OVARIAN EGG MORPHOLOGY IN CHALCIDOID WASPS (HYMENOPTERA: CHALCIDOIDEA) PARASITIZING GALL WASPS (HYMENOPTERA: CYNIPIDAE)

H. Vårdal¹, J. F. Gómez² & J. L. Nieves-Aldrey³ 

¹Swedish Museum of Natural History, Department of Zoology, Box 50007, SE-1004 05 Stockholm, Sweden. e-mail: hege.vardal@nrm.se

²Facultad de Ciencias Biológicas, Departamento de Zoología y Antropología Física, Universidad Complutense de Madrid, Jose Antonio Novais, 2. E-28040 Madrid, Spain. e-mail: jf.gomez@bio.ucm.es

³Museo Nacional de Ciencias Naturales, Departamento de Biodiversidad y Biología Evolutiva, José Gutiérrez, Abascal 2, 28006 Madrid, Spain. e-mail: aldrey@mncn.csic.es (corresponding author)

IntroductionTOP

The most diverse group of parasitic wasps is probably the superfamily Chalcidoidea (Hymenoptera) with its 22000 described species and an immense variety of life modes (Noyes, 1978, 1990a; Gordh et al., 1979; Gibson, 1993). Most chalcidoids are entomophagous and attack 339 arthropod families (Clausen, 1940; Noyes, 2002). The great majority are egg and larval parasitoids, while some groups are, at least in part, phytophagous gall inhabitants on as much 444 plant families (Noyes, 2002) or even true gall inducers (La Salle, 2005).

The gall wasps or cynipids (Hymenoptera: Cynipidae) is a large group of Hymenoptera representing one of the more speciose radiations of gall-inducing insects with more than 1400 species described (Nieves-Aldrey, 2001). The classification of the family has been recently revised into twelve tribes which are strongly supported as monophyletic (Ronquist et al., 2015), nine of which are represented in the western Palaeartic. In addition of their intrinsic biological interest, plant galls induced by Cynipidae host large communities of insects which are composed mainly of other cynipid inquiline species and parasitoids which attack all the gall inhabitants. These micro communities support intrincate food webs which have been focus of many ecological studies (Askew, 1961; Schönrogge et al., 1995, 1996; Bailey et al., 2009).

The parasitoids inhabitant cynipid galls in western Palaearctic belong mainly to six chalcidoid families: Euytomidae, Torymidae, Ormyridae, Pteromalidae, Eupelmidae and Eulophidae. This parasitoid fauna has been catalogued in recent years (Askew et al., 2006, 2013).

Egg and eggshell can be useful for identification of chalcidoid species (Askew, 1961). For instance Claridge & Askew (1960) showed clear differences in the egg between species of Eurytoma rosae Nees, 1834 species-group. Extensive morphological variability in egg structure has been observed between and within families of Chalcidoidea. The egg is short and oval or slender and elongate (Parker, 1924a; Clausen, 1940; Iwata, 1962). A summary of external features of the chalcidoid egg from literature is given in Table 1.

The eggshell ultrastructure has been described for a handful of chalcidoids including Nasonia vitripennis (Walker, 1836) (Hymenoptera: Pteromalidae) (King et al., 1968; Richards, 1969) and Eurytoma amygdali Enderlein, 1907 (Hymenoptera: Eurytomidae) (Mouzaki & Margaritis, 1994; Zarani & Margaritis, 1994). It typically consists of a thin vitelline membrane, adjacent and often attached by interlocking ridges to the oocyte (King et al., 1968) and an outer chorion. The chorion is divided into an inner electron-translucent endochorion and an outer electron-dense exochorion (King et al., 1968; Richards, 1969; Mouzaki & Margaritis, 1994). The endochorion has a uniform smooth structure, whereas the exochorion may have sublayers of granular and columnar structure (Mouzaki & Margaritis, 1994). Spines originating from the endochorion and extending through the exochorion and onto the egg surface were found in Catolaccus (Hymenoptera: Pteromalidae) (King et al., 1968). The structure and elasticity of the eggshell facilitate the stretching of the egg through the narrow and long ovipositor. Thus the chorion appears to be reduced in structure, i.e. fewer layers, compared with insects with different oviposition requirements (i.e. diameter of the ovipositor relative to egg width) like the reduviid Rhodnius prolixus Stahl that has seven layers in its chorion (Beament, 1946; King et al., 1968; Richards, 1969). The endochorion has been shown to contain peroxidase in Eurytoma amygdali (Hymenoptera: Eurytomidae) to provide elasticity (Mouzaki & Margaritis, 1994; Zarani & Margaritis, 1994). The anterior micropylar region of the egg of Nasonia vitripennis (Hymenoptera: Pteromalidae) is marked by an opening or by several grooves (King, 1962) on the external surface of the chorion. In Eurytoma amygdali (Hymenoptera: Eurytomidae), it is situated at the end of a short posterior filament, the micropylar appendage in the posterior end of the egg (Zarani & Margaritis, 1994). The structure of the eggshell often reflects mode of parasitism. Endoparasitic species often have a thin hydropic eggshell that can take up nutrients from the surrounding host tissue (Margaritis, 1985).

The aim of the study was to provide new data on the egg morphology of chalcidoids by analysing species associated with cynipid galls. Descriptive data on immature stages of species inhabitant cynipid galls are potentially important for studies such as phylogenetic analyses, the ecology of chalcidoid communities, as well as studies of food webs. In recent years some works have been published on comparative morphology of terminal-instar larvae of species of several chalcidoid families associated to gall wasps in Europe (Nieves-Aldrey et al., 2008; Gómez et al., 2008, 2011, 2013; Gómez & Nieves-Aldrey, 2012). However there are not similar comprehensive studies on egg morphology of the same or related species. We give here scanning electron micrographs showing the eggs of 3 species of Eulophidae, 1 Eupelmidae, 8 Eurytomidae, 3 Ormyridae, 4 Pteromalidae and 7 Torymidae as well as transmission electron micrographs showing the eggshell ultrastructure of 1 Eurytomidae (Sycophila biguttata) and 1 Pteromalidae (Cecidostiba fungosa). Data on egg shape variation, as well as aspects of egg and eggshell properties and oviposition habits are also investigated. Furthermore the ultrastructure of the eggshells of these species is described for the first time. Finally the micropyle and eggshell surface structure are shown for a number of the species.

Material and methodsTOP

SELECTED TAXA

We studied the eggs of 26 species of Chalcidoidea collected in Spain and Sweden between 2000 and 2007 (Table 2). The females were reared from cynipid galls on Asteraceae, Fagaceae, Lamiaceae, Papaveraceae, Rosaceae and Sapindaceae induced by species of the tribes Aylacini, Aulacideini, Diastrophini, Diplolepidini, Pediaspidini and Cynipini (Table 2). Taxonomy of cynipids follows Nieves-Aldrey (2001) and Ronquist et al. (2015).

REARING AND DISSECTION

Adult females were reared from galls stored in rearing cages or extracted after dissection of galls. Eggs were dissected from the ovaries of the females and counted. It is often difficult to count the number of eggs in the ovaries as the egg may be covered with ovarian sheets and be in varying degree of maturity, thus the egg count given in Table 4 for each species is approximate. Potential difficulties by using only ovarian eggs for descriptions may be that in some species the eggs tend to transform dramatically after oviposition, and sometimes ovarian tissue may conceal the surface structure of each individual egg (HV, personal observation). In the present study only ovarian eggs have been observed with the exception of the very few cases when we happened to find deposited eggs inside galls (see Table 2, Fig. 11). The most mature eggs in the ovaries were selected for the study. We leave the study of post-oviposition eggs for later studies.

PREPARATIONS FOR MORPHOLOGICAL STUDIES

Prior to scanning electron microscopy the adult females were preserved in absolute alcohol and ovarian eggs were dissected and put directly on SEM stubs and into the scanning electron microscope (Zeiss Supra35VP and FEI Quanta 200) on low vacuum without critical point drying or gold coating.

For transmission electron microscopy, the reared females were fixed in PAGF (Stefanini et al., 1967) for 1-2 months. Eggs were dissected and fixed in osmium-tetroxide and dehydrated through an ethanol series, finishing with acetone. The specimens were then infiltrated with 1:1 solution of acetone and epoxy resin (Epon 812) over night and then in pure resin at 60 degrees Celsius for 48 hours before sectioning (60 nm thin sections on copper grids), triple-dyeing in lead citrate- uranyl acetate-lead citrate, and examination in an LEO 912 AB transmission electron microscope.

TERMINOLOGY AND MEASUREMENTS

General terminology used in egg and eggshell descriptions follows King (1962) and Margaritis (1985).

The length of the egg body is measured from the posterior tip to the transition between the egg body and the peduncle (Table 3). The width measurement is taken at the maximum width of the egg body. Only external measurements of the ovipositor were taken. The length of the ovipositor was measured from the side. The ovipositor width was measured from the ventral side at the narrowest point at the apical tip and at the broadest point at the basal articulation (Table 3).

To examine how the egg shape varies within the Chalcidoidea, the different egg shapes were drawn onto a recent phylogeny (Fig. 12) (Heraty et al., 2013). Redrawings from previous authors’ illustrations were included (Silvestri, 1916; Parker, 1924a; Grandi, 1929; Dowden, 1935; Cendaña, 1937; Clausen, 1940; Jackson, 1961; La Salle & LeBeck 1983; Noyes, 1990a; Rosen et al., 1992).

ResultsTOP

Egg and ovipositor measurements are given in Table 3 and egg shape characters, approximate egg counts as well as environment at egg deposition site are given in Table 4.

Pedunculate eggs were observed in 5 families (Figs. 1-3, 5-6; Table 4) and non-pedunculate egg in Eulophidae (Fig. 1), Ormyridae (Fig. 4), Pteromalidae (Fig. 5) and Torymidae (Fig. 6).

For species in our material (Fig. 11), the peduncle does not appear to be attached to the host tissue, but aiding the egg in passing through the ovipositor. The diameter of the eggs with peduncle (mean: 60.85 μm) is narrower than the eggs without peduncle (mean: 62.15 μm) (Table 3). The eggs with a peduncle have a shorter egg body (mean: 205.66 μm) than the ones without a peduncle (mean: 353.69 μm) (Table 3). The non-pedunculate egg may be short and pear-shaped (Eulophidae), oval/subrectangular (Mesopolobus mediterraneus and Torymus rubi), but most often elongate and rather long egg body.

The ovipositor length is positively correlated with egg body length. In 75 % of the species, a short egg body is associated with short ovipositor and long egg body with a long ovipositor (Table 4). There are 3 exceptions in which the egg body is short and the ovipositor is long (Sycophila biguttata, Megastigmus aculeata and Torymus rubi) and 3 species in which the egg body is long and the ovipositor is short (Eupelmus microzonus, Eurytoma infracta, Ormyrus wachtli) and this may in some cases be attributed to the fact that either the ovipositor or the egg body length was in the vicinity of the limit between what we defined as short and long or perhaps, in a few cases, the eggs may not have been fully mature when examined.

Egg body width is always wider than external ovipositor width measurements (Table 3) meaning that the egg shell must be flexible to facilitate the oviposition through the egg canal.

EULOPHIDAE

The eggs of Aprostocetus epicharmus (Walker, 1839) (Tetrastichinae) and Dichatomus acerinus (Eulophinae) are elongate and somewhat wider in the anterior end (Fig. 1A) than at the posterior end. The specimens of Dichatomus acerinus contained around 50 eggs of approximately the same size (Table 4), The surface is mostly smooth, but has tiny papillae in the anterior half in Dichatomus acerinus (Figs. 1C, 7A).

The eggs of Aulogymnus skianeuros and Aprostocetus epicharmus have a smooth surface, peduncles equal in length to the egg body (Fig. 9A).

EUPELMIDAE

The egg of Eupelmus microzonus Förster, 1860 is elongate with a peduncle that is slightly longer than the egg body at the anterior end and a short process at the posterior end (Fig. 2). The micropyle is clearly visible at the end of the peduncle (Fig. 9B). The surface of the egg is entirely smooth.

EURYTOMIDAE

Four species each of the genera Eurytoma and Sycophila represent Eurytomidae in the present study (Table 2). Eurytoma eggs have an elongate egg body with a peduncle at one end and a short process at the other end (Figs. 3A-D). The latter is more pronounced in E. brunniventris Ratzeburg, 1852 (Fig. 3A) and E. strigifrons Thomson, 1876 (Fig. 3D) than in E. infracta Mayr, 1904 (Fig. 3B) and E. rosae (Fig. 3C). The peduncle is generally between 1 and 1 1/2 times as long as the egg body. Sometimes the eggs are of unequal size and immature eggs can be seen inside the females.

The micropyle was observed at the end of the peduncle in E. brunniventris and E. strigifrons (Fig. 9C), but not in the other two species. Rounded papillae (* in Fig. 7ABC) and/or small depressions (white arrow Figs. 7B, 8C) (and sometimes both intermixed (Fig. 7B) can be seen on the egg body surface of E. rosae (Fig. 7B) and E. infracta (Fig. 3B) and E. brunniventris (Fig. 8C), whereas the E. strigifrons (Fig. 3D) egg surface is entirely smooth.

Observations were done of post-oviposition eggs of E. brunniventris in the gall of Cynips quercus (agamic generation). What can be seen as small rounded structures in the depressions (white arrow in Fig. 8C) in the ovarian egg, apparently develop into hooks/spines (Fig. 8D) in the post-oviposition egg and the egg expands to at least double the size of the ovarian egg after being deposited in the gall.

The eggs of Sycophila biguttata (Swederus, 1795), S. binotata (Fonscolombe, 1832), S. mayri (Erdös, 1959) and S. submutica (Thomson, 1876) (Fig. 3E-H) are very similar to each other and are oval and tapered at both ends.

They carry a long slender peduncle at one end and a shorter process at the other end. It is difficult to measure the length of the peduncle as it is almost impossible to disentangle from the rest of the peduncles in the egg mass without breaking and it appears to be very flexible and is easily stretched out, but it appear to be several times as long as the egg body. The micropyle was seen at the end of the peduncle of S. submutica. The surface of the egg body is entirely without structure and appears flaky and soft.

Sections were made of the eggs of S. biguttata and examined in the transmission electron microscope (Fig. 10B). The eggshell consists of an inner vitelline membrane (VM) forming a firm layer around the oocyte, and the outer chorion. The vitelline membrane is the thinnest layer of the eggshell (about 0.03 μm in thickness) and is electron dense and uniform in structure. The chorion is separated in the inner endochorion (EN) and the outer exochorion (EX). The endochorion is further divided into an electron dense inner part and a more electron translucent outer part. It is slightly narrower (about 0.070 μm in thickness) than the exochorion (about 0.076 μm in thickness), which appears uniform in structure.

ORMYRIDAE

The eggs of Ormyrus (Fig. 4) are highly elongate and slightly broader in the anterior end than in the posterior end. Neither of the 3 examined species have peduncles. The micropyle was seen as quite a large 2 or 3-lobed opening at the anterior end of the eggs of O. wachtli Mayr, 1904 (Fig. 4C) and O. papaveris (Perris, 1840) (Fig. 4B). The surface appears more or less smooth, but in O. papaveris several of the examined species have eggs with irregular ridges on the surface, which appears as a grid pattern (marked by r in Fig. 7D).

PTEROMALIDAE

The examined eggs of Pteromalidae are elongate and tapered at both ends as in Pteromalus bedeguaris (Linnaeus, 1758) (Fig. 5C) or somewhat broader in the anterior end as in Stinoplus lapsanae Graham, 1969 (Fig. 5D) and Cecidostiba fungosa (Geoffoy, 1785) (Fig. 5A). None of the studied species have pedunculated eggs.

The micropyle was not observed in any of the pteromalid eggs. The surface of the egg is smooth in all the species except Pteromalus bedeguaris, in which it is covered in small spine-like structures (marked by s in Fig. 7E).

The eggshell of Cecidostiba fungosa was examined using TEM (Fig. 10A). It consists of an inner electron dense vitelline membrane (about 0.3 μm in thickness), a middle endochorion (about 2 μm in thickness) and an outer relatively thin electron dense exochorion (about 0.4 μm in thickness). The exochorion may contain vesicle-like structures (marked by * on Fig. 10A) as well as crystalline structures (black arrows on Fig. 10A), whereas the endochorion is uniform and apparently gelatinous in structure. The innermost layer, the vitelline membrane is electron dense and appears to be anchored by extensions (white arrows on Fig. 10A) into the oocyte.

TORYMIDAE

The shape of the examined torymid eggs varies from elongate and slightly broader at the anterior end in Adontomerus impolitus (Askew & Nieves-Aldrey, 1988) (Fig. 6A), Chalcimerus borceai Steffan & Andriescu, 1962 (Fig. 6B), Glyphomerus stigma (Fabricius, 1793) (Fig. 6C) and Torymus nobilis Boheman, 1834 (Fig. 6E) to short and subrectangular as in Torymus rubi (Schrank, 1781) (Fig. 6F) or oval with a long peduncle at the posterior end and a short process at the posterior end as in Megastigmus aculeatus (Fig. 6D).

The micropyle was not observed for any of these species. The egg surface is smooth in all the examined torymid species except in Adontomerus impolitus (Fig. 7C) in which the entire surface is covered in papilla-like structure and in Glyphomerus stigma (marked by * in Fig. 7F) in which at least parts of the egg are covered with small spines.

DiscussionTOP

EXTERNAL EGG FEATURES

Several functions have been suggested and shown for the peduncle of eggs of parasitic wasps. Fulton (1933) described the oviposition procedure for Pteromalus (as Habrocytus) cerealellae and explained how the egg body, which was 0,14-0,16 mm in diameter distributed its content along the whole length of the peduncle when the egg body enters the egg canal which has a diameter of only 0,04 mm. In other groups, such as Exenterus (Hymenoptera: Ichneumonidae), the peduncle varies from a simple stalk with a knob-like anchor to a highly specialized double stalk. The former simply attaches the eggs to the host larva’s integument, whereas the latter is firmly inserted into the host larva’s cuticle, which makes these eggs much less sensitive to desiccation than the former (Mason, 1967). In the eggs of the eucharitid Golumiella longipetiolata, the typical eucharitid peduncle is absent and a posterior anchor attach the egg to the leaf petiole where the eggs are deposited vertically onto the surface (Heraty et al., 2004). At the apical end a pseudostalk covered with secretion, which is believed to attract ants that act as host for the larvae of this species.

Respiration is a documented function of the peduncle in some groups of wasps. In several encyrtid species the egg has been shown to leave a portion of its peduncle on the outside of the host larva, so the encyrtid larva has access to oxygen when it hatches (Maple, 1947). Similarly, for Eurytoma amygdali (Hymenoptera: Eurytomidae) a respiratory function was suggested for the tip of the peduncle as this part of the egg apparently remained on the outside of the almond fruit after oviposition (Mouzaki & Margaritis, 1994).

Pedunculate eggs are found in most chalcid families (Fig. 12) in species ovipositing both inside host larvae in a fluid environment as well as in the drier environment on the host larvae inside galls on different plants (Table 4), so pedunculate egg are apparently not restricted to certain oviposition habits.

The sperm entrance or micropyle is usually at the anterior end of the eggs for most parasitic wasps including Chalcidoidea (Rotheram, 1973) or at the tip of the peduncle when present (Maple, 1947; Wishart & Monteith, 1954).

In eggs of at least some species of Eurytomidae that carry a long peduncle at the anterior end as well as a shorter process at the posterior pole, the micropyle has been reported at the end of the egg that carries the short posterior process (Claridge & Askew, 1960; Mouzaki & Margaritis, 1994; Zarani & Margaritis, 1994). We found, however, an opening at the tip of the long anterior peduncle of both Eurytoma brunniventris and Eurytoma strigifrons (Hymenoptera: Eurytomidae) and assume that this structure is equivalent with the micropyle of the other chalcidoidoid eggs with peduncle.

EGG LOAD

The approximate egg number varies considerably (Table 4). The size of the egg load may be indicative of whether the species is pro-ovigenic (female emerging with a full complement of mature eggs) or synovigenic (maturing eggs continuously). Compared with many other koinobiont parasitoid wasps and oak gall wasps that are considered pro-ovigenic and carry hundreds or more than a thousand ovarian eggs (Quicke, 1997; Vårdal et al., 2003), our result show a generally relatively low egg count, which may be an indication of synovigeny in some species (Table 4).

EGG SURFACE

Papillae or hook-like structures, spines and ridges are on the surface of eggs of some ectoparasitoid species (Fig. 7, Table 4). These structures may help to attach the egg to the host larva or the substrate where it is deposited, (Claridge & Askew, 1960; Arthur, 1961; Quicke, 1997). No surface structures were observed in eggs of endoparasitoids like Aprostocetus (Eulophidae) and Sycophila (Eurytomidae). All the studied endoparasitoids have a smooth chorion (Table 4), and this appears to be the most common state for eggs that are deposited inside another insect (Parker, 1924a; Hagen, 1964).

The surface structure of the mymarid egg is smooth, and this is also the case for the eggs of other outgroup taxa like Scelionidae and Diapriidae (Clausen, 1940) (Fig. 12; Tables 1, 4). This appears to be the most common state for egg surface throughout the superfamily. Surface structures like spines and papillae possibly evolved independently in Eurytomidae, Leucospidae, Torymidae and Pteromalidae and in Ormyridae. The eggshell of some Perilampidae, Trichogrammatidae, Eurytomidae, Encyrtidae and Ormyridae are heavily sculptured with irregular ridges (Table 1, Fig. 12). All the species, for which these surface structures are observed, are ectoparasitoids.

The surface sculpture of the ovarian and oviposited egg is different in Eurytoma brunniventris. In the ovarian egg, the surface is covered by depressions (Fig. 8C). In the depressions small projections with blunt tips can be seen in the ovarian egg, that apparently extend into spines or hooks in the post-oviposition egg (Fig. 8D). Claridge & Askew (1960) also observed that the spines were not present in the ovarian egg of Eurytoma brunniventris (Hymenoptera: Eurytomidae). Development of spines after oviposition appears to be a good strategy as they would probably delay the passage of the egg through the narrow egg canal, yet several species have ovarian eggs with well-developed spines (Fig. 7E-F). King et al. (1968) observed that the spines of Catolaccus sp. (Pteromalidae) are an integral part of the flexible translucent layer (endochorion) and protrude through the exochorion. Presumably the flexibility of the endochorion will allow the spines to fold when passing through the egg canal.

The egg surface in Ormyrus papaveris has irregular ridges (Fig. 2D). In several insect orders similar patterns are believed to be caused by imprints of the follicle cells that have participated in eggshell formation (Hinton, 1981).

THE EGGSHELL

Endoparasitoids often have hydropic eggshells, which have the ability to take up substances through the chorion (Quicke, 1997). To facilitate this action as well as the swelling of the egg after nutrion-uptake, the eggshell is often highly convoluted and flexible and thinner than the eggshell of anhydropic eggs, which contains all the yolk necessary for the embryo development (Quicke, 1997). A thicker impermeable anhydropic eggshell as well as enough yolk to nurture the embryo during the entire egg stage is expected for species exposing their eggs to desiccation (Quicke, 1997). Depending of the environment of the host larva, this may be the case for several ectoparasitic species, which may thus be expected to have thicker eggshells than endoparasitoids into which the eggs are normally deposited directly into the body tissue of the host. The egg of the ectoparasitic Cecidostiba fungosa has a thick chorion (2.5 μm) in which the endochorion is about 4 times as thick as the exochorion (Fig. 10A). In the endoparasitic Sycophila biguttata (Fig. 10B) the endochorion is as thick as the exochorion and 15 times thicker than in Cecidostiba fungosa (0.15 μm). In the examined material it is clear that the eggs of the endoparasitoids are narrower (mean: 44.1 μm) and shorter (mean: 136.5 μm) than the eggs of the ectoparasitoids (width mean: 65.2 μm, length mean: 345.25 μm) (Tables 2, 3) and one reason for this is probably that in our material the female ectoparasitoids in most cases are much larger the female endoparasitoids.

ConclusionsTOP

The chalcidoid egg is extremely variable in size, shape and surface structure. Certain families like polyphyletic Pteromalidae and monophyletic Torymidae exhibit all of the egg types, whereas only one egg type with relatively little variation is observed in certain families like Eurytomidae, Ormyridae, Perilampidae and Eucharitidae. In general, eggs of endoparasitoids have unsculptured eggshell, whereas ectoparasitoid often have egg surface structure, which might be involved in the attachment of the egg on the cuticle of the host.

The perception that the endoparasitoid eggshell (endochorion) is thin, whereas the ectoparasitoid eggshell (endochorion) is several times thicker is supported by our results.

AcknowledgmentsTOP

We are greatly indebted to the European Union´s Programme Structuring the European Research Area under SYNTHESYS for financing a visit for the senior author to the Museo Nacional de Ciencias Naturales (MNCN) in Madrid. We are grateful to Josefina Cabarga for administrating the SYNTHESYS visit. JLN-A was supported by projects CGL2009-10111 and CGL-2010-15786 of the Spanish PLAN NACIONAL DE I+D+i.

Gary Wife and Stefan Gunnarsson at the Biological Structural Analysis facility at Evolutionary Biology Centre at Uppsala University, Sweden and Laura Tormo at the electron microscope facility at MNCN, Madrid helped with the scanning electron microscope. Carina Svensson and Lena Gustavsson at the Department of Zoology at the Swedish Museum of Natural History in Stockholm, Sweden helped us prepare the egg sections and use the transmission electron microscope. We thank Carlo Polidori who critically reviewed and made useful comments on the manuscript. Johan Nylander (Bioinformatics Infrastructure for Life Sciences (BILS) and Swedish Museum of Natural History) is also acknowledged for technical support.

ReferencesTOP