MORPHOLOGY,HISTOLOGY, AND FINE STRUCTURE Morphology and Development of Immature Stages of Fidiobia dominica (: : Sceliotrachelinae)

1 2 2 JOSEP-ANTON JACAS, JORGE E. PEN˜ A, AND R. E. DUNCAN

Ann. Entomol. Soc. Am. 100(3): 413Ð417 (2007) ABSTRACT The external morphology and development of immature stages of Fidiobia dominica Evans & Pen˜ a (Hymenoptera: Platygastridae), a Neotropical solitary endoparasitoid of the eggs of Diaprepes spp. (Coleoptera: Curculionidae), are reported. This species behaves as a solitary idiobiont endoparasitoid. It has a stalked egg and two instars. The Þrst instar is cyclopoid and seven-segmented, whereas the second instar is hymenopteriform and 11-segmented. Mandibulae are conspicuous in both instars. On completion of the larval development, the host egg turns amber transparent, making parasitized eggs easily recognizable. The pupa is exarate. F. dominica is a protandrous species and once emerged, males help females to emerge.

KEY WORDS Diaprepes abbreviatus, biological control, egg

Fidiobia dominica Evans & Pen˜ a (Hymenoptera: (Clausen 1940, Askew 1971, Masner 1995, Austin et al. Platygastridae) is a solitary egg endoparasitoid that 2005). The genera of this subfamily that parasitize attacks at least two species of root weevils, Diaprepes coleopteran eggs are solitary idiobiont endoparasi- doublerii Gue´rin and Diaprepes abbreviatus (L.) (Co- toids and are thought to develop in a way similar to leoptera: Curculionidae), indigenous to the Carib- that of Scelionidae (Masner 1995). Although the Þrst bean region (Evans and Pen˜ a 2005). D. abbreviatus instar of some platygastrids is more or less hymenop- was introduced into Florida in 1964 (Woodruff 1968) teriform, Þrst instars of many species are extraordinary and since has become a serious pest of citrus through- in appearance and superÞcially resemble cyclopoid out much of central and southern Florida, and it is copepods (Clausen 1940, Askew 1971). When fully currently spreading in the citrus-growing regions of grown, these larvae are extremely bloated and ovoid. Texas and California (Grafton-Cardwell et al. 2004). A The number of instars seems to vary from one to three, lack of native egg for this weevil in citrus depending on species (Clausen 1940, Masner 1995, orchards in Florida (Hall et al. 2001), and past failures Sampson et al. 2006). The incomplete knowledge on of classical biological control (Beavers et al. 1980), this important group of natural enemies compelled us have triggered renewed efforts to introduce, release, to study and provide basic information on the external and evaluate candidate egg parasitoids from the Ca- morphology and development of immature stages of F. ribbean region into Florida (Pen˜ a et al. 1998, Pen˜ a and dominica. Amalin 2000, Hall et al. 2002, Jacas et al. 2005, Castillo et al. 2006). One of these candidate species, F. do- Materials and Methods minica, a platygastrid collected in Dominica from D. doublerii egg masses, was subsequently introduced Specimens used in this study were obtained from into Florida in April 2003, and it has been reared a colony initiated from individuals of F. dominica through Ϸ50 generations on D. abbreviatus eggs in the imported from Dominica (Evans and Pen˜ a 2005). quarantine facility in Homestead, FL (Evans and Pen˜ a The colony was maintained on D. abbreviatus eggs 2005). The release of this parasitoid was approved in in the insectary facilities of the University of Florida June 2006, and releases are under way at various sites at the Tropical Research and Education Center in Florida as part of the classical biological control (TREC) at 25.6 Ϯ 1ЊC, Ϸ65% RH, and a photoperiod program against this pest. of 12:12 (L:D) h. Voucher specimens of F. dominica Little is known about the biology and the immature are deposited in the Florida Collection of , morphology of Platygastridae and even less so about Gainesville, FL, U.S. National Museum of Natural His- that of species in the subfamily Sceliotrachelinae tory (USNM), and in the Canadian National Collec- tion, Ottawa, Canada. 1 Corresponding author: Universitat Jaume I, Departament de Stock Colonies. Adult D. abbreviatus root weevils Cie`ncies Agra`ries i del Medi Natural, Campus del Riu Sec, E-12071- were collected from the foliage of ornamental trees in Castello´ de la Plana, Spain (e-mail: [email protected]). nurseries around TREC, Homestead, FL (25Њ30 N, 2 Department of Entomology and Nematology, Tropical Research and Education Center, University of Florida, 18905 SW 280th St., 80Њ30, 1-m altitude). The weevils were placed in 30- by Homestead, FL 33031. 30- by 30-cm Plexiglas cages with water and foliage of

0013-8746/07/0413Ð0417$04.00/0 ᭧ 2007 Entomological Society of America 414 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 3

a food source, and the open end of the test tube was covered with two-ply Kimwipe (Kimwipes EX-L, Kimberly-Clarke, Neehah, WI) secured with rubber tubing to allow ventilation. Adult were removed from the vials 6 h later. The latter were kept in a climatic chamber at 25ЊC and a photoperiod of 12:12 (L:D) h until dissected. Eggs, presumably parasitized by F. dominica, were dissected at different times and checked under a ste- reomicroscope (Leica MZ6, Leica, Wetzlar, Ger- many) with a cold light source (Leica CLS100). Non- parasitized hosts were rejected and the inspection Þnished once 15Ð20 parasitized eggs had been ob- tained. These specimens were mounted on a glass microscope slide in PVA mounting medium (BioQuip Products Co., Rancho Dominguez, CA) for observa- tion by using a light microscope. Immature stages were measured by use of the Eclipse Net software (Nikon 2003). The number of instars was determined using the width of the mandibles (Dyar 1890).

Results Ϯ Њ Fig. 1. Egg. em, embryo; pd, pedicel; hc, host chorion. Under laboratory conditions (25 1 C and a pho- toperiod of 12:12 [L:D] h), F. dominica reared on eggs of D. abbreviatus has a developmental time of 18Ð22 d Conocarpus erectus L. (Myrtales: Combretaceae) that from oviposition to adult emergence. Parasitized eggs provided a food source and an oviposition substrate. could be easily recognized by their transparent whit- Wax paper strips (3 by 10 cm) stapled together also ish appearance and unorganized structure when dis- were used as an oviposition substrate (Castillo et al. sected as early as 24 h after exposure to the parasitoid. 2006). Both foliage and paper strips were replaced Conversely, unparasitized eggs have a more opaque every 2Ð3 d. Paper strips containing D. abbreviatus white aspect and show some tissue/organ organization eggs were removed and placed inside a similar cage. when examined. As time progresses, differences be- Adults of F. dominica were introduced into the cage come more conspicuous, and the D. abbreviatus em- and provided honey and water. Presumably, parasit- bryo can be easily observed in the unparasitized egg. ized egg masses were removed from the cage 4Ð5 d Eggs. F. dominica eggs are almost transparent, and later and placed in emergence cages (same dimen- the newly formed larvae become visible as hatching sions as above) supplied with both water and a honey approaches (Fig. 1). Young eggs average 120 Ϯ 3 ␮m food source until adult emergence (Ϸ12 d). in length by 65 Ϯ 3 ␮m in width at the widest point Experimental Specimens. Wax paper strips with (n ϭ 17). The egg is lemon shaped, with a pedicel Ͻ1-d-old eggs were carefully separated from each extending from the anterior end. The maximum length other and cut into one by 0.5-cm strips containing a of the pedicel observed was 176 ␮m. However, this single egg mass, introduced into a glass vial (Fisher- length was highly variable (72 Ϯ 47 ␮m; n ϭ 17) in part brand ßint glass printed white 12- by 75-mm dispos- because of pedicel breakage during dissection. The able culture tubes, Fisher, Pittsburgh, PA) with three pedicel is internally attached to the host egg chorion, naive F. dominica adults Ͻ1 d old. A smear of honey which prevents F. dominica egg from freely moving was provided on the inner surface of each test tube as within the host.

Table 1. Measurements (mean ؎ SE in micrometers) of F. dominica immature stages at specified times (in days) after parasitization at 25°C

Body Body Tail Mandible Mandible Time Instar n n n n n length width length length width 1 First 17 297 Ϯ 10a 17 107 Ϯ 2aa 17 61 Ϯ 2a 15 30 Ϯ 1a 15 15 Ϯ 5a 2 First 17 694 Ϯ 11b 17 241 Ϯ 8b 15 67 Ϯ 4a 13 31 Ϯ 1a 13 11 Ϯ 1a 3 First 17 884 Ϯ 18c 17 288 Ϯ 17c 17 63 Ϯ 5a 13 31 Ϯ 1a 13 12 Ϯ 1a 4 Second 21 1,064 Ϯ 27d 21 437 Ϯ 13d 14 47 Ϯ 1b 14 10 Ϯ 1a ANOVA F ϭ 398.10 F ϭ 346.15 F ϭ 0.55 F ϭ 17.25 F ϭ 1.34 df ϭ 3, 71 df ϭ 3, 71 df ϭ 2, 48 df ϭ 3, 51 df ϭ 3, 51 P Ͻ 0.00001 P Ͻ 0.00001 P ϭ 0.5789 P Ͻ 0.00001 P ϭ 0.2710

Within columns, values followed by the same letter are not signiÞcantly different (P Ͻ 0.05). a Cephalothorax width. May 2007 JACAS ET AL.: MORPHOLOGY AND DEVELOPMENT OF F. dominica 415

Fig. 2. Neonate Þrst instar. Dorsal view. md, mandibles; mx, maxillae; lg, spiked ligula.

Approximately 1 d after oviposition, the embryo the anterior margin of the cephalothorax at this stage increases to 158 Ϯ 6 ␮m in length by 88 Ϯ 4 ␮min of development (Fig. 2). At the end of the Þrst instar, width at the widest point (n ϭ 15) as it completes its which takes 2Ð3 d, the larvae average 884 ␮m in length development, at which time the neonate larva breaks and 288 ␮m in width (Fig. 3). through the egg. Only in two eggs of Ͼ400 host eggs The second instar is hymenopteriform (Fig. 4a), examined were two parasitoid eggs observed in the with an elongated oval body (1064 ␮m in length and same host egg, and only once were two larvae found 437 ␮m in width), and it has a small head bearing within the same host egg. Correspondingly, no more relatively large mandibles and a suctorial mouth (Fig. than one adult emerged from each of these hosts. 4b). Ten body segments, in addition to the head, are Larvae. Mandible measurements (Table 1) indicate clearly visible, and the cauda tapers posteriorly to a two instars in F. dominica. The neonate, or the Þrst single point. By the end of its development, the para- instar, is whitish, almost transparent, and cyclopoid sitoid larva consumes the contents of its host, leaving (Clausen 1940, Askew 1971). The cephalothorax is only the egg chorion, which gradually takes an amber- enlarged (Ϸ107 ␮m in width) and bears relatively like appearance that makes parasitized eggs clearly enormous, sickle-shaped mandibles (30 Ϯ 1 and 13 Ϯ recognizable. Just before pupation, the larva, which 1 ␮m in length and width at the base, respectively; n ϭ occupies the entire host egg, shrinks, and a free space 41) and a spiked ligula with short, rasping teeth (Figs. forms in the interior of the host (Fig. 4c); this process 2 and 3). The abdomen tapers posteriorly and is re- is completed in 2Ð3 d. duced to seven segments. The caudal segment is bi- Pupa. The pupa of F. dominica, as in all Hymenop- lobed with each lobe bearing one long spine-like pro- tera, is exarate. Initially, the pupa is whitish and does cess that is armed with a few smaller spines. When the not occupy the whole inner volume of the host (Fig. larva is dislodged from the host egg, it often moves 5a). Later, it becomes darker with red eyes, and the vigorously by ventral or backward thrusts of the cau- meconium is expelled. Subsequently, the pupa elon- dal processes and the posterior portion of the abdo- gates and occupies almost all available space within men. It also moves its mandibles vigorously when the host (Fig. 5b). Eventually, the eyes darken and the disturbed. The ligula and maxillae are clearly visible on thorax turns uniformly black. Mature pupae average 997 Ϯ 100 ␮m in length (n ϭ 11) (Fig. 5c), and adult characteristics can be easily observed through the amber-colored egg. Upon completion of pupal development, the adult chews an emergence hole in the host egg chorion by using a dotted rim existing at the anterior end of D. abbreviatus eggs. Emerged adults (mainly males) were observed helping later emerging adults (females) to chew the exit hole (Fig. 6).

Discussion As is typical of idiobiont parasitoids (Pennacchio Fig. 3. Late Þrst instar. md, mandibles; bc, bifurcated and Strand 2006), female F. dominica probably inject cauda. some kind of venom at oviposition that prevents fur- 416 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 100, no. 3

Fig. 4. Second instar. (a) Prepupa. (b) Shrinking prepupa within egg. (c) Mandibulate suctorial mouth. md, mandibles; sm, suctorial mouth. ther host development. This behavior could account two categories: the Þrst involves marking host eggs for the distinctive features observed in parasitized either externally or internally, and the second involves eggs as early as 24 h after oviposition. Females also female wasps defending egg batches from competitors might be injecting host-marking substances during (Austin et al. 2005). No defending behavior has been oviposition. Our results indicate that both multipara- observed in either our stock colony or the assays; sitism and superparasitism are rare in this solitary therefore, host marking may be the strategy used by F. species. Many platygastrid wasps exploit hosts that lay dominica. clumps of eggs, and strategies to prevent superpara- Stalked eggs are not rare among platygastrids, and, sitism are common. These strategies fall mostly into as in other species, the pedicel of F. dominica egg is longer than the egg body (Clausen 1940, Sampson et al. 2006). The location of the egg in the host egg content for platygastrid egg parasitoids is fairly con- stant for any given species (Clausen 1940, Masner 1995). However, we could not determine a precise location of a F. dominica egg in the host embryo. F. dominica has two instars, and this number Þts within the range given by Masner (1995) for platyg- astrids. The Þrst instar larva is cyclopoid and the sec- ond instar is hymenopteriform (Clausen 1940, Masner 1995, Sampson et al. 2006). burrelli Muese- beck, another Sceliotrachelinae described by Clancy (1944), has a single instar, which is hymenopteriform and has Þne pointed mandibles. However, A. burelli parasitizes nymphs of Pseudococcus comstocki (Ku- wana) (: Pseudococcidae), and its biology is completely different from that of F. dominica. Free- living Þrst instars of platygastrids usually feed directly upon the body ßuids of the host, and their mandibles seem to be designed for Þghting, rather than for feed- ing (Clausen 1940). Whether this is true for F. do- minica deserves further investigation. As larval development progresses, F. dominica par- asitized eggs turn amber. Such a change in color is

Fig. 5. Exarate pupae. (a) Young white pupa. (b) Elon- Fig. 6. Recently emerged adult male chewing exit hole gated white pupa. (c) Mature pupa. ey, eye; mc, meconium. for a sister. May 2007 JACAS ET AL.: MORPHOLOGY AND DEVELOPMENT OF F. dominica 417 common in eggs parasitized by platygastrids (Austin et prepes doubleirii (Coleoptera: Curculionidae) from Do- al. 2005). Other species of egg parasitoids of D. ab- minica. Fla. Entomol. 88: 61Ð66. breviatus released in Florida do not cause this color Godfray, H.C.J. 1994. Parasitoids. Behavioral and evolution- change. Therefore, this trait may provide a tool to ary ecology. Princeton University Press, Princeton, NJ. quickly estimate the level of parasitism in the Þeld Grafton-Cardwell, E., K. Godfrey, J. E. Pen˜ a, C. McCoy, and when this species is released from quarantine, pro- R. Luck. 2004. Diaprepes root weevil. University of Cal- ifornia, Agricultural and Natural Resources Publ. 8131. vided that this trait does not change under Þeld con- Hall, D., J. E. Pen˜ a, R. Franqui, R. Nguyen, P. Stansly, C. ditions. Mccoy, S. Lapointe, R. Adair, and B. Bullock. 2001. Sta- Mating at the emergence site is very common tus of biological control by egg parasitoids of Diaprepes among species that attack gregarious hosts or hosts abbreviatus (Coleoptera: Curculionidae) in citrus in Flor- living in discrete, well-deÞned patches (Godfray ida and Puerto Rico. BioControl 46: 61Ð70. 1994). In such cases, it is not uncommon that males Hall, D. G., J. Eger, J. E. Pen˜ a, R. Duncan, C. O’Brien, G. emerge Þrst and wait for females to emerge. Males of Evans, and C. McCoy. 2002. Exploration in Belize for F. dominica not only waited but also helped females parasitoids attacking eggs of citrus weevils and an eval- exit the weevil egg. Such altruistic behavior also has uation of Pediobious irregularis and Horismenus bennetti (Hymenoptera: ) as potential biological con- been observed in other systems. Suzuki and Hiehata trol agents of Diaprepes abbreviatus and Pachnaeus litus (1985) and Kurosu (1985) reported males of another (Coleoptera: Curculionidae). Fla. Entomol. 85: 663Ð666. egg parasitoid, Trichogramma papilionis Nagarkatti Jacas, J. A., J. E. Pen˜ a, and R. E. Duncan. 2005. Successful (Hymenoptera: ), helping females oviposition and reproductive biology of Aprostocetus va- emerge by pulling their bodies through a hole chewed quitarum (Hymenoptera: Eulophidae): a predator of Dia- in the chorion. prepes abbreviatus (Coleoptera: Curculionidae) Biol. Control 33: 352Ð359. Kurosu, U. 1985. Male altruism and wing polymorphism in Acknowledgments a parasitic wasp. J. Ethol. 3: 11Ð19. Masner, L. 1995. The proctotrupoid families, pp. 209Ð265. In We thank G. Evans (USDAÐAPHISÐPPQ-Systematic En- P. E. Hanson and I. D. Gauld [eds.], The Hymenoptera of tomology Laboratory, Beltsville, MD) for critically reviewing Costa Rica. Oxford University Press, Oxford, United King- an earlier version of this manuscript. Special thanks are ex- dom. tended to D. Long, J. Alegria, and Z. Alegria (TREC) for Pennacchio, F., and M. R. Strand. 2006. Evolution of devel- providing technical assistance throughout the investigation. opmental strategies in parasitic Hymenoptera. Annu. The research was supported by University of Florida-Insti- Rev. Entomol. 51: 233Ð258. tute of Food and Agricultural Sciences, USDA, and Coop- Pena, J. E., J. E´ tienne, R. Duncan, and J. Pinto. 1998. In- erative State Research, Education, and Extension Service. ˜ troduction of Ceratogramma etiennei (Hymenoptera: Tri- chogrammatidae) for biological control of Diaprepes ab- References Cited breviatus in Florida, USA, pp. 145Ð148. In S. Hassan [ed.], Egg parasitoids, 5th International Symposium, 4Ð7 April Askew, R. R. 1971. Parasitic . Elsevier, New York. 1998, IIBC, Cali, Colombia. Parey Buchverlag, Berlin, Austin, A. D., N. F. Johnson, and M. Dowton. 2005. System- Germany. atics, evolution, and biology of scelionid and platygastrid Pen˜ a, J. E., and D. M. Amalin. 2000. Biological control of wasps. Annu. Rev. Entomol. 50: 553Ð582. Diaprepes abbreviatus by parasitoids, pp. 66Ð76. In S. H. Beavers, J. B., S. A. Lovestrand, and A. G. Selhime. 1980. Futch [ed.], Diaprepes short course, 22 March 2000, Co- Establishment of the exotic parasite Tetrastichus haitiensis operative Extension Service Florida Agricultural Exper- (Hym: Eulophidae) and recovery of a new Trichogramma iment Station. Citrus Research & Education Center, Lake (Hym: Trichogrammatidae) from root weevil egg masses Alfred, FL. in Florida. Entomophaga 25: 91Ð94. Sampson, B. J., T. A. Rinehart, O. E. Liburd, S. J. Stringer, and Castillo, J., J. A. Jacas, J. E. Pen˜ a, B. J. Ulmer, and D. G. J. M. Spiers. 2006. Biology of parasitoids (Hymenop- Hall. 2006. Effect of temperature on life history of tera) attacking Dasineura oxycoccana and Prodiplosis vac- Quadrastichus haitiensis (Hymenoptera: Eulophidae), cinii (Diptera: ) in cultivated blueberries. an endoparasitoid of Diaprepes abbreviatus (Coleoptera: Ann. Entomol. Soc. Am. 99: 113Ð120. Curculionidae). Biol. Control 36: 189Ð196. Suzuki, Y., and K. Hiehata. 1985. Mating systems and sex Clancy, D. W. 1944. Biology of Allotropa burrelli, a gregar- ratios in the egg parasitoids, Trichogramma dendrolimi ious parasite of Pseudococcus comstocki. J. Agric. Res. 69: and T. papilionis (Hymenoptera: Trichogrammatidae). 159Ð167. Anim. Behav. 33: 1223Ð1227. Clausen, C. C. 1940. Entomophagous insects. McGraw-Hill, Woodruff, R. E. 1968. The present status of a West Indian New York. weevil (Diaprepes abbreviatus (L.) in Florida. Fla. Dep. Dyar, H. G. 1890. The number of moults of lepidopterous Agric. Div. Plant Ind. Entomol. No. 77. larvae. Psyche 5: 420Ð422. Evans, G. A., and J. E. Pen˜ a. 2005. A new Fidiobia species (Hymenoptera: Platygastridae) reared from eggs of Dia- Received 10 July 2006; accepted 18 December 2006.