Hymenoptera: Pteromalidae) on Ceratitis Capitata (Wiedemann) (Diptera: Tephritidae)

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Micron 40 (2009) 646–658

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Micron

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The preimaginal stages and development of Spalangia cameroni Perkins (Hymenoptera: Pteromalidae) on Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

Jose´ Tormos a,*, Francisco Beitia b, Elias A. Bo¨ckmann a, Josep D. Ası´s a a A´rea de Zoologıá, Facultad de Biologıá, Universidad de Salamanca, 37071 Salamanca, Spain b Instituto Valenciano de Investigaciones Agrarias, Unidad Asociada de Entomologıá IVIA/CIB-CSIC, Apartado Oficial, 46113 Montcada, Valencia, Spain

ARTICLE INFO ABSTRACT

Article history: The development and morphology of the immature phases of Spalangia cameroni Perkins, 1910 Received 9 January 2009 (Hymenoptera, Pteromalidae) are described from a laboratory rearing culture maintained on Ceratitis Received in revised form 3 February 2009 capitata (Wiedemann, 1824) (Diptera, Tephritidae), using microscopic techniques, including light and Accepted 5 February 2009 scanning electron microscopy. The surface of the chorion of the egg is smooth and the micropyle occurs at the anterior end. The immature larvae are similar to the mature larva, differing mainly in the size of the Keywords: head capsule and mandibles. The mature larva displays tubercules on the body segments as well as a Pteromalidae pleurostoma and superior and inferior mandibular processes. On completion of its larval development, Immature phases an adecticous and exarate pupa is produced. The mandibles of the pupa, as for the adult, are toothed. Scanning electron microscopy Tephritidae Three larval instars are recorded based on statistical analyses of the sizes of the larval mandibles and Management purposes head capsules, in combination with such characters as the number of exuviae and excretion of the meconium. There are significant positive correlations between mandible length and width of larval head capsule with the number of instars, thus indicating that the mandible length and width of larval capsule are good predictors of the number of instars in this parasitoid. Developmental time from egg to adult emergence was 33–34 days for females and 28–29 days for males at 21–26 8C, 55–85 RH and a L16:D8 photoperiod. Our results show that the eggs and different instars of S. cameroni can be unambiguously identified only by SEM. Therefore, characterization of the immature stages of Spalangia species using SEM should be done before subsequent routine identifications using a binocular microscope or stereomicroscope. ß 2009 Elsevier Ltd. All rights reserved.

1. Introduction As indicated above, most studies addressing S. cameroni have focused on its potential use for biological control of Diptera Spalangia cameroni Perkins, 1910 (Hymenoptera, Pteromalidae) is pests. Little attention has been paid to its developmental biology a solitary primary ectoparasitoid of the pupae of various Diptera (although see Gerling and Legner, 1968). Developmental biology pests. Currently it is one of the parasitoids most used worldwide for studies, including morphological characterization of the pre- biological control of Musca domestica Linnaeus, 1758 – the ‘‘housefly’’ imaginal stages, can be important for the identification of an –andStomoxys calcitrans (Linnaeus, 1758) – the ‘‘stable fly’’ – species insect at species level before adult emergence and can simplify that are harmful in the intensive (confined) raising of livestock and quantification of the impact of natural enemies in biological birds(NovartisAnimalHealthInc.,PerkinsLtda.;Protecnet(2009)).In control programs (Bellows and Van Driesche, 1999; Lla´ cer et al., this respect, it is being used in countries such as Denmark, the United 2005; Onagbola and Fadamiro, 2007). Little is known about States, Australia, Costa Rica and Colombia in inundative releases, larval morphology in pteromalid wasps (Grassberger and Frank, sometimes reaching parasitoidism rates of 40% (Geden and Hogsette, 2003; Rojas-Go´ mez and Bonet, 2003; Onagbola and Fadamiro, 2006; Steenberg et al., 2001). In Spain, the species has been bred since 2007; Tormos et al., 2007). The present study addresses the 2003 on a semi-massive basis using Ceratitis capitata (Wiedemann, characterization of the developmental biology and morphology 1824), the ‘‘Mediterranean Fruit Fly’’, as host with a view to testing its of the preimaginal stages of this species because the character- usefulness as a biological weapon against this Diptera. ization and description offered by Gerling and Legner (1968) are brief and lacking in detail. Descriptions of the preimaginal morphology of another species of this genus, S. endius Walker, * Corresponding author. Tel.: +34 923 294463; fax: +34 923 294515. 1839 have been provided by Handschin (1934) and Zhang and E-mail address: [email protected] (J. Tormos). Zhang (1990). Nevertheless, these descriptions are also extre-

Table 1 Measurements (mean S.E.M., in mm) of the body sizes of the instars and pupae of S. cameroni at speciﬁc intervals after parasitization (mature larva: third instar + prepupaa).

Age (days) First instar Second instar Mature larva Pupa

After N Length Width N Length Width N Length Width N Length parasitization (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.)

3 4 0.31 0.02 0.10 0.00 4 5 0.57 0.10 0.23 0.05 5 2 0.65 0.00 0.27 0.03 6 8 0.86 0.30 0.39 0.15 7 2 0.92 0.07 0.47 0.01 13 1.31 0.66 0.66 0.37 8 4 1.10 0.20 0.52 0.14 9 10 1.61 0.75 0.75 0.36 10 1 1.62 0.00 0.75 0.00 6 2.31 0.66 1.08 0.31 11 10 2.42 0.59 1.19 0.27 12 7 2.48 0.52 1.14 0.28 13 11 2.64 0.39 1.22 0.21 14 3 2.11 0.82 1.12 0.30 15 5 2.17 0.62 1.02 0.28 16 7a 2.23 0.64 1.08 0.33 17 5a 2.29 0.64 1.12 0.31

17/33–34 20 2.01 0.26 mely brief and cannot be used for comparative purposes. In this of the parasitoid at the Instituto Valenciano de Investigaciones study morphological parameters, including measurements of Agrarias (Vale` ncia, Spain), in a climatic chamber (Sanyo the width of the head capsule and mandible length and plotting MLR350) at 21–26 8C, 55–85% RH, and a L16:D8 photoperiod. of the frequency distribution of these data, followed by Pupae of the Mediterranean Fruit Fly were used as hosts, and the statistical analyses were used in conjunction with reliable parasitoid, conﬁned in a plastic cage (30 cm 20 cm 20 cm) characters, such as the presence of exuvia, excretion of the with ventilation, were fed with honey impregnated on strips of meconium, and the initiation of the prepupal period (especially blotting paper, plus sugar and water. These conditions were the initiation of the ‘‘pronymph’’) to determine the number of used throughout the study, except that a RH of 95% was used instars (Muesebeck and Parker, 1933; Lo¨ hr et al., 1989; Lla´ cer (following Gerling and Legner, 1968) in the experiments aimed et al., 2005; Onagbola and Fadamiro, 2007; Wright, 1986,). at determining the moment of eclosion and the presence of Characterization of the developmental biology of this parasitoid different moults. on C. capitata is of particular relevance because this is the ﬁrst time it has been reared on this host since being recorded as a 2.2. Development, morphology and characterization of the parasitoid (Falco´ et al., 2006). preimaginal stages

2. Materials and methods To study the development of the immature stages, 10 recently parasitized pupae of C. capitata were dissected to 2.1. Insects completely expose the eggs, and were placed in a chamber with 95% RH. Periodic observations were made of the develop- The stages of the preimaginal phases and data on the ment through eclosion and production of exuviae. The slowing developmental biology of S. cameroni were obtained by rearing down of movement and expulsion of the meconium were

Table 2 Length (in mm) of the mandible and width (in mm) of the head capsule of the instars of S. cameroni at speciﬁc intervals after parasitization (mature larva: third instar + prepupaa). Intervals of presence of non-melanized (NM) and melanized (M) pupae.

Age (days) First instar Second instar Third instar + prepupaa Pupae

After N Length N Width N Length N Width N Length N Width N NM << (M) ,, (M) parasitization (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.) (mean S.E.M.)

3 4 29.75 0.95 6 0.14 0.05 4 5 35.00 2.54 5 0.14 0.05 5 2 38.00 0.00 7 0.14 0.03 6 8 44.50 2.07 3 0.14 0.00 7 2 47.00 1.41 7 0.15 0.00 13 70.53 3.12 1 0.31 0.00 8 4 70.00 2.44 6 0.31 0.00 9 10 77.90 3.21 5 0.31 0.00 10 1 80.00 0.00 8 0.32 0.00 6 96.66 1.86 1 0.40 0.00 11 10 97.20 3.61 5 0.41 0.00 12 7 102.00 6.95 5 0.41 0.01 13 11 107.45 8.59 6 0.43 0.03 14 3 110.00 13.07 4 0.41 0.01 15 5 114.60 9.52 4 0.41 0.01 16 7a 111.00 8.08 5 0.41 0.01 17 5a 114.00 8.03 5 0.40 0.01 17–27 20 20 28–29 812 0

33–34 00 8 648 J. Tormos et al. / Micron 40 (2009) 646–658 used to determine the start of the prepupal period. The 2.3. Histological methods, stereomicroscope, and SEM pupal phase was considered to be that lasting from the transformation of the ‘‘pronymph’’ in the pupa to the emergence For light microscopic examination of the eggs and instars, the of the adults. methods of Tormos et al. (2003, 2004) were employed. Photos, and To study the morphology of the immature stages, 15 pairs of measurements to the nearest 0.01 mm, of different structures of adult parasitoids of 8 days of age were taken from a terrarium the preimaginal phases were made with a Leica MZ125 microscope where large numbers of females and males had cohabited equipped with a Cannon S50 camera using the IM50 Leica software for those 8 days, and were placed individually in a Petri (Leica Microsystems Imaging Solutions) (Instituto Valenciano de dish (9 cm 1.5 cm). The parasitoids were provided daily, Investigaciones Agrarias). For scanning electron microscopy, the until the appearance of the first parasitoid pupae, with 10 samples were frozen in slush N2 and attached to the specimen pupae of C. capitata of 2 days of age (optimum conditions to holder of a CT-1000C Cryo-transfer system (Oxford Instruments, obtain a good solitary parasitoidism, unpublished data). The Oxford, UK) interfaced with a JEOL JSM-5410 scanning electron parasitized pupae were periodically dissected every 24 h in microscope (SEM) (Universidad Politećnica de Valencia, Vale`ncia, order to examine the morphological differences of the different Spain). The samples were then transferred from cryostage to the stages. To determine the volume of the eggs, the formula Vh = 1/ microscope sample stage, where the condensed surface water was 6(a b2 p) was used, which is commonly employed in the sublimed by controlled warming to 90 8C. Then, the samples determination of volume of ovoid arthropod eggs (Corey and were transferred again to the cryostage and sputter-coated with Reid, 1991). gold. Finally the samples were returned to the microscope sample Two hundred eggs, 400 immature larvae, 200 mature larvae and stages for examination at an accelerating voltage of 15 keV. Images 200 pupae of S. cameroni were fixed and preserved in 70% ETOH for were also taken using Oxford Instruments. subsequent study and description. The ensuing descriptions essentially employ the terminology and organization used by 2.4. Statistical analyses Finlayson (1987) and Tormos et al. (2004). Voucher specimens are deposited at the Instituto Valenciano de Investigaciones Agrarias In an attempt to detect a possible relationship between the (Vale`ncia, Spain). length of the mandibles (maximum length: measured from the

Figs. 1–4. Spalangia cameroni Perkins: Egg: 1. Egg on host pupa. 2. SEM of egg. 3. Desiccated egg, showing the translucent chorion. 4. SEM of egg showing the micropylar area (m) at the anterior end and the posterior end (p): (4a) detail of posterior end; (4b) detail of micropylar area showing the micropyle (mi). J. Tormos et al. / Micron 40 (2009) 646–658 649

Figs. 5 and 6. Spalangia cameroni Perkins: ﬁrst instar larva: 5. Ventral view on host pupa with details of cuticular differentiations: 5a and 5a0. 6. Lateral view on host pupa with details of: 6a. Head capsule showing the prominent circular structure containing a mandibulate mouth (m), tegumental differentiations (td), and papilliform antenna. 6b. Detail of last abdominal segment where a transversal slit (d) and a microtrichial ring (e) can be observed.

Fig. 7. Spalangia cameroni Perkins: ﬁrst instar larva: 7. Larva on the host abdomen, showing a detail of the cephalic skeleton (tentorium, epistoma, pleurostoma and hypostoma), the sensorial structures (sensilla, setae and antennae), and mandibles (7a, drawing) and a detail of the papilliform antenna under SEM (7b). 650 J. Tormos et al. / Micron 40 (2009) 646–658

Figs. 8–10. Spalangia cameroni Perkins: second instar larva: 8. General aspect from a dorsal view under stereomicroscope (note orange gut occupying most of larva). 9. General aspect from a ventral view under SEM. 10. General aspect from a dorsal view under SEM; detail of spiracle (10a, arrow). points of articulation with the head to the pointed tip) and the formed prior to analysis since the assumption of normality was number of days of development, both variables were related by a generally met. ‘‘Scatter plot’’. The linear relationship between both variables was The different groupings of the mandible and head capsule sizes determined by ﬁtting a straight line. The same procedure was with respect to the time of development obtained with the carried out between the width of the head capsule (maximum previous analysis were combined with such reliable characters as width) and the time of development, as well as between the length the number of exuviae on the body of the parasitoid larva and the of the mandibles and the width of the head capsule. The same data excretion of the meconium, in order to establish the time of were also subjected to linear regression analysis in order to development of the different immature stages and the number of estimate the regression coefﬁcients. The data were not trans- instars.

Figs. 11–13. Spalangia cameroni Perkins: mature larva: 11. Inside the host puparium 12. Beside remains of host pupa 13. Showing the ‘‘swelling’’ (s) and head (h), with a detail of the latter (13a, drawing). J. Tormos et al. / Micron 40 (2009) 646–658 651

Fig. 14. Spalangia cameroni Perkins: mature larva: 14. General aspect from a dorsal view under SEM showing the head (h) and the swelling (s), with a detail of both structures (14a), and details of a spiracle (14c, drawing) and of the last segment of the body (14b).

Finally, in order to determine the possible underlying implicit mesothoracic segment): 0.09–0.75 mm ((mean) S.E.M. = 0.29 grouping in the instars, with respect to head width and mandible 0.15, n = 21)] (Table 1) broader anteriorly, more or less conical in length, a cluster analysis was performed using the hierarchical shape, with head capsule and 13 body segments; vermiform, clustering method. whitish or almost transparent, slightly curved to the ventral side. The statistical analysis was performed using the SPSS (12.0) Nine pairs of spiracles become apparent on the sixth day after package. parasitization. Last abdominal segment notched transversely (Fig. 6b). Segments 1–13 with short cuticular differentiations of 3. Results microtrichiae type, forming rings around the division between the segments (Figs. 5a, 5a0,6b). Cranium (Figs. 6, 6a, 7a) ([width = 0.14– 3.1. Description and characterization of the preimaginal stages of S. 0.16 mm ((mean) S.E.M. = 0.14 0.00 mm, n = 28]) (Table 2) cameroni (Figs. 1–20) with the cephalic skeleton well developed, showing, apart from the tentorium, the following distinct and sclerotized sclerites: 3.1.1. Egg epistoma, pleurostoma and hypostoma. The cranium has a prominent The egg (Figs. 1–4b) (length = 0.24–0.40 mm [(mean) more or less circular structure containing a mandibulate mouth S.E.M. = 0.32 0.005), maximum width = 0.08–0.11 mm ( (mean) (Fig. 6a). Around this structure there are three pairs of sensilla, S.E.M. = 0.09 0.0009), n = 72)] is waxy white, elongate, cres- two setae and two papilliform antennae (Figs. 6a, 7a, 7b). Mouthparts: cent-shaped, has pointed anterior and posterior ends with an enlarged posterior-medial portion (hymenopteriform), and has a thin transparent chorion. Three regions are visible through the chorion: (a) an anterior transparent region, (b) a central opaque area, and (c) a posterior transparent region. Observed under the stereomicroscope, the surface of the chorion lacks apparent sculpturing (Figs. 1 and 3). Under SEM, the surface of the chorion also appears smooth (Figs. 2 and 4), but the micropyle can be seen (Fig. 4b) at the anterior end of the egg, surrounded by a distinctly pigmented micropyle region. The egg is more pointed at one end (Fig. 4a) than the other, being almost round at one end and narrow at the opposite end (stalked type) (Fig. 2). Prior to hatching, the eggs neither change shape nor increase substantially in size as deduced from a comparison of the volume of 72 eggs [36 of 24 h of age/36 of 48 h of age (prior to hatching)] (F1,70 = 2,026, P 0, 159). In all cases observed (n = 89) the eggs were deposited on the abdomen of the host, and hatched between 48 and 72 h after oviposition at 21–26 8C, 55–85% RH and a L16:D8 photoperiod (Table 1).

3.1.2. Larva. 1st instar Fig. 15. Spalangia cameroni Perkins: mature larva: detail of head under SEM showing General aspect (Figs. 5–7). Body [length = 0.29–1.5 mm several sensorial structures (ss) and possible labial palpi (lp) and maxillary palpi ((mean) S.E.M. = 0.67 0.29, n = 21), width (at the level of the (mp). 652 J. Tormos et al. / Micron 40 (2009) 646–658

Mandibles (Fig. 7a) ([length = 0.29–48 mm((mean) S.E.M. = 3.1.4. Larva. 3rd instar (mature larva) 39.04 6.53, n = 21] (Table 2) well defined, with an oblong General aspect. Body (Figs. 11–14) (length = 0.92–3.43 mm molar lobe and a single sharp, slightly curved, and well sclerotized ((mean) S.E.M. = 2.21 0.67 mm, n = 54), maximum width = blade. In observed cases (n = 30), 23 larvae were situated on 0.48–1.59 mm ((mean) S.E.M. = 1.06 0.32, n = 54) (Table 1) the abdomen, 6 on the thorax and one on the head. This broader in mid region, with a reduced head, with distinct segmenta- instar lasted for 5 days. At the end of the first instar the larvae tion, and with 3 thoracic and 10 abdominal segments; whitish; averaged 0.92 0.07 mm in length and 0.47 0.01 mm in width weakly sclerotized. Pleural lobes (Figs. 13, 14, 16, 16b–d, 17a) very (Table 1). well developed on 3 thoracic and first 8 abdominal segments. Tegument with several sensorial structures (Fig. 16), situated close to 3.1.3. Larva. 2nd instar the pleural lobes (swellings), among which two setae are prominent, General aspect (Figs. 8–10a). Body [length = 0.62–2.81 mm one located on the anterodorsal margin of each swelling (Figs. 16, 16c, ((mean) S.E.M. = 1.39 0.64, n =28),width(atthelevelofthe 17b) and the other located on the swelling (Figs. 16, 16b, 17a), and mesothoracic segment): 0.27–1.32 mm ((mean) S.E.M.=0.67 microtrichiae (Fig. 16d, 16d0) distributed around the protuberances. 0.33, n =28)](Table 1) similar to the first instar but with spiracles Anal segment (Fig. 14b) with a tenuous transverse slit. Spiracles more apparent (Fig. 10a), funnel-shaped, with atrium, subatrium (Figs. 14c, 16, 16a) on mesothorax, metathorax, and on first 7 with three chambers well differentiated and several scarcely abdominal segments; atrium (length = 80 mm, d maximum = 40 mm) apparent and closing apparatus; without microtrichiae and with a with peritreme and asperites well differentiated, funnel-shaped, with circle of small setae in the middle of each segment, and with an three well differentiated chambers; closing apparatus orange gut occupying most of the body (Fig. 8). The cranium (length = 47 mm; width = 7 mm) adjacent to atrium. Cranium (Figs. 9 and 10) is broader that of the first instar ([width = 0.31– (Figs. 13, 13a, 14, 14a, 15, 18) [width = 0.40–0.43 mm ((mean) 0.33 mm ((mean) S.E.M.=0.31 0.00 mm, n =18],themand- S.E.M. = 0.41 0.01, n = 37)] (Table 2) wider than high (from apex ibles are longer ([length = 0.64–80 mm((mean) S.E.M. = of cranium to base of mandibles) = 100–105 mm, narrower than the 73.42 4.81, n =28](Table 2), and the cranium has more sensilla first thoracic segment, indented dorsally along its middle line (d =2–3mm) and setae (length = 6–7 mm). In observed cases (Fig. 14a), very weakly sclerotized, with 7 pairs of setae (n = 71), 28 larvae were situated on the abdomen, 29 on the (length = 2–3 mm) and sensilla (d = 1–2 mm) (Fig. 13a). Antennae thorax, and 14 on the head. This instar lasted for 4days.Atthe scarcely developed (Fig. 14a). Mandibles (Fig. 13a) [length = 94– end of the 2nd instar, the single larva measured was 1.62 mm long 123 mm((mean) S.E.M. = 105.51 9.56, n = 54) (Table 2) scler- and 0.75 mm wide (Table 1). otized, triangular in shape, each with sharp, toothless blade. Maxillae

Fig. 16. Spalangia cameroni Perkins: mature larva: lateral zone of thorax, under SEM, showing sensorial structures (ss) and a detail of: (a) spiracle (16a); (b) a seta on the swelling (ses) (16b) and (c and d) seta (se) (16c) and microtrichiae (m) (16d, 16d0 detail) around the swelling. J. Tormos et al. / Micron 40 (2009) 646–658 653

Fig. 20. Puparia of C. capitata showing emergence holes of S. cameroni.

situated on the abdomen, 55 on the thorax and 6 on the head. This instar lasted for 8 days. At the end of the third instar the larvae averaged 2.29 0.64 mm in length and 1.12 0.31 mm in width (Table 1). The last 2 days correspond to postdefecant mature larva (prepupa) which starts with the deposition of the meconium. The prepupa, which stops feeding and shows little movement, has two well differentiated ‘‘types’’, corresponding to the so-called eonym- phal and pronymphal stages (Morris, 1937; Hagen, 1964). The morphology of the ‘‘pronymph’’ (Fig. 18), which precedes the pupal stage, is very peculiar owing to the lengthening of the body, the narrowing of the thorax in relation to the width of the abdomen, and the beginning of the indication of a head capsule.

Fig. 17. Spalangia cameroni Perkins: mature larva: detail of the seta on (17a) and at the anterodorsal margin (17b) of the swelling. 3.1.5. Pupa General aspect (Fig. 19a–c). Naked (length = 1.70–2.40 mm and labium indistinguishable, but under SEM the maxillary (mp) and ((mean) S.E.M. = 2.01 0.26 mm, n = 20) (Table 1). Adecticous labial (lp) palpi are visible (Fig. 15). Epistoma, pleurostoma, superior and exarate. Initially, this immature stage is white (Fig. 19a) but it and inferior mandibular processes, hypostoma and tentorium well gradually darkens, ﬁrst to a deep creamy white (Fig. 19b) and then, developed (Fig. 13a). In observed cases (n = 82), 21 larvae were before the appearance of the adult, to black (Fig. 19c). The pupa of the

Figs. 18 and 19. Spalangia cameroni Perkins: mature larva (pronymph): 18. General aspect (cephalic capsule (cc), constricted thorax segments (cts)). 19. Pupa in three different stages of melanization: (a) white (19a), (b) cream (19b), (c) black (19c). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of the article.) 654 J. Tormos et al. / Micron 40 (2009) 646–658

Fig. 21. Box-plot depicting the box diagrams representing the median, quartiles and Fig. 23. Box-plot showing the box diagrams representing the median, quartiles and superior and inferior whiskers of the mandible length as a function of the instar. No superior and inferiors whiskers of the head capsule width as a function of the instar. outliers are seen in any case. No outliers are seen in any case. male (n = 12) is sclerotized and melanized 12 days after pupation whereas that of the female (n = 8) required an additional 5 days to reach this stage (Table 2). The adult uses its mandibles to chew a small exuviae, the excretion of the meconium, and the appearance of the irregular circular (Fig. 20) hole through the puparial wall for ‘‘pronymph’’. These initial data, taken from an experiment emergence, usually at the anterior end or near the middle of the designed speciﬁcally to study the development of the immature puparium. stages [climatic chamber (Sanyo MLR350) at 21–26 8C, 95% RH, and L16:D8 photoperiod], served as reference to separate the different 3.2. Total developmental time preimaginal phases and the different instars, obtained from parasitized pupae of the offspring [climatic chamber (Sanyo Under the environmental conditions described above, the eggs of MLR350) at 21–26 8C, 55–85% RH, and L16:D8 photoperiod]. S. cameroni hatch into neonate ﬁrst instar larvae within 2–3 days. The data relating to mandible length (obtained from host pupae The total larval developmental period is between 16 and 17 days parasitized by S. cameroni offspring) of the larvae (the larvae have under the same conditions (Table 1). Males and females spend about simple, tusk-like mandibles, whereas the mandibles of the pupae 12 and 17 days, respectively, as pupae before their emergence. Thus, and the adults are toothed at the tip) were subjected to a ‘‘box- complete development of male S. cameroni takes about 29 days, and plot’’ (Fig. 21) and a ‘‘scatter plot’’ (Fig. 22) that revealed both the the female emerges about 5 days later (Table 2). effects of age (the time period between oviposition and removal of the larva from the host) on the length of the mandibles (m-lengths) 3.3. Characterization and separation of immature stages of larvae of S. cameroni of different ages. In particular, using this variable it is possible separate all larvae into three groups (instar The immature stages of S. cameroni described above were groupings) that are well differentiated (different instars as shown separated ‘‘a priori’’ on the basis of their age, by the presence of by the previous experiment) (Figs. 21 and 22), and a linear

Fig. 22. Scatterplot showing the relationship between mandible length, larval age Fig. 24. Scatter plot showing the relationship between the width of the head and instar (the fourth larva instar does not exist. This is the mature postdefecant capsule, larval age and instar (the fourth larva instar does not exist. This is the larva (prepupa)). mature postdefecant larva (prepupa)). J. Tormos et al. / Micron 40 (2009) 646–658 655

Fig. 25. Dendrogram from the cluster analysis using the head width variables (a, b, c Fig. 26. Dendrogram from the cluster analysis using the mandible length variable (a, correspond to the ﬁrst, second and third instars, respectively). b, c correspond to the ﬁrst, second and third instars, respectively). 656 J. Tormos et al. / Micron 40 (2009) 646–658

relationship was established between length of the mandible and the time of development (Fig. 22). A subsequent one-way factorial ANOVA revealed signiﬁcant effects of age (instars) on mandibular

length (F2,100 = 480.19, P < 0.001). An additional Tukey’s HSD test revealed significant differences at the 95% confidence level between the pairs of means of three well differentiated groups (instars) (Fig. 21). Finally, linear regression analysis confirmed positive correlations between mandibular lengths and instar groupings (Fig. 22), suggesting that larval mandible length is a good predictor of the number of instars in S. cameroni. An identical treatment was performed on width of the head capsules (hc- width) (Figs. 23 and 24) with similar results and conclusions. A one-way factorial ANOVA also revealed significant effects of age

(instar) on head capsule width (F2,80 = 8773.76, P < 0.0001), and an additional Tukey’s HSD test revealed significant differences (95%) between the pairs of means of three well differentiated groups (instars) (Fig. 23). The results of these analyses and of the experiment reported at the beginning of this section were used to compile Tables 1 and 2. The cluster analysis carried out with the head width and mandible length variables, both when treated separately (Figs. 25 and 26) and together (Fig. 27), revealed a perfect delimitation of three groups, corresponding to the three instars shown by the larval phase of S. cameroni. The two groups into which, in the first step, the larva of the first and third stages are subdivided as regards mandible length, could be due to sexual dimorphism.

4. Discussion and conclusions

The data concerning the biology (solitary parasitoidism/ facultative superparasitoidism (Tormos et al., unpublished data), the location of the egg and larvae on the host, the movements of the larvae on the host, the presence of protandry), and developmental aspects (number of instars, development rates of the different phases and immature instars) of S. cameroni using C. capitata as host and under the described environmental conditions are similar to those obtained previously for this parasitoid on M. domestica Linnaeus (Gerling and Legner, 1968). Characters that have commonly been employed to determine instars in parasitoids, such as measurements of the widths of larval head capsules and mandible length (Dyar, 1890; Lo¨hr et al., 1989; Muesebeck and Parker, 1933; Odebiyi and Bokonon-Ganta, 1986; Onagbola and Fadamiro, 2007; Tschinkel et al., 2003; Wen et al., 1995), allowed us to corroborate the number of instars that had been established previously from the presence of exuviae. Our results showed that the width of the larval head capsule and the length of the mandible correlated positively to instar groupings suggesting that both may also be reliable characters for determining the number of instars in parasitoids. Mandible length has also been used in morphometric studies (Kawano, 2000; Manzoor and Akhtar, 2006; Onagbola and Fadamiro, 2007). The chorion of the egg of S. cameroni, as in the case of eggs that are deposited externally, should be sculptured (Tormos et al., 2007). However, they are smooth, as for the eggs of Spalangia drosophilae Ashmead, 1887 (Simmonds, 1953). In both cases, the egg, even though deposited on the host’s body and therefore externally, is deposited within the host’s puparium. Its micropylar region, as for the relatively few parasitic Hymenoptera for which this has been reported, is at the anterior end of the egg. Although there are very few detailed morphological studies of this region of the egg in parasitic wasps, the considerable differences that have been reported suggest that the region may be of potential use in Fig. 27. Dendrogram from the cluster analysis using the head width and mandible length variables (a, b, c correspond to the first, second and third instars, phylogenetic studies addressing parasitic Hymenoptera (Quicke, respectively). 1997). The immature larvae are similar to those of S. drosophilae (Simmons, 1953), differing from them in the presence, number, J. Tormos et al. / Micron 40 (2009) 646–658 657 and position of sensory structures on the head and body. The first out this study. Financial support for this paper was provided from two instars are also similar to the mature larva, differing mainly in the Junta de Castilla y Leo´ n, project SA012A05. the last instar by the size of the head capsule and mandibles, in the presence, number, and position of sensory structures on the head References and body, and in the differentiation of the notch of the last body segment. Bellows, T.S., Van Driesche, R.G., 1999. Life table construction and analysis for evaluating biological control agents. In: Bellows, T.S., Fisher, T.W. (Eds.), The mature larva of S. cameroni shares the following characters Handbook of Biological Control. Principles and Applications of Biological states with other Chalcidoidea: (a) a reduced head; 3 thoracic and Control. Academic Press, San Diego, CA, USA, pp. 199–223. 10 abdominal segments, and 9 pairs of spiracles, (b) a cephalic Cals-Usciati, J., Cals, P., Pralavorio, R., 1985. Adaptations foctionelles des structures skeleton, although present, is not very well developed, (c) lieés a` la prise alimentaire chez la larva primaire de Trybliographa daci Weld (Hymenoptera, Cynipoidea) endoparasite de la Mouche des fruits Ceratitis mandibles well developed, sclerotized, and triangular, a short capitata. C. R. Acad. Sci. III 300, 103–108. blade without teeth, and maxillae and labium completely fused Corey, S., Reid, D.M., 1991. Comparative fecundity of decapod crustaceans. I. The and salivary orifice absent. fecundity of thirty tree species of nine families of caridean shrimps. Crustaceana 60, 270–294. With the mature larvae of other Pteromalidae it shares the Dyar, H.G., 1890. The number of moults of lepidopterous larvae. Psyche 5, 420–422. presence of tubercules upon the body segments as well as of a Falco´ , J.V., Garzo´ n-Luque, E., Pe´rez-Hinarejos, M., Tarazona, I., Malago´ n, J., Beitia, F., pleurostoma and superior and inferior mandibular processes. 2006. Two native pupal parasitoids of Ceratitis capitata (Diptera, Tephritidae) found in Spain. IOBC/WPRS Bull. 29, 71–74. However, the presence of a completely differentiated epistoma Finlayson, T., 1987. Ichneumonoidea. In: Stehr, F.W. (Ed.), Immature Insects. separates the larva of S. cameroni from other described larvae of Kendall/Hunt Publishing Company, Dubuque, IA, USA, pp. 649–664. Pteromalidae and at the same time approximates it to that of S. Geden, C.J., Hogsette, J.A., 2006. Suppression of house flies (Diptera: Muscidae) in Florida poultry houses by sustained releases of Muscidifurax raptorellus and drosophilae (Simmonds, 1953), and to that recently described of Spalangia cameroni (Hymenoptera: Pteromalidae). Environ. Entomol. 35, 75– Trichomalopsis peregrina (Graham, 1969) (Tormos et al., 2007). The 82. larva of S. cameroni differs of other species described in the genus in Gerling, D., Legner, E.F., 1968. Developmental history and reproduction of Spalangia cameroni parasite of Synanthropic flies. Ann. Entomol. Soc. Am. 61, 1436–1443. the position and number of the tegumental differentiations Grassberger, M., Frank, C., 2003. Temperature-related development of the para- (sensilla and setae). The distribution and number of these sensorial sitoid wasp, Nasonia vitripennis as forensic indicator. Med. Vet. Entomol. 17, structures likely is important for discriminating species in the 257–262. family, as shown for the Eurytomidae (Roskam, 1982). Handschin, E., 1934. Studien an Lyperosia exigua Meijere und ihren Parasiten. III. Teil. Die Anziehung von Spalangia zu ihrem Wirte. Rev. Suisse Zool. 41, 267–297. From the aspect of functional morphology, the mouth of the Hagen, K.S., 1964. Developmental stages of parasites. In: Debach, P. (Ed.), Biological three instars, as in other ectoparasitoids, is transformed into a Control of Insects, Pests and Weeds. Chapman & Hall, Ltd., London, pp. 168–246. suctorial tube (Quicke, 1997; Lla´ cer et al., 2005). The config- Kawano, K., 2000. Genera and allometry in the stag beetle family Lucanidae, Coleoptera. Ann. Entomol. Soc. Am. 93, 198–207. uration includes a preoral sucker, and feeding is achieved by Llaćer, E., Urbaneja, A., Garrido, A., Jacas, J., 2005. Morphology and development of pharyngeal pumping and continuous suction of host tissue immature stages of Galleosomyia fausta (Hymenoptera: Eulophidae: Tetrasti- fragments that are shred into tiny fragments by the mandibles chinae). Ann. Entomol. Soc. Am. 98, 747–753. Lo¨hr, B., Santos, B., Varela, A.M., 1989. Larval development and morphometry of (Cals-Usciati et al., 1985; Lla´ cer et al., 2005; Thompson, 1986). Epidinocarsis lopezi (DeSantis) (Hymenoptera: Encyrtidae), a parasitoid of the Therefore, the only function of the mandibles is to puncture the cassava mealybug, Phenacoccus manihoti Mat.-Ferr. (Homoptera: Pseudococci- host tegument and allow the larva to imbibe the fluids oozing dae). J. Appl. Entomol. 107, 334–343. Manzoor, F., Akhtar, M.S., 2006. Morphometirc analysis of population samples of from the resulting wound. This is why the larva changes its soldier caste of Odontotermes obesus (Rambur) (Isoptera, Termitidae, Macro- feeding position many times during its development period, termitinae). Anim. Biodivers. Conserv. 29, 91–107. why no solid host tissues are attacked, and the remnant of the Morris, K.R.S., 1937. The prepupal stage in Ichneumonidae, illustrated by the life- history of Exenterus abruptorius Thb. Bull. Entomol. Res. 28, 525–534. host is very similar in size to the initially parasitized host Muesebeck, C.F.W., Parker, L., 1933. Hyposoter disparis Viereck, and introduced (Gerling and Legner, 1968). ichneumonid parasite of the gypsy moth. J. Agric. Res. 46, 335–347. 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Protecnet ‘Comunidad Internet para proteccio´ n Fito y Zoosanitaria of S. drosophilae. costarricense’, http://www.protecnet.go.cr/general/boletin/pleglaboratorio/ plectrllbiol.htm. Although the instars of S. cameroni can be differentiated, Quicke, D., 1997. Parasitic Wasps. Chapmann & Hall, London, UK. grossly, from those of other pteromalid species, and even from Rojas-Go´ mez, C.V., Bonet, A., 2003. Life cycle and development of immature stages those described for other species of Spalangia, such as S. of Dinarmus basalis (Rondani, 1877) (Hymenoptera, Chacidoidea: Pteromali- dae). Folia Entomol. Mex. 42, 359–370. drosophilae, by using a stereomicroscope and by the presence/ Roskam, J.C., 1982. Larval characters of some eurytomid species (Hymenoptera, absence and distribution of sensory structures, our results show Chalcidoidea). Proc. K. Ned. Akad. Wetensc. 85, 293–305. that the eggs and different instars of S. cameroni only can be Simmonds, F.J., 1953. 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