because parasitoid females were used only once in each replicate in both assays and many parasi toids were needed, replicates were carried out in different days. Parasitoids were 5-10 d old and they were randomly taken from the colony in any replicate. Fruits were remove d after exposure, and held in ventilated containers until the emergence of insects. Chi-square analysis (PROC FREQ, SAS Institute 2000) was used to test if parasitism was independent of age. Role of the Oviposition Plug in Host Location Choice and no-choice tests were conducte d during May and June 2004 to determine the importance of the oviposition plug to T. eugenii for finding and parasitizing host eggs. Previous observations showed that the ovipos ition plug indicated the presence of an egg in 91% of all cases (Chapter 2). Host eggs for these tests were obtained from groups of three weevil females, 7-10 d old, placed in 250 mL plastic cups. Each group was provided with a ‘Jalapeo’ pepper (4.65 + 0.48 cm length, 1.93 + 0.29 cm width, and 7.40 + 1.47 g weight, n=50) for 24 h. Subsequently, fruits were held at room temperature (22 + 2C and 60-70% RH), before exposure to the parasitoid the next day. Only pepper fruits with 10 eggs were used. Excess plugs and eggs (Fig. 3) were removed from the fruits using fine-tipped forceps. Each experimental unit was considered as one or two pepper fruits, depending on the test, offered to an individual T. eugenii female 4-6 d old. Subject parasitoids were randomly selected from the colony and used only once. Each female was placed individually in 1.9 L plastic containers (12x11x15 cm) with two males to ensure mating.

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48 Water and honey were provided as previously described. Infested frui ts were exposed to the parasitoids for 24 h. Figure 3. Longitudinal section of an infested ‘Jalapeo’ fruit showing a pepper weevil egg and oviposition plug (ov-p). Choice tests were conducted by offering two pepper fruits (10 weevil eggs each) to individual T. eugenii females. The oviposition plugs were removed from half of the eggs of each pepper 24 h before the test. Plugs we re removed under a stereoscopic microscope using forceps, and a cotton swab was used to clean any visible re sidue. No-choice tests were conducted by offering a single pepper frui t with ten weevil eggs, either with or without oviposition plugs. Weevil eggs were later removed, placed in a drop of tap water on a microscope slide (4 or 5 at once), and crushed under a glass cover slide. A binocular composed microscope was then used to obs erve parasitoid eggs (100X magnification). Fourteen replicates were run for each treatmen t, and data were analyzed using chi-square

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49 tests to determine the differences between observed and expected values (PROC FREQ, SAS Institute 2000). Effect of Weevil: Fruit Ratio on Parasitoid Production Cages (3.8 L) containing 20-30 T. eugenii females and 20-25 males, 1-3 d old, were used as the experimental unit. The percen tage of parasitism was compared between ‘Jalapeo’ fruits exposed 24 h to unsexed w eevils (ratio 10:1 fruit), or exposed 24 h to mated females (ratio 1:1 fruit) Daily during 10 consecutive da ys 13 infested fruits (4.44 + 0.72 cm length, 1.47 + 0.23 cm width, and 4.83 + 1.10 g weight, n = 30) of each pepper weevil ratio, exposed to weevil adults as above, were placed in the corresponding parasitoid cage. Fruits were exposed for 24 h to the parasitoids the first and the last day, but only 2.5 h the remaining 8 d. Fruits were re moved and held in plastic containers until emergence of insects. Three replications were run for each treatment during September and October, 2004. Data were evaluated for normality (PROC CAPABILITY, SAS Institute 2000) before the analysis, and stat istical differences were determined using student’s t-distribution (PRO C TTEST, SAS Institute 2000). Life Cycle ‘Jalapeo’ pepper fruits c ontaining pepper weevil eggs 18-40 h old were obtained by exposing the fruits 24 h to a dens ity of 10 weevils per fruit (5.29 + 0.80 cm length, 1.67 + 0.24 cm width, and 5.90 + 1.40 g weight, n = 10). An experimental unit consisted of eight infested fruits exposed 2 h to T. eugenii in cages each containing 15 females and 20 males 5-7 d old. Fruits were then removed and incubated in ventilated containers at 27 + 1C, 50-60% RH, and 14:10 (L:D) photoperiod Eight experimental units were run

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50 concurrently. In addition, 10 infested pepper fruits from the same batch, but not exposed to the parasitoids, served as control for the whole experiment. To determine incubation time of T. eugenii 40 to 60 weevil eggs, usually one egg per fruit of each experimental unit, were re moved and dissected from fruits at 18, 20, 22, and 24 h after parasitoid exposure. In additi on, ten to 15 control eggs were dissected each time to confirm the absence of parasitoids. Length and greatest width of parasitoid eggs 18-20 h old (n=33) were measured using a binocular microscope fitted with an ocular micrometer calibrated with a stage micrometer (parallel lines at 10 ). The remaining pepper fruits (64 expose d and 10 unexposed) were held under conditions described above for 9 days after parasitoid exposure. Peppers were then dissected and all prepupae were removed, t ogether with the completed or partially completed pupal cell, and held in individual polystyrene cells (Mul tiwell, 24 well plates with lid, Becton Dickinson and Co.). Cells were filled with paper towel packed into the cells with no water to suppor t the pupal cells, which were observed every 4 h during the first 3 d, and then twice a day (9:00 AM and 6:00 PM) afterwards unt il the emergence of adults. The Multiwell cells with the host and parasitoids were maintained at 27 + 1C, 50-60% RH, and 14:10 (L:D) photoperiod, but the observations were made at room temperature (22C). Twenty unexposed and 160 exposed pepper we evil larvae were isolated as above, but only data from those parasitoids that were observed emerging from the host or spinning their cocoons and successfully reaching the adult stage are reported. Development times were calculated for the egg, endoparasitic and ectoparasitic larval stages, prepupa (spinning cocoon), and pupa. Stages were considered completed when

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51 50% plus 1 individuals reached the next st age. The prepupal stage was considered to occur from the cessation of larval feeding through the completion of the cocoon. Means and standard deviations were calculated for each stage. Fecundity During September to December 2004, 10 adult T. eugenii females 18 h old or younger were placed individually in 3.8 L plasti c containers. Three males were included in each cage to ensure mating. Each cage wa s provided daily with ‘Jalapeo’ fruits previously exposed for 24 h to pepper weevil females at the density of one pepper weevil per fruit (4.60 + 0.45 cm length, 1.94 + 0.30 cm width, and 7.37 + 1.52 g weight, n = 46). Weevil adults were provided water and honey as previous ly described. Five of the T. eugenii females were each provided every day from 9:00 AM to 6:00 PM with 12 fresh peppers, each containing 4-8 pepper weevil eggs. The other five females were each provided with two peppers fruits replaced ever y 1.5 h during the same period, so that the total number exposed was also 12 fruits per fe male. Every day, half of the pepper weevil eggs of each fruit were removed and dissected to count the number of T. eugenii eggs per host using the methodology described above. The remaining eggs and fruits were held until the emergence of T. eugenii to estimate survival and sex ratio. The program LIFETABLE.SAS (Maia et al. 2000) was used to estimate net reproductive rate (Ro), generation time (T), doubling time (D t), intrinsic rate of increase (rm), and finite rate of increase ( ) (Chapter 3) (Birch 1948, Maia et al. 2000, Southwood and Henderson 2000). Frequency of superparasitism between treatments was compared using chi-square analysis (PROC FREQ, SAS Institute 2000).

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52 Adult Longevity T. eugenii adults 12 h old or less were caged in two 1.8 L containers as follows: 1) 10 females and 10 males provided with wate r only, and 2) 10 females and 10 males provided with honey and water. The wasps we re observed twice a day (9:00 AM and 7:00 PM) and mortality was recorded until all die d. The two treatments were replicated three times. A comparison of longevity was also made between these females and ovipositing females from the fecundity experiment. The Student’s t test (PROC TTEST, SAS Institute 2000) was used to make all comparisons. Results Surveys About 50 kg of pepper fruits infested by pepper weevil larvae were collected under natural conditions from Nayarit, Mexico. W eevil and parasitoid recovery varied with location (Table 11), although fruits from a ll sites were severely infested. Although a mean of 1.40 + 0.97 weevil per fruit was observed in the field from a random sample of 57 fruits, only an average of 0.73 weevils per fruit emerged in quarantine. Two genera of Braconidae (including Triaspis eugenii ) and one each of Pteromalidae, Eurytomidae and Eupelmidae were recovered, accounting for onl y 2.85% of total parasitism (Table 11). The dominant species recovered from both collecting trips was C. hunteri which represented about 83% of emerged parasitoids.

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53Table 11. A. eugenii and parasitoids emerged from approxi mately 50 kg of infested hot peppers collected in the state of Nayarit, Mexico, April and May, 2003. Location (sites visited) Anthonomus eugenii Catolaccus hunteri Triaspis eugenii Eurytoma sp. Eupelmus sp. Bracon sp. A p r i l 2 0 0 3 Caada del Tabaco (3) 1260 1 1 Los Corchos (2) 427 7 4 1 1 Puerta de Mangos (1) 1484 1 M a y 2 0 0 3 Caada del Tabaco (1) 151 11 1 1 1 2 1 Los Corchos (5) 698 32 12 2 3 2 Los Medina(3) 251 15 4 1 1 Palma grande (1) 237 4 1 4 1 1 1 Villa Jurez (1) 226 19 3 Total 4734 88 27 5 4 5 2 6 1 1 0

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54 Host-Age Range Heat units were calculated for each age class to precisely determine which ones could be parasitized (Table12, 13). T. eugenii preferred younger eggs to older eggs in choice tests (X2 = 5.88, df = 1, P= 0.015) (Table 12). Nevertheless, T. eugenii was equally able to parasitize eggs over f our age classes when given no choice (X2 = 1.53, df = 1, P = 0.674) (Table 13). The eyes and mandi bles of pepper weevil larvae were visible through the chorion in th e most mature eggs before wasp exposure. Table 12. Parasitism of pepper weevil eggs by T. eugenii when given the choice of two ages of eggs Host maturity Heat units1 Age (h) Parasitism Not parasitized Parasitized Number Percentage2 12.66 28.66 24 – 48 51 64 55.6 23.66 – 39.66 48 – 72 98 68 40.9 1Accumulated heat units based on 11C threshold temperature 2X2 = 5.88, df = 1, P = 0.015 Table 13. Parasitism of pepper weevil eggs by T. eugenii when given no choice of egg age Host maturity Heat units1 Age (h) Parasitism Not parasitized Parasitized Number Percentage2 0.10-4.66 0 7 6 13 68.4 12.45-15.66 24 – 31 10 29 74.3 23.45-26.66 48 – 55 16 26 61.9 34.45-37.66 72 – 79 16 30 65.2 1Accumulated heat units based on 11C threshold temperature 2X2 = 1.53, df = 1, P = 0.674 Role of the Oviposition Plug in Host Location Parasitism of pepper weevil eggs by T. eugenii was clearly affected by the presence or absence of the oviposition plugs (Table 14). Over two and a half more eggs were

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55 parasitized when covered normally by an ovi position plug, compared to eggs over which the plugs were removed in choice tests. The predominance of parasitism in plugge d eggs was just as evident under no choice conditions (Table 15). Given no choice, T. eugenii parasitized three times more eggs covered normally by an ovipositi on plug, compared to unplugged eggs. Table 14. Parasitism of pepper weevil eggs by T. eugenii when given the choice of eggs covered with an oviposition plug and eggs with no plug Oviposition plug Parasitism Not parasitize Parasitized Percentage1 Normal 69 71 50.7 Removed 114 26 18.6 1X2 = 31.94 df = 1, P < 0.0001 Table 15. Parasitism of pepper weevil eggs by T. eugenii when given no choice of eggs covered with an oviposition plug and eggs with no plug Oviposition plug Parasitism Not parasitize Parasitized Percentage1 Normal 51 89 63.6 Removed 111 29 20.7 1X2 = 52.73, df = 1, P < 0.0001 Effect of Weevil: Fruit Ratio on Parasitoid Production When T. eugenii females were exposed to ‘Jalape o’ pepper fruits infested with pepper weevil eggs by mated weevil females at a ratio of one weevil per fruit more parasitoids (t= 5.26, df = 58, P < 0.001) and fewer weevil adults (t= 5.26, df = 58, P < 0.001) emerged per fruit, compared to when T. eugenii were exposed to fruits infested by unsexed weevils at a ratio of 10 weevils per fruit (Table 16). Percentage of arasitism was similarly affected (t= 4.40, df = 58, P < 0.0001). Therefore, infesting pepper fruits with

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56 one weevil female per fruit made more effici ent use of weevils, weevil eggs, and fruits than infesting pepper fruits w ith 10 weevils per fruit. Table 16. T. eugenii produced with two pepper weevil densities Weevil adult density Insects per fruit Parasitism (%) T. eugenii Weevils Unsexed weevils (10/ fruit) 0.81 + 0.26 0.94 + 0.35 45.5 + 7.1 Mated weevil females (1/ fruit) 1.25 + 0.48 0.52 + 0.30 71.2 + 12.3 Life Cycle The eggs of T. eugenii were translucent and elongat ed with no evident surface sculpturing. They were 246 + 33.6 m long by 73.4 + 16 m wide. The length included a small pedicel of 31.2 + 5.7 m long (Fig 4A). The majority of the eggs hatched 23.0 + 1 h after the 2 h exposure to T. eugenii (Table 17). Emergence wa s completed (41 larvae of 42 eggs) after 25 + 1 h. After emergence and for a few more hours, first instar T. eugenii were translucent, except for the large sclero tized head and large mandibles. The segments of the body were nearly of equal size, ex cept for the last one which was rounded and slightly larger than the rest (Fig 4B). So me of the larvae observed on the microscope slides appeared to be attacking unhatche d parasitoid eggs within the host-egg. The endoparasitic phase lasted almost 10 d and comprised more than half of the total parasitoid developmental time (Table 17). Parasitized pepper weevil larvae, even late 3rd instars, appeared to develop and to molt normally and had no evidence of decreased activity, compared to the observed control. The parasitoid larva emerged from the pepper weevil prepupa, usuall y after the completion of th e pupal cell. The parasitoid larva emerged through a hole made in the distal end of the abdomen of the host. Bending the base of the head forward, it began to f eed immediately on the we evil prepupa (Figure

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57 5A). The last few abdominal segments of th e parasitoid remained in the host at the beginning of the ectoparasitic phase (Figure 5B ). In about 8 h the parasitoid completely consumed the host, except for the cephalic capsu le (Table 17). The parasitoid then spun its cocoon and pupated inside the pupal cell of th e host, where it remained for 6-7 d. Some parasitoid larvae removed from the pupal cell of the host and placed directly into the polystyrene cells made pa rtial cocoons; however, they fa iled to complete the cocoon, and eventually died. Possibly, the lack of the pupal cell of the host caused dehydration. The developmental times of males and fema les did not differ (Table 17), but it is common to see in the laboratory that males emerge before females. Table 17. Developmental times of T. eugenii in days (Mean SD) at 27 1C Sex Egg n= 197 Parasitic development Endo Ecto n= 8 n= 9 Prepupa1 or n=14 Pupa n= 25 n= 35 Total n= 25 n= 35 Female 9.87 0.30 0.27 0.22 0.40 0.17 6.63 0.82 16.63 0.88 Male 0.96 0.2 0.42 0.18 6.40 0.52 16.36 0.88 1Spinning cocoon Adults emerged by chewing through the co coon then through the fruit wall. The emergence from infested peppers could be noticed as a small rounded hole. Adults were no longer than 2.4 mm and completely black. The only evident difference between sexes observed was the presence of the oviposito r in the female (Figure 6 C, D).

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58 Figure 4. Immature stages of T. eugenii : (A) Egg 22 h after ovipos ition, (B) First instar, 30-33 h after oviposition, with well developed mandibles.

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59 Figure 5. Larva of T. eugenii (A) Emerging from the poste rior end of the host, (B) Beginning to feed on the host even before emerging completely.

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60 Figure 6. T. eugenii adults. (A) Female, (B) Male.

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61 Fecundity The preoviposition period was shorter than 24 h for six of ten females, between 2448 h for three females, and between 48-72 h for one female. Little difference in the oviposition pattern was observed between hos t exposure treatments (Fig. 7). Maximum fecundity was reached on the 4th day after emergence and then gradually declined, reaching zero by day 18. Females deposited 95% of their eggs by day 12 when hosts were changed every 1.5 h, and 92% when hosts were left for 9 h. During that period, females laid 29.63 + 9.8 and 33.13 + 12.2 eggs per day, respectively, for the 1.5 h and 9 h host exposure periods. All females died by day 21 (Figure 7). When hosts were changed every 1.5 h, females produced an average of 372.40 223.14 eggs, compared to 433.20 193.80 when hosts were left for 9 h; however, the difference was not significan t (t= 0.5, df = 164, P = 0.901). Superparasitism was common with both treatments, although the proportion of superparasitized hosts was significantly greater when T. eugenii females were exposed to infested fruits for 9 h, compared to when fe males were exposed to infested fruits every 1.5 h (64.4% vs 55.3%, X2= 7.95, df = 1, P = 0.0048). Superparasitism ranged from two to nine eggs, and averaged 2.24 + 1.34 for the 9 h exposure period compared to 2.10 + 1.40 for the 1.5 h exposure treatment. These valu es were not significantly different (t= 1.76, df = 924, P = 0.0779).

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62 Figure 7. Fecundity of T. eugenii females at 27 + 2C either exposed to hosts changed every 1.5 h (n=5) or exposed to hosts per 9 h (n=5). The proportion of superparasitism and the numbers of emerged T. eugenii and pepper weevils suggested str ongly that more than half of the host eggs were not dissected. Consequently, the proportion of supe rparasitism from the dissected host eggs, and the survivorship of pepper weevil (90% ) estimated in Chapter 2, were used to estimate survivorship of the parasitoid. Th e estimate was made using the proportion of superparasitism observed from the dissected hosts each day. Demographic parameters for T. eugenii were estimated for the 9 h and 1.5 h tr eatments (Table 18). Values were not significantly different between the two host exposure intervals according to Student’s ttest comparison, P < 0.05 (Maia et al. 2000). Adult Longevity Males and mated females with ac cess only to water lived 2.3 + 0.7 d and 2.3 + 0.6 d, respectively, with no significant differe nce between the sexes (t=0.15, df = 58, P=0.881). Mated females provided with hone y lived significantly longer (24.7 + 10.6 d) 0 5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 A ge in daysEggs per female Host changed every 1.5 h Host available per 9 h

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63 that did males (15.8 + 9.2 d) (t=3.47, df = 58, P < 0.001). Finally, females laying eggs and provided with honey lived 16.5 + 3 d, which was significa ntly less than the 24.7 + 10.6 d for females not laying eggs (t=3.26, df = 38, P < 0.002). Table 18. Demographic parameters and confidence limits (+ 95%) for T. eugenii reared on pepper weevil eggs in ‘Jalapeo’ fr uits when parasitoid females were offered hosts for different exposure intervals at 27 + 2C Parameter1 Host changed every 1.5 h Host available for 9 h Net reproductive rate (Ro) 102. 83 (8.47 197.18) 106.15 (26.89 185.40) Generation time in days (G) 19.22 (18.13 20.31) 19.95 (18.90 21.00) Intrinsic rate of increase (rm) 0.24 (0.20 – 0.29) 0.24 (0.20 – 0.28) Doubling time (Dt) 2.82 (2.24 – 3.40) 2.92 (2.37 3.47) Finite rate of increase ( ) 1.28 (1.22 1.34) 1.27 (1.21 – 1.32) Sex ratio 1:1 1:1 1 Difference between treatments was not significantl y (Student’s t-test for pairwise comparison, P < 0.05, Maia et al. 2000). Discussion Surveys The total number of paras itoids recovered from both trips was 139, most of which emerged from weevil infested fruits collected in May. C. hunteri and T. eugenii represented 83% and 6.5%, respectively, but parasitism reached only 2.85%. These

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64 numbers were lower than those previously reported from that region: 865 and 1 210 parasitoids (recovered of 45 and 80 kg of infe sted fruit by pepper weevil, respectively) according to Mariscal et al (1988) and Toapanta (2001). T. eugenii was the dominant species (84% and 88%, respectively), with le vels of parasitism ranging from 29 to 40%, respectively. The destruction by hurricane Kenna and subsequent reduced acreage of the growing pepper area in Nayarit during 2003, th e possible damage to alternative hosts by the hurricane, and the intensifie d use of insecticides during la te months in the season, are possible explanations for low levels of pa rasitism of the peppe r weevil observed during the surveys in 2003 (2.85%) compared to prev ious reports (29-40%) (Mariscal et al. 1988, Toapanta 2001). Nevertheless, the 115 C. hunteri observed in the present study was not very different from 87 and 102 individua ls observed in the previous studies. C. hunteri is a generalist ectoparasitoid of more than 17 species of Curculionidae and Bruchidae (Cross and Mitchell 1969, Cross and Chesnut 1971). It can survive as an adult for more than 40 days and, in addition to carbohydrates, can feed on host larvae or, sometimes, pupae to survive (Cate et al. 1990, Rodriguez et al. 2000). Thus, C. hunteri is well equipped to survive uns table environmental conditions In contrast, the pepper weevil is the only known host of T. eugenii It has not been observed to host feed and cannot survive as long as C. hunteri especially without a carbohydrate source. Thus, T. eugenii would have fewer resources to draw upon under rapidly changing conditions. Host-Age Range During choice tests, T. eugenii preferred younger eggs to older eggs. Age may be related to host suitability (Godfray 1994). Because eggs may lack a cellular defensive response to foreign intrusion (Askew 1971) or because, with the exception of eggs in

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65 diapause, eggs rapidly change from storag e nutrients to chemically more complex embryonic tissue (Sander et al. 1985), pa rasitoids may prefer young eggs. These explanations have been supported by author s who found that parasitism by some egg parasitoids declines as the host ages (Strand and Vi nson 1983a, b, Ruberson et al. 1987, Reznik and Umarova 1990, Castillo et al. 2005). Nevertheless, this information has been supported mainly in observations with parasi toids from Trichrogrammatidae, Scelionidae, Mymaridae, and Eulophidae. They are idiobiont s that complete thei r development in the eggs, and they may have differences with pa rasitoids which develop as koinobionts, such as egg-larval or egg-pupal parasitoid s (Strand 1989, Vinson 1998, Godfray 1994, Quicke 1997). In contrast to many idiobiont egg parasito ids, egg-larval or egg-pupal parasitoids may lay eggs in a more advanced stage of host development (Clausen cited by Quicke 1997, Abe 2001, Quimio and Walter 2001, Kaeslin et al. 2005). Strand and Pech (1995) suggested that egg parasitoids may ev ade the immune response of the host by preferentially ovipositing in pregastrula embr yos in which the immune system has not yet become functional. However, Kaeslin et al. (2005) observed th at all stages of Spodoptera littoralis (Boisduval) eggs can be parasitized by Chelonus inanitus (L.), because the behavior of the parasitoid larva or adult a ssure that the larva w ill be located in the haemocoel of the host. When freshly deposited host eggs are parasitized, the parasitoid hatches in the yolk and enters the host em bryo either after wai ting or immediately through the dorsal opening. When host eggs ar e parasitized at 1 or 2 d old, the host embryo is covered by an embryonic cuticle th rough which the hatching parasitoid larva

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66 bores with its abdominal tip. When old e ggs (2.5-3.5 d) are parasitized, the female parasitoid oviposits directly into the haemocoel of the host embryo. T. eugenii was able to parasitize young and old eggs, an ability which is shared with other koinobiont species that attack eggs of their hosts but later emerge from larvae or pupae (Clausen cited by Quicke 1997, Ab e 2001, Quimio and Walter 2001, Kaeslin et al. 2005). The physiological intera ctions between host and para sitoid are not completely understood, but the ability to use either young or mature hos ts represents a desirable characteristic that increases probabi lity of locating an appropriate host. Role of the Oviposition Plug in Host Location Host habitat location by pa rasitoids often involves ch emical cues, as well as physical and electromagnetic signals (Q uicke 1997, Vinson 1976, 1998, Turlings et al. 1998, Havill and Raffa 2000). These signals include odors emanating from host frass, symbiotic fungi, or host-damaged plants (Quicke 1997, Vinson 1976, 1998, Turlings et al. 1998). Parasitoids attacking concealed larvae, such as fruit flies or wood borers, can locate their hosts by detection of movement transmitted as vibrations in the substrate (Godfray 1994, Quicke 1997). Such stimuli are no t available to paras itoids of concealed eggs. Such parasitoids could re ly on olfactory signals from chemicals such as kairomones of the host (e.g. accessory gland secretions), or from tactile and chemical signals emanating from mechanical damage such as oviposition wounds (Strand 1989, Godfray 1994). Once a probable site of a host has been lo cated, parasitoids res pond to less volatile substances, or to tactile cues where closer antennation and the ovipositor might play more important roles (Godfray 1994, Quicke 1997).

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67 This study demonstrated that the host ovipos ition plug is important for parasitoid host location. A high proportion, around 80%, of pe pper weevil eggs were not parasitized by T. eugenii when the oviposition plug was removed. The oviposition plug, then, provided means for the parasitoid to detect and, subsequently, to parasitize its host. It is possible that the antennae were used to detect host kairomones found in the plugs, because drumming was observed to increase as soon as the female was close to the plugs. The response of some parasitoids to these subs tances has been demonstrated in more than one species (Quicke 1997, Turlings et al 1990, 1998, Vinson 1998). However, the design of the present study did not permit the evalua tion of other sources of stimuli, such as physical or visual characteri stics of the plugs, which may be important in the host detection and oviposition by T. eugenii More studies need to be done in this area. When the pepper weevils were reared unde r crowded conditions, the proportion of eggs lacking ovipositions plugs increased (Chapter 2). For this reason, it was more efficient to rear the pepper weevil by exposing pepper fruit to one female weevil per fruit rather than 10 unsexed weevils per fruit. Additionally, less feeding damage on pepper fruits by weevils helped to diminish the number of rotten fruits in the colony (E. Rodriguez, unpublished data). Life Cycle T. eugenii is a member of the tribe Brachistini in the subfamily Helconinae and the family Braconidae. The members of Brach istini are cosmopolitan and are known as egglarval endoparasitoids of different families of Curculionidae and Bruchidae (Beirne 1946, Martin 1956, Shaw and Huddleston 1991, Sharke y 1997). Additionally, there is at least one species, Nealiolus curculionis (Fitch), which has been reported parasitizing early larvae of the sunflower stem weevil, Cylindrocopturus adspersus (LeConte) (Charlet and

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68 Seiler 1994, Charlet et al. 2002). Early reports confirmed and described the endoand ectoparasitic phases of the im mature stages of some Triaspis species, such as T. vestiticida and T. caudatus (Berry 1948, Obrtel 1960); howev er, few species of this genus have been studied in detail (Sharkey 1997). Females of T. eugenii were able to lay eggs from the first day of life. This behavior is characteristic of many koinobiont parasi tic wasps which do not need to consume protein-rich host hemolymph to initiate ooge nesis (Quicke 1997). The parasitoid egg was deposited in the host egg, where it hatche d in less than 24 h. No parasitoids were recovered from larvae (4-5 d af ter infestation). The endoparasi tic phase thus initiated in the egg and continued until the prepupal stage of the host. Only one parasitoid emerged from each host egg. The first larval instar possesses large mandibles. The presence of such mandibles in the early inst ars of solitary paras itoids has been suggested as useful for physical defense against either the same or di fferent species in case of superparasitism or multiple parasitism, respectively (Claus en 1940, Godfray 1994, Quicke 1997, Ming et al. 2003). In contrast, physiological supp ression by asphyxiation may be a common mechanism by which established larvae eliminate younger competitors (Fisher 1971, Godfray 1994, Quicke 1997). Egg-larval parasitoids occur in several subfamilies of Braconidae (Alysiinae, Helconinae, Ichneutinae), but members of th e subfamily Cheloninae more often use this strategy (Clausen 1940, Shaw 1997). The potential as biological control agents of some members of this subfamily could be one r eason for the availability of information on species such as Chelonus sp. nr. curvimaculatus Cameron, an egg-larval parasitoid of the pink bollworm, Pectinophora gossypiella (Saunders) (Hentz et al. 1997), and Chelonus

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69 inanitus an egg-larval parasitoid of Spodoptera littoralis (Kaeslin et al. 2005). The information reported in the present study c onfirms similarities in the life cycle of T. eugenii with that of these species of Cheloninae, and with that of Triaspis vestiticida (Berry 1947). In addition, th e present study describes li fe table parameters of T. eugenii information which is needed to establish the potential of this species. Potential of T. eugenii as a Biological Agent of the Pepper Weevil Despite the fact that five species and at le ast five other genera of parasitoids have been collected from the pepper weevil (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut 1971, Mariscal et al. 1998, Toapanta 2001), T. eugenii and Urosigalphus sp. are the only known species that parasitize eggs of this pest. Urosigalphus sp. (14 females and 9 males) was collected by P. Stansly during July 2003 in Oaxaca, Mexico, and was held in quarant ine facilities and reared in the same conditions that T. eugenii However, the parasitoids produced only males in its offspring, making it impossible to establish a col ony (E. Rodriguez, unpublished data). The egg of the pepper weevil could be c onsidered the stage more ecologically susceptible to be parasitized, because it is defenseless and is deposited close to the surface in the pepper fruits. Therefore, the ab ility to parasitize pepper weevil eggs could be one of the most importan t biological advantages of T. eugenii. Because T. eugenii can reach the host before the larva migrates deep inside into the fru it, it could attack the pepper weevil in any cultivar, regardless of fruit size. The reproductive capacity of T. eugenii is another important biological characteristic of the parasitoid. Females were able to produce an average of 402.8 + 199.5 eggs. Females laid more than 90% of their e ggs during the first 12 days of life, depositing 31.4 + 22.6 eggs per female per day.

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70 Superparasitism was common, although the pr oportion of superparasitized hosts observed was greater where all infested fruits were left for 9 h rather than being changed at 1.5 h intervals (64% versus 55%). T. eugenii lays eggs individually in only 28 sec (E. Rodriguez, unpublished data). The short time required for oviposition coupled with the longer exposure period may explain the higher rate of superparasitism. However, it does not explain the apparent inability of the paras itoid to distinguish parasitized hosts, even in the presence of nonparasitized hosts. Decreasing exposure time from 9 h to 1.5 h reduced superparasitism. Further reduction of exposure time would be impractical within the context in the rearing system developed here. Additional manipulations ma y be necessary such as more hosts per female, fewer hosts per fruit, and larger cag e size. On the other ha nd, superparasitism is relatively common in laboratory conditions in other rearing sy stems of parasitoids, such as Chelonus sp. nr. curvimaculatus (Hentz et al. 1997), or Catolaccus grandis Burks (Morales-Ramos and Cate 1993); however, supe rparasitism does not occur often in the field according to the same authors. Give n these considerations, the reproductive potentials of the pest and parasitoid were compared using fecundity and demographic parameters of the pepper w eevil (Chapter 2) and of T. eugenii using the data set of the combined 10 T. eugenii females. It was assumed that superparasitism does not occur under field conditions (Table 19). The fecundity of the pepper weevil (355.28 + 167.51) and that of T. eugenii (402.8 + 199.55) with the same sex ratio (0.5) and surv ivorship (0.9) indicated no differences in the net reproductive rate (Ro) (P = 0.82). Nevertheless, the remaining parameters favored T. eugenii (P < 0.00001). The intrinsic rate of increase (rm), which might be one of the

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71 most important parameters to compare populations because it combines Ro and generation time (T) (rm = loge Ro / T, Birch 1948, Southwood and Henderson 2000), was almost twice as great for T. eugenii as for the pepper weevil. Therefore, the important pest/parasitoid difference was generation time. Table 19. Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and 14 pepper weevil adults at 27 + 2C Parameter Pepper weevil1 T. eugenii1 Net reproductive rate (Ro) 158.10 (115.1 – 201.1) 167.1 (92.7 – 241.3) Intrinsic rate of increase (rm) 0.14 (0.13 – 0.16) 0.26 (0.24 – 0.28) Generation time (T) 35.4 (30.4 – 40.4) 19.6 (19.0 – 20.2) Doubling time (Dt) 4.8 (4.3 – 5.4) 2.6 (2.4 – 2.8) Finite rate of increase ( ) 1.2 (1.1 – 1.2) 1.3 (1.2 – 1.3) 1The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively T. eugenii has been cited as the most abundant parasitoid of the pepper weevil in field samples from Nayarit, Mexico, a region where the pepper weevil occurs naturally (Mariscal et al 1988, Schuster et al. 1999, To apanta 2001). It parasitizes the egg of its host and has almost twice the potential fo r increase than the pepper weevil. These attributes make T. eugenii an excellent candidate for bi ological control of the pepper weevil.

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72 CHAPTER 4 RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA: BRACONIDAE) IN FIELD CAGES AND IN THE FIELD Introduction The pepper weevil is considered on e of the key pests in peppers ( Capsicum spp.) in many growing regions of the United States Mexico, and Central America, and only cultural and chemical tactics are presently available for it s control (Elmore et al. 1934, Riley and King 1994, Mariscal et al. 1998, Schus ter et al. 1999). Since the pepper weevil was reported in Mexico (Cano and Alcacio 1894) and in the United States (Walker 1905, Pratt 1907, Elmore et al. 1934), no viable biolog ical control strategy has been developed to manage the pest. Some reasons for the lack of successful biological control of the pepper weevil may include: (1) a paucity of natura l enemies recorded from this pest (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut 1971, Clausen 1978), (2) limited impact of those parasitoids (Elmore and Campbell 1954, Genung and Ozaki 1972), (3) incompatibility of natural enemies with inse cticides used to cont rol pepper weevil, and (4) limited biological control research b ecause of the relatively small economic importance of pepper compared with other crops (Riley and King 1994). Recently Mariscal et al. (1998) identified nine different species of parasitoids attacking pepper weevil larvae in the state of Nayarit, Mexico, which is located in the central pacific coast and which is a likely center of origin of the pest. The most abundant of these parasitoids was the braconid Triaspis eugenii Wharton and Lopez-Martinez,

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73 which attained 18-40% parasitism in the fi eld (Mariscal et al. 1998, Schuster et al.1999, Toapanta 2001). These relatively high leve ls of parasitism, complemented with the continual presence of T. eugenii during surveys between 1997 and 2003, and with its ability to attack pepper weevil eggs, renewed in terest in biological c ontrol to combat the pepper weevil (Mar iscal et al. 1998, Schuster et al. 1999, Toapanta 2001). T. eugenii is a solitary egg-pre pupal parasitoid of the pepper weevil. Eggs are deposited into pepper weevil eggs, and hatchi ng larvae feed as endoparasitoids; however, larvae later emerge from the host prepupa a nd complete development as ectoparasitoids (Chapter 3). The habit of attacking the egg, loca ted close to the fruit surface, rather than the larva that burrows deep into the fruit, in addition to its repr oductive superiority to pepper weevil, make T. eugenii a good candidate for biological control of the pest. Nevertheless, the reproductive potential in the field could be influen ced by many factors, including the ability of the parasitoid to locate host and food, and the influence of environmental conditions. Therefore, the evalua tion of field releases is an indispensable step in assessing the effectiveness of the pa rasitoid as a biological control agent. T. eugenii cannot survive for more than two or three days at 27 + 1C without a carbohydrate supplement such as honey. Under field conditions, many parasitoids use nectar or honeydew for this purpose (House 1977, Powell 1989, Baggen and Gurr 1998). It cannot be assumed that these sources woul d always be adequate in or near every pepper crop. Many parasitoids a nd predators also use plant vol atiles to locate the host plants of their hosts or preys (Vins on 1976, Alphen and Vet 1989, Godfray 1994, Quicke 1997). The volatiles often are re leased from damaged plants (Turlings et al. 1990, 1995, 1998, Powell et al. 1998, Takabayashi et al. 1998, Havill and Raffa 2000, James and

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74 Grasswitz 2005). There is no information abou t the importance of weevil damage in host location or foraging behavior of T. eugenii within a patch. This information could be used to improve the effectiveness of establishing th e parasitoid in the field. The objectives of these studies were to assess the ability of T. eugenii to survive and pa rasitize the pepper weevil in large field cages and in the open field, and to evaluate the effect of honey supplements and weevil damage on this ability. Materials and Methods Insects Pepper weevil and T. eugenii adults were obtained from colonies maintained at the SWFREC in Immokalee, FL, as described in Chapters 2 and 3. Mated weevil females, 8 to 10 d old, and parasitoids 3 d old or younger were randomly chosen from the colonies for release into field cages. Parasitoids that we re released in the open field were 4 d old or younger. Field Cages Experiments were conducted in two scr eenhouses 7.3 x 3.6 x 3.65 m, with sides of 50 x 24 mesh screen (266 x 818 m openings) and roofs of polyeth ylene (Figure 8A). Each screenhouse was divided into 2 larg e cages 3.65 m in length by an organdy cloth partition. A zipper was sewn into the partition to serve as a door. Each cage represented an experimental unit (Figure 8B). Temperatur e and humidity were r ecorded during assays using HOBO data loggers (Onset Comput er Corporation, Bourne, MA).

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75 Figure 8. Field cages used at the SWFREC. (A) General view of a unit containing two cages. (B) Organdy partition and zipper between two experimental units.

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76 Effect of Honey Supplement Five pots 7.8 L, each containing two ‘Jalap eo’ pepper plants, were placed in each cage. Plants were 20.6 + 5.2 cm tall (n=10) and were spaced 35 cm apart. At the time of the evaluations, plants had 4.0 + 2.4 fruits smaller than 4 cm in length. To provide natural host finding conditions for T. eugenii two pepper weevil adults were confined in organdy sleeves on floral buds on each of two plants (in the same pot) per cage two days before experiments commenced. Honey was offered in lines and replaced every other day on two plastic yellow cards (5 x 8 cm) which were protected fr om sunlight by a cork roof (15 x 22 cm) supported by wire mesh (Figure 9A). A pe pper flush damaged by pepper weevil adults was placed in a 28 mL plastic cup filled with water (Chapter 3) and attached to the wire under the cork roof to attract parasitoids to the source of carbohydrates (Figure 9B). One of these devices (wasp refuge) was suspende d 30 cm above the plants in each cage. The experiment had two treatments, a re fuge with honey and a refuge without honey, which were replicated four times. B ecause replications were developed at different times, each screenhouse containing bot h treatments was considered a block. The two weevil damaged plants per cage of each block were replaced with newly damaged plants prior to the release of T. eugenii Using a fine brush, 10 T. eugenii females 3 d old or younger were placed onto the pepper flush of the refuge of each cage (Figure 9B), which was located 1.6 m from the infested fruits Refuges and pepper flushes were observed for 5 sec at 1, 2, 3, 8, 24, and 48 h af ter release to check for the presence of parasitoids. Parasitism was evaluated daily in each cage for 3 d by hanging two infested fruits per each of the damaged plants. The fr uits contained a total of 20 weevil eggs 24-40 h old.

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77 Figure 9. Honey-wasp-refuge. (A) General vi ew of the set up. (B) Using a unit as T. eugenii release point. After 8 hours (9:00 AM to 5:00 PM), th e exposed fruits were removed and dissected in the laboratory to assess the percentage of parasitism and superparasitism (Chapter 3). A new group of infested fruits was hung in the same location at the same time the next morning. Each block was replicated four times and each cage received the

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78 alternative treatment for the following re plication to nullify an influence of cage orientation. Chi-square analysis (PROC FR EQ, SAS Institute 2000) was used to test whether parasitism was independent of the presence of food (honey) Survival Survival of T. eugenii was evaluated using females confined in 250 mL plastic cups that had 3 cm diameter organdy sealed holes in the tops for ventilation (Chapter 2, Fig. 1). Cups were placed inside the canopy of po tted plants to avoid direct sunlight. Five T. eugenii females 3 d old or younger per cup represen ted an experimental unit. Treatments were cups without honey or cups with honey st reaked every 2 or 3 d on the internal side of the cups. Survival was checked every 1, 3, 9, 24, and 32 h, and then was checked each 24 h thereafter until all parasitoids were dead. Five replicates were performed in a single block. Student’s t-test (PRO C TTEST, SAS Institute 2000) was used to evaluate differences between treatments. Effect of Host-Damaged Plants Twenty 7.8 L pots, each containing two ‘Jalapeo’ pepper plants were placed in each cage. The pots were spaced 30 cm apart and plants were 40 + 14.3 cm tall with 15 + 7.8 fruits less than 4 cm long at the beginni ng of evaluations. Treatments in this assay were non-damaged plants and weevil damage d plants replicated five times. Because replications were performe d at different times, each screenhouse containing both treatments was considered a block. Plants we re noticeably larger and more mature during the last two blocks, and the presence of open flowers which could be a source of nectar was estimated from five plants per cage. At the same time, plants infested with aphids and leafminers in these two blocks were redistributed among cages to maintain a homogenous environment. At the end of each replication, plants used to support fruits

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79 with hosts were replaced, and cages received the alternative treatment A “Jalapeo’ plant was damaged by using an organdy sleeve to c onfine three mated females (8-10 d old) on one terminal with floral buds 48 h prior to releasing T. eugenii The damaged plant was used to hang infested fruits with pepper w eevil eggs daily as above. The same procedure without weevils was followed for the control. Five T. eugenii females 3 d old or younger were then introduced into each cage, using a fine brush to place the parasitoids onto the foliage of the potted plants furthest (3.6 m) from infested fruits. Previous observations indicated that this number of wasps wa s sufficient to detect a response. Parasitism was evaluated daily in e ach cage for two days by hanging five ‘Jalapeo’ pepper fruits cont aining a total of 25 pepper weevil eggs, 24-40 h old, in a random pattern on the previously infested plan t or in the previously designated plant, which was placed in an equivalent po sition among the 20 plants, in undamaged treatments. Infested fruits were held for 9 h as in the previous experiment and dissected to check parasitism. In addition, these fruits were observed for 20 sec at 10, 20, 40, 60, 120, and 180 min after releasing the pa rasitoids. Cage walls were also observed for parasitoids for 30 sec at 1, 2, 3, 8, 24, 32, and 48 h after re lease. A chi-square test (PROC FREQ, SAS Institute 2000) was used to compare para sitism in cages with and without weevil damaged plants. The numbers of parasitoids obser ved in infested fruits were compared in cages with and without weevil damaged plants using student’s t test (PROC TTEST, SAS Institute 2000). Before analysis, data were transformed using the square root of y + 0.5 to satisfy assumptions of normality (Sokal and Ro hf 1969). Student’s t test was applied also to data on wasps observed on cage walls afte r verifying normality of the data (PROC CAPABILITY, SAS Institute 2000). All means are reported in the original scale.

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80 Field Release and Establishment On June 17, 2004, 100 T. eugenii females and 70 males 4 d old or younger were released at the SWFREC in Immokalee, FL. Parasitoids were released from 7:30 to 8:30 AM using a fine brush to place them on the folia ge of pepper plants that were at the end of the fruiting cycle. Parasitoids were releas ed on six 100 m rows of ‘Jalapeo’ pepper. Three hundred randomly selected terminals were observed to assess the adult pepper weevil population. No flowers or buds were seen and fruits were large (7.68 + 1.46 cm length, 2.77 + 0.73 cm width, n=10) and mature. Fruits (n=10) were dissected to estimate the infestation of weevil larvae. The crop was destroyed a week after the release. Before eliminating plants, 6 kg of fruits were collect ed and held in an organdy fabric cage (60 x 60 x 60 cm) at 27 + 2C to allow weevil or parasitoid adults to emerge. The number of collected fruits was estimated by weighting 20 fruits. During January to April of 2005, T. eugenii was released five times on 90 m row of approximately 400 ‘Jalapeo’ plants of differe nt ages maintained at the SWFREC (Table 20). Parasitoids 4 d old or younger were placed with a fine brush on floral buds, or foliage, in groups of 5-10 females at 10 m intervals along the row. Table 20. Releases of T. eugenii during 2005 at the SWFREC in Immokalee, FL. Date T. eugenii January 10 100 80 February 8 80 40 February 18 35 March 25 60 50 April 20 80 60 Total 355 230 Before each release, 10 immature pepper fru its with weevil feeding punctures were collected and dissected to confirm the presence of weevil eggs. The pepper weevil

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81 infestation was also estimated by checking 50 te rminals of pepper plants just prior to each parasitoid release. Fruits were harvested ev ery 12 wk, and peppers with weevil feeding punctures or chlorotic calyxes were held in the laboratory until em ergence of weevil or parasitoid adults. On March 25, three honey-wa sp-refuges were established in the field and 60 parasitoid females and 50 males, 4 d ol d or younger, were released at those points at 5:00 PM. Five second obser vations at those honey-wasp-refuges were made at 1, 15, and 24 h after releasing in each refuge to check for th e presence of parasitoids. During the summer of 2005 at the SWFREC 10 potted pepper plants 5-6 months old and with mature fruits present were tr ansplanted 10-20 m from a ‘Jalapeo’ plot severely infested by the pe pper weevil during the previous spring. These plants were watered and fertilized during that season. In August 8, 2005, 70 fruits from those plants, and 15 more from a greenhouse nearby, were coll ected by P. Stansly. Fruits were held at 27 + 2C until the emergence of weevil or pa rasitoid adults. During the fall of 2005, experimental ‘Jalapeo’ peppe rs were destroyed at the SWFREC by hurricane Wilma. No peppers were grown in the field during sp ring of 2006 at the SWFREC, but 25-30 potted (7.8 L) ‘Jalapeo’ plants were kept outdoor s during spring 2006 and any pepper fruit with feeding punctures or chlorotic calyxes were collected to attempt further recovery of T. eugenii. Results Effect of Honey Supplement T. eugenii found and parasitized pepper weevil eggs in the presence or absence of honey in the wasp-refuge during the first day of release. Eggs from the second and third days were not parasitized. No difference in parasitism was found between refuges with

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82 honey (94.3%) or without honey (82.3%) (X2 = 2.29, df = 1, P = 0.1299). After releases, some parasitoids remained a few minutes in the wasp-refuge searching the pepper flushes, whether honey was present or not but soon left. No parasitoids were subsequently observed at any of the refuges. Survival Parasitoid females in cups with honey survived longer (3.65 + 2.06 d) than parasitoids without honey (0.62 + 0.56 d) (t = 7.11, df = 24, P < 0.0001). In the presence of honey, more than 50% of the females were a live at day four, and the last females died at day seven. In contrast, fe males without honey died in an average of 1.3 days (Fig. 10). Figure 10. Survival of T. eugenii adults in plastic cups with and without honey, when the cups were placed inside the canopy of pepper plants, 25.4 + 4.9C, 67 + 19% RH. Effect of Host-Damaged Plants T. eugenii females were able to find and to parasitize pepper weevil eggs in pepper fruits hung in damaged and undamaged ‘Jalap eo’ plants; however, the level of parasitism was higher in fruits hung in damage d plants (57.6%) than th at in fruits hung in 0 1 2 3 4 5 6 0.04 0.13 0.37 1 1.3 2 3 4 5 6 7 8 D a y s Live wasps Honey No honey

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83 undamaged plants (26.4%) (X2 = 24.97, df = 1, P = 0.001). No parasitism was observed on eggs in fruits hung in cages a day after the release of parasitoids. Overall, the numbers of T. eugenii adults observed on fruits hung on plants damaged by the pepper weevil decrease d with time (Table 21). More T. eugenii females were observed on fruits hung in plants dama ged by the pepper weevil 10 and 20 min after release, compared to the number observe d on fruits hung on undamaged plants (t= 2.45, df = 8, P = 0.040). Fewer parasitoids were obser ved at 40 and 60 min after release, with no differences between treatments. No T. eugenii adults were obser ved on fruits hung on undamaged plants after 60 min, or hung on any pl ant after 120 min. Ho wever, parasitoids were seen on the upper wall of cages duri ng the entire 48 h observation period. The number decreased with time; 3.4 + 0.9 versus 3.6 + 0.5, respectively for damaged and undamaged plants at 1 h; 2.2 + 0.8 versus 2.0 + 1.2, respectively at 24 h; and 0.4 + 0.5 versus 0.6 + 0.9 respectively at 48 h. There were not differences between treatments at any time (t-test, P < 0.05). Table 21. Number of T. eugenii adults released in field cag es and subsequently observed on pepper fruits (n=5) hung on pepper plants either damaged or undamaged by the pepper weevil, 28.2 + 4.7C, 55 + 9% RH Time after releases (min) Adults observed on fruits of pepper plants damaged by weevil undamaged t-test (P < 0.05) 10 2.0 + 1.22 0.4 + 0.55 20 2.0 + 1.22 0.4 + 0.55 40 1.4 + 0.89 0.8 + 0.84 ns 60 0.6 + 0.89 0.6 + 0.89 ns 120 0.2 + 0.45 0 180 0 0

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84 Field Release and Establishment At the time of the first releas e of parasitoids on 17 June 2004, 3.3 weevil adults were observed per 100 terminals, and 1.1 + 0.57 weevil larvae were seen per fruit (n=10), although no weevil eggs were detected. An estim ated of 731 pepper fruits were collected before the crop was destroyed, from whic h 380 adult weevils but no wasps emerged. Environmental conditions during T. eugenii releases at SWFREC during 2005 are shown in Figure 11. Pepper weevil adults we re found in the crop in excess of one adult per 100 hundred terminals at the time of four of the five releases of T. eugenii and more larvae than eggs were detected in the fru its (Table 22). Overall, the number of fruits damaged by pepper weevil larvae and the numbe r of emerged weevil adults increased through time (Table 22). No T. eugenii adults were observe d in honey-wasp-refuges following releases of the parasitoid during March 25. Figure 11. Daily temperatures and humidity during T. eugenii field releases on ‘Jalapeo’ pepper at the SWFREC in Immokalee, FL, 2005. 0 10 20 30 40 50 60 70 80 90 100 JanFeb Ma r A p r May M o n t h 2 0 0 5 Temperature( C)HR(%)

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85 T. eugenii emerged for the first time on April 14 from infested fruits collected on April 6. Infested fruits were then collected almost every week, and parasitoids were recovered in low numbers th rough May 2 (Table 23). A few Catolaccus hunteri adults also emerged. T. eugenii and C. hunteri together caused 6.7% parasitism. The pepper row was eliminated during the last week of May, 2005. Table 22. Adults per 50 terminals, and the num ber of weevil lifestages per 10 fruits, 1 or 2 d before field releases of T. eugenii on ‘Jalapeo’ pepper plots at SWFREC, Immokalee, FL, 2005 T. eugenii release (date) No. of pepper weevils Adults Eggs Larvae Emerged adults January 10 0 0 0 0 February 8 2 0 12 12 February 18 1 0 2 148 March 25 3 2 5 300 April 20 1 2 7 Table 23. Parasitoid and pe pper weevil adults recovered from ‘Jalapeo’ pepper following field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005 Emergence Triaspis eugenii Catolaccus hunteri Pepper weevil April 1 180 April 14 6 10 4 1 147 April 19 1 1 April 25 5 8 2 April 30 20 5 535 May 2 9 5 206 Total 41 28 7 1 1068 Pepper weevil adults emerged from the field sample and greenhouse samples collected by P. Stansly during the summer 2005 at the rate 0.9 and 0.53 weevils per fruit, respectively. However, no T. eugenii emerged. No pepper weevils were detected in the

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86 potted ‘Jalapeo’ plants esta blished at the SWFREC by the time sampling was terminated on April 14, 2006. Discussion Effect of Honey Supplement T. eugenii searched the pepper flushes offere d in the wasp refuges for a few minutes but left, apparently not to retur n. Parasitism did not differ between honey and no honey treatments, and was observed only during the first day of exposure. As expected, survival of T. eugenii adults was greater when adults were caged in plastic cups with honey. Neverthe less, longevity of adults was drastically reduced to 3.7 d in the plastic cups, compared to 16.5 d for ovipositing females in the laboratory at 27 + 1C (Chapter 3). The difference on survivorship may be due to heat st ress that might have occurred in the cups or the field cages. It is known that floral and extraflora l nectars are indispensable sources of carbohydrates for most adults of Hymenoptera (House 1977, Powell 1989, Baggen and Gurr 1998, Kogan et al. 1999). Availability of these foods will affect longevity, fecundity, and parasitism (Powell 1989, Id ris and Grafius 1995, Baggen and Gurr 1998). It is expected that efficienc y, biological potential and surv ival of parasitoids that are dependent upon these sources of energy to surv ive could be limited in monocultures that provide those resources only for a limited time (Altieri 1992, Kogan et al. 1999). In the field cages, T. eugenii in the plastic cups died in less than 48 h without a carbohydrate source. Offering honey in the wasp-refuges di d not appear to incr ease foraging time. However, it is possible that food was not a lim iting factor in the field cages. Parasitoids might have used honey or left the patc h before any carbohydrate was required.

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87 Effect of Host-Damaged Plants More T. eugenii adults and higher pa rasitism were observed on infested fruits on damaged plants compared to fruits on undamaged plants. Thus, the difference in parasitism could be explained by the greater portion of parasitoids that found hosts in damaged plants. T. eugenii found infested fruits in less th an 10 min in both treatments; however, less than 50% of the released parasitoids were observed on fruits hung in damaged plants, and less than 10% were obser ved on fruits in undamaged plants (Table 22). On the other hand, the majority of paras itoids left the foragi ng patch within 120 min or less, regardless of treatment. No parasiti sm was observed 24 h after the first exposure, but T. eugenii adults were observed on the cage walls where they apparently spent the rest of their lives. Parasitoids use chemical, visual, and/or electromagnetic cues from the host habitat to locate their hosts (Vinson 1976, 1998, Alphen and Vet 1989, Godfray 1994, Quicke 1997). The oviposition plug is important for T. eugenii to identify and to parasitize hosts (Chapter 3), but might also serve as a cu e for short distance host location. In this experiment both treatments offered similar c onditions and plants damaged by the pepper weevil attracted more parasitoids. T. eugenii then was able to dete ct and to recognize a host patch (infested fruits in damaged plants). It is possible that the more likely cues used by T. eugenii to find their hosts from a distance are volatiles produced by damaged plants or left by the pest. Volatiles produced by pest -damaged plants have been suggested as important cues for other parasitoids to find their hosts. In wind tunne ls plant volatiles, particularly those produced by aphid feeding, were important in long-range cues in the initial stage of host location by the braconid Aphidius ervi Haliday (Powell et al. 1998). Poplar tree leaves damaged by the gypsy moth ( Lymantria dispar L.) were three times

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88 more attractive to its braconid parasitoid, Glyptapanteles flavicoxis (Marsh), than undamaged leaves (Havill and Raffa 2000). The studies on pest-damaged emissions clearly suggest that these volatiles are importa nt for parasitoids to locate hosts. These volatiles, many times related to terpenoids, ma y be produced in defense against pests, but may also serve in attracting the natural en emies of these pests (Turlings et al. 1995, 1998). It is possible that T. eugenii was able to recognize pepper weevil damaged plants from a distance, and that volatile chemicals th en might play an important role in a host patch selection. Even with the presence of 25 host eggs and a pepper plant damaged by pepper weevils, T. eugenii did not remain more than 120 min on the host patch. The hypothesis of host marking by an ovipositing parasitoid can be proposed to explain the observed behavior. Some parasitoids mark thei r hosts once the hosts are parasitized, thus reducing the acceptability of the patch to themselves or to other females. After ovipositing and marking a host, parasitoids may disperse from the immediate vicinity of the host (Vinson 1984, Van Dijken et al. 1992, Potting et al. 1997). In support of this hypothesis, the rate of superparasitism observe d in the field cages (28%) was lower than that reported in the laboratory (55%) (Chapter 3). These obser vations are consistent with the presence of chemi cal marking of parasitized hosts, inducing females to reject and to leave a patch already explored. If that is true, it could explain why T. eugenii females left the patch in less than 120 min, and why supe rparasitism was less often detected in the field cages. On the other hand, chemical markings may be less effective in small cages in the laboratory because females cannot leave the patch, and because females may become habituated to the chemical in the confined space.

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89 Field Release and Establishment T. eugenii completes its life cy cle in 16.6 d at 27 + 1C, requiring about 265 heat units based on a development threshold of 11C (Chapter 3). The first successful recovery of parasitoids from the field occurred on Ap ril 14, 2005. Considering field temperatures and the time that fruits were held in the laboratory, these parasitoids could have originated from releases made on February 18 (267 heat units). Parasitoids emerging April 19 could have originated from the rel ease on March 25 (272 heat units). However, T. eugenii that emerged on April 25 do not correspond to a precise date of release, and parasitoids that emerged on April 30 and May 2 should have originated around the second week of April, possibly on days 13-14 (266 heat units) when there were no releases. It appears likely, then, that T. eugenii was able to complete at least one generation in the field. No parasitoids were r ecovered subsequently from the field, but the sampling was very limited because the lack of pepper crops during the summer of 2005, and because of the destruction of crops in the fall of 2005 by hurricane Wilma. Pepper plants do not set fruit at temper atures above 32C (Bosland and Votava 1999). Therefore, the high temperatures dur ing late spring and summer in southwest Florida preclude commercial production of pe ppers. Nevertheless, ad ults of the pepper weevil survive the temperatures of summers in Florida (Toapanta 2001) or the winters of California (Elmore et al. 1934), apparently f eeding and ovipositing on alternative hosts such as nightshade (Elmore et al. 1934, Wilson 1986, Genung and Ozaki 1972, Patrock and Schuster 1992). Pepper weevil adults f eed on buds and immature fruits but, when these are not available, adults can feed on foliage (Elmore et al. 1934, Goff and Wilson 1937). As result, adults can live at least two or three months during the hot season of the year (Elmore et al. 1934, Toapanta 2001), or maybe longer in less extreme conditions.

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90 Elmore et al. (1934) reported that overwintering weevils may live as long as 10 months. The amount of time between peaks of repr oductive structures, which offer the best quality of fruits for ovipositing, does not seem to represent a problem for the survivorship of this species. However, the lack of eggs of the pepper weevil in some seasons in the year, or even between peaks of commerci al production within the same crop, may represent one of the most importa nt barriers for establishment of T. eugenii in Florida. Considering these facts, at le ast two factors may explain th e unsuccessful parasitism by T. eugenii during the first release in 2004, and th e inconsistent parasitism in some releases in 2005. First, there may have been few or no peppe r weevil eggs present at the time of the releases. Only pepper weevil larvae were observed in fruits in the field during the first release in 2004. The presence of pepper weevil e ggs was confirmed by sampling on only two of five release da tes in the spring of 2005. Second, T. eugenii may be inefficient in locating host eggs. Females were able to find hosts in the field cages at release rates equivalent to 3,753 and 7,507 fe males per hectare. In the open field parasitoid females were rel eased at rates equivalent to 3,500 (Feb 18) and 6,000 (March 25) per hectare. In two other releases, the equivalent release rates of females per hectare was 10,000 (January 10) and 8,000 (February 8, April 8) respectiv ely. Thus, it would seem that the lack of host e ggs during wasp releases was more important in explaining no or inconsistent parasitism in the field releases. Considering (1) that pepper weevil adu lts can survive without laying eggs during unfavorable seasons or between peaks of production in the pepper cycle, (2) that T. eugenii leaves an explored patch in a short time and survives less than two weeks as an adult, and (3) that pepper crops do not consis tently provide hosts a nd nectars, different

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91 methods of ensuring the presence of pepper weevil eggs at the mo ment of parasitoid releases must be developed in order to estab lish this parasitoid in the field in Florida.

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92 CHAPTER 5 CONCLUSIONS Rearing Methodology and Influence of Food Quality on Biology of Pepper Weevil The pepper weevil preferred f eeding on the anthers of flor al buds rather than on fruits. This behavior may be related to the hi gher nitrogen content of anthers compared to fruits. The pepper weevil is able to obtain nutrients for egg production by feeding only on peppers. However, because of the nitrogen content and, maybe, the balance of amino acids, weevils fed on floral buds and immature ‘Jalapeo’ peppers had higher fecundity than weevils fed only on immature ‘Jalapeo’ fruits. Pepper weevil females preferred to oviposit in immature pepper fruits compared to floral buds and mature fruits. Females may pr efer immature fruits over floral buds for oviposition because fruits provide a greater resource to support larval development. Actually, weevils developing in floral buds ar e usually smaller than weevils developing in immature fruits (Patrock and Schuster 1992 ). On the other hand, immature fruits could be preferred to mature fruits not only becau se the nitrogen content of the pericarp is higher, but also because the thinner pericarp offers a shorter distance for the offspring to reach the immature placenta and seeds where nitrogen content is higher yet. Overcrowding of pepper weevil females by males or other females resulted in reduced fecundity of females and increased incidence of eggs lacking the normal oviposition plug. A single mated female per im mature fruit appeared to be the most efficient combination to maximize fecundity, and to maximize the number of eggs with oviposition plugs.

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93 Life History of Triaspis eugenii T. eugenii is a solitary egg-pre pupal parasitoid of the pepper weevil. Eggs are deposited in young or mature eggs of th e pepper weevil. Hatc hing larvae feed as endoparasitoids, but emerge from the prepupa of the host to complete development as ectoparasitoids. At 27 + 1C, females developed in 16.6 + 0.9 d, just a few hours less than males. Adults of T. eugenii were not observed to feed on the host and needed a carbohydrate source to survive. Mated female s provided with honey and water lived 24.7 + 10.6 d and females without honey lived 2.3 + 0.7 d. Females ovipositing constantly lived 16.5 + 3 d and were able to produce 402.8 + 199.5 eggs. About 90% of the eggs were laid during the first 12 d of life at the rate of 31.4 + 22.7 eggs per female per day. The oviposition plug of the pepper weevil played an important role in the ability of T. eugenii to find and to parasitize pepper weevil eggs. Further research is necessary to determine if the cues utilized by the wasp to recognize and/or locate the plugs are chemical, physical or both. When pepper fruits with weevil eggs were exposed for 2 h or more to T. eugenii in the laboratory, less than 50% of the parasitoid eggs reached the adul t stage because of a high level of superparasitism. Exposing fru its for 1.5 h to single ovipositing females slightly reduced superparasitism. Demographic parameters, such as the intrinsic rate of increase (rm), estimated without considering superparasitism, indica ted the greater reproductive potential of T. eugenii (rm = 0.26) compared to the pepper weevil (rm = 0.14). So T. eugenii could build upon pepper weevil populations, and superpar asitism must be reduced to improve efficiency in the rearing system.

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94 Field Cage and Field Release T. eugenii was able to find pepper weevil e ggs in fruits hung on weevil-damaged and undamaged plants in the field cages; how ever, more parasitoids were observed in weevil-damaged plants. Thus, volat ile chemicals (plant or weevil plus plant) might play a role during the location of hos t patches by the parasitoid. Wasps did not remain more than 120 min in the host patch and did not co me back to a new gr oup of hosts provided 24 h later. Superparasitism was observed at a lower frequency in the field cages (28%), compared to the laboratory (55 to 64%).This may indicate that T. eugenii marks parasitized hosts, inducing the parasitoid to l eave the host patch in 2 h or less. If that is true, it is possible that in confined conditions (laborat ory cages 3.8 L) host marking would have less of an effect for at least two reasons. First, fe males cannot leave the patch, thus increasing the number of times th at they locate the same host. Second, the reduced space and abundant stimuli might cause habituation to the substance that helps to prevent superparasitism by T. eugenii Parasitoids died in less than 48 h wit hout honey as a carbohydrate source, although a device offering honey in the field cages wa s not efficient in attracting them. This observation strongly suggests the importance of further research to develop a better method of providing an attractiv e source of car bohydrates to T. eugenii in the field. T. eugenii was recovered in low numbers from th e field after releases in the spring of 2005 at the SWFREC in Immokalee, FL. Although at least one generation was completed in the field, no parasitoids were recovered subsequently. However, subsequent sampling also was limited.

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95 The pepper weevil has the ability to survive as an adult for more than two or three months by feeding on foliage or flowers of di fferent hosts. Thus, the pepper weevil can survive even without the presence of pepper fruit or floral buds for oviposition. Conversely, the longevity of T. eugenii adults is short. Thus, in order for the parasitoid population to survive longer periods, weevil eggs are needed for reproduction. Therefore, it might be difficult for this parasitoid to survive during summers or winters when pepper is not present, or to surviv e during the pepper season when plants may not have fruits acceptable for pepper weevil oviposition. Even if T. eugenii fails to become established in the pepper agroecosystem as it currently exis ts in Florida, this parasitoid is still an excellent prospect for biological control of the pepper weevil by augmentation. It is highly host specific, it parasitizes weevil eggs, and it has an intrinsic rate of increase that is almost twice that of the pepper weevil. Further Research T. eugenii may be the parasitoid wi th more potential for bi ological control of the pepper weevil, but there is no information to support that it ma y colonize and self perpetuate under annual pepper crop producti on systems or environmental conditions different from Nayarit, Mexico. The possibility of establishment of T. eugenii in different locales will be greater if th e ecology of the parasitoid is studied in the natural pepper weevil-host plant-parasitoid system in indigenous sites in Mexico. How does T. eugenii survive in Nayarit when there are no peppers in the field? Are there any alternative host plants for the pepper weevil that can offer flowers or fruits for oviposition? How does T. eugenii survive when there is a l acking of pepper weevil eggs? Are there any other insect hosts for T. eugenii ?

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96 Mexico is the center of or igin and domestication of Capsicum annuum L., the most important cultivated species of chile-peppers (Vavilov 1951, MacMeish 1964, Pickersgill 1969, Eshbaugh 1976, Loaiza-Figue roa 1989). Wild and semi-domesticated peppers grow everywhere in the country. With this base we may propose at least four different ways for T. eugenii to survival when no comme rcial peppers are grown in Nayarit. First, the alternative plant hosts that offer floral buds and/or acceptable fruits for constant pepper weevil oviposition are w ild or semi-domesticat ed peppers. Second, the alternative host plant that plays the same role is a weed ( Solanum spp.?). Third, wild or semi-domesticated peppers and weeds may serve as alternative host pl ants. Four, there is an alternative insect host of T. eugenii that develops in the same or different plants. All of these options need to be studi ed before the survival of T. eugenii can be understood. Even if T. eugenii does not become established in Florida, its host specificity and reproductive potential make it an excellent pr ospect for augmentati ve biological control of the pepper weevil. However, as with any exotic or indigenous natural enemy with potential to combat a pest, a large number of parasitoids would be needed to determine the parasitoid/host ratio that would provide e ffective control in the open field. Therefore, improved and economical rearing systems need to be developed for both the pepper weevil and T. eugenii. Research is needed to develop a nd artificial diet for pepper weevil larvae in order to eliminate the need of immature peppers. An artificial means of collecting and then offeri ng pepper weevil eggs to T. eugenii and different ways of diminishing superparasitism are also needed. The system developed for mass rearing Catolaccus grandis (Hymenoptera: Pteromalidae) with cotton boll weevil larvae reared in an artificial diet (Cate 1987, Cate

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97 et al. 1990) might aid in the development of a rearing system for T. eugenii In the former system third instars of the boll weevil are encapsulated in Parafilm bubbles for presentation to C. grandis. The Parafilm bubble method wa s satisfactory for rearing Bracon mellitor Say, which also attacks boll weevil larvae developing concealed inside fruits and floral buds (Cate 1987). Hopefully, the rearing system for the pepper weevil and T. eugenii could be enhanced using inform ation gained in the present study, including the use of nitrogen to increase the fecundity of the pepper weevil, and the use of signals (chemical or physical) from the ovi position plug to increase parasitization of pepper weevil eggs by T. eugenii. On the other hand, if an alternative host for T. eugenii could be identified, that host might be reared more easily in the labor atory thus leading to an improved rearing system for the parasitoid.

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98 LIST OF REFERENCES Abe, Y. 2001. Egg-pupal and egg-larval parasitism in the parasitoid Gronotoma micromorpha (Hymenoptera: Eucoilidae). A ppl. Entomol. Zool. 36: 479-482. Abreu, E., and C. Cruz. 1985. The occurrence of th e pepper weevil Anthonomus eugenii Cano (Coleoptera: Curculionid ae) in Puerto Rico. J. Ag ri. Univ. Puerto Rico. 59: 223-224. Agee, H. R. 1964. Characters for determination of sex of the boll weevil. J. Econ. Entomol. 57: 500-501. Aguilar, R., and R. Servn. 2000. Altern ative wild host of the pepper weevil Anthonomus eugenii Cano in Baja California Sur, M xico. Southwest. Entomol. 25: 153-154. Alphen, J. J. M., and L. E. M. Vet. 1989. An evolutionary approach to host finding and selection. pp. 23-61. In: J. Waage, and D. Greathead, (eds.), Insect parasitoids. Academic Press, London. Altieri, M. A. 1992. Biodiversidad, agroecologa y manejo de plagas. Consorcio latinoamericano sobre agroecologa y desa rrollo (CLADES). CETAL, Valparaso, Chile. Anderson, R. S. 1992. Origin and biogeogra phy of the weevils of southern Florida (Coleoptera: Curculionidae). Can. Entomol. 126: 819-839. Andrews, K. L., A. Rueda, G. Gandini, S. Evans, A. Arango, and M. Avedillo. 1986. A supervised control program for the pepper weevil, Anthonomus eugenii Cano, in Honduras, Central America. Trop. Pest. Manag. 32: 1-4. Anonymous. 1997. Elemental analysis by Micr o-Dumas combustion. Stable Isotope/Soil Biology Laboratory, Institute of Ecology, Un iversity of Georgia, Athens GA, USA http://www.uga.edu/~sisbl/udumas.html last accessed November 23, 2005. Arcos, G., J. Hernndez, D. E. Uriza, O. Pozo, y A. Olivera. 1998. Tecnologa para producir chile jalapeo en la planicie costera del Golfo de Mxico. INIFAPPRODUCE. Secretara de Agricultura, Mxico. Folleto Tcnico No. 24. 206 p. Askew, L. L. 1971. Parasitic insects. Elsevier, New York. Awmack, C. S., and S. L. Leather. 2000. Host plant quality and fecundity in herbivorous insects. Annu. Rev. Entomol. 47: 817-844.

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99 Baggen, L. R., and G. M. Gurr. 1998. The influence of food on Copidosoma koehleri (Hymenoptera: Encyrtidae), and the use of floweri ng plants as a habitat management tool to enhance biol ogical control of potato moth, Phthorimaea operculella (Lepidoptera: Gelechiidae). Biological Control 11: 9-18. Beirne, B. P. 1946. Notes on the biology of so me hymenopterous parasites of the beech weevil ( Rhynchaenus fagi L.) (Col.) Proc. Royal Ento mol. Soc. London 21: 7-11. Bell, N. L., T. A. Jackson, and T. L. Nelson. 2000. The potential of entomopathogenic nematodes as biocontrol agen ts for Clover Root Weevil ( Sitona lepidus ). New Zealand Plant Protection 53: 48-53. Berdegue, M., M. K. Harris, D. Riley, and B. Villalon. 1994. Host plant resistance of pepper to the pepper weevil, Anthonomus eugenii Cano. Southwest. Entomol. 19: 265-271. Berry, P. 1947. Anthonomus vestitus and its natural enemies in Peru, and their importation into the United St ates. J. Econ. Entomol. 40:801-804. Birch, L. C. 1948. The intrinsic rate of natura l increase of an insect population. J. Anim. Ecol. 17:15-26. Bosland, P. W., and E. J. Votava. 1999. Peppe rs: vegetable and spice capsicums. CABI Publishing, New York. Bruton, B. D., L. D. Cha ndler, and M. E. Miller. 1989. Plant Disease 73: 170-173. Burke, H. R. 1968. Pupae of the weevil tribe Anthonominae (Coleoptera: Curculionidae). Tech. Monog. 5. Texas Agri. Exp. St a. College Station, Tex. 92 p. Burke, H. R. 1976. Bionomics of the Ant honomine weevil. Annu. Rev. Entomol. 21: 283-303. Burke, H. R., and R. E. Woodr uff. 1980. The pepper weevil ( Anthonomus eugenii Cano) in Florida (Coleoptera: Curc ulionidae). Fla. Dept. Agr. Consumer Serv. Entomol. Cir. 219. 4p. Burke, R. H., W. E. Clark, J. R. Cate, and P. A. Fryxell. 1986. Origin and dispersal of the boll weevil. Bull. of the ESA. 228-238. Cabanillas, H. E. 2003. Susceptib ility of the boll weevil to Steinernema riobravis and other entomopathogenic nematodes. J. Invert. Pathol. 82: 188-197. Campbell, R. E. 1924. Injuries to pepper in California by Anthonomus eugenii Cano. J. Econ. Entomol. 17: 645-647. Cano y Alcacio, D. 1894. El barrenillo. La Naturaleza. 2: 377-379.

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110 BIOGRAPHICAL SKETCH Esteban Rodrguez-Leyva was born on November 28, 1966, in Mexico City, to Benito Rodrquez and Pilar Leyva. He earned hi s Bachelor of Science in plant science in 1992, from Universidad Autnoma Chapi ngo, Chapingo, Mxico. Upon graduation he worked as a research assistant in the Fruit Crops Center at Colegi o de Postgraduados en Ciencias Agrcolas (Graduate College of Agriculture Sciences) at Chapingo, Mxico. In the fall of 1993 he started his Master of Science in entomology. Before completion of his M.S. degree, in spring of 1996, he was offe red an opportunity to work as research assistant at the Entomology and Acarology De partment, Colegio de Postgraduados at Montecillo, Mxico. Under the supervision of Drs. Jorge L. Leyva and Nina M. Brcenas, he began to work with parasito ids of the pepper weevil and boll weevil in 1997. He started his Ph.D. research under the supe rvision of Drs. P. A. Stansly and D. J Schuster in August 2001. His research focused on Triaspis eugenii, a parasitoid described from Mexico, and its potential for biological control of the pepper weevil. He will receive his degree in spring 2006, and he will be jo ining the Entomology program in the Colegio de Postgraduados at Montecillo -Texcoco, Mxico, as a research associate. E-mail address is esteban669@hotmail.com.


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LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
BRACONIDAE) AND EVALUATION OF ITS POTENTIAL FOR BIOLOGICAL
CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA:
CURCULIONIDAE)















By

ESTEBAN RODRIGUEZ-LEYVA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Esteban Rodriguez-Leyva
























This work is dedicated to my parents, Benito Rodriguez and Pilar Leyva, and brothers,
Martin, Ver6nica, and Victor, who have been teaching me the importance of having
resolutions in life, social gathering and education included, within an
admirable familial strength. Without their support it would be
harder to complete this study, to my nieces and nephew.

Este trabajo lo dedico a mis padres, Benito Rodriguez y Pilar Leyva, y a mis hermanos,
Martin, Ver6nica, y Victor, quienes me han mostrado la importancia de tener
metas en la vida, incluido el convivir y la educaci6n, en el seno de una
extraordinaria fuerza familiar. Sin su apoyo completar este trabajo
hubiese sido mas dificil. A mis sobrinas y sobrino.















ACKNOWLEDGMENTS

Many people and institutions contributed to the success of this dissertation. I am

extremely grateful to Consejo Nacional de Ciencia y Tecnologia (National Council of

Science and Technology) and Colegio de Postgraduados from Mexico which supported

tuitions, living expenses, and laborer situations through all the process for getting the

goals of this study. My gratitude and appreciation go to my advisor, Dr. Philip A. Stansly,

he found resources for conducting and assisting my dissertation and gave me

independence to express ideas and objectives. Additionally, for the many hours that he

spent reading, correcting, and re-reading drafts of this dissertation. I am grateful to Dr.

David J. Schuster who offered encouragement, support, and always friendship. I would

like to recognize the rest of my committee, Drs. Norman C. Leppla, Ru Nguyen, and

Steven A. Sargent, for being available and offering experiences to improve my academic

development. Special thanks go to Dr. Susan Webb for allowing me to use resources in

her laboratory in Gainesville, and Mike Miller for being such an amiable person.

Special recognition goes to Debbie Hall, Myrna Litchfield, and Nancy Sanders for

providing constant support, always with professionalism and amiability.

I want to thank also the personnel in the methods and quarantine facilities at the

Division of Plant Industry, Gainesville, FL, and many people of the South West Florida

Research and Education Center (SWFREC) at Immokalee, which supported me in the

implementation of a bunch of activities. I thank James M. Conner, Carmen G6mez, Jaime

Martinez, Alexandra Delgado, and Ronald D. French. At the same time, I thank those









who helped me have a nice time in the center, C. Vavrina, F. Jaber, R. Pluke, M. Ozores,

P. Roberts, R. Muchovej, S. Sukla, K. Cushman, J. Knowles, K. Jackson, C. Pandey, M.

Triana, J. Castillo, F. Roka, J. & K. Hill, K. & R. Systma, B. & B. Hyman, A. Garcia, D.

Spencer, and L. Rigby. I also thank Emily Vasquez and Steven Davis of the entomology

group at Gulf Coast Research and Education Center (GCREC) in Bradenton.

I thank Eugenio Mariscal for his help during the field trips to Nayarit, Mexico. I

thank my friends in Gainesville, I will always remember academic, cultural, and

gastronomical moments, excursions and of course "intoxicating moments of life" that I

spent with all of them.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ......................................... .................. ix

LIST OF FIGURES ......... ......................... ...... ........ ............ xi

ABSTRACT ........ .............. ............. .. ...... .......... .......... xii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

L literature R review ................................................................... ......... ..... ....... .. .4
Origin of Capsicum spp. and Distribution of Pepper Weevil.............................4
E conom ic Im portance................................... ......................... ............... 5
D e sc rip tio n ....................................................... ................ 6
B iology and L ife H history ............................................ .................. ...............
Host Plant Association ............................ ...... ..... .... ...............
Capsicum spp. ............................................................9
Solanum spp. .................................................. ................ 11
M anagem ent Strategies ............................................... ............................ 12
H ost plant resistance ................................. ...... ......... ......... ... ...... .....12
Cultural and chem ical control ............................. ..... ....... ............... 13
B biological control ...................................... .......... .... .............. ..14
O bjectiv es .................................................... ........... .. ..... ........ 19

2 REARING METHODOLOGY AND INFLUENCE OF FOOD QUALITY ON
BIOLOGY OF PEPPER W EEVIL............ ...................................... ........ ...... 20

Introduction............ .. ....... ................................ .......................... ..... 20
M materials an d M eth od s .................................................................... .....................22
Laboratory W eevil Colony .............. ...................................................... ... 22
Crowding Effects on Oviposition................................................24
Fruit Q quality ..............................................................25
A dult Feeding and Oviposition ........................................ ....................... 26
L ife T able ........................................27
Nitrogen Content of Fruits and Buds ................. ................ ............. 28
R results ............. ...... ..... ......... .............................................29









Crowding Effects and Oviposition ............................ ................................... 29
Fruit Q quality ...................................... .............. ..................30
Feeding and Oviposition in Peppers versus Floral Buds (7d) ...........................30
Oviposition with or without Floral Buds (7d)................... ................................31
L ife T ab le ................. ......................................................... 3 1
Nitrogen Content of Fruits and Buds.................................. .................. 32
D discussion ................ .. .... .... .............. ............... ............ 33
Crowding Effects and Oviposition ............................ ................................... 33
F ruit Q quality .......................................................................34
A dult Feeding and Oviposition ........................................ ....................... 35
L ife T ab le .........................................................................3 7

3 LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez
(HYMENOPTERA: BRACONIDAE) ................................ ......................... 40

In tro du ctio n ...................................... ................................................ 4 0
M materials and M methods ....................................................................... ..................43
S u rv e y s ................................................................4 3
P arasitoid R hearing .............................. .... ...................... .. ........ .... ............44
H ost-A ge Range ................ ... ......... .... ........ .. .. ...... ... 46
Role of the Oviposition Plug in Host Location ................................................47
Effect of Weevil: Fruit Ratio on Parasitoid Production .................................. 49
L ife C y cle .........................................................................4 9
F e cu n d ity .............................................. .. ................ ................ 5 1
A dult L longevity ............. ................. .......... ...... ......................... 52
R e su lts ................................................................................................................... 5 2
S u rv e y s ................................................................5 2
H ost-A ge R ange ................ ... ......... .... ........ .. .. ...... ... 54
Role of the Oviposition Plug in Host Location ..........................................54
Effect of Weevil: Fruit Ratio on Parasitoid Production .............................. 55
L ife C y cle .........................................................................56
F e cu n d ity ....................................................... ................ 6 1
A dult Longevity ....................................... ....... ........ .. ........ .... 62
D isc u ssio n ............................................................................................................. 6 3
S u rv e y s ................................................................6 3
H ost-A ge Range ................ ... ......... .... ........ .. .. ...... ... 64
Role of the Oviposition Plug in Host Location ................................................66
L ife C y cle .......................................... ........... ....... ...... ..... ............... 6 7
Potential of T eugenii as a Biological Agent of the Pepper Weevil .................69

4 RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez
(HYMENOPTERA: BRACONIDAE) IN FIELD CAGES AND IN THE FIELD ...72

Introdu action ...................................... ................................................. 72
M materials and M methods ....................................................................... ..................74
In se c ts ......................................................................................................7 4
F field C ag e s ................................................................7 4









Effect of Honey Supplement .................................. .......................... 76
Survival ............................................................................... 78
Effect of H ost-D am aged Plants.................................... .................................... 78
Field Release and Establishm ent ......................................................................80
R esu lts ............................................................................................... 8 1
Effect of Honey Supplement .................................. .......................... 81
Survival ............................................................................... 82
Effect of H ost-D am aged Plants.................................... .................................... 82
Field Release and Establishm ent ......................................................................84
D iscu ssio n ........................... ............ ....................................................... ............... 8 6
Effect of Honey Supplement .................................. .......................... 86
Effect of H ost-D am aged Plants.................................... .................................... 87
Field Release and Establishm ent ......................................................................89

5 CON CLU SION S .................................. .. .......... .. .............92

Rearing Methodology and Influence of Food Quality on Biology of Pepper
W e e v il ....................................................................................................9 2
Life H history of Triaspis eugenii ............................... ................................. 93
Field C age and Field R release ........................................ ......................... 94

LIST OF REFERENCES ..... ........... ......................................... ................ 98

BIOGRAPHICAL SKETCH ............. .............................................................110





























viii
















LIST OF TABLES


Table page

1 Biological data of pepper weevil, selected references until 2005 ............................9

2 Non Capsicum Solanaceae used by pepper weevil* ..........................................12

3 Natural enemies of the pepper weevil, references until 2005 .............................17

4 Characteristics of 'Jalapefio Mitla' used for nitrogen content determinations.........28

5 Numbers of eggs per female per day for different numbers of females and males
per single 'Jalapefio' pepper fruit....................................... .......................... 30

6 Feeding and oviposition of pepper weevil adults when given a choice of
'Jalapefio' pepper fruit or floral buds (7d) .................................... ............... 30

7 Oviposition of pepper weevil females fed with 'Jalapefio' pepper fruits only or
w ith pepper fruits plus floral buds....................................... ......................... 31

8 Life table parameters for A. eugenii females (n =14) reared on 'Jalapefio'
peppers at 27 + 20C, and 14:10 (L:D) photoperiod.................... ...............32

9 Nitrogen content (Mean + SD) of anthers in floral buds and 'Jalapeio' fruits at
three m aturation stages ............... ............................................ .. .. ........ ...... 33

10 Life table parameters for the pepper weevil............. .. ........... .............. 38

11 A. eugenii and parasitoids emerged from approximately 50 kg of infested hot
peppers collected in the state of Nayarit, Mexico, April and May, 2003 ..............53

12 Parasitism of pepper weevil eggs by T. eugenii when given the choice of two
ages of eggs ........................................... ............................ 54

13 Parasitism of pepper weevil eggs by T. eugenii when given no choice of egg age .54

14 Parasitism of pepper weevil eggs by T. eugenii when given the choice of eggs
covered with an oviposition plug and eggs with no plug...................................55

15 Parasitism of pepper weevil eggs by T. eugenii when given no choice of eggs
covered with an oviposition plug and eggs with no plug...................................55









16 T eugenii produced with two pepper weevil densities ........................................ 56

17 Developmental times of T eugenii in days (Mean SD) at 27 1C .................57

18 Demographic parameters (+ 95% CL) for T eugenii reared on pepper weevil
eggs in 'Jalapefio' fruits for different exposure intervals at 27 + 2C ...................63

19 Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and
14 pepper w eevil adults at 27 + 2 C ............................. ... ......................... 71

20 Releases of T eugenii during 2005 at the SWFREC in Immokalee, FL.................80

21 Number of T eugenii adults released in field cages and subsequently observed
on pepper fruits (n=5) hung on pepper plants at 28.2 + 4.7C, 55 + 9% RH...........83

22 Adults per 50 terminals, and the number of weevil lifestages per 10 fruits, 1 or 2
d before field releases of T eugenii at SWFREC, Immokalee, FL, 2005 ...............85

23 Parasitoid and pepper weevil adults recovered from 'Jalapefio' pepper following
field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005 ......................85















LIST OF FIGURES


Figure pge

1 Device for exposing different densities of weevil adults to 'Jalapefio' peppers.....24

2 Mean fecundity of adult pepper weevil females (n = 14) reared on 'Jalapefio'
peppers at 27 + 2 C and 14:10 (L:D) photoperiod. ............................................32

3 Longitudinal section of an infested 'Jalapefio' fruit showing a pepper weevil egg
and oviposition plug (ov-p). ..... ........................... ......................................48

4 Immature stages of T. eugenii: (A) Egg 22 h after oviposition, (B) First instar,
30-33 h after oviposition, with well developed mandibles. ....................................58

5 Larva of T. eugenii. (A) Emerging from the posterior end of the host, (B)
Beginning to feed on the host even before emerging completely..........................59

6 T. eugenii adults. (A) Female, (B) M ale. ...................................... ............... 60

7 Fecundity of T eugenii females at 27 + 2C either exposed to hosts changed
every 1.5 h (n=5) or exposed to hosts per 9 h (n=5). ...........................................62

8 Field cages used at the SWFREC. (A) General view of a unit containing two
cages. (B) Organdy partition and zipper between two experimental units .............75

9 Honey-wasp-refuge. (A) General view of the set up. (B) Using a unit as T
eug enii release point...................... ..................................... .. ........ ......77

10 Survival of T eugenii adults in plastic cups with and without honey, when the
cups were placed inside the canopy of plants, 25.4 + 4.90C, 67 + 19% RH ...........82

11 Daily temperatures and humidity during T. eugenii field releases on 'Jalapeio'
pepper at the SWFREC in Immokalee, FL, 2005. ...............................................84















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
BRACONIDAE) AND EVALUATION OF ITS POTENTIAL FOR BIOLOGICAL
CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA:
CURCULIONIDAE)

By

Esteban Rodriguez-Leyva

May 2006

Chair: Philip A. Stansly
Cochair: David J. Schuster
Major Department: Entomology and Nematology

The pepper weevil, Anthonomus eugenii Cano, is considered the most important

pest of peppers (Capsicum spp.) in Tropical and Subtropical America. Juvenile stages

develop inside buds and immature fruits, so that only adults are susceptible to insecticidal

control. No viable biological tactic has been developed to combat this pest but Triaspis

eugenii, a braconid recently collected in Mexico that attacks pepper weevil eggs, could

offer an important addition to its management. The parasitoid was collected from Nayarit,

Mexico, during 2003, and a rearing methodology was developed using pepper weevil

reared on immature 'Jalapefio' peppers. Low levels of parasitism and high levels of

superparasitism hampered the rearing and reliable estimation of parasitoid demography

until the weevil rearing system was improved. Confining individual mated females to a

single immature 'Jalapefio' fruit maximized weevil fecundity, and also the number of

oviposition punctures that were plugged by female. Weevils fed with pepper floral buds,









a high nitrogen source, and immature 'Jalapefios' laid 5.5 eggs per female per day over a

life span of 64.5 d at 27 2C. The net reproductive rate (Ro=158.1) and intrinsic rate of

increase (rm = 0.14) obtained in a life table study were higher than any previous report.

T. eugenii is a solitary egg-prepupal parasitoid of the pepper weevil. At 27 1C,

T eugenii eggs hatched at 23 1 h. Females developed in 16.6 0.9 d and males in 16.4

0.9 d. Females (n=10) laid 402 199 eggs of which less than 50% reached the adult

stage because of high levels of superparasitism in the laboratory. Ovipositing females

lived 16.5 3.02 d, and parasitoids died in fewer than 48 h without carbohydrates

(honey). Net reproductive rate (Ro) was estimated at 106 and 167, and intrinsic rate of

increase (rm) at 0.24 and 0.26 with and without superparasitism respectively.

The oviposition plug deposited by the pepper weevil played a decisive role in the

ability of T eugenii to find and to parasitize weevil eggs. Crowded rearing conditions

inhibited deposition of oviposition plugs and consequently efficiency of parasitism.

Wasps were able to find hosts in field cages, and semiochemicals from weevil-damaged

plants appeared to play a role in host patch location. However, T eugenii did not stay

more than 120 min in the patch regardless of the presence of weevil damaged plants.

Superparasitism was less frequent in the field cages (28%) compared to the laboratory

(55-64%). The relatively short duration of foraging on large patch size compared to the

rearing environment might explain why.

T. eugenii was recovered in low numbers from field releases in spring 2005 at

Immokalee, FL, and at least one generation was completed in the field. No wasps have

been recovered after that although sampling has been limited. Even if T. eugenii does not

establish in Florida, its host specificity, its ability to parasitize weevil eggs, and its almost









double intrinsic rate of increase, compared to the pest, make this parasitoid an excellent

prospect for biological control by augmentation against the pepper weevil.














CHAPTER 1
INTRODUCTION

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is

considered one of the most important insect pests of all cultivated varieties of chile =

pepper (Capsicum spp.) in the New World. Pepper weevil is considered a key pest of this

crop in the southern United States, Hawaii, Mexico, Central America, and Puerto Rico

(Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz 1985, Riley and King

1994, Coto 1996, Arcos et al. 1998).

Pepper weevil biology, control tactics, distribution, and sampling methods have

been studied (Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992, Riley et al.

1992a,b, and Toapanta 2001). Nevertheless, insecticidal control is necessary once the

pest is present in pepper crops (Riley and King 1994, Riley and Sparks 1995, Arcos et al.

1998, Mariscal et al. 1998). Unfortunately, the use of insecticides as the principal method

of control can provoke many adverse effects such as marketing restrictions, exposure of

non-target organisms, environmental contamination, pesticide resistance, and secondary

pest outbreaks within the same crop (Doutt and Smith 1971, Van Driesche and Bellows

1996).

Biological control could be an alternative strategy to incorporate into the integrated

pest management (IPM) of pepper weevil (Riley and King 1994, Mariscal et al. 1998,

Schuster et al.1999). However, little is known of the natural enemies of this pest. Pepper

weevil arrived or was incidentally introduced from Mexico to the United States at the

beginning of the last century (Walker 1905, Pratt 1907). Early reports indicated two









species of ants, three pteromalids, one braconid, and one mite that attack this pest in the

United States (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut

1971). However, the effect of those enemies on pepper weevil populations was not

considered important (Pratt 1907, Elmore et al. 1934, Elmore and Campbell 1954).

A few decades ago, Wilson (1986) reported on the ectoparasitoid Catolaccus

hunter Crawford (Hymenoptera: Pteromalidae) attacking pepper weevil in Florida. C.

hunter is the dominant parasitoid attacking this pest in Florida and Puerto Rico (Wilson

1986, Schuster et al. 1999), and one of the most abundant in some states of Mexico

(Mariscal et al. 1998, Schuster et al. 1999, Rodriguez et al. 2000).

C. hunter has been collected from a closely related pest of cotton, the cotton boll

weevil Anthonomus grandis Boheman (Coleoptera: Curculionidae), in several countries

in Central and South America, Mexico, and the United States (Pierce et al. 1912,

Towsend 1912, Cross and Mitchell 1969). Biological studies showed that C. hunter has a

higher fecundity than that of the pepper weevil (Rodriguez et al. 2000, Seal et al. 2002).

Those findings stimulated and supported the evaluation of C. hunter for controlling this

pest (Schuster et al. 1999, Rodriguez et al. 2000).

Releasing C. hunter in field plots in Mexico and Florida produced equivocal

results; in Mexico this parasitoid was not effective for combating pepper weevil on bell

pepper (Corrales 2002). However, some augmentative releases of this parasitoid prior to

and during the pepper season suggested that this parasitoid might have potential to reduce

weevil damage (D. Schuster, UFL, personal communication). These contradictory results

may be due, in part, to the fact that C. hunter attacks the 3rd instar host that is usually

inaccessible deep within the pepper fruit. Therefore, augmentative releases of C. hunter









prior to the crop cycle could reduce pepper weevil populations in alternative host plants

like nightshade, while releases in early infestations of pepper weevil, or in small fruited

varieties, might be effective because floral buds and small fruits allow C. hunter to reach

its host. Once the pepper fruits increase in size, there is no way to expect parasitism. In

fact, Riley and Schuster (1992) indicated that C. hunter was not detected in fallen fruits

larger than 2.5 cm in diameter. According to this information, an effective biological

control agent for pepper weevil might be one that attacks earlier and more accessible life

stages.

The search for biological control agents in the native region of the target pest is one

of the first steps in a successful biological control program (DeBach 1964, Hagen and

Franz 1973, Huffaker and Messenger 1976). Mexico and Central America have been

indicated as a center of origin and domestication of Capsicum annuum L., the most

important cultivated species of chile = pepper (Vavilov 1951, MacNeish 1964, Pickersgill

1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, 1980, Loaiza-Figueroa et al.

1989). Furthermore, it was in Mexico that the pepper weevil was first reported as a

pepper pest (Cano and Alcaciol894).

Mariscal et al. (1998) collected nine different genera of hymenopteran parasitoids

from pepper weevil in Nayarit, Mexico, including a subsequently described species

Triaspis eugenii Wharton & Lopez-Martinez 2000 (Hymenoptera: Braconidae). Only

Mexico appears to have this diversity of parasitoids of pepper weevil, and additional

search would likely yield more potential species.

T. eugenii was the most abundant parasitoid emerging on pepper weevil in Nayarit.

Incidence of parasitism ranged from 18 to 40%, making it the most important parasitoid









of this pest in the field (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This

level of parasitism in unsprayed fields suggests that T. eugenii could be a good candidate

for inclusion in an IPM program against pepper weevil in the United States, Mexico, and

Central America. Nevertheless, information on the biology and ecology of this braconid

is needed to evaluate its potential as a biological control agent.

Literature Review

Origin of Capsicum spp. and Distribution of Pepper Weevil

Central and southern Mexico and part of Central America, a region called

Mesoamerica according to anthropologists, has been indicated as a center of origin and

domestication of the most important cultivated species of chile = pepper, Capsicum

annuum L. (Vavilov 1951, MacNeish 1964, Pickersgill 1969, 1971, Eshbaugh 1976,

Loaiza-Figueroa et al. 1989, Conciella et al. 1990). The other four important species of

cultivated chiles (or peppers) are Capsicum frutescens "Tabasco"; C. chinense Jacquin

"Habanero or Scotch Bonnet"; C. pubescens Ruiz and Pavon "Manzano or Pera"; and C.

baccatum L. All are endemic to South America (Heiser and Smith 1951, Smith and

Heiser 1957, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976,

McLeod et al. 1982).

A Mesoamerican origin of the pepper weevil, Anthonomus eugenii Cano

(Coleoptera: Curculionidae), is suggested by the following facts: a) the origin of the most

important species of cultivated pepper (C. annuum) is probably Mesoamerica; b) the

pepper weevil has not been reported from South America (Clark and Burke 1996); and c)

this insect was found for the first time damaging peppers in Central Mexico in

Guanajuato (Cano and Alcacio 1894).









A few years after the report from Guanajuato, Mexico, the pepper weevil was

reported from Texas (Walker 1905). According to Pratt (1907) this insect was introduced

from Mexico on infested fruit shipments. After affecting pepper crops in Texas, the insect

was reported from California in 1923, New Mexico and Arizona in 1927, and Hawaii in

1933 (Elmore et al. 1934). It was reported from the west coast of Florida in 1935 (Goff

and Wilson 1937), and the east coast in 1972 (Genung and Ozaki 1972). During 1935-

1972, when high levels of organochlorine insecticides were used on the east coast, there

were no records of pepper weevil in the zone, but in 1972 it was a widespread pest

(Burke and Woodruff 1980).

Presently, the pepper weevil is a pest in all pepper growing areas of the United

States including North Carolina and New Jersey (Burke and Woodruff 1980). It was once

reported in greenhouses in Canada, probably introduced on seedling plants from

California (Riley and King 1994). Mexico, Guatemala, El Salvador, Honduras,

Nicaragua, and more recently Puerto Rico, Costa Rica, Jamaica, and Panama have been

added to the list of countries where this insect is considered a serious problem on peppers

(Cano and Alcacio 1894, Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz

1985, Andrews et al. 1986, Riley and King 1994, Clark and Burke 1996, Coto 1996).

Economic Importance

The pepper weevil lays eggs, feeds, and develops completely inside the fruits,

which contribute to the difficulty of controlling the insect. The damage to flowers and/or

young pods causes abscission and diminishes yield up to 30 to 90% if treatment is not

implemented (Campbell 1924, Elmore et al. 1934, Goff and Wilson 1937, Velasco 1969,

Genung and Ozaki 1972, Riley and Sparks 1995). In 1990, losses in the United States due

to the pepper weevil were estimated at 23 million dollars on the 31,000 ha grown in









California, New Mexico, Florida and Texas (Riley and King 1994). By 2003, the growing

area of the crop in the United States had increased to close to 32,000 ha and the value of

production reached 550 million dollars (National Agriculture Statistics Service 2004).

Using the same proportion of damage (10%) estimated by Riley and King (1994), the

economic damage in 2003 could have reached more than 50 million dollars. In Mexico,

Central America, and the Caribbean islands, serious economic impact is indicated but not

quantified (Riley and King 1994). Additional costs include the many adverse affects of

pesticides such as exposure of non-target organisms, environmental contamination,

pesticide resistance, and secondary pest outbreaks within the same crop (Doutt and Smith

1971, Van Driesche and Bellows 1996).

The pepper weevil shares many similarities with its congener the cotton boll

weevil: Mesoamerica as the likely origin, the damage to their hosts by feeding and

developing inside the fruits, and the history of dispersal in the United States (Burke et al.

1986). However, the difference in acreage and, consequently, economic importance of

cotton and pepper in the United States, explains why pepper weevil has never received

the same attention as the boll weevil, and why even a hundred years after the original

detection, there is relatively little information about biogeography, evolution, and

biological control of the pepper weevil (Riley and King 1994).

Description

The pepper weevil was described from specimens collected from Guanajuato,

Central Mexico, by Cano and Alcacio (1894). The author mentioned as a motive the

importance of the insect as a pest of pepper crops in that region. This weevil belongs to

the subfamily Anthonominae and shares many characteristics with the other 330

described species of the genus Anthonomus (Burke 1976, Anderson 1992, Clark and









Burke 1996): basically robust and convex body, elongation of anterior part of the head to

form a rostrum longer than broad, reduced mandibles on the tip of this rostrum, and flat

scales like hair covering elytra, scutellum, and to some extent other parts of the body

(Burke 1976).

Detailed descriptions of immature lifestages and adults have been written by

Elmore et al. (1934), Burke (1968), and Clark and Burke (1996). Pepper weevil adults are

usually not longer that 3 mm, and males possess larger metatibial mucrones than females,

a characteristic useful to determine sex (Eller 1995). As with boll weevils, pepper weevil

males have a notch visible on the 8th tergum of the abdomen, which females do not have

(Agee 1964, Sappington and Spurgeon 2000). However, the small size of the pepper

weevil makes the last characteristic difficult to see (Eller 1995).

Biology and Life History

The biology of the pepper weevil was first described by Cano and Alcacio (1894),

Walker (1905), and Pratt (1907); however, Elmore et al. (1934) made the first complete

biological study. The pepper weevil feeds and develops on several species of Solanaceae,

but it is only a pest on peppers, Capsicum spp. (Elmore et al. 1934, Wilson 1986, Patrock

and Schuster 1992). Adults feed on buds, flowers, fruits, and even leaves. Larvae feed

and develop completely inside floral buds and immature fruits. Premature abscission is a

consequence of feeding and developing inside buds and fruits resulting in loss of

production (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al. 1934).

Even if there were no abscission of fruits, there could be loss, as the fruits have

larvae inside and are often spoiled. Actually, abscission of fruits could be considered an

advantage because fallen fruits can be gathered to avoid reinfestation of the crop

(Berdegue et al. 1994) and because fewer damaged fruits make it to the market.









There are three instars and the number of generations per year depends only of

temperature and availability of food resources (Elmore et al. 1934). Pepper weevil

develops within a wide range of temperature (10-30C), but the ideal range is from 27 to

30C (Toapanta et al. 2005).

Other reports that included information on generation time, oviposition, fecundity,

fertility, and host plant associations have advanced our understanding of the biology of

this species (Genung and Ozaki 1972, Burke and Woodruff 1980, Wilson 1986, Gordon

and Armstrong 1990, Patrock and Schuster 1992, Arcos et al. 1998). In addition, a life

table study by Toapanta et al. (2005) provided additional demographic parameters such

as net reproductive rate, intrinsic rate of increase, and finite rate of increase at different

temperatures. With this information the biology of the pepper weevil is reasonably well

known (Table 1), but is not complete. Various factors that were not included in fecundity

experiments could affect fitness of this species, including type and quality of food. It is

well known that the boll weevil needs to feed on floral buds, because of their high

nitrogen content, for ovarian development and oviposition (Hunter and Hinds 1905, Isley

1928). Other studies demonstrated that fecundity was optimal in boll weevil fed with a

diet containing high nitrogen content (10% cottonseed flour as the amino acid nitrogen

source) but drastic nitrogen reduction (2.5% cottonseed flour) reduced egg production

(Hilliard 1983). Oogenesis and oviposition in pepper weevil proceeds only on peppers

(almost any reference in Table 1). However, adults prefer to feed on floral buds instead of

fruits (Patrock and Schuster 1992), so the importance of this type of food and its effect on

fecundity should be considered for future studies.










Table 1. Biological data of pepper weevil, selected references until 2005
Parameter (average in days) Conditions Reference


Generation time
25
20.9
32.1
17-18

17.5
14.2
22.7, 13.9, 12.9

Longevity of adults
78.7
90
31.7
Oviposition period
72
30
76, 50, 52

Oviposition rate
(eggs/ female/ day)
4.7
6.6
6.0
7.1
8
1.9, 1.7, 3.1

Fecundity (eggs/female)
341
198
253
144, 85, 161


Summer
Fall
25.7-27.7C; 70% RH; on an
artificial diet
23-27C
25.7-27.70C; 40-100% RH
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively

Laboratory reared
Insectary
22-27C; 60-70% RH

Laboratory reared
Insectary
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively


Laboratory reared
Insectary
23-27 C
25.7-27.70C; 40-100% RH
22-27 oC; 60-70% RH
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively


Insectary
22-27oC; 60-70% RH
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively


Walker 1905
Elmore et al. 1934

Toba et al. 1969

Genung & Ozaki 1972
Wilson 1986
Toapanta et al. 2005


Elmore et al. 1934
Goff & Wilson 1937
Gordon & Armstrong 1990

Elmore et al. 1934
Goff & Wilson 1937
Toapanta et al. 2005



Elmore et al. 1934
Goff & Wilson 1937
Genung & Ozaki 1972
Wilson 1986
Gordon & Armstrong 1990
Toapanta et al. 2005


Elmore et al. 1934
Goff & Wilson 1937
Gordon & Armstrong 1990
Toapanta et al. 2005


Population values
a R =25.15, 11.76, 33.57 21.0, 27.0, and 300C, 60% RH; Toapanta et al. 2005
b rm= 0.06, 0.07, 0.11 14:10 (L:D), respectively
ST =52.51, 35.79, 32.39
a Net reproductive rate (Ro); b Intrinsic rate of increase (rm); c Generation time (T)

Host Plant Association

Capsicum spp.

Host plants utilized by pepper weevil for reproduction are confined to the genera

Capsicum and Solanum, both in the family Solanaceae. All of the five species of pepper









grown as crops (C. annuum, C. frutescens, C. chinense, C. pubescens or C. baccatum) are

suitable for oviposition and development of the pepper weevil (Elmore et al. 1934,

Wilson 1986, Patrock and Schuster 1992). The majority of studies describing feeding,

oviposition, survival and reproduction have been developed in these hosts.

Pepper weevil adults feed on floral buds, flowers, fruits, and sometimes leaves of

pepper plants; they lay eggs inside floral buds and young pepper pods. For feeding,

females and males bore a small hole with the mandibles, which are located at the end of

the rostrum, to reach internal tissue (Walker 1905, Elmore et al. 1934).

For oviposition, females spend a short time in selecting a site on the fruits, after

which they bore a small hole with the mandibles, turn completely around and deposit an

individual egg into the hole. Once the egg is in the cavity, the female deposits a yellowish

or brownish substance that turns black on drying and seals the hole (=oviposition plug)

(Elmore et al. 1934, Goff and Wilson 1937, Genung and Ozaki 1972). After hatching, the

larva bores into the interior of the fruit until reaching the placenta and seeds. Once

development is completed, the insect pupates inside the fruit (Pratt 1907, Elmore et al.

1934, Goff and Wilson 1937, Burke and Woodruff 1980, Riley and Sparks 1995). The

particular feeding and reproduction behavior of this insect make it inaccessible to many

natural enemies, and also to most insecticides (Pratt 1907, Elmore et al. 1934, Elmore and

Campbell 1954).

The size, quality, and availability of buds, flowers, and fruits could be factors that

affect the selection of oviposition sites for pepper weevil (Walker 1905, Elmore et al.

1934, Riley and King 1994). More eggs were laid in fruits than in flowers (Patrock and

Schuster 1992). For feeding and oviposition, adults prefer small fresh fruits rather than









mature fruits (Walker 1905, Wilson 1986). According to Bruton et al. (1989), the pepper

weevil preferred developing bell pepper fruits from 1.3 to 5.0 cm in diameter rather than

smaller (less than 1.3 cm in diameter) or larger mature fruits (bigger than 5 cm in

diameter) for oviposition.

Solanum spp.

The suitability for feeding, oviposition and development of the pepper weevil has

been confirmed for at least 10 species of the genus Solanum (Table 2). Six additional

species of the family Solanaceae served as food, but were not suitable for oviposition in

non-choice tests (Wilson 1986, Patrock and Schuster 1992). Nevertheless, laboratory

results do not necessary apply to the field. For example, the pepper weevil was able to

develop in eggplant (S. melongena) in the laboratory, but rarely is observed to attack this

crop in the field.

The developmental time of pepper weevil reared on bell peppers was not different

from those reared in American black nightshade (S. americanum) or eastern black

nightshade (S. ptycanthum), but the dry weight of adults from pepper was greater than

those from the other hosts (Patrock and Schuster 1992). A hypothesis of restriction of

resources, where bigger fruits could offer more food for the developing larva, could help

to explain why pepper weevil females prefer fruits instead of flowers for oviposition. The

same hypothesis could explain the greater dry weight of pepper weevil adults reared from

bell pepper fruit than those from nightshade fruit (Patrock and Schuster 1992). The

qualitative differences in suitability among hosts of pepper weevil have not been

evaluated.










Table 2. Non Capsicum Solanaceae used by pepper weevil*
Plant species Feeding Oviposition Development
Solanum americanum Mill. L, F, Fr1 Fr S4
S. carolinense L. L, F, Fr F, Fr S
S. dimidiatum Fav. L, F, Fr F, Fr S
S. eleganifolium Cav. L, F, Fr F, Fr S
S. melongena L. L, F, Fr F, Fr S
S. pseudocapsicum L. L, F, Fr Fr S
S. pseudogracile Heiser L, F, Fr Fr S
S. ptycanthum Dun L, F, Fr Fr S
S. rostratum Dunal L, F F S
S. triquetrum Cav. L, F2 F2 S
S. tuberosum L. L, F2 N3
Datura stramonium L. L, F N
Lycopersicon esculentum Mill. L, F, Fr N
Nicotiana alata Link and Otto L, F N
Petunia parviflora Vilm F N
Physalis pubescens L. L, F, Fr N
*Source: Wilson (1986), Patrock and Schuster (1992)
L= Leaves, F = Flowers, Fr = Fruits. 2Fruit unavailable. 3No oviposition observed on flowers or fruits.
4Successful development.

Management Strategies

Host plant resistance

Host plant resistance is an important component of IPM (Painter 1951, NAS 1969).

Berdegue et al. (1994) indicated that some types of pepper exhibited less damage from

pepper weevil because of an escape mechanism -concentrated production before the

presence of the pest instead of antibiosis. Quifiones and Lujan (2002) indicated that

some 'Jalapefio' lines might have some tolerance mechanism against damage from this

pest. Seal and Bondari (1999) indicated that only two commercial varieties of peppers,

one of them Habanero, had lower rates of infestation by pepper weevil than the remaining









nine varieties tested in field evaluations. Unfortunately, there is not a single cultivated

variety known to provide any important resistance characteristic against this pest.

Cultural and chemical control

Current management practices against pepper weevil consist of a combination of

cultural and chemical control. According to Riley and King (1994), control practices

against pepper weevil focused on cultural control during almost the first 40 years

following introduction into the United States. The lack of other tactics was explained by

the difficulty of reaching the insect inside the fruit (Walker 1905). The inorganic

insecticides, available during those years, especially calcium arsenate, could not be used

because of human toxicity (Elmore et al. 1934, Elmore and Campbell 1954) and were

observed to provoke secondary pest problems in the crop (Folsom 1927, Elmore et al.

1934). Insecticides have improved in selectivity for other pests, but they have not

improved in efficacy against pre-imaginal stages of the pepper weevil.

Recommendations for avoiding damage included establishing a pepper free period

-some months if possible- to reduce populations by food deprivation. Other

recommended practices included 1) using weevil-free seedlings to establish new crops, 2)

removing alternative hosts inside and around the fields, 3) collecting and destroying

fallen fruits to reduce reinfestation, and 4) destroying crop residues immediately after

harvest (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore at al. 1934, Goff and

Wilson 1937, Watson and Lobdell 1939).

From the mid 1940's to 1980's, chemical control gained favor due to the

availability of synthetic insecticides (Elmore and Campbell 1954, Riley and King 1994).

Increasing knowledge of dispersal and sampling techniques (Andrews et al. 1986, Riley

et al. 1992a b, Riley and Schuster 1994) aided in managing this pest without calendar









spraying of pesticides (Riley et al. 1992a b, Riley and King 1994). When 5% of the

terminals were damaged (Cartwright at al. 1990) or when one adult pepper weevil was

present per 400 terminal buds (Riley and Schuster 1992 b), significant economic loss in

highly productive crops resulted. These criteria could serve as action thresholds.

Azadirachtin, bifenthrin, cyfluthrin, cryolite, esfenvalerate, oxamyl, permethrin,

acetamiprid, and thiamethoxam are some of the insecticides labeled for combating the

pepper weevil in Florida (Olson et al. 2005, P. Stansly, personal communication).

Biological control

Known natural enemies of the pepper weevil are summarized in Table 3. Although

biological control is usually considered an integral part of the IPM of any pest, natural

enemies of pepper weevil in Florida have not been shown to play an important role in

suppression of this pest (Elmore and Campbell 1954, Wilson 1986, Schuster et al. 1999).

For this reason, although efforts to introduce natural enemies against pepper weevil have

been considered, no specific parasitoid had been introduced and released for this pest in

the United States until the present study (Elmore et al. 1934, Elmore and Campbell 1954,

Clausen 1978, Schuster et al. 1999).

Entomopathogenic nematodes (Heterorhabditis sp. and Steinernema sp.) have

proved effective against certain insect pests that develop some part of their life cycle in

the soil (Glaser 1931, Bell at al. 2000). Studies in Texas have shown that S. riobravis can

kill boll weevil larvae inside abscised squares and bolls of cotton if applied to the soil

under ideal conditions of moisture (Cabanillas 2003). In a similar way, entomophagous

nematodes might attack the pepper weevil inside buds or pods that are on the ground, and

could thus diminish pest populations; however, there is little information in regard to

entomopathogenic fungi, as well as viruses or bacteria that might attack this pest. In a









recent work in Florida, a commercial formulation ofBeauveria bassiana (Balsamo) was

used against pepper weevil adults, but proved to be ineffective in the field (Schuster et al.

1999).

Three pteromalids, one braconid, two species of ants, and one mite were identified

attacking the pepper weevil in the United States (Pratt 1907, Pierce et al. 1912, Elmore et

al. 1934, Cross and Chesnut 1971). Nevertheless, their impact on the pest was not

considered important (Elmore and Campbell 1954, Genung and Ozaki 1972). The cited

natural enemies that are endemic to the United States would be unlikely to act effectively

against an exotic pest.

Before 1950, two attempts of classical biological control of this pest were made in

the United States. Eupelmus cushmani Crawford and Catolaccus (Heterolaccus) hunter

Crawford were collected from Guatemala and released in Hawaii during 1934-37, where

they established, but did not provide encouraging results (Clausen 1978). The other

attempt during 1942-1943 occurred in California with the release of Triaspis vestiticida

Viereck, a parasitoid ofAnthonomus vestitus Boheman, the Peruvian cotton boll weevil in

Peru (Berry 1947, Clausen 1978). However, the parasitoid was never recovered from

peppers (Clausen 1978). These three natural enemies were originally obtained for

biological control programs against the boll weevil (Anthonomus grandis Boheman) and

evaluated against pepper weevil in the hope that similarities between these two pests,

might make possible the use of the same natural enemies (Clausen 1978). Nevertheless,

there are no reports of E. cushmani or T. vestiticida ever parasitizing pepper weevil.

C. hunter has the widest distribution of any parasitoid of boll weevil or pepper

weevil in the United States, Mexico, and Central America (Pierce et al. 1912, Cross and









Mitchell 1969, Cross & Chesnut 1971, Mariscal et al. 1998, Schuster et al. 1999,

Rodriguez et al. 2000). It is the most important parasitoid attacking pepper weevil in

Florida (Wilson 1986, Schuster et al. 1999), and one of the most widely distributed in

Mexico (Mariscal et al. 1998, Schuster et al. 1999, Aguilar and Servin 2000, Rodriguez et

al. 2000). It is also present in Honduras (Riley and Schuster 1992) Costa Rica (Schuster

et al. 1999), and it is the only one known from Puerto Rico (Schuster et al. 1999).

C. hunter is a generalist that is known to attack at least 17 species of Curculionidae

and 2 of Bruchidae (Cross and Mitchell 1969, Cross and Chesnut 1971); it usually

develops on the last instar of its host (Wilson 1986, Cate et al. 1990, Rodriguez et al.

2000). The particular biology of this species was useful for mass rearing at a moderate

scale on the cowpea weevil, Callosobruchus maculatus F. (Rodriguez et al. 2002,

Vasquez et al. 2005). Those findings, and some biology studies that showed that C.

hunter has greater fecundity and intrinsic rate of increase than pepper weevil (Rodriguez

et al. 2000, Seal et al. 2002), stimulated and supported the plan of evaluating C. hunter

as a biological control agent. Releases of 1050 C. hunter per hectare in Sinaloa, Mexico,

were not effective in combating pepper weevil on bell pepper (Corrales 2002).

Nevertheless, in Florida results of weekly releases of the equivalent of 7900 C. hunter

per hectare suggested that this parasitoid has potential to reduce damage of pepper weevil

on peppers (D. Schuster, personal communication). One of the most important

disadvantages of using C. hunter to combat pepper weevil is the fact that it attacks the

3rd instar host, which is usually inaccessible deep within the fruit of commercial pepper

varieties.









Table 3. Natural enemies of the pepper weevil, references until 2005
Natural enemies Reference


Predators
Hymenoptera: Formicidae
Solenopsis geminata Fab.
Tetramonium guinense Fab.
Passeriformes: Icteridae
Sturnella magna (Easter meadowlark bird)

Parasitoids
Pteromalidae
Zatropis incertus Ashmead
(=Catolaccus incertus Ashmead)
Catolaccus hunter Crawford

Habrocytus pierce Crawford

Braconidae
Bracon mellitor Say

Triaspis eugenii Wharton & Lopez-Martinez


Urosigalphus sp.
Bracon sp.
Aliolus sp.
Eulophidae
Euderus sp.
Syempiesis sp.
Eupelmidae
Eupelmus sp.

Eurytomidae
Eurytoma sp.


Pratt 1907, Hinds 1907
Wilson 1986

Genung & Ozaki 1972



Pierce 1907, Cross & Chesnut
1971
Pierce et al. 1912, Cross &
Chesnut 1971, Mariscal et al. 1998
Cross & Chesnut 1971

Pratt 1907, Pierce et al. 1912,
Cross & Chesnut 1971

Mariscal et al. 1998, Wharton &
Lopez-Martinez 2000, Toapanta
2001
Mariscal et al. 1998, Toapanta
2001
Mariscal et al. 1998




Mariscal et al. 1998, Toapanta
2001

Mariscal et al. 1998


Mites
Acarina: Pyemotidae
Pyemotes ventricosus Newport Pierce et al. 1912, Cross &
Chesnut 1971

Even though Mexico and Central America have been indicated as the center of

origin and domestication of the most important cultivated species of peppers (Vavilov

1951, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, Loaiza-









Figueroa et al. 1989, Conciella et al. 1990), and the pepper weevil was first described as a

pest of that crop in Mexico (Cano and Alcacio 1894), no significant surveys for natural

enemies were undertaken until 1997 (Mariscal et al. 1998). A survey by these authors in

the west central coastal state of Nayarit, Mexico, detected four species of Braconidae, one

of Pteromalidae, two of Eulophidae, and one each from Eupelmidae and Eurytomidae

(Table 3). Of these, the most abundant was a new species, later described as Triaspis

eugenii Wharton and Lopez-Martinez (Hymenoptera: Braconidae) (Wharton and Lopez-

Martinez 2000), which was reported to reach incidences of 18-40% parasitism under field

conditions (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This is a solitary

parasitoid which was reported to parasitize the egg, and to complete its life cycle in less

than three weeks with a life span of less than four (Mariscal et al. 1998, Toapanta 2001,

Rodriguez et al. 2004).

The genus Triaspis Haliday (Hymenoptera: Braconidae) is placed in the tribe

Brachistini of the subfamily Helconinae (Martin 1956, Sharkey 1997). This genus could

be represented by 100 species in the New World, but just a few of them have been

described from this region (Sharkey 1997). In general, biology and life history of the

genus Triaspis is poorly known. Some species of Brachistini are egg-larval parasitoids of

weevils, bruchids, and anthribids, and the last larval instar has an ectoparasitic phase

(Clausen 1940, Berry 1947, Saw and Huddleston 1991, Sharkey 1997). In spite of the fact

that the biology of the genus Triaspis is not completely known, some species of this

genus have been used in biological control programs in North America.

T. thoracicus was imported to Canada and the United States to combat species of

Bruchidae, especially the pea weevil, Bruchuspisorum L. Apparently this species failed









to establish because it has a preference for laying eggs in plant tissue instead of dry seeds

(Martin 1956, Clausen 1978). Efforts to combat the cotton boll weevil in USA with

Triaspis vestiticida Viereck, were previously mentioned. T. vestiticida is a solitary

parasitoid which attacks the egg of the host (peruvian cotton boll weevil) and develops as

an endoparasitoid in early instars. At the end of the last instar, the larva emerges from the

host to feed as an ectoparasitoid, leaving only the host cephalic capsule (Berry 1947).

Although these habits may prevail throughout the tribe, more detailed studies of T

eugenii are necessary to understand its habits and potential use.

Objectives

The high incidences of parasitism by T. eugenii under natural conditions in Nayarit,

Mexico and the recent information that confirms that this species attacks the weevil egg

make it a good candidate for biological control of pepper weevil. Attempts to use this

species for biological control will require more specific information on host/parasitoid

relationships. Therefore, the objectives of the present study were the following:

1. Clarify key aspects of the biology of the pepper weevil, including demographic
parameters, to improve rearing methodologies

2. Evaluate oviposition behavior and population dynamics of T. eugenii to optimize
rearing procedures and to assess control potential

3. Test the ability of T. eugenii to control pepper weevil














CHAPTER 2
REARING METHODOLOGY AND INFLUENCE OF FOOD QUALITY ON
BIOLOGY OF PEPPER WEEVIL

Introduction

Adult pepper weevils, Anthonomus eugenii Cano, feed on floral buds, flowers,

fruits, and even pepper leaves, but the larvae feed and develop completely inside floral

buds and immature fruits (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al.

1934). When feeding in floral buds, weevil larvae damaged especially the anthers

(Walker 1905, Elmore et al. 1934, Patrock and Schuster 1992). During the oviposition

process on fruit, females spend a short time in selecting a site on the pod, usually close to

the calyx. They then bore into the pod, turn completely, and deposit an individual egg in

the hole. Once the egg is in the cavity, the female deposits a brownish substance that

turns black on drying and seals the hole (oviposition plug) (Walker 1905, Elmore et al.

1934, Goff and Wilson 1937). The pepper weevil laid more eggs in pepper fruits than in

flowers (Patrock and Schuster 1992), and adults preferred small and immature fruits to

mature fruits for feeding and oviposition (Elmore et al. 1934, Wilson 1986, Bruton et al.

1989).

Although the pepper weevil pepper weevil has been considered the major pest of

peppers for more than eight decades in Mexico and the United States (Cano and Alcacio

1894, Walker 1905, Pratt 1907, Elmore et al. 1934, Riley and King 1994), no artificial

rearing system has been developed to facilitate biological studies. Consequently, most

studies have depended on weevils emerging of infested peppers collected from the field









(Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992). An attempt to rear pepper

weevil on artificial diet can be excluded from this generalization (Toba et al. 1969).

These authors were able to rear 6 generations of weevils using diet. However, females

would not lay eggs in the diet, so the processes of collection and manipulation of eggs are

laborious and ineffective (Toapanta 2001).

Toapanta et al. (2005) described a methodology to produce weevils in peppers, but

these authors did not define the quality of fruits offered. A similar methodology with a

few variations is practiced at the Golf Coast Research and Education Center (GCREC),

Wimauma, FL. Both methodologies consist basically of exposing peppers for 48-72 h to

weevil adults in cages 50x40x40 cm. Fruits are subsequently removed and held in

ventilated plastic containers until the emergence of weevils. Toapanta (2001) used mainly

'Serrano' peppers, whereas 'Jalapefio' peppers from the supermarket were used at

GCREC. Toapanta (2001) also indicated that the largest number of weevils, 1.7 adults per

fruit, was obtained using 5 weevils per fruit. At GCREC a ratio of 15 adults per fruit is

used, although an evaluation of efficiency has not been conducted (D. Schuster, personal

communication). These methodologies have been useful to maintain dozens of

generations in the laboratory, but efficiency could be improved by considering factors

such as competition within fruits, fruit decay, and effect of food quality. Therefore,

modifying fruit quality and decreasing competition could improve the laboratory rearing

of the weevil.

Even though there is a basic understanding of the biology of pepper weevil, there

are no studies that refer to food quality and its effect on fitness of this species. For

example, there are no reports that explain why weevils prefer feeding on floral buds









instead of fruits. This might be related to food quality (nitrogen concentration or protein

content). That is suggested because the ovarian development and oviposition of the boll

weevil, Anthonomus grandis Boheman, are biological processes that depend upon the

presence of cotton squares, a high nitrogen content food (Hunter and Hinds 1905, Hunter

and Pierce 1912, Isley 1928). The pepper weevil does not need floral buds for ovarian

development as has been proved by authors who reared the weevil in the laboratory with

only peppers available for feeding and oviposition (Toapanta et al. 2005, D. Schuster,

personal communication). However, studies that include feeding on floral buds are

necessary to understand nutritional factors in this species.

The biology of the pepper weevil has been studied by several authors (as

summarized in Riley and King 1994), but most, including Toapanta et al. (2005),

reported a fecundity of no more than 150 or 160 eggs per female. Only Elmore et al.

(1934) reported a fecundity as high as 340 eggs per female. Possibly a better

understanding of the effects of food quality and the availability of immature fruits and

floral buds could allow a further realization of the reproductive potential of this species

and thereby improve the rearing system. The objectives of this chapter were to determine

the effects of crowding on oviposition and the effects of fruit quality on rearing of the

pepper weevil, and to provide demographic parameters when immature fruits and floral

buds are offered to adults.

Materials and Methods

Laboratory Weevil Colony

A colony of the pepper weevil was established in a laboratory at the Entomology

and Nematology Department in Gainesville, UFL, with 200-250 insects that emerged









from infested fruits collected during the Spring of 2003 at the Southwest Florida

Research and Educational Center (SWFREC), Immokalee, FL. Genetic enrichment was

provided by addition of insects collected periodically from the same site, particularly

during Spring 2004 and 2005. The insects were reared continuously on fresh peppers,

'Jalapefio M' (Harris Seeds Rochester, NY), and 'Jalapefio Mitla' (Otis S. Twilley Seed

Co. Hodges SC), but all the experiments were conducted with 'Mitla' fruits.

The colony was moved in April 2004 to a rearing room at the SWFREC maintained

at 27 + 20C, 60-70% RH, and 14:10 (L:D) h photoperiod, and the experiments with the

pepper weevil were conducted at those conditions, unless otherwise indicated. Pepper

fruits were exposed to unsexed adult weevils using a ratio of 10:1 insects per fruit. Plastic

jars 3.8 L (14x14x25 cm Rubbermaid Home Products Wooster, OH), with four lateral

holes 3 cm in diameter covered with polyethylene screen, were used as oviposition cages.

The cages had a knit sleeve (Kimberly-Clark Co. Roswell, GA) 50 cm long taped to the

opening of the jar to prevent escape of insects during manipulations. Each cage held ca.

100 unsexed weevil adults. Water was provided in a cotton wick placed in 28-mL plastic

cups, and lines of honey were dispensed every day on the upper side of the cage. Every

24 h, 10 small fresh 'Jalapefios' (ca. 5 cm long) were placed in the cages with the

weevils, and 10 fruits placed 24 h previously were removed. The fruits were then held in

plastic containers, 60-70 fruits each, until the emergence of adults. These containers

(33x21x11 cm Rubbermaid Home Products Wooster, OH) had six lateral holes 3 cm in

diameter covered with polyethylene screen to ensure ventilation. Weevils that emerged

within the same week were collected and confined in a new oviposition cage. Each cage









was used for four or five weeks, because fecundity was assumed to decline thereafter and

because many weevils died during daily manipulations.

Crowding Effects on Oviposition

Production of eggs and presence or absence of oviposition plugs was evaluated for

different numbers of mated females alone and for different numbers of males and females

together. Weevils were held in clear plastic cups 250 mL, each provided with a single

fresh 'Jalapefio' pepper (4.95 + 0.61 cm long, 1.86 + 0.24 cm wide, and 8.57 + 2.02 g

weight, n=49). Individual peppers were vertically oriented using adhesive poster putty

(Henkel Consumer Adhesives, Inc. OH) on the tip of the fruits. Water was provided by

daily saturating a cotton wick placed in the bottom of each cup (Fig. 1).


















Figure 1. Device for exposing different densities of weevil adults to 'Jalapefio' peppers.

The treatments were 1 female, 1 female/ 1 male, 2 females, 2 females/ 2 males, 5

females, 5 females/ 5 males, and 10 females. The large metatibial mucrones of males

were used to sex weevils (Eller 1995). To facilitate sexing, groups of 10-12 weevil adults

were placed in Petri dishes over a white background and were observed through a

dissection microscope at 16X magnification. An average of 22 females and 16 males









could be sexed in 10 min using this method (n=4). These seven treatments were arranged

in a randomized complete block design with three replications. Because it is assumed that

fecundity of pepper weevil is higher in the first weeks of its life (Patrock and Schuster

1992) and because of the availability of insects, each block was set up at different time

using insects 7-10 days old. 'Jalapefio' fruits were replaced daily for 7 d and the number

of eggs per day, and number of eggs with or without an oviposition plug, was evaluated.

To compare the number of eggs per female per day, data were analyzed using an

analysis of variance (PROC ANOVA, SAS Institute 2000) and means were compared by

Least Significant Difference (LSD) (P < 0.05). Data were normalized prior to the analysis

using the square root transformation (PROC CAPABILITY, SAS Institute 2000), but

data are presented in the original scale. The presence or absence of oviposition plugs by

treatment was analyzed using a chi-square test (df = 1, P < 0.05) to determine differences

between observed and expected (1:1) values (PROC FREQ, SAS Institute 2000). When

one of the cells had expected counts less than 5, the Fisher's exact test to contrast the null

hypothesis was used (PROC FREQ, SAS Institute 2000).

Fruit Quality

The number of adult weevil progeny obtainable using the ratio of 10 unsexed

weevils per fruit was compared for two fruit qualities: immature green and green mature

(marketable fruits). Prior to evaluation oviposition cages (3.8 L) containing 100 + 10

unsexed weevils 1-5 d old were provided with 10 immature fruits and 5 or 10, depending

on availability, floral buds. The fruits and buds were replaced daily for 3 days. After that,

one cage was assigned to one of two treatments: 10 immature green fruits (4.75 + 0.61

cm long, 1.77 + 0.25 cm wide, and 7.66 + 2.01 g weight, n=37), harvested from potted

plants grown outdoors, or 10 mature green 'Jalapefio' fruits (6.67 + 0.77 cm long, 2.06 +









0.41 cm wide, and 14.96 + 3.58 g weight, n=37), bought at the supermarket. Both

qualities of fruit were washed with liquid dish detergent and rinsed up with tap water

before offering to the weevils. Fruits were replaced every 24 h (5:00 PM to 5:00 PM) and

exposed fruits were held until adult emergence in the ventilated plastic containers used in

colony maintenance. Each cage of weevils was considered an experimental unit and was

evaluated for 22 days. Because of the availability of peppers three replicates were

conduced at different times during July and September of 2003. After checking the data

for normality (PROC CAPABILITY, SAS Institute 2000), statistical differences were

determined using the student's t-distribution (PROC TTEST, SAS Institute 2000).

Adult Feeding and Oviposition

One pepper weevil female and one male both 18 h old or younger were placed in

individual 250 mL plastic cups. A lid closed each cup and a 3 cm diameter organdy-

sealed hole provided ventilation (Fig 1). Each weevil pair was considered an

experimental unit and assigned to one of two treatments: a) peppers only or b) peppers

plus floral buds. Every day during the first 7 days of life, each weevil pair was provided a

cotton wick saturated in water, as well as either a single young pepper (length 3.23 +

0.40, width 1.07 + 0.15, n = 15) or a single young pepper plus three floral buds before

anthesis. The peppers were vertically oriented in the cups, as indicated above, and floral

buds were placed in the bottom of the cups. Each treatment had 8 replicates. Because

there were not enough floral buds for doing all replicates at the same time, the replicates

were performed in groups at different times (3, 3, and 2). After 24 h of exposure, food

items were replaced and observed under a stereoscopic microscope to evaluate number of

feeding and/or oviposition punctures. Punctures were dissected to determine the presence

or absence of eggs. Before the analysis, the data were normalized by the arcsine square









root transformation (PROC CAPABILITY, SAS Institute 2000), but means are presented

in the original scale. Statistical differences were determined using the student's t-test

(PROC TTEST, SAS Institute 2000). Results from weevils that received both floral buds

and fruits in the same cup were further analyzed to compare the preference of feeding and

ovipositing in floral buds or fruits using student's t-test (PROC TTEST, SAS Institute

2000).

Life Table

Fourteen weevil pairs 18 h old or younger were caged individually in plastic cups

as previously described. Males were removed after 7 d to avoid possible interference with

oviposition behavior (Elmore et al. 1934). A male was subsequently introduced into each

cage for 2 d every 4 wk for mating. The females, or pairs when the males were present,

received one fresh 'Jalapefio' pepper daily (length 3.23 + 0.40, width 1.07 + 0.15, n =

25). The pepper was removed and dissected to count the number of eggs. Two floral buds

were offered every day in addition to the fruits for the lifetime of the female, except

during week 2-4 due to short supply. The procedures were followed until death of the

females.

The sex ratio of the species was estimated from field and laboratory samples, and

survivorship was evaluated from a sample of 102 peppers with only one weevil egg per

fruit from the laboratory colony. In addition, fertility was estimated from a sample of 171

eggs laid in immature peppers from the same colony. Previous observations indicated that

an oviposition plug ensured the presence of an egg in 91% of all cases (SE = 0.018, n =

216). Therefore, removing oviposition plugs to check for the presence of eggs was not

necessary. A permanent marker was used to mark the oviposition plugs and the fruits

were dissected 4 d later to look for larvae.









Using LIFETABLE.SAS (Maia et al. 2000), the whole data set was used to

estimate net reproductive rate (Ro), defined as the number of females produced by one

adult female during its mean life span; generation time (T), the period between the birth

of parents and the birth of the offspring; doubling time (Dt), the time necessary to double

the initial population; intrinsic rate of increase (rm), the potential growth of a population

under given conditions; and finite rate of increase (k), the daily rate of increase of each

cohort (Birch 1948, Maia et al. 2000, Southwood and Henderson 2000, Toapanta et al.

2005). Data reported by Elmore et al. (1934) and Toapanta et al. (2005) were subjected to

the same analysis and compared to results obtained in the present study.

Nitrogen Content of Fruits and Buds

The nitrogen content of anthers, pericarp and a mix of placenta and seeds of three

maturation stages of 'Jalapefios Mitla' was evaluated. These maturation stages were

classified according to size and appearance as: a) immature green, b) mature green, c)

mature red (Table 4). Four samples were collected from plants 2-4 months old which

were grown in a greenhouse. During each sampling, 70-90 floral buds just prior

dehiscence and 6-7 peppers of each maturation stage were collected. Anthers were

removed with forceps, and fruits were dissected to separate pericarp from placenta and

seed. Because of possible variation among harvests (June and July 2005), each sampling

was considered as a block.

Table 4. Characteristics of 'Jalapefio Mitla' used for nitrogen content determinations
Pepper quality (n=10) Length, cm Width, cm Wall thickness
Immature Green 4.24 + 0.24 1.12 + 0.14 0.18 + 0.02
Mature Green' 6.76 + 0.60 2.42 + 0.24 0.39 + 0.03
Mature Red 7.72 + 0.67 2.57 + 0.20 0.41 + 0.03
1Similar to fresh market quality









The samples of each botanical structure were placed in individual paper bags, dried

at 50 C for three days, and ground to a powder. Nitrogen content was determined by the

soil laboratory at the SWFREC using the alternative dry Micro-Dumas combustion

analysis for total nitrogen in solid phase samples. This method is based on transformation

to the gas phase by extremely rapid and complete flash combustion of the sample

material (Matejovic 1993, 1995, Anonymous 1997). There were four replications

(samples) in a randomized block design. The data were normalized by square root

transformation (Sokal and Rohlf 1969, Ott and Longnecker 2001) prior to ANOVA

(PROC ANOVA, SAS Institute 2000); however, the means and standard deviations are

presented in the original scale. Means were compared by the LSD test (LSD) (P < 0.05)

when the F value was significant.

Results

Crowding Effects and Oviposition

The number of weevils per fruit had a significant influence on the numbers of eggs

per female per day (F = 12.47; df= 8,138; P < 0.0001). The largest number of eggs per

female per day was obtained with a single female per fruit followed by one female and

one male (Table 5). There were no differences among the remaining treatments (LSD test

P < 0.05). Increasing the number of weevils from 1 female to 2 females/2 males or even 5

females had no effect on the number of plugged eggs (Fisher's exact test P = 0.266 and

0.139, respectively). However, the percentage of unplugged eggs increased 5 or 6 fold at

densities of either 5 females/5 males or 10 females (Table 5). There was no differences

between these treatments (X2 = 2.61, P = 0.1064).









Table 5. Numbers of eggs per female per day for different numbers of females and males
per single 'Jalapefio' pepper fruit
Weevil density Eggs/female/day (Mean + SD)1 Unplugged eggs (%)
1 5.0 + 2.43 a 5 (4.76)

1 1 3.38 + 1.99b 2 (2.82)

2$ 2.05 +1.37c 1 (1.16)

2 92 1.97 +0.8c 3 (3.61)

5 $ 1.68 + 0.54 c 4 (2.26)

5 9 5 1.74 + 0.82 c 25 (13.66)

10 Y 1.57 + 0.48 c 30 (9.10)

Means with the same letter are not significantly different (LSD P < 0.05)

Fruit Quality

Immature green and green marketable fruits were both able to support the rearing

of pepper weevil, although there were significant differences. Using the 10 weevils per

fruit density 2.65 + 1.16 weevil adults emerged per immature fruit, compared to only 1.11

+ 0.81 weevils per marketable fruit (t = 8.81, df= 65, P < 0.0001).

Feeding and Oviposition in Peppers versus Floral Buds (7d)

When floral buds and peppers were offered at the same time, females fed and laid

eggs in both (Table 6). Nevertheless, weevils preferred feeding on floral buds instead of

fruits (t = 13.39, df = 55, P < 0.0001). In contrast, females preferred to lay eggs in pepper

fruits rather than floral buds (t = 9.59, df = 55, P < 0.0001).

Table 6. Feeding and oviposition of pepper weevil adults when given a choice of
'Jalapefio' pepper fruit or floral buds (7d)
Parameter Pepper fruits Floral buds t-test (P < 0.05)
Feeding punctures per day 3.27 + 4.99 18.75 + 8.64 *
Eggs per day 12.73 + 10.46 0.16+0.49 *









Oviposition with or without Floral Buds (7d)

Differences in oviposition were observed between females that were provided with

pepper fruits only, compared to those that were provided with both floral buds and fruits

(Table 7). Females provided with fruits and buds laid many more eggs the first day of

oviposition (t = 2.81, df = 14, P = 0.0261), and over the entire 7 d test period (t = 5.08, df

= 14, P = 0.0002) than females provided only with fruits. Pepper weevil females laid only

1.25% of eggs in the floral buds when both floral buds and fruits were provided at the

same time.

Table 7. Oviposition of pepper weevil females fed with 'Jalapefio' pepper fruits only or
with pepper fruits plus floral buds
Parameter Peppers Pepper plus floral t-test (P < 0.05)
buds
Days to begin oviposition 2.87 + 0.99 2.62 + 0.52 ns

Eggs at first day of 3.50 + 1.41 11.87 + 8.30 *
oviposition
Total number of eggs (7 d) 23.75 + 11.16 89.12 + 34.65 *


Life Table

The 14 females survived 28 to 103 d, with an average of 64.5 + 25.8 d. During this

time 8 of 14 females (57%) began oviposition on the second day, and 13 of 14 females

were depositing eggs by the third day (Fig. 2). The remaining female took seven days to

begin oviposition. Fecundity ranged from 0 to 32 eggs per female per day with an

average of 5.51 + 5.31 eggs per female per day, and declined to near zero by 86 d. The

fertility of eggs deposited in immature fruits was 97.66% (n = 171, SD = 0.15) and the

survivorship of all preimaginal stages was 89% (n = 102, SD = 0.31). These data and the

sex ratio of the species, that was not significantly different from 1:1 either in the field or









in the laboratory (X2 =0.95, P < 0.05, X2 =0.35, P < 0.05, respectively), were used to

estimate the demographic parameters of pepper weevil (Table 8).


18
16
14
0 12
10
E 8
4-
c,)
LJ
2

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96101
Age in days

Figure 2. Mean fecundity of adult pepper weevil females (n = 14) reared on 'Jalapeio'
peppers at 27 + 2 C and 14:10 (L:D) photoperiod.

Table 8. Life table parameters for A. eugenii females (n =14) reared on 'Jalapefio'
peppers at 27 + 20C, and 14:10 (L:D) photoperiod
Parameter Value (+ 95% confidence limits)
Net reproductive rate (Ro)a 158.1 (115.1 201.1)
Intrinsic rate of increase (rm)b 0.14 (0.12 0.15)
Generation time (T)c 36.04 (31.26 41.22)
Doubling time (Dt)' 4.93 (4.42 5.49)
Finite rate of increase (X) d 1.15 (1.13 1.17)

aFemale/female, bloge (Ro)/T, cDay, dFemale/female/day.

Nitrogen Content of Fruits and Buds

Nitrogen content was different among botanical structures and maturation stages of

peppers (F = 41.61; df = 6, 27; P < 0.0001). Highest nitrogen content occurred in the

anthers of floral buds. An intermediate concentration occurred in placentas and seeds at









any maturation stage, and in pericarp of the immature green fruits. The lowest nitrogen

concentration was detected in pericarp of mature green and mature red fruits (Table 9).

Table 9. Nitrogen content (Mean + SD) of anthers in floral buds and 'Jalapefio' fruits at
three maturation stages
Maturation stage Botanical structure N content (%) dry weight
Floral buds Anthers 5.23 + 0.09 a

Immature green Placenta and seeds 3.39 + 0.04 b
Mature red Placenta and seeds 3.21 + 0.25 bc
Immature green Pericarp 3.19 + 0.38 bc
Mature green Placenta and seeds 2.96 + 0.09 c
Mature green Pericarp 2.25 + 0.42 d
Mature red Pericarp 2.01 + 0.35 d

Means followed by the same letter are not significantly different (LSD, P < 0.05)

Discussion

Crowding Effects and Oviposition

Increasing density of female pepper weevils per fruit reduced oviposition and

increased the number of eggs lacking their normal oviposition plugs. An individual

female produced more eggs than a female and a male together, and both treatments had

higher number of eggs per female than two females together. The first comparison

confirmed a previous study by Elmore et al. (1934) that suggested that the presence of

males could inhibit oviposition behavior of females. However, the second comparison

indicated that inhibition of oviposition also could occur with the presence of more

females. Similar results with unsexed weevils were reported by Toapanta (2001), who

indicated that a ratio of five unsexed weevils per fruit were more efficient for producing

adults than larger ratios (10:1, 15:1, 20:1).









Crowding by either males or females interfered with the oviposition process. Males

may interfere by seeking to mate with females, and females may interfere by contending

with each other for oviposition resources. The final phase of oviposition is deposition of

the plug, so the same factors might have influenced the presence of eggs lacking

oviposition plugs.

Feeding could also contribute to the reduction of oviposition plugs by crowding.

Pepper weevil prefers both to oviposit and to feed close to the calyx (Walker 1905,

Elmore et al. 1934, Wilson 1986, Toapanta et al. 2005). More weevils per fruit would

mean more feeding punctures and, consequently, more mechanical damage to tissue close

to the calyx, including the plugs.

These results demonstrated why rearing the pepper weevil by using large numbers

of weevils per fruit is inefficient. A small number of insects per fresh 'Jalapefio', even a

single mated female, may be the most efficient combination. Additionally, this system

maximizes the number of oviposition plugs which may prevent dehydration of eggs as

well as exposure to opportunistic predators, such as mites or ants.

Fruit Quality

Quality of the host can exert an important influence on fecundity and survival of

insects (Awmack and Leather 2002). Exposing pepper weevil females to immature green

peppers resulted in more offspring than exposing females to marketable fruits (= mature

green). The nitrogen content in the pericarp of immature fruits was higher than in mature

green fruits (Table 9). Partitioning of nitrogen by fruit age and tissue could affect the

capacity of both larvae and adults to acquire nitrogen.

Eggs are laid generally in the pericarp of peppers (Walker 1905, Elmore et al. 1934,

Goff and Wilson 1937) due to the small size of the pepper weevil proboscis (Clark and









Burke 1996). Eggs laid in older fruits will produce larvae that have to bore through a

thicker pericarp, which is lower in nitrogen compared to an immature fruit (Tables 4, 9).

Additionally, the maturation process in peppers includes substituting soft tissues with

fiber, the clearest example being the seed coat on mature fruits (Bosland and Votava

1999). The high nitrogen content in mature seeds may not be accessible to the larva.

Thus, thickening of the pericarp and redistribution of nitrogen to seeds during maturation

of pepper fruits could explain why the pepper weevil prefers to feed and to oviposit on

immature fruits, and why the rearing process of weevils could be improved if immature

fruits are used.

Adult Feeding and Oviposition

Early observations of pepper weevil adults in the field indicated a feeding

preference for floral buds compared to fruits (Walker 1905, Elmore et al. 1934). These

observations were later corroborated with laboratory tests (Wilson 1986, Patrock and

Schuster 1992). The data presented here corroborate this behavior in 'Jalapefio' floral

buds and fruits, and demonstrate the likely cause. Many more feeding punctures were

made per floral bud compared to 'Jalapefio' fruit. Patrock and Schuster (1992) reported a

mean of 0.74 and 0.75 feeding punctures for floral bud and fruit, respectively, produced

by a single female. This contrasted with 3.27 feeding punctures in fruits against 18.75

feeding punctures on floral buds using a pair of weevils in this study. Patrock and

Schuster (1992) used 10-20 d old females in bell pepper fruits ('Early Calwonder') at 22

+ 4C, while 1-7 d old weevils in 'Jalapefio' fruits at 27 + 2C were used in the present

study. More information related to the quality of the varieties of peppers and the vigor of

the insects used could help to better understand those differences.









Patrock and Schuster (1992) indicated that a female laid more eggs on immature

fruits than floral buds (0.96 and 0.36 eggs, respectively), because fruits offered more

resources to support larvae. Data collected here (0.16 eggs per floral bud and 12.73 eggs

per fruit) followed the same trend, although the number of eggs laid per fruit was 10

times greater.

The seven day oviposition experiment provided information on the ability of floral

buds to increase fecundity. The preoviposition period was not affected by access to floral

buds, and a similar value (2 or 3 days) was reported by Elmore et al. (1934), Wilson

(1986), Gordon and Armstrong (1990), and Toapanta et al. (2005). Nevertheless, the

number of eggs at the first day of oviposition (3.5 vs 11.9) and the total number of eggs

during the first seven days after emergence (23.7 vs 89.1) was always higher for females

that received anthers. The total number of eggs deposited in the first seven days of life of

females provided with immature 'Jalapefio' fruits and floral buds was greater than any

number reported before for this species in previous works (Elmore et al. 1934, Wilson

1986, Gordon and Armstrong 1990, Patrock and Schuster 1992, Toapanta et al. 2005),

probably because of feeding on anthers. An effect from feeding on floral buds (= cotton

squares) has been reported for the boll weevil, which needs to feed on floral buds for the

ovaries to develop (Hunter and Hinds, 1905, Hunter and Pierce 1912, Isley 1928, 1932).

The pepper weevil is able to obtain sufficient nutrients for egg production from

pepper fruits alone, as the majority of references in Table 1 indicate, and it is common

practice to rear the weevils on a strict diet of fruit. The present comparison was

conducted for only 7 days, so it is not possible to say that total fecundity would be lower

on a strict fruit diet. Possibly, the resulting nitrogen deficiency could be compensated by









increasing consumption, as has been observed for other species including Celerio

euphorbiae L. (Lepidoptera: Sphingidae), and Pieris rapae L. (Lepidoptera: Pieridae)

(House 1965, Slansky and Feeny 1977, Awmack and Leather 2002).

In addition to nitrogen content, a proper balance of amino acids is critical for

growth and reproduction in insects (Linding 1984, Weis and Berenbaum 1989, Nation

2002). Therefore, the increased fecundity observed for the pepper weevil could also be

due to the balance of amino acids that might be present in floral buds. Suitable diets for

the boll weevil were developed only after developing a basic knowledge of the proteins

and free amino acids in anthers of young cotton flowers (Earle et al. 1966, Hilliard 1983).

Further studies that identify the proteins and amino acids of anthers of peppers could be

useful in the development of a better artificial diet for pepper weevil adults.

Life Table

Pepper weevils laid a mean of 341 eggs per female with an average of 4.7 eggs per

day over an average life span of 78.7 d (Elmore et al. 1934). No temperature was

recorded in that study. Goff and Wilson (1937) reported a fecundity of 198 eggs with an

average of 6 eggs per day. Gordon and Armstrong (1990) reported 253.6 eggs over a life

span of 31.7 d, with an average of 8 eggs per day at 22 to 27C. Recently, Toapanta et al.

(2005) reported a wide range of fecundity for this species according to the number of

fruits offered to the weevils and temperature. Using eight females per group, these

authors reported 3.1 eggs per day at 300C and 158 eggs per female in single peppers over

51 days. Offered 3 peppers per day at 270C, females were able to lay 3.9 eggs per day or

281 eggs over 72 d. The fecundity of pepper weevil females fed with floral buds observed

in this study was 355 eggs per female, with an average of 5.5 eggs per day and a life span

of 64.5 d.









During the adult stage of insects, the reproductive potential is fully realized only if

the females have an adequate supply of energy for surviving and of nutrients for egg

production (Hilliard 1983, Awmack and Leather 2002). The biology of the pepper weevil

has been studied for more than a hundred years, but just three studies (Elmore et al. 1934,

Gordon and Armstrong 1990, and the present) provided floral buds along with fruits to

females when estimating fecundity. Not surprisingly, these studies reported the highest

fecundity for this species. Fecundity is the major component determining population

dynamics of a species and, along with survivorship, influences all demographic

parameters (Birch 1948, Southwood and Henderson 2000). Consequently, the

demographic parameters based on fecundity were higher for data reported by Elmore et

al. (1934) and the present study (Table 10).

Table 10. Life table parameters for the pepper weevil
Parameter Elmore et al. (1934)' Toapanta et al. This study
(2005) 300C 27 + 2C
Net reproductive rate (Ro) 153.45 33.57 158.10

Intrinsic rate of increase (rm) 0.09 0.11 0.14

Generation time (T) 53.22 32.39 36.04

Doubling time (Dt) 7.33 6.35 4.93

Finite rate of increase (k) 1.10 1.11 1.15

'The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively.
No temperature data provided.

The Ro value for pepper weevil females fed with immature 'Jalapefio Mitla' and

floral buds was 4.6 times greater than the highest value reported by Toapanta et al.

(2005). These authors fed the insects only with 'Serrano' or 'Jalapefio' peppers, but no

floral buds. Furthermore, the condition or quality of fruits was not indicated. The









Generation time (T), and Doubling time (Dt) reported by Elmore et al. (1934) were larger

than those observed in this study. This could be a consequence of distributing the eggs

evenly over the entire oviposition period. It was not possible to know the true distribution

of eggs over time to make a better estimation. Therefore, both parameters might be

overestimated. If the generation time of this study is used with the approximation

indicated by Birch (1948) to calculate the intrinsic rate of increase (rm = loge Ro / T), the

rm for Elmore et al. (1934) data set is similar to this study (rm = 153.45/36.04 = 0.14).

The difference in results in this study versus Elmore's data are due to generation

time, with fecundity (as indicated by Ro) being the same. These values represented the

potential of this species when feeding on floral buds and immature fruits, a situation that

would occur in the field. It might explain in part the success of this species in infesting a

young pepper crop, when floral buds and immature fruits are abundant. These

demographic parameters could be useful for: a) comparisons of different rearing

conditions, b) comparisons of population dynamics with natural enemies, and c)

predictions of the potential of biological control agents to control this pest.














CHAPTER 3
LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
BRACONIDAE)

Introduction

The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is

considered one of the key pests of peppers in the United States, Mexico, Central

America, and some Caribbean Islands (Elmore et al. 1934, Riley and King 1994,

Mariscal et al.1998, Schuster et al.1999). Adults lay eggs in floral buds and immature

peppers, and larvae develop inside beyond the reach of insecticides. Consequently,

chemical control targets only adults. An action threshold of a single adult weevil per 400

pepper terminals has been proposed (Riley et al. 1992 a,b). This low action threshold, and

the use of insecticides as the principal method of control, can result in adverse effects

including marketing restrictions, exposure of non-target organisms, environmental

contamination, pesticide resistance, and secondary pest outbreaks (NAS 1969, Luckmann

and Metcalf 1982, Doutt and Smith 1971). Unfortunately, no other viable control strategy

for the pepper weevil has been developed during the one hundred years that have elapsed

since the pest was first reported in Mexico (Cano and Alcacio 1894) and the United

States (Walker 1905, Pratt 1907).

Biological control could be an alternative tactic to incorporate into integrated pest

management (IPM) of the pepper weevil; however, sufficiently effective natural enemies

have yet to be discovered. One of the natural enemies of pepper weevil which has

received attention as a potential biological control agent is the generalist and









cosmopolitan parasitoid Catolaccus hunter Crawford (Hymenoptera: Pteromalidae). This

ectoparasitoid of the last instar of the pepper weevil has a greater fecundity than its host

(Rodriguez et al. 2000, Seal et al. 2002). It has been recorded as one of the most common

parasitoids of pepper weevil in some states of Mexico (Mariscal et al. 1998, Rodriguez et

al. 2000), Central America, and Florida (Cross and Mitchell 1969, Wilson 1986, Riley

and Schuster 1992, Schuster et al. 1999). However, releasing the equivalent of 1150 C.

hunter adults per hectare per week in field plots at Sinaloa, Mexico, was not effective

against this pest (Corrales 2002). On the other hand, results of releasing 7900 C. hunter

per hectare, prior to and during the early pepper season, indicated that this parasitoid

could reduce damage by the pepper weevil in Florida (D. Schuster, personal

communication). Divergent results may be due, in part, to the fact that C. hunter attacks

the 3rd instar host that is inaccessible deep within the pepper fruit. Augmentative releases

of this parasitoid prior to the crop cycle on the alternative host plant nightshade, which

has small fruits, could reduce pepper weevil populations. Releases early in the crop cycle

when fruits are small, or in small fruited varieties, might be effective because the host is

more accessible to C. hunter. Unfortunately, once the fruits increase in size the effect of

the parasitoid is limited. In fact, Riley and Schuster (1992) indicated that C. hunter was

not detected in fallen fruits larger than 2.5 cm in diameter. Therefore, it is desirable to

find species that may be effective as biological control agents of this pest, preferably

those that attack earlier and more accessible life stages.

A candidate parasitoid was recently reported in Mexico, where pepper, pepper

weevil, and parasitoids likely evolved. This species is considered the most important

parasitoid of the pepper weevil in Nayarit, Mexico, where it causes the highest incidence









of parasitism on pepper weevil (from 18 to 40%) in field samples (Mariscal et al. 1998,

Schuster et al. 1999, Toapanta 2001). This solitary endoparasitic wasp was described in

2000 as Triaspis eugenii Wharton & Lopez-Martinez (Hymenoptera: Braconidae).

However, there is still little information available on its biology and behavior (Sharkey

1997, Mariscal et al. 1998, Toapanta 2001).

This Chapter focuses on the biology and life history of T. eugenii. Initial efforts to

maintain the parasitoid colony were hampered by low levels of parasitism and high levels

of superparasitism. These factors limited reproductive potential and, thus, reliable

estimates of demographic parameters. In addition, inefficient rearing demanded excessive

resources (peppers and hosts). Preliminary observations in the laboratory suggested two

possible causes for low levels of parasitism: 1) a limited acceptable range of host-age for

parasitizing, and 2) the inability of females to find or recognize the host. One hypothesis

to explain this latter difficulty was that exposing pepper fruit to too many weevil adults in

the colony resulted in fewer eggs sealed with an oviposition plug (Chapter 2). The lack of

acceptable hosts could then result in either retention of eggs or in excessive

superparasitism. These questions required a solution before demographic parameters

could be reliably estimated and before the potential of the parasitoid as a biological

control agent could be evaluated. The objectives of this study then were to determine the

host lifestage suitable for parasitization by T. eugenii, to determine the parasitoid's ability

to recognize and to parasitize plugged and unplugged host eggs, to describe the life cycle

of the parasitoid, and to estimate demographic parameters for this species.









Materials and Methods

Surveys

Based on results of Mariscal et al. (1988) and Toapanta (2001), two surveys for T.

eugenii were conducted in pepper growing regions at Nayarit, Mexico. In April and May

of 2003, sampling was conducted in Puerta de Mangos, Caiada del Tabaco, Villa Juarez,

Los Corchos, Los Medina, and Palma Grande, all located in Santiago Ixcuintla county,

Nayarit, between 21 36' and 22 17' north latitude, and between 104 53' and 1050 40'

west longitude (INEGI 1999).

Hurricane Kenna made land fall in San Blas on October 25, 2002, just 30 km from

Santiago Ixcuintla and many pepper fields were destroyed. Late replanting resulted in

delayed harvest, with the result that fields included in the first survey in April 2003 were

still being managed for weevils with insecticides. During the second survey in May 2003,

the majority of peppers were collected from abandoned pepper crops. Pepper fruits with

signs of damage of the weevil (including presence of feeding and/or oviposition

punctures) were collected from the plants and/or from the ground. A random sample

(n=57) of these fruits was dissected to confirm weevil presence. Around 25 kg of fruit or

about 3,300 peppers were collected in each survey. All were Capsicum annuum L. The

dominant variety was 'Serrano' but local cultivars such as 'Serranillo', 'Caloro', and a

few 'Jalapefio' were also collected. Fruits were held in plastic nets of 2 kg in the field and

later at room temperature to avoid water condensation. Within 3 d the plastic nets

containing the peppers were placed in individual Ziploc plastic bags (S.C. Johnson &

Son, Inc. WI) provided with a paper towel to absorb moisture, and transported by air to

the United States in two insulated coolers provided with ice substitute (Rubbermaid Inc.,









Wooster, OH). The material was allowed to enter in the United States under the permit

46799 of the Animal and Plant Health Inspection Service (APHIS), USDA.

Upon arrival in Miami, the material was brought immediately to the quarantine

facilities at the Florida Department of Agriculture and Consumer Services (FDACS),

Division of Plant Industry (DPI), in Gainesville, Florida. In the presence of the quarantine

officer, the peppers were distributed into lidded plastic containers (30x23x10 cm). Each

container had 4 lateral holes, 5 cm in diameter, covered with fine mesh screen for

ventilation. A paper towel was placed at the bottom of each container to absorb excess

moisture. These emergence containers were held in the maximum security room in

quarantine at 27 + 30C, 50 to 70% RH, and 12:12 (L:D) h photoperiod. All the

parasitoids except T. eugenii were preserved in 70% ethanol and labeled and identified

with the aid of available literature. Identifications of braconids and chalcids were verified

by R. Wharton and R. Lomeli, respectively, both of Texas A&M University, College

Station. Voucher specimens were deposited at the Florida State Collection of Arthropods

at DPI.

Parasitoid Rearing

Wasps were held in 3.8 L plastic cages (Chapter 2) with the lateral holes sealed

with organdy. Parasitoids had free access to water in a cotton wick placed in 28-mL

plastic cups, and to lines of honey dispensed every day on the upper side of the cage. In

addition, a 7-8 cm long pepper flush (of leaves and floral buds) was inserted in a 28 mL

plastic cup filled with water and placed in each cage to simulate natural host finding

conditions. The flush had been exposed for 48 h to three pepper weevil adults.

Pepper weevil hosts were obtained from a colony held in Florida Reach-In

Incubation chambers (Walker et al. 1993) maintained at 27 + 1IC and 70-80% RH, and









14:10 (L:D) h photoperiod. Immature 'Jalapefio' fruits (4.7 + 0.77 cm length, 1.56 + 0.27

cm width, and 5.57 + 1.07 g weight, n = 25) were exposed to weevils for 24 h at a ratio of

10 weevils per fruit. Under these conditions, pepper weevil eggs take 2.9 + 0.1 d to hatch,

and an additional 1.7 + 0.1 d to reach the second instar (Toapanta et al. 2005).

Accordingly, two age ranges of eggs (2-24 h and 26-48 h after weevil exposure) and first

or early second instars (4 to 5 d after infestation), were offered to the wasps. Each cage

contained one or two T. eugenii females. At least two pepper fruits of each age category

were offered daily to the wasps beginning the second day after emergence. Following

exposure, fruits were held in ventilated containers until emergence of insects.

When it became apparent that parasitoids were only recovered from peppers that

had weevil eggs, the exposure period of fruits to weevils in the colony was limited to 24

h. Fruits were replaced between 4:00 to 6:00 PM, held at 22 + 20C until 10:00 AM the

next day, exposed to parasitoids for 24 h, and then held as above.

After the second generation of parasitoids, 10-15 females and 10-12 males were

included per cage to maintain a ratio of one parasitoid female per infested fruit per day.

Each cage was used 10-12 d. Females emerging from one cage were used to mate with

males emerging from a different cage to prevent inbreeding within the colony. Using this

rearing system, 80-100 females and 100 males were held each generation until the colony

could be moved from quarantine during October and November, 2003. The colony was

then held at the Entomology and Nematology Department, University of Florida,

Gainesville, at 27 + 1C and 70-80% RH, and 14:10 (L:D) h photoperiod for five months.

The colony was moved in April 2004 to the South West Florida Research and Education

Center (SWFREC) at Immokalee FL, and was maintained in a rearing room at 27 + 20C,









60-70% RH, and 14:10 (L:D) h photoperiod. Experiments were conducted under these

environmental conditions, unless otherwise indicated. 'Jalapefio Mitla' was the variety

used most in rearing the colony and in conducting experiments, but when this cultivar

was not available, 'Jalapefio Ixtapa' was used.

Host-Age Range

Choice tests were conducted during December 2003 to compare the acceptance of

eggs either 24-40 h or 48-64 h old. 'Jalapefio' fruits 5.25 + 0.53 cm length, 1.68 + 0.15

cm width, and 7.41 + 0.70 g weight, (n = 15), were exposed the required time to 10

weevil females per fruit. Fruits with eggs were offered to the parasitoids for 8 h.

A no-choice test was conducted during June 2005 and included eggs either 3.5,

27.5, 51.5 or 75.5 + 3.5 h old. 'Jalapefio' fruits (4.73 + 0.68 cm length, 1.48 + 0.26 cm

width, and 5.26 + 0.67 g weight, n = 10) were exposed seven hours to female weevils at a

density of one weevil per fruit. Fruits with eggs of the first age class were used without

storage while fruits in the other age classes were held at 22 + 20C and 50-60% RH for 24-

72 h before being offered to T. eugenii. Infested fruits were exposed to parasitoids for 2

h. Three randomly selected weevil eggs were removed under a dissecting microscope

from peppers in each age class to observe development. Heat units for each age,

including the time exposed to the wasp, were proportionally estimated using the average

temperature for each period. Heat units = (maximum temperature + minimum

temperature/2) threshold temperature. The threshold temperature was considered to be

11C.

For the choice test, an experimental unit was considered a group of 10 infested

peppers, five for each egg age class, offered at the same time to 20 T. eugenii females.









Five replicates were run. For the no-choice test, the experimental unit was five infested

peppers of each egg age class, which were offered simultaneously to 10 T eugenii

females, there were three replicates. Because parasitoid females were used only once in

each replicate in both assays and many parasitoids were needed, replicates were carried

out in different days. Parasitoids were 5-10 d old and they were randomly taken from the

colony in any replicate. Fruits were removed after exposure, and held in ventilated

containers until the emergence of insects. Chi-square analysis (PROC FREQ, SAS

Institute 2000) was used to test if parasitism was independent of age.

Role of the Oviposition Plug in Host Location

Choice and no-choice tests were conducted during May and June 2004 to determine

the importance of the oviposition plug to T. eugenii for finding and parasitizing host eggs.

Previous observations showed that the oviposition plug indicated the presence of an egg

in 91% of all cases (Chapter 2). Host eggs for these tests were obtained from groups of

three weevil females, 7-10 d old, placed in 250 mL plastic cups. Each group was

provided with a 'Jalapefio' pepper (4.65 + 0.48 cm length, 1.93 + 0.29 cm width, and

7.40 + 1.47 g weight, n=50) for 24 h. Subsequently, fruits were held at room temperature

(22 + 20C and 60-70% RH), before exposure to the parasitoid the next day. Only pepper

fruits with 10 eggs were used. Excess plugs and eggs (Fig. 3) were removed from the

fruits using fine-tipped forceps.

Each experimental unit was considered as one or two pepper fruits, depending on

the test, offered to an individual T. eugenii female 4-6 d old. Subject parasitoids were

randomly selected from the colony and used only once. Each female was placed

individually in 1.9 L plastic containers (12x11x15 cm) with two males to ensure mating.









Water and honey were provided as previously described. Infested fruits were exposed to

the parasitoids for 24 h.

























Figure 3. Longitudinal section of an infested 'Jalapefio' fruit showing a pepper weevil
egg and oviposition plug (ov-p).

Choice tests were conducted by offering two pepper fruits (10 weevil eggs each) to

individual T eugenii females. The oviposition plugs were removed from half of the eggs

of each pepper 24 h before the test. Plugs were removed under a stereoscopic microscope

using forceps, and a cotton swab was used to clean any visible residue. No-choice tests

were conducted by offering a single pepper fruit with ten weevil eggs, either with or

without oviposition plugs. Weevil eggs were later removed, placed in a drop of tap water

on a microscope slide (4 or 5 at once), and crushed under a glass cover slide. A binocular

composed microscope was then used to observe parasitoid eggs (100X magnification).

Fourteen replicates were run for each treatment, and data were analyzed using chi-square









tests to determine the differences between observed and expected values (PROC FREQ,

SAS Institute 2000).

Effect of Weevil: Fruit Ratio on Parasitoid Production

Cages (3.8 L) containing 20-30 T. eugenii females and 20-25 males, 1-3 d old, were

used as the experimental unit. The percentage of parasitism was compared between

'Jalapefio' fruits exposed 24 h to unsexed weevils (ratio 10:1 fruit), or exposed 24 h to

mated females (ratio 1:1 fruit). Daily during 10 consecutive days 13 infested fruits (4.44

+ 0.72 cm length, 1.47 + 0.23 cm width, and 4.83 + 1.10 g weight, n = 30) of each pepper

weevil ratio, exposed to weevil adults as above, were placed in the corresponding

parasitoid cage. Fruits were exposed for 24 h to the parasitoids the first and the last day,

but only 2.5 h the remaining 8 d. Fruits were removed and held in plastic containers until

emergence of insects. Three replications were run for each treatment during September

and October, 2004. Data were evaluated for normality (PROC CAPABILITY, SAS

Institute 2000) before the analysis, and statistical differences were determined using

student's t-distribution (PROC TTEST, SAS Institute 2000).

Life Cycle

'Jalapefio' pepper fruits containing pepper weevil eggs 18-40 h old were obtained

by exposing the fruits 24 h to a density of 10 weevils per fruit (5.29 + 0.80 cm length,

1.67 + 0.24 cm width, and 5.90 + 1.40 g weight, n = 10). An experimental unit consisted

of eight infested fruits exposed 2 h to T. eugenii in cages each containing 15 females and

20 males 5-7 d old. Fruits were then removed and incubated in ventilated containers at 27

+ 1IC, 50-60% RH, and 14:10 (L:D) photoperiod. Eight experimental units were run









concurrently. In addition, 10 infested pepper fruits from the same batch, but not exposed

to the parasitoids, served as control for the whole experiment.

To determine incubation time of T. eugenii 40 to 60 weevil eggs, usually one egg

per fruit of each experimental unit, were removed and dissected from fruits at 18, 20, 22,

and 24 h after parasitoid exposure. In addition, ten to 15 control eggs were dissected each

time to confirm the absence of parasitoids. Length and greatest width of parasitoid eggs

18-20 h old (n=33) were measured using a binocular microscope fitted with an ocular

micrometer calibrated with a stage micrometer (parallel lines at 10 [a).

The remaining pepper fruits (64 exposed and 10 unexposed) were held under

conditions described above for 9 days after parasitoid exposure. Peppers were then

dissected and all prepupae were removed, together with the completed or partially

completed pupal cell, and held in individual polystyrene cells (Multiwell, 24 well plates

with lid, Becton Dickinson and Co.). Cells were filled with paper towel packed into the

cells with no water to support the pupal cells, which were observed every 4 h during the

first 3 d, and then twice a day (9:00 AM and 6:00 PM) afterwards until the emergence of

adults. The Multiwell cells with the host and parasitoids were maintained at 27 + 1IC,

50-60% RH, and 14:10 (L:D) photoperiod, but the observations were made at room

temperature (220C).

Twenty unexposed and 160 exposed pepper weevil larvae were isolated as above,

but only data from those parasitoids that were observed emerging from the host or

spinning their cocoons and successfully reaching the adult stage are reported.

Development times were calculated for the egg, endoparasitic and ectoparasitic larval

stages, prepupa (spinning cocoon), and pupa. Stages were considered completed when









50% plus 1 individuals reached the next stage. The prepupal stage was considered to

occur from the cessation of larval feeding through the completion of the cocoon. Means

and standard deviations were calculated for each stage.

Fecundity

During September to December 2004, 10 adult T eugenii females 18 h old or

younger were placed individually in 3.8 L plastic containers. Three males were included

in each cage to ensure mating. Each cage was provided daily with 'Jalapefio' fruits

previously exposed for 24 h to pepper weevil females at the density of one pepper weevil

per fruit (4.60 + 0.45 cm length, 1.94 + 0.30 cm width, and 7.37 + 1.52 g weight, n = 46).

Weevil adults were provided water and honey as previously described. Five of the T

eugenii females were each provided every day from 9:00 AM to 6:00 PM with 12 fresh

peppers, each containing 4-8 pepper weevil eggs. The other five females were each

provided with two peppers fruits replaced every 1.5 h during the same period, so that the

total number exposed was also 12 fruits per female. Every day, half of the pepper weevil

eggs of each fruit were removed and dissected to count the number of T. eugenii eggs per

host using the methodology described above. The remaining eggs and fruits were held

until the emergence of T eugenii to estimate survival and sex ratio. The program

LIFETABLE.SAS (Maia et al. 2000) was used to estimate net reproductive rate (Ro),

generation time (T), doubling time (Dt), intrinsic rate of increase (rm), and finite rate of

increase (k) (Chapter 3) (Birch 1948, Maia et al. 2000, Southwood and Henderson 2000).

Frequency of superparasitism between treatments was compared using chi-square

analysis (PROC FREQ, SAS Institute 2000).









Adult Longevity

T. eugenii adults 12 h old or less were caged in two 1.8 L containers as follows: 1)

10 females and 10 males provided with water only, and 2) 10 females and 10 males

provided with honey and water. The wasps were observed twice a day (9:00 AM and 7:00

PM) and mortality was recorded until all died. The two treatments were replicated three

times. A comparison of longevity was also made between these females and ovipositing

females from the fecundity experiment. The Student's t test (PROC TTEST, SAS

Institute 2000) was used to make all comparisons.

Results

Surveys

About 50 kg of pepper fruits infested by pepper weevil larvae were collected under

natural conditions from Nayarit, Mexico. Weevil and parasitoid recovery varied with

location (Table 11), although fruits from all sites were severely infested. Although a

mean of 1.40 + 0.97 weevil per fruit was observed in the field from a random sample of

57 fruits, only an average of 0.73 weevils per fruit emerged in quarantine. Two genera of

Braconidae (including Triaspis eugenii) and one each of Pteromalidae, Eurytomidae and

Eupelmidae were recovered, accounting for only 2.85% of total parasitism (Table 11).

The dominant species recovered from both collecting trips was C. hunter, which

represented about 83% of emerged parasitoids.












Table 11. A. eugenii and parasitoids emerged from approximately 50 kg of infested hot peppers collected in the state of Nayarit,
Mexico, April and May, 2003.
Location (sites visited) Anthonomus eugenii Catolaccus hunter Triaspis eugenii Eurytoma sp. Eupelmus sp. Bracon sp.


Cafiada del Tabaco (3)
Los Corchos (2)
Puerta de Mangos (1)

Cafiada del Tabaco (1)
Los Corchos (5)
Los Medina(3)
Palma grande (1)
Villa Juarez (1)


Total


A p r il 2 0 0 3

1


1260
427
1484

151
698
251
237
226

4734


May
1
2


2003


1 2
2
1 1
4 1 1 1


4 5 2 6 1 1 0









Host-Age Range

Heat units were calculated for each age class to precisely determine which ones

could be parasitized (Tablel2, 13). T eugenii preferred younger eggs to older eggs in

choice tests (X2 = 5.88, df = 1, P= 0.015) (Table 12). Nevertheless, T eugenii was

equally able to parasitize eggs over four age classes when given no choice (X2 = 1.53, df

= 1, P = 0.674) (Table 13). The eyes and mandibles of pepper weevil larvae were visible

through the chorion in the most mature eggs before wasp exposure.

Table 12. Parasitism of pepper weevil eggs by T eugenii when given the choice of two
ages of eggs
Host maturity Parasitism
Heat units' Age (h) Not parasitized Parasitized
Number Percentage2
12.66 28.66 24 48 51 64 55.6

23.66 39.66 48 72 98 68 40.9

'Accumulated heat units based on 11 C threshold temperature
2X2 = 5.88, df= 1, P = 0.015

Table 13. Parasitism of pepper weevil eggs by T eugenii when given no choice of egg
age
Host maturity Parasitism
Heat units1 Age (h) Not parasitized Parasitized
Number Percentage2
0.10-4.66 0 7 6 13 68.4

12.45-15.66 24 31 10 29 74.3

23.45-26.66 48 55 16 26 61.9

34.45-37.66 72 79 16 30 65.2

'Accumulated heat units based on 11 C threshold temperature
2X2 = 1.53, df= 1, P = 0.674

Role of the Oviposition Plug in Host Location

Parasitism of pepper weevil eggs by T eugenii was clearly affected by the presence

or absence of the oviposition plugs (Table 14). Over two and a half more eggs were









parasitized when covered normally by an oviposition plug, compared to eggs over which

the plugs were removed in choice tests.

The predominance of parasitism in plugged eggs was just as evident under no

choice conditions (Table 15). Given no choice, T eugenii parasitized three times more

eggs covered normally by an oviposition plug, compared to unplugged eggs.

Table 14. Parasitism of pepper weevil eggs by T eugenii when given the choice of eggs
covered with an oviposition plug and eggs with no plug
Oviposition plug Parasitism
Not parasitize Parasitized Percentage1
Normal 69 71 50.7

Removed 114 26 18.6

'X2 = 31.94, df= 1, P < 0.0001

Table 15. Parasitism of pepper weevil eggs by T eugenii when given no choice of eggs
covered with an oviposition plug and eggs with no plug
Oviposition plug Parasitism
Not parasitize Parasitized Percentage1
Normal 51 89 63.6

Removed 111 29 20.7

'X2 = 52.73, df= 1, P < 0.0001

Effect of Weevil: Fruit Ratio on Parasitoid Production

When T eugenii females were exposed to 'Jalapefio' pepper fruits infested with

pepper weevil eggs by mated weevil females at a ratio of one weevil per fruit more

parasitoids (t= 5.26, df= 58, P < 0.001) and fewer weevil adults (t= 5.26, df= 58, P <

0.001) emerged per fruit, compared to when T eugenii were exposed to fruits infested by

unsexed weevils at a ratio of 10 weevils per fruit (Table 16). Percentage of arasitism was

similarly affected (t= 4.40, df = 58, P < 0.0001). Therefore, infesting pepper fruits with









one weevil female per fruit made more efficient use of weevils, weevil eggs, and fruits

than infesting pepper fruits with 10 weevils per fruit.

Table 16. T eugenii produced with two pepper weevil densities
Weevil adult density Insects per fruit Parasitism (%)
T. eugenii Weevils

Unsexed weevils (10/ fruit) 0.81 + 0.26 0.94 + 0.35 45.5 + 7.1

Mated weevil females (1/ fruit) 1.25 + 0.48 0.52 + 0.30 71.2 + 12.3


Life Cycle

The eggs of T eugenii were translucent and elongated with no evident surface

sculpturing. They were 246 + 33.6 [tm long by 73.4 + 16 [tm wide. The length included a

small pedicel of 31.2 + 5.7 utm long (Fig 4A). The majority of the eggs hatched 23.0 + 1

h after the 2 h exposure to T eugenii (Table 17). Emergence was completed (41 larvae of

42 eggs) after 25 + 1 h. After emergence and for a few more hours, first instar T. eugenii

were translucent, except for the large sclerotized head and large mandibles. The segments

of the body were nearly of equal size, except for the last one which was rounded and

slightly larger than the rest (Fig 4B). Some of the larvae observed on the microscope

slides appeared to be attacking unhatched parasitoid eggs within the host-egg.

The endoparasitic phase lasted almost 10 d and comprised more than half of the

total parasitoid developmental time (Table 17). Parasitized pepper weevil larvae, even

late 3rd instars, appeared to develop and to molt normally and had no evidence of

decreased activity, compared to the observed control. The parasitoid larva emerged from

the pepper weevil prepupa, usually after the completion of the pupal cell. The parasitoid

larva emerged through a hole made in the distal end of the abdomen of the host. Bending

the base of the head forward, it began to feed immediately on the weevil prepupa (Figure









5A). The last few abdominal segments of the parasitoid remained in the host at the

beginning of the ectoparasitic phase (Figure 5B). In about 8 h the parasitoid completely

consumed the host, except for the cephalic capsule (Table 17). The parasitoid then spun

its cocoon and pupated inside the pupal cell of the host, where it remained for 6-7 d.

Some parasitoid larvae removed from the pupal cell of the host and placed directly into

the polystyrene cells made partial cocoons; however, they failed to complete the cocoon,

and eventually died. Possibly, the lack of the pupal cell of the host caused dehydration.

The developmental times of males and females did not differ (Table 17), but it is

common to see in the laboratory that males emerge before females.

Table 17. Developmental times of T. eugenii in days (Mean SD) at 27 1C
Sex Egg Parasitic development Prepupa1 Pupa Total
Endo Ecto 9 or o 9 n= 25 9 n= 25
n= 197 9 n= 8 0 n= 9 n=14 6 n= 35 6 n= 35
Female 0.96 + 0.2 9.87 0.30 0.27 0.22 0.40 + 0.17 6.63 + 0.82 16.63 0.88

Male 0.42 + 0.18 6.40 + 0.52 16.36 + 0.88

Spinning cocoon

Adults emerged by chewing through the cocoon then through the fruit wall. The

emergence from infested peppers could be noticed as a small rounded hole. Adults were

no longer than 2.4 mm and completely black. The only evident difference between sexes

observed was the presence of the ovipositor in the female (Figure 6 C, D).




















































Figure 4. Immature stages of T eugenii: (A) Egg 22 h after oviposition, (B) First instar,
30-33 h after oviposition, with well developed mandibles.






















































Figure 5. Larva of T. eugenii. (A) Emerging from the posterior end of the host, (B)
Beginning to feed on the host even before emerging completely.


























0*


E~3


Figure 6. T. eugenii adults. (A) Female, (B) Male.


'9


S-L'
.ryl






~ ~i~i~iiil


~ ~ii~iliiil









Fecundity

The preoviposition period was shorter than 24 h for six often females, between 24-

48 h for three females, and between 48-72 h for one female. Little difference in the

oviposition pattern was observed between host exposure treatments (Fig. 7). Maximum

fecundity was reached on the 4th day after emergence and then gradually declined,

reaching zero by day 18. Females deposited 95% of their eggs by day 12 when hosts were

changed every 1.5 h, and 92% when hosts were left for 9 h. During that period, females

laid 29.63 + 9.8 and 33.13 + 12.2 eggs per day, respectively, for the 1.5 h and 9 h host

exposure periods. All females died by day 21 (Figure 7).

When hosts were changed every 1.5 h, females produced an average of 372.40 +

223.14 eggs, compared to 433.20 193.80 when hosts were left for 9 h; however, the

difference was not significant (t= 0.5, df = 164, P = 0.901).

Superparasitism was common with both treatments, although the proportion of

superparasitized hosts was significantly greater when T eugenii females were exposed to

infested fruits for 9 h, compared to when females were exposed to infested fruits every

1.5 h (64.4% vs 55.3%, X2= 7.95, df = 1, P = 0.0048). Superparasitism ranged from two

to nine eggs, and averaged 2.24 + 1.34 for the 9 h exposure period compared to 2.10 +

1.40 for the 1.5 h exposure treatment. These values were not significantly different (t=

1.76, df = 924, P = 0.0779).











50

45

40 \ / \ X
5\ \ Host changed every 1.5 h
35 -/ \
\ --*-- Host available per 9 h
30)




15 \

10 I A
0 / \

0 ..--------------------'- ^ *-. a--. --, -, -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Age in days


Figure 7. Fecundity of T. eugenii females at 27 + 2C either exposed to hosts changed
every 1.5 h (n=5) or exposed to hosts per 9 h (n=5).

The proportion of superparasitism and the numbers of emerged T. eugenii and

pepper weevils suggested strongly that more than half of the host eggs were not

dissected. Consequently, the proportion of superparasitism from the dissected host eggs,

and the survivorship of pepper weevil (90%) estimated in Chapter 2, were used to

estimate survivorship of the parasitoid. The estimate was made using the proportion of

superparasitism observed from the dissected hosts each day. Demographic parameters for

T. eugenii were estimated for the 9 h and 1.5 h treatments (Table 18). Values were not

significantly different between the two host exposure intervals according to Student's t-

test comparison, P < 0.05 (Maia et al. 2000).

Adult Longevity

Males and mated females with access only to water lived 2.3 + 0.7 d and 2.3 + 0.6

d, respectively, with no significant difference between the sexes (t=0.15, df= 58,

P=0.881). Mated females provided with honey lived significantly longer (24.7 + 10.6 d)









that did males (15.8 + 9.2 d) (t=3.47, df = 58, P < 0.001). Finally, females laying eggs

and provided with honey lived 16.5 + 3 d, which was significantly less than the 24.7 +

10.6 d for females not laying eggs (t=3.26, df = 38, P < 0.002).

Table 18. Demographic parameters and confidence limits ( 95%) for T. eugenii reared
on pepper weevil eggs in 'Jalapefio' fruits when parasitoid females were
offered hosts for different exposure intervals at 27 + 2C
Parameter1 Host changed every 1.5 h Host available for 9 h

Net reproductive rate (Ro) 102. 83 106.15

(8.47- 197.18) (26.89- 185.40)

Generation time in days 19.22 19.95

(G) (18.13-20.31) (18.90-21.00)

Intrinsic rate of increase 0.24 0.24

(rm) (0.20 0.29) (0.20 0.28)

Doubling time (Dt) 2.82 2.92

(2.24- 3.40) (2.37- 3.47)

Finite rate of increase (k) 1.28 1.27

(1.22 1.34) (1.21- 1.32)

Sex ratio 1:1 1:1

Difference between treatments was not significantly (Student's t-test for pairwise comparison,
P < 0.05, Maia et al. 2000).

Discussion

Surveys

The total number of parasitoids recovered from both trips was 139, most of which

emerged from weevil infested fruits collected in May. C. hunter and T. eugenii

represented 83% and 6.5%, respectively, but parasitism reached only 2.85%. These









numbers were lower than those previously reported from that region: 865 and 1 210

parasitoids (recovered of 45 and 80 kg of infested fruit by pepper weevil, respectively)

according to Mariscal et al. (1988) and Toapanta (2001). T eugenii was the dominant

species (84% and 88%, respectively), with levels of parasitism ranging from 29 to 40%,

respectively.

The destruction by hurricane Kenna and subsequent reduced acreage of the

growing pepper area in Nayarit during 2003, the possible damage to alternative hosts by

the hurricane, and the intensified use of insecticides during late months in the season, are

possible explanations for low levels of parasitism of the pepper weevil observed during

the surveys in 2003 (2.85%) compared to previous reports (29-40%) (Mariscal et al.

1988, Toapanta 2001). Nevertheless, the 115 C. hunter observed in the present study was

not very different from 87 and 102 individuals observed in the previous studies. C.

hunter is a generalist ectoparasitoid of more than 17 species of Curculionidae and

Bruchidae (Cross and Mitchell 1969, Cross and Chesnut 1971). It can survive as an adult

for more than 40 days and, in addition to carbohydrates, can feed on host larvae or,

sometimes, pupae to survive (Cate et al. 1990, Rodriguez et al. 2000). Thus, C. hunter is

well equipped to survive unstable environmental conditions. In contrast, the pepper

weevil is the only known host of T. eugenii. It has not been observed to host feed and

cannot survive as long as C. hunter, especially without a carbohydrate source. Thus, T

eugenii would have fewer resources to draw upon under rapidly changing conditions.

Host-Age Range

During choice tests, T. eugenii preferred younger eggs to older eggs. Age may be

related to host suitability (Godfray 1994). Because eggs may lack a cellular defensive

response to foreign intrusion (Askew 1971) or because, with the exception of eggs in









diapause, eggs rapidly change from storage nutrients to chemically more complex

embryonic tissue (Sander et al. 1985), parasitoids may prefer young eggs. These

explanations have been supported by authors who found that parasitism by some egg

parasitoids declines as the host ages (Strand and Vinson 1983a, b, Ruberson et al. 1987,

Reznik and Umarova 1990, Castillo et al. 2005). Nevertheless, this information has been

supported mainly in observations with parasitoids from Trichrogrammatidae, Scelionidae,

Mymaridae, and Eulophidae. They are idiobionts that complete their development in the

eggs, and they may have differences with parasitoids which develop as koinobionts, such

as egg-larval or egg-pupal parasitoids (Strand 1989, Vinson 1998, Godfray 1994, Quicke

1997).

In contrast to many idiobiont egg parasitoids, egg-larval or egg-pupal parasitoids

may lay eggs in a more advanced stage of host development (Clausen cited by Quicke

1997, Abe 2001, Quimio and Walter 2001, Kaeslin et al. 2005). Strand and Pech (1995)

suggested that egg parasitoids may evade the immune response of the host by

preferentially ovipositing in pregastrula embryos in which the immune system has not yet

become functional. However, Kaeslin et al. (2005) observed that all stages of Spodoptera

littoralis (Boisduval) eggs can be parasitized by Chelonus inanitus (L.), because the

behavior of the parasitoid larva or adult assure that the larva will be located in the

haemocoel of the host. When freshly deposited host eggs are parasitized, the parasitoid

hatches in the yolk and enters the host embryo either after waiting or immediately

through the dorsal opening. When host eggs are parasitized at 1 or 2 d old, the host

embryo is covered by an embryonic cuticle through which the hatching parasitoid larva









bores with its abdominal tip. When old eggs (2.5-3.5 d) are parasitized, the female

parasitoid oviposits directly into the haemocoel of the host embryo.

T. eugenii was able to parasitize young and old eggs, an ability which is shared

with other koinobiont species that attack eggs of their hosts but later emerge from larvae

or pupae (Clausen cited by Quicke 1997, Abe 2001, Quimio and Walter 2001, Kaeslin et

al. 2005). The physiological interactions between host and parasitoid are not completely

understood, but the ability to use either young or mature hosts represents a desirable

characteristic that increases probability of locating an appropriate host.

Role of the Oviposition Plug in Host Location

Host habitat location by parasitoids often involves chemical cues, as well as

physical and electromagnetic signals (Quicke 1997, Vinson 1976, 1998, Turlings et al.

1998, Havill and Raffa 2000). These signals include odors emanating from host frass,

symbiotic fungi, or host-damaged plants (Quicke 1997, Vinson 1976, 1998, Turlings et

al. 1998). Parasitoids attacking concealed larvae, such as fruit flies or wood borers, can

locate their hosts by detection of movement transmitted as vibrations in the substrate

(Godfray 1994, Quicke 1997). Such stimuli are not available to parasitoids of concealed

eggs. Such parasitoids could rely on olfactory signals from chemicals such as kairomones

of the host (e.g. accessory gland secretions), or from tactile and chemical signals

emanating from mechanical damage such as oviposition wounds (Strand 1989, Godfray

1994). Once a probable site of a host has been located, parasitoids respond to less volatile

substances, or to tactile cues where closer antennation and the ovipositor might play more

important roles (Godfray 1994, Quicke 1997).









This study demonstrated that the host oviposition plug is important for parasitoid

host location. A high proportion, around 80%, of pepper weevil eggs were not parasitized

by T eugenii when the oviposition plug was removed. The oviposition plug, then,

provided means for the parasitoid to detect and, subsequently, to parasitize its host. It is

possible that the antennae were used to detect host kairomones found in the plugs,

because drumming was observed to increase as soon as the female was close to the plugs.

The response of some parasitoids to these substances has been demonstrated in more than

one species (Quicke 1997, Turlings et al. 1990, 1998, Vinson 1998). However, the design

of the present study did not permit the evaluation of other sources of stimuli, such as

physical or visual characteristics of the plugs, which may be important in the host

detection and oviposition by T eugenii. More studies need to be done in this area.

When the pepper weevils were reared under crowded conditions, the proportion of

eggs lacking ovipositions plugs increased (Chapter 2). For this reason, it was more

efficient to rear the pepper weevil by exposing pepper fruit to one female weevil per fruit

rather than 10 unsexed weevils per fruit. Additionally, less feeding damage on pepper

fruits by weevils helped to diminish the number of rotten fruits in the colony (E.

Rodriguez, unpublished data).

Life Cycle

T. eugenii is a member of the tribe Brachistini in the subfamily Helconinae and the

family Braconidae. The members of Brachistini are cosmopolitan and are known as egg-

larval endoparasitoids of different families of Curculionidae and Bruchidae (Beirne 1946,

Martin 1956, Shaw and Huddleston 1991, Sharkey 1997). Additionally, there is at least

one species, Nealiolus curculionis (Fitch), which has been reported parasitizing early

larvae of the sunflower stem weevil, Cylindrocopturus adspersus (LeConte) (Charlet and









Seiler 1994, Charlet et al. 2002). Early reports confirmed and described the endo- and

ectoparasitic phases of the immature stages of some Triaspis species, such as T

vestiticida and T caudatus (Berry 1948, Obrtel 1960); however, few species of this genus

have been studied in detail (Sharkey 1997).

Females of T eugenii were able to lay eggs from the first day of life. This behavior

is characteristic of many koinobiont parasitic wasps which do not need to consume

protein-rich host hemolymph to initiate oogenesis (Quicke 1997). The parasitoid egg was

deposited in the host egg, where it hatched in less than 24 h. No parasitoids were

recovered from larvae (4-5 d after infestation). The endoparasitic phase thus initiated in

the egg and continued until the prepupal stage of the host. Only one parasitoid emerged

from each host egg. The first larval instar possesses large mandibles. The presence of

such mandibles in the early instars of solitary parasitoids has been suggested as useful for

physical defense against either the same or different species in case of superparasitism or

multiple parasitism, respectively (Clausen 1940, Godfray 1994, Quicke 1997, Ming et al.

2003). In contrast, physiological suppression by asphyxiation may be a common

mechanism by which established larvae eliminate younger competitors (Fisher 1971,

Godfray 1994, Quicke 1997).

Egg-larval parasitoids occur in several subfamilies of Braconidae (Alysiinae,

Helconinae, Ichneutinae), but members of the subfamily Cheloninae more often use this

strategy (Clausen 1940, Shaw 1997). The potential as biological control agents of some

members of this subfamily could be one reason for the availability of information on

species such as Chelonus sp. nr. curvimaculatus Cameron, an egg-larval parasitoid of the

pink bollworm, Pectinophora gossypiella (Saunders) (Hentz et al. 1997), and Chelonus









inanitus, an egg-larval parasitoid of Spodoptera littoralis (Kaeslin et al. 2005). The

information reported in the present study confirms similarities in the life cycle of T

eugenii with that of these species of Cheloninae, and with that of Triaspis vestiticida

(Berry 1947). In addition, the present study describes life table parameters of T. eugenii,

information which is needed to establish the potential of this species.

Potential of T. eugenii as a Biological Agent of the Pepper Weevil

Despite the fact that five species and at least five other genera of parasitoids have

been collected from the pepper weevil (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934,

Cross and Chesnut 1971, Mariscal et al. 1998, Toapanta 2001), T. eugenii and

Urosigalphus sp. are the only known species that parasitize eggs of this pest.

Urosigalphus sp. (14 females and 9 males) was collected by P. Stansly during July 2003

in Oaxaca, Mexico, and was held in quarantine facilities and reared in the same

conditions that T eugenii. However, the parasitoids produced only males in its offspring,

making it impossible to establish a colony (E. Rodriguez, unpublished data).

The egg of the pepper weevil could be considered the stage more ecologically

susceptible to be parasitized, because it is defenseless and is deposited close to the

surface in the pepper fruits. Therefore, the ability to parasitize pepper weevil eggs could

be one of the most important biological advantages of T. eugenii. Because T. eugenii can

reach the host before the larva migrates deep inside into the fruit, it could attack the

pepper weevil in any cultivar, regardless of fruit size.

The reproductive capacity of T eugenii is another important biological

characteristic of the parasitoid. Females were able to produce an average of 402.8 + 199.5

eggs. Females laid more than 90% of their eggs during the first 12 days of life, depositing

31.4 + 22.6 eggs per female per day.









Superparasitism was common, although the proportion of superparasitized hosts

observed was greater where all infested fruits were left for 9 h rather than being changed

at 1.5 h intervals (64% versus 55%). T eugenii lays eggs individually in only 28 sec (E.

Rodriguez, unpublished data). The short time required for oviposition coupled with the

longer exposure period may explain the higher rate of superparasitism. However, it does

not explain the apparent inability of the parasitoid to distinguish parasitized hosts, even in

the presence of nonparasitized hosts.

Decreasing exposure time from 9 h to 1.5 h reduced superparasitism. Further

reduction of exposure time would be impractical within the context in the rearing system

developed here. Additional manipulations may be necessary such as more hosts per

female, fewer hosts per fruit, and larger cage size. On the other hand, superparasitism is

relatively common in laboratory conditions in other rearing systems of parasitoids, such

as Chelonus sp. nr. curvimaculatus (Hentz et al. 1997), or Catolaccus grandis Burks

(Morales-Ramos and Cate 1993); however, superparasitism does not occur often in the

field according to the same authors. Given these considerations, the reproductive

potentials of the pest and parasitoid were compared using fecundity and demographic

parameters of the pepper weevil (Chapter 2) and of T. eugenii using the data set of the

combined 10 T. eugenii females. It was assumed that superparasitism does not occur

under field conditions (Table 19).

The fecundity of the pepper weevil (355.28 + 167.51) and that of T eugenii (402.8

+ 199.55) with the same sex ratio (0.5) and survivorship (0.9) indicated no differences in

the net reproductive rate (Ro) (P = 0.82). Nevertheless, the remaining parameters favored

T. eugenii (P < 0.00001). The intrinsic rate of increase (rm), which might be one of the









most important parameters to compare populations because it combines Ro and

generation time (T) (rm = loge Ro / T, Birch 1948, Southwood and Henderson 2000), was

almost twice as great for T eugenii as for the pepper weevil. Therefore, the important

pest/parasitoid difference was generation time.

Table 19. Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and
14 pepper weevil adults at 27 + 2C
Parameter Pepper weevil' T. eugenii1

Net reproductive rate (Ro) 158.10 167.1

(115.1 201.1) (92.7- 241.3)

Intrinsic rate of increase (rm) 0.14 0.26

(0.13 -0.16) (0.24 0.28)

Generation time (T) 35.4 19.6

(30.4 40.4) (19.0 20.2)

Doubling time (Dt) 4.8 2.6

(4.3 5.4) (2.4- 2.8)

Finite rate of increase (k) 1.2 1.3

(1.1- 1.2) (1.2- 1.3)

1The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively

T. eugenii has been cited as the most abundant parasitoid of the pepper weevil in

field samples from Nayarit, Mexico, a region where the pepper weevil occurs naturally

(Mariscal et al 1988, Schuster et al. 1999, Toapanta 2001). It parasitizes the egg of its

host and has almost twice the potential for increase than the pepper weevil. These

attributes make T eugenii an excellent candidate for biological control of the pepper

weevil.














CHAPTER 4
RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez
(HYMENOPTERA: BRACONIDAE) IN FIELD CAGES AND IN THE FIELD

Introduction

The pepper weevil is considered one of the key pests in peppers (Capsicum spp.) in

many growing regions of the United States, Mexico, and Central America, and only

cultural and chemical tactics are presently available for its control (Elmore et al. 1934,

Riley and King 1994, Mariscal et al. 1998, Schuster et al. 1999). Since the pepper weevil

was reported in Mexico (Cano and Alcacio 1894) and in the United States (Walker 1905,

Pratt 1907, Elmore et al. 1934), no viable biological control strategy has been developed

to manage the pest.

Some reasons for the lack of successful biological control of the pepper weevil may

include: (1) a paucity of natural enemies recorded from this pest (Pratt 1907, Pierce et al.

1912, Elmore et al. 1934, Cross and Chesnut 1971, Clausen 1978), (2) limited impact of

those parasitoids (Elmore and Campbell 1954, Genung and Ozaki 1972), (3)

incompatibility of natural enemies with insecticides used to control pepper weevil, and

(4) limited biological control research because of the relatively small economic

importance of pepper compared with other crops (Riley and King 1994).

Recently Mariscal et al. (1998) identified nine different species of parasitoids

attacking pepper weevil larvae in the state of Nayarit, Mexico, which is located in the

central pacific coast and which is a likely center of origin of the pest. The most abundant

of these parasitoids was the braconid Triaspis eugenii Wharton and Lopez-Martinez,









which attained 18-40% parasitism in the field (Mariscal et al. 1998, Schuster et al.1999,

Toapanta 2001). These relatively high levels of parasitism, complemented with the

continual presence of T. eugenii during surveys between 1997 and 2003, and with its

ability to attack pepper weevil eggs, renewed interest in biological control to combat the

pepper weevil (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001).

T. eugenii is a solitary egg-prepupal parasitoid of the pepper weevil. Eggs are

deposited into pepper weevil eggs, and hatching larvae feed as endoparasitoids; however,

larvae later emerge from the host prepupa and complete development as ectoparasitoids

(Chapter 3). The habit of attacking the egg, located close to the fruit surface, rather than

the larva that burrows deep into the fruit, in addition to its reproductive superiority to

pepper weevil, make T eugenii a good candidate for biological control of the pest.

Nevertheless, the reproductive potential in the field could be influenced by many factors,

including the ability of the parasitoid to locate host and food, and the influence of

environmental conditions. Therefore, the evaluation of field releases is an indispensable

step in assessing the effectiveness of the parasitoid as a biological control agent.

T. eugenii cannot survive for more than two or three days at 27 + 1IC without a

carbohydrate supplement such as honey. Under field conditions, many parasitoids use

nectar or honeydew for this purpose (House 1977, Powell 1989, Baggen and Gurr 1998).

It cannot be assumed that these sources would always be adequate in or near every

pepper crop. Many parasitoids and predators also use plant volatiles to locate the host

plants of their hosts or preys (Vinson 1976, Alphen and Vet 1989, Godfray 1994, Quicke

1997). The volatiles often are released from damaged plants (Turlings et al. 1990, 1995,

1998, Powell et al. 1998, Takabayashi et al. 1998, Havill and Raffa 2000, James and









Grasswitz 2005). There is no information about the importance of weevil damage in host

location or foraging behavior of T. eugenii within a patch. This information could be used

to improve the effectiveness of establishing the parasitoid in the field. The objectives of

these studies were to assess the ability of T. eugenii to survive and parasitize the pepper

weevil in large field cages and in the open field, and to evaluate the effect of honey

supplements and weevil damage on this ability.

Materials and Methods

Insects

Pepper weevil and T. eugenii adults were obtained from colonies maintained at the

SWFREC in Immokalee, FL, as described in Chapters 2 and 3. Mated weevil females, 8

to 10 d old, and parasitoids 3 d old or younger were randomly chosen from the colonies

for release into field cages. Parasitoids that were released in the open field were 4 d old or

younger.

Field Cages

Experiments were conducted in two screenhouses 7.3 x 3.6 x 3.65 m, with sides of

50 x 24 mesh screen (266 x 818 [tm openings) and roofs of polyethylene (Figure 8A).

Each screenhouse was divided into 2 large cages 3.65 m in length by an organdy cloth

partition. A zipper was sewn into the partition to serve as a door. Each cage represented

an experimental unit (Figure 8B). Temperature and humidity were recorded during assays

using HOBO data loggers (Onset Computer Corporation, Bourne, MA).












































































-


Figure 8. Field cages used at the SWFREC. (A) General view of a unit containing two

cages. (B) Organdy partition and zipper between two experimental units.


_r_
--
-rS

L-









Effect of Honey Supplement

Five pots 7.8 L, each containing two 'Jalapefio' pepper plants, were placed in each

cage. Plants were 20.6 + 5.2 cm tall (n=10) and were spaced 35 cm apart. At the time of

the evaluations, plants had 4.0 + 2.4 fruits smaller than 4 cm in length. To provide natural

host finding conditions for T eugenii, two pepper weevil adults were confined in organdy

sleeves on floral buds on each of two plants (in the same pot) per cage two days before

experiments commenced.

Honey was offered in lines and replaced every other day on two plastic yellow

cards (5 x 8 cm) which were protected from sunlight by a cork roof (15 x 22 cm)

supported by wire mesh (Figure 9A). A pepper flush damaged by pepper weevil adults

was placed in a 28 mL plastic cup filled with water (Chapter 3) and attached to the wire

under the cork roof to attract parasitoids to the source of carbohydrates (Figure 9B). One

of these devices (wasp refuge) was suspended 30 cm above the plants in each cage.

The experiment had two treatments, a refuge with honey and a refuge without

honey, which were replicated four times. Because replications were developed at

different times, each screenhouse containing both treatments was considered a block. The

two weevil damaged plants per cage of each block were replaced with newly damaged

plants prior to the release of T. eugenii. Using a fine brush, 10 T. eugenii females 3 d old

or younger were placed onto the pepper flush of the refuge of each cage (Figure 9B),

which was located 1.6 m from the infested fruits. Refuges and pepper flushes were

observed for 5 sec at 1, 2, 3, 8, 24, and 48 h after release to check for the presence of

parasitoids. Parasitism was evaluated daily in each cage for 3 d by hanging two infested

fruits per each of the damaged plants. The fruits contained a total of 20 weevil eggs 24-40

h old.

















































Figure 9. Honey-wasp-refuge. (A) General view of the set up. (B) Using a unit as T
eugenii release point.

After 8 hours (9:00 AM to 5:00 PM), the exposed fruits were removed and

dissected in the laboratory to assess the percentage of parasitism and superparasitism

(Chapter 3). A new group of infested fruits was hung in the same location at the same

time the next morning. Each block was replicated four times and each cage received the









alternative treatment for the following replication to nullify an influence of cage

orientation. Chi-square analysis (PROC FREQ, SAS Institute 2000) was used to test

whether parasitism was independent of the presence of food (honey).

Survival

Survival of T. eugenii was evaluated using females confined in 250 mL plastic cups

that had 3 cm diameter organdy sealed holes in the tops for ventilation (Chapter 2, Fig.

1). Cups were placed inside the canopy of potted plants to avoid direct sunlight. Five T

eugenii females 3 d old or younger per cup represented an experimental unit. Treatments

were cups without honey or cups with honey streaked every 2 or 3 d on the internal side

of the cups. Survival was checked every 1, 3, 9, 24, and 32 h, and then was checked each

24 h thereafter until all parasitoids were dead. Five replicates were performed in a single

block. Student's t-test (PROC TTEST, SAS Institute 2000) was used to evaluate

differences between treatments.

Effect of Host-Damaged Plants

Twenty 7.8 L pots, each containing two 'Jalapefio' pepper plants were placed in

each cage. The pots were spaced 30 cm apart and plants were 40 + 14.3 cm tall with 15 +

7.8 fruits less than 4 cm long at the beginning of evaluations. Treatments in this assay

were non-damaged plants and weevil damaged plants replicated five times. Because

replications were performed at different times, each screenhouse containing both

treatments was considered a block. Plants were noticeably larger and more mature during

the last two blocks, and the presence of open flowers which could be a source of nectar

was estimated from five plants per cage. At the same time, plants infested with aphids

and leafminers in these two blocks were redistributed among cages to maintain a

homogenous environment. At the end of each replication, plants used to support fruits









with hosts were replaced, and cages received the alternative treatment. A "Jalapefio' plant

was damaged by using an organdy sleeve to confine three mated females (8-10 d old) on

one terminal with floral buds 48 h prior to releasing T. eugenii. The damaged plant was

used to hang infested fruits with pepper weevil eggs daily as above. The same procedure

without weevils was followed for the control. Five T eugenii females 3 d old or younger

were then introduced into each cage, using a fine brush to place the parasitoids onto the

foliage of the potted plants furthest (3.6 m) from infested fruits. Previous observations

indicated that this number of wasps was sufficient to detect a response.

Parasitism was evaluated daily in each cage for two days by hanging five

'Jalapefio' pepper fruits containing a total of 25 pepper weevil eggs, 24-40 h old, in a

random pattern on the previously infested plant or in the previously designated plant,

which was placed in an equivalent position among the 20 plants, in undamaged

treatments. Infested fruits were held for 9 h as in the previous experiment and dissected to

check parasitism. In addition, these fruits were observed for 20 sec at 10, 20, 40, 60, 120,

and 180 min after releasing the parasitoids. Cage walls were also observed for parasitoids

for 30 sec at 1, 2, 3, 8, 24, 32, and 48 h after release. A chi-square test (PROC FREQ,

SAS Institute 2000) was used to compare parasitism in cages with and without weevil

damaged plants. The numbers of parasitoids observed in infested fruits were compared in

cages with and without weevil damaged plants using student's t test (PROC TTEST, SAS

Institute 2000). Before analysis, data were transformed using the square root of y + 0.5 to

satisfy assumptions of normality (Sokal and Rohf 1969). Student's t test was applied also

to data on wasps observed on cage walls after verifying normality of the data (PROC

CAPABILITY, SAS Institute 2000). All means are reported in the original scale.









Field Release and Establishment

On June 17, 2004, 100 T eugenii females and 70 males 4 d old or younger were

released at the SWFREC in Immokalee, FL. Parasitoids were released from 7:30 to 8:30

AM using a fine brush to place them on the foliage of pepper plants that were at the end

of the fruiting cycle. Parasitoids were released on six 100 m rows of 'Jalapefio' pepper.

Three hundred randomly selected terminals were observed to assess the adult pepper

weevil population. No flowers or buds were seen and fruits were large (7.68 + 1.46 cm

length, 2.77 + 0.73 cm width, n=10) and mature. Fruits (n=10) were dissected to estimate

the infestation of weevil larvae. The crop was destroyed a week after the release. Before

eliminating plants, 6 kg of fruits were collected and held in an organdy fabric cage (60 x

60 x 60 cm) at 27 + 2C to allow weevil or parasitoid adults to emerge. The number of

collected fruits was estimated by weighting 20 fruits.

During January to April of 2005, T eugenii was released five times on 90 m row of

approximately 400 'Jalapefio' plants of different ages maintained at the SWFREC (Table

20). Parasitoids 4 d old or younger were placed with a fine brush on floral buds, or

foliage, in groups of 5-10 females at 10 m intervals along the row.

Table 20. Releases of T eugenii during 2005 at the SWFREC in Immokalee, FL.
Date T. eugenii

January 10 100 80
February 8 80 40
February 18 35
March 25 60 50
April 20 80 60
Total 355 230

Before each release, 10 immature pepper fruits with weevil feeding punctures were

collected and dissected to confirm the presence of weevil eggs. The pepper weevil









infestation was also estimated by checking 50 terminals of pepper plants just prior to each

parasitoid release. Fruits were harvested every 1- 2 wk, and peppers with weevil feeding

punctures or chlorotic calyxes were held in the laboratory until emergence of weevil or

parasitoid adults. On March 25, three honey-wasp-refuges were established in the field

and 60 parasitoid females and 50 males, 4 d old or younger, were released at those points

at 5:00 PM. Five second observations at those honey-wasp-refuges were made at 1, 15,

and 24 h after releasing in each refuge to check for the presence of parasitoids.

During the summer of 2005 at the SWFREC, 10 potted pepper plants 5-6 months

old and with mature fruits present were transplanted 10-20 m from a 'Jalapefio' plot

severely infested by the pepper weevil during the previous spring. These plants were

watered and fertilized during that season. In August 8, 2005, 70 fruits from those plants,

and 15 more from a greenhouse nearby, were collected by P. Stansly. Fruits were held at

27 + 2C until the emergence of weevil or parasitoid adults. During the fall of 2005,

experimental 'Jalapefio' peppers were destroyed at the SWFREC by hurricane Wilma. No

peppers were grown in the field during spring of 2006 at the SWFREC, but 25-30 potted

(7.8 L) 'Jalapefio' plants were kept outdoors during spring 2006 and any pepper fruit with

feeding punctures or chlorotic calyxes were collected to attempt further recovery of T.

eugenii.

Results

Effect of Honey Supplement

T. eugenii found and parasitized pepper weevil eggs in the presence or absence of

honey in the wasp-refuge during the first day of release. Eggs from the second and third

days were not parasitized. No difference in parasitism was found between refuges with









honey (94.3%) or without honey (82.3%) (X2 = 2.29, df = 1, P = 0.1299). After releases,

some parasitoids remained a few minutes in the wasp-refuge searching the pepper

flushes, whether honey was present or not, but soon left. No parasitoids were

subsequently observed at any of the refuges.

Survival

Parasitoid females in cups with honey survived longer (3.65 + 2.06 d) than

parasitoids without honey (0.62 + 0.56 d) (t = 7.11, df = 24, P < 0.0001). In the presence

of honey, more than 50% of the females were alive at day four, and the last females died

at day seven. In contrast, females without honey died in an average of 1.3 days (Fig. 10).


6

5 --Honey
S- -A-- No honey

U)
S3 \3- *



1 \

0 A
0.04 0.13 0.37 1 1.3 2 3 4 5 6 78
Days

Figure 10. Survival of T. eugenii adults in plastic cups with and without honey, when the
cups were placed inside the canopy of pepper plants, 25.4 + 4.90C, 67 + 19%
RH.

Effect of Host-Damaged Plants

T. eugenii females were able to find and to parasitize pepper weevil eggs in pepper

fruits hung in damaged and undamaged 'Jalapefio' plants; however, the level of

parasitism was higher in fruits hung in damaged plants (57.6%) than that in fruits hung in









undamaged plants (26.4%) (X2 = 24.97, df = 1, P = 0.001). No parasitism was observed

on eggs in fruits hung in cages a day after the release of parasitoids.

Overall, the numbers of T eugenii adults observed on fruits hung on plants

damaged by the pepper weevil decreased with time (Table 21). More T. eugenii females

were observed on fruits hung in plants damaged by the pepper weevil 10 and 20 min after

release, compared to the number observed on fruits hung on undamaged plants (t= 2.45,

df = 8, P = 0.040). Fewer parasitoids were observed at 40 and 60 min after release, with

no differences between treatments. No T. eugenii adults were observed on fruits hung on

undamaged plants after 60 min, or hung on any plant after 120 min. However, parasitoids

were seen on the upper wall of cages during the entire 48 h observation period. The

number decreased with time; 3.4 + 0.9 versus 3.6 + 0.5, respectively for damaged and

undamaged plants at 1 h; 2.2 + 0.8 versus 2.0 + 1.2, respectively at 24 h; and 0.4 + 0.5

versus 0.6 + 0.9 respectively at 48 h. There were not differences between treatments at

any time (t-test, P < 0.05).

Table 21. Number of T eugenii adults released in field cages and subsequently observed
on pepper fruits (n=5) hung on pepper plants either damaged or undamaged by
the pepper weevil, 28.2 + 4.7C, 55 + 9% RH
Time after releases Adults observed on fruits of pepper plants t-test
(min) damaged by weevil undamaged (P < 0.05)
10 2.0 + 1.22 0.4 + 0.55 *
20 2.0 + 1.22 0.4 + 0.55 *
40 1.4 + 0.89 0.8 + 0.84 ns
60 0.6 + 0.89 0.6 + 0.89 ns
120 0.2 + 0.45 0
180 0 0









Field Release and Establishment

At the time of the first release of parasitoids on 17 June 2004, 3.3 weevil adults

were observed per 100 terminals, and 1.1 + 0.57 weevil larvae were seen per fruit (n=10),

although no weevil eggs were detected. An estimated of 731 pepper fruits were collected

before the crop was destroyed, from which 380 adult weevils but no wasps emerged.

Environmental conditions during T eugenii releases at SWFREC during 2005 are

shown in Figure 11. Pepper weevil adults were found in the crop in excess of one adult

per 100 hundred terminals at the time of four of the five releases of T eugenii, and more

larvae than eggs were detected in the fruits (Table 22). Overall, the number of fruits

damaged by pepper weevil larvae and the number of emerged weevil adults increased

through time (Table 22). No T eugenii adults were observed in honey-wasp-refuges

following releases of the parasitoid during March 25.




100
90
80
70
60
050
40
30
S20A
E
I)10


Month 2005


Figure 11. Daily temperatures and humidity during T. eugenii field releases on 'Jalapefio'
pepper at the SWFREC in Immokalee, FL, 2005.









T. eugenii emerged for the first time on April 14 from infested fruits collected on

April 6. Infested fruits were then collected almost every week, and parasitoids were

recovered in low numbers through May 2 (Table 23). A few Catolaccus hunter adults

also emerged. T. eugenii and C. hunter together caused 6.7% parasitism. The pepper row

was eliminated during the last week of May, 2005.

Table 22. Adults per 50 terminals, and the number of weevil lifestages per 10 fruits, 1 or
2 d before field releases of T. eugenii on 'Jalapefio' pepper plots at SWFREC,
Immokalee, FL, 2005
T. eugenii release No. of pepper weevils
(date) Adults Eggs Larvae Emerged adults
January 10 0 0 0 0
February 8 2 0 12 12
February 18 1 0 2 148
March 25 3 2 5 300
April 20 1 2 7


Table 23. Parasitoid and pepper weevil adults recovered from 'Jalapefio' pepper
following field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005
Emergence Triaspis eugenii Catolaccus hunter Pepper weevil

April 1 180
April 14 6 10 4 1 147
April 19 1 1
April 25 5 8 2 535
April 30 20 5
May 2 9 5 206
Total 41 28 7 1 1068


Pepper weevil adults emerged from the field sample and greenhouse samples

collected by P. Stansly during the summer 2005 at the rate 0.9 and 0.53 weevils per fruit,

respectively. However, no T. eugenii emerged. No pepper weevils were detected in the









potted 'Jalapefio' plants established at the SWFREC by the time sampling was terminated

on April 14, 2006.

Discussion

Effect of Honey Supplement

T. eugenii searched the pepper flushes offered in the wasp refuges for a few

minutes but left, apparently not to return. Parasitism did not differ between honey and no

honey treatments, and was observed only during the first day of exposure.

As expected, survival of T eugenii adults was greater when adults were caged in

plastic cups with honey. Nevertheless, longevity of adults was drastically reduced to 3.7 d

in the plastic cups, compared to 16.5 d for ovipositing females in the laboratory at 27 +

1C (Chapter 3). The difference on survivorship may be due to heat stress that might have

occurred in the cups or the field cages.

It is known that floral and extrafloral nectars are indispensable sources of

carbohydrates for most adults of Hymenoptera (House 1977, Powell 1989, Baggen and

Gurr 1998, Kogan et al. 1999). Availability of these foods will affect longevity,

fecundity, and parasitism (Powell 1989, Idris and Grafius 1995, Baggen and Gurr 1998).

It is expected that efficiency, biological potential and survival of parasitoids that are

dependent upon these sources of energy to survive could be limited in monocultures that

provide those resources only for a limited time (Altieri 1992, Kogan et al. 1999). In the

field cages, T. eugenii in the plastic cups died in less than 48 h without a carbohydrate

source. Offering honey in the wasp-refuges did not appear to increase foraging time.

However, it is possible that food was not a limiting factor in the field cages. Parasitoids

might have used honey or left the patch before any carbohydrate was required.