Histology and ultrastructure of glands associated with the sting apparatus of the swarm-founding wasp Protopolybia exigua (Hymenoptera: Vespidae: Polistinae).

Social insects present a great diversity of exocrine glands, which are linked to fundamental roles of social life, and their morphological characterization represents the first step toward the knowledge of their function. We here describe the exocrine glands present in the sting apparatus of queens and workers of the wasp Protopolybia exigua. Histological analysis of serial sections under light microscopy revealed a glandular epithelium in the spiracular plate, the quadrate plate, and the gonostyli, while the last two in addition also contain clusters of class-3 cells. A big cluster of class-3 cells was also found in the oblong plate of some workers and queens. These findings reveal that a variety of glands, in addition to the common venom gland and Dufour gland, are present in association with the sting apparatus, of which three glands are novel reports for the exocrine repertoire of social insects. The position of the glands suggests a role in lubrication to provide mobility of the sting and venom ejection and/or participation in alarm behavior, while a function related to reproduction is also possible. The peculiar characteristics displayed by swarm-founding wasps are linked with exocrine gland secretions, making their investigation in Epiponini wasps a vast and interesting field.

A Behavior-Manipulating Virus Relative as a Source of Adaptive Genes for Drosophila Parasitoids.

Some species of parasitic wasps have domesticated viral machineries to deliver immunosuppressive factors to their hosts. Up to now, all described cases fall into the Ichneumonoidea superfamily, which only represents around 10% of hymenoptera diversity, raising the question of whether such domestication occurred outside this clade. Furthermore, the biology of the ancestral donor viruses is completely unknown. Since the 1980s, we know that Drosophila parasitoids belonging to the Leptopilina genus, which diverged from the Ichneumonoidea superfamily 225 Ma, do produce immunosuppressive virus-like structure in their reproductive apparatus. However, the viral origin of these structures has been the subject of debate. In this article, we provide genomic and experimental evidence that those structures do derive from an ancestral virus endogenization event. Interestingly, its close relatives induce a behavior manipulation in present-day wasps. Thus, we conclude that virus domestication is more prevalent than previously thought and that behavior manipulation may have been instrumental in the birth of such associations.

Connecting biology, optics and nanomechanical properties in micro-wasps.

Coloration in insects provides a fruitful opportunity for interdisciplinary research involving both physics and biology, and for a better understanding of the design principles of biological structures. In this research we used nanometric and micrometric analyses to investigate the morphological and mechanical properties of the black-orange-black (BOB) color pattern in scelionid wasps, which has never been studied. The primary objective of the present investigation was to explore the structural and mechanical differences in the mesoscutum of four species: Baryconus with an orange mesosoma (i.e. BOB pattern), all black Baryconus, Scelio with an orange mesosoma (i.e. BOB pattern), and all black Scelio. The most outstanding findings include the absence of multilayer structures that generate structural color, a pigment concentrated in the upper surface of the epicuticle, and surprising differences between the four species. Three of the four species showed an accordion-like structure in the furrow (notaulus), whereas the adjacent mesoscutum was different in each species. Moreover, the normalized color component spectra for blue, green and red colors of the black mesoscutum of each genus showed the same spectral dependence while the orange color manifested small changes in the dominant wavelength, resulting in slightly different orange tones.

Sperm morphology of Elasmus polistis Burks, 1971 (Hymenoptera: Chalcidoidea: Eulophidae).

The sperm morphology of the parasitoid Elasmus polistis (Eulophidae) has been investigated with light and transmission electron microscopy. The sperm were filiform and spiraled, with 165.6 (± 4.6) µm in length, and showed a distinctive head, formed by a one-layered small acrosome and a nucleus, and a flagellar region. An extracellular sheath from which many long filaments radiated out covered the acrosome and part of the nucleus. The spiral nucleus, with 24.1 (± 1.3) µm in length, was filled with homogeneously compact chromatin. In the nucleus-flagellum transition, the centriole adjunct extended posteriorly from the nuclear base in a spiral around the basal body, which has two central microtubules, and axoneme for approximately 1.1 µm. The two mitochondrial derivatives began roughly at the same level and at the base of the centriole adjunct. In cross-section, they were symmetrical, with a slightly oval shape and a smaller diameter in comparison to the axoneme. The latter, also spiraled, consisted of 9 + 9 + 2 microtubules that was formed from the basal body situated just below and aligned with the nucleus. The E. polistis sperm showed the same basic structures and morphological characteristics as observed in other Chalcidoidea. However, it was possible to distinguish the sperm of this species from those of other Eulophidae by (i) the long length of the centriole adjunct on the flagellum, and (ii) the presence of two central microtubules within the basal body. The sperm characteristics suggest that Eulophidae is closely related to Trichogrammatidae and both families are more similar to Eurytomidae, Pteromalidae, and Torymidae than Agaonidae.

Functional morphology of the sting in two digger wasps (Hymenoptera: Crabronidae) with different types of prey transport.

Digger wasps of the family Crabronidae (Insecta: Hymenoptera) are generally known to use their sting to paralyze or kill a prey. However, only a few species of digger wasps transport their prey to the nest impaled on the sting. How sting morphology correlates with this peculiar type of prey carriage is still unclear. We examined the sting morphology of two phylogenetically closely-related species of digger wasps of similar size, which hunt for similar preys but use different types of prey transportation. Data from light microscopy (LM), scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were analyzed to find possible correlations between shape, material composition, and function of the stings. The similarity of the material composition in the stings of the two species suggests that the material of stings does not play a dominant role in their functional differences. On the contrary, differences in the curvature and surface sculpture of sting elements likely result in different stress distributions under mechanical loading.

Three-dimensional ultrastructural organization of the ommatidium of the minute parasitoid wasp Trichogramma evanescens.

Existing information on insect compound eyes is mainly limited to two-dimensional information derived from histological or ultrathin sections. These allow a basic description of eye morphology, but are limited in z-axis resolution because of the section thickness or intervals between sections, so that accurate volumetric information cannot be generated. Here we use serial-sectioning transmission electron microscopy to present a 3-D reconstruction at ultrastructural level of a complete ommatidium of a miniaturized insect compound eye. Besides the general presentation of the three dimensional arrangement of the different cell types within the ommatidium, the reconstruction allowed volumetric measurements and numerical analyses to be undertaken, revealing new insights into the number, size and distribution of cell organelles in insect ommatidia. Morphological features that can be related to miniaturization, namely the dimensions and displacement of nuclei, reduction of average pigment granule volume and loss of pigment granules in the terminals of the cone cells, the impact of metabolic activity of cell types on miniaturization, as well as maintenance of rhabdomere volume and limits to its miniaturization, are all discussed.

Ultrastructural Alterations of Midgut Epithelium, But Not Greater Wing Fluctuating Asymmetry, in Paper Wasps (Polistes dominula) from Urban Environments.

Polistes paper wasps can be used to monitor trace metal contaminants, but the effects of pollution on the health of these insects are still unknown. We evaluated, in a south-eastern area of Spain, whether workers of Polistes dominula collected at urban and rural sites differ in health of midgut tissue and in fluctuating asymmetry, an estimate of developmental noise. We found that wasps collected at the urban sites had abundant lead (Pb)-containing spherites, which were less visible in wasps from the rural sites. Evident ultrastructural alterations in the epithelium of the midgut of the wasps collected at the urban sites included broken and disorganized microvilli, a high amount and density of heterochromatin in the nucleus of epithelial cells, cytoplasmic vacuolization and mitochondrial disruptions. Altogether, these findings suggest a negative effect on the transmembrane transport and a less efficient transcription. On the contrary, a healthy epithelium was observed in wasps from the rural sites. These differences may be preliminarily linked with levels of lead pollution, given that wasps from urban sites had double the Pb concentrations of wasps from rural sites. Level of fluctuating asymmetry was unrelated to wasp origin, thus suggesting no link between developmental noise and Pb-driven pollution.

First extensive characterization of the venom gland from an egg parasitoid: structure, transcriptome and functional role.

The venom gland is a ubiquitous organ in Hymenoptera. In insect parasitoids, the venom gland has been shown to have multiple functions including regulation of host immune response, host paralysis, host castration and developmental alteration. However, the role played by the venom gland has been mainly studied in parasitoids developing in larval or pupal hosts while little is known for parasitoids developing in insect eggs. We conducted the first extensive characterization of the venom of the endoparasitoid Ooencyrtus telenomicida (Vassiliev), a species that develops in eggs of the stink bug Nezara viridula (L.). In particular we investigated the structure of the venom apparatus, its functional role and conducted a transcriptomic analysis of the venom gland. We found that injection of O. telenomicida venom induces: 1) a melanized-like process in N. viridula host eggs (host-parasitoid interaction), 2) impairment of the larval development of the competitor Trissolcus basalis (Wollaston) (parasitoid-parasitoid interaction). The O. telenomicida venom gland transcriptome reveals a majority of digestive enzymes (peptidases and glycosylases) and oxidoreductases (laccases) among the most expressed genes. The former enzymes are likely to be involved in degradation of the host resources for the specific benefit of the O. telenomicida offspring. In turn, alteration of host resources caused by these enzymes may negatively affect the larval development of the competitor T. basalis. We hypothesize that the melanization process induced by venom injection could be related to the presence of laccases, which are multicopper oxidases that belong to the phenoloxidases group. This work contributed to a better understanding of the venom in insect parasitoids and allowed to identify candidate genes whose functional role can be investigated in future studies.

From two to three dimensions: The importance of the third dimension for evaluating the limits to neuronal miniaturization in insects.

Most studies dealing with the limits to miniaturization in insect brains have until now relied on information based on data collected in two dimensions: either histological sections imaged by light microscopy, or electron micrographs of single ultrathin sections imaged by transmission electron microscopy (TEM). To test the validity of transferring information gained from two-dimensional images to the third dimension, we examined a 3D image stack from serial-section TEM (ssTEM) of the optic neuropiles of the miniature parasitic wasp Trichogramma brassicae (Bezdenko, 1968). We reinvestigated the proposed lower limit of 2 µm for the diameters of neuronal somata and found average volumes of 6.5 µm3 for lamina cells and 3.8 µm3 for medulla cells. We likewise found a limiting factor for the volume of nuclei, which averages 41.9% and 49.2% of the cell body volume, respectively, but that in turn the compactness of heterochromatin was not a limiting factor in the minimal volume of the nuclei. Finally, we also found a minimum axon diameter of 98 nm that could nevertheless accommodate axoplasmic mitochondria. Incorporating the third dimension thus proves critically important in avoiding volumetric misinterpretations of these values. We discuss the limitations of analyzing the effects of miniaturization from profile data of neurons and demonstrate that miniaturization within the nervous system can lie beyond previously described limits and in some cases is already present in the optic lobe neurons of T. brassicae.

Egg-laying behavior and morphological and chemical characterization of egg surface and egg attachment glue of the digger wasp Ampulex compressa (Hymenoptera, Ampulicidae).

For providing their offspring females of the digger wasp species Ampulex compressa hunt cockroaches, paralyze them and attach as a rule one egg to the coxa of one of the mid legs of their prey. We observed the egg-laying behavior and examined with light- and scanning microscopy (i) nearly mature eggs from ovaries of freshly dissected females and (ii) eggs immediately after their deposition on the coxae of their prey. The length of the white bean-shaped eggs varied between 2.2 and 3.0 mm, their diameter between 0.66 and 0.72 mm, and their weight between 345 and 832 µg. The surface of fresh, untreated eggs shows even at higher magnifications (>20.000×) a smooth appearance. However, after conventional fixation, dehydration with ethyl-alcohol and critical-point drying the egg-surface exhibited a little bit texture. The eggs are at two-third of their underside glued to the coxa of the prey. With the naked eye the glue appears as a compact mass. The eggs may be mechanically removed from the substrate (their attachment site); however, in doing so the viscous attachment glue appears in a more fibrous consistence. The polypeptide composition washed off the egg surface and the glue revealed no similarities, whereas the molecular mass of two polypeptides were similar between glue and the Dufour's gland contents.

A metatranscriptomic analysis of diseased social wasps (Vespula vulgaris) for pathogens, with an experimental infection of larvae and nests.

Social wasps are a major pest in many countries around the world. Pathogens may influence wasp populations and could provide an option for population management via biological control. We investigated the pathology of nests of apparently healthy common wasps, Vespula vulgaris, with nests apparently suffering disease. First, next-generation sequencing and metatranscriptomic analysis were used to examine pathogen presence. The transcriptome of healthy and diseased V. vulgaris showed 27 known microbial phylotypes. Four of these were observed in diseased larvae alone (Aspergillus fumigatus, Moellerella wisconsensis, Moku virus, and the microsporidian Vavraia culicis). Kashmir Bee Virus (KBV) was found to be present in both healthy and diseased larvae. Moellerella wisconsensis is a human pathogen that was potentially misidentified in our wasps by the MEGAN analysis: it is more likely to be the related bacteria Hafnia alvei that is known to infect social insects. The closest identification to the putative pathogen identified as Vavraia culicis was likely to be another microsporidian Nosema vulgaris. PCR and subsequent Sanger sequencing using published or our own designed primers, confirmed the identity of Moellerella sp. (which may be Hafnia alvei), Aspergillus sp., KBV, Moku virus and Nosema. Secondly, we used an infection study by homogenising diseased wasp larvae and feeding them to entire nests of larvae in the laboratory. Three nests transinfected with diseased larvae all died within 19 days. No pathogen that we monitored, however, had a significantly higher prevalence in diseased than in healthy larvae. RT-qPCR analysis indicated that pathogen infections were significantly correlated, such as between KBV and Aspergillus sp. Social wasps clearly suffer from an array of pathogens, which may lead to the collapse of nests and larval death.

The insect ovipositor as a volatile sensor within a closed microcosm.

We show that the insect ovipositor is an olfactory organ that responds to volatiles and CO2 in gaseous form. We demonstrate this phenomenon in parasitic wasps associated with Ficus racemosa where ovipositors, as slender as a human hair, drill through the syconium (enclosed inflorescences) and act as a guiding probe to locate highly specific egg-laying sites hidden inside. We hypothesize that olfaction will occur in the ovipositors of insects such as parasitic fig wasps where the hosts are concealed and volatile concentrations can build up locally. Relevant stimuli such as herbivore-induced fig volatiles and CO2 elicited electrophysiological responses from the ovipositors. Silver nitrate staining also revealed pores in ovipositor sensilla, indicating their olfactory nature. Insects could use volatile sensors on their ovipositors to evaluate ecologically relevant stimuli for oviposition. Further investigations on the sensory nature of ovipositors can provide designs for development of ovipositor-inspired micro-chemosensors.

The unusual eyes of Xenos peckii (Strepsiptera: Xenidae) have green- and UV--sensitive photoreceptors.

The highly specialized evolution of Strepsiptera has produced one of the most unusual eyes among mature insects, perhaps in line with their extremely complex and challenging life cycle. This relatively rare insect order is one of the few for which it has been unclear what spectral classes of photoreceptors any of its members may possess, an even more apt question given the nocturnal evolution of the group. To address this question, we performed electroretinograms on adult male Xenos peckii: we measured spectral responses to equi-quantal monochromatic light flashes of different wavelengths, and established VlogI relationships to calculate spectral sensitivities. Based on opsin template fits, we found maximal spectral sensitivity (λmax) in the green domain at 539 nm. Application of a green light to 'bleach' green receptors revealed that a UV peak was contributed to by an independent UV opsin with a λmax of 346 nm. Transcriptomics and a phylogenetic analysis including 50 other opsin sequences further confirmed the presence of these two opsin classes. While these findings do not necessarily indicate that these unorthodox insects have color vision, they raise the possibility that UV vision plays an important role in the ability of X. peckii males to find the very cryptic strepsipteran females that are situated within their wasp hosts.

The anatomy of the thrips Heliothrips haemorrhoidalis (Thysanoptera, Thripidae) and its specific features caused by miniaturization.

A new set of data on the internal and external structure of the adult and larva of the thrips Heliothrips haemorrhoidalis (Bouché, 1833) is presented. The structure of the internal systems of this thrips was revealed using modern methods of 3D computer modelling. The changes in shape and relative size are discussed as an outcome of miniaturization in comparison to the supposed ancestor of this species. The layout of the internal systems of thrips is compared to those of other insects similar in size: beetles of the families Ptiliidae and Corylophidae and wasps of the families Mymaridae and Trichogrammatidae.

Morphology and ultrastructure of the allomone and sex-pheromone producing mandibular gland of the parasitoid wasp Leptopilina heterotoma (Hymenoptera: Figitidae).

Chemical communication by the parasitoid wasp Leptopilina heterotoma is based largely on (-)-iridomyrmecin. The female wasps use (-)-iridomyrmecin as a defensive allomone, a chemical cue to avoid competition with con- and heterospecific females, and as a major component of their sex pheromone to attract males. Males of L. heterotoma produce (+)-isoiridomyrmecin, which is also used for chemical defense. In this study we show that females and males of L. heterotoma produce the iridomyrmecins in a pair of mandibular glands. Each gland consists of a secretory part composed of class 3 gland cells and their accompanying duct cells, as well as a reservoir bordered by a thin intima. The gland discharges between the mandible base and the clypeus. Males have considerably smaller glands than females, which corresponds to the lower amount of iridomyrmecins produced by males. Chemical analyses of the mandibular gland contents showed that the gland of females contained mainly (-)-iridomyrmecin, as well as low amounts of the other previously described iridoid pheromone compounds, while the glands of males contained only (+)-isoiridomyrmecin. The morphology and sizes of the mandibular glands of males and females of L. heterotoma have evolved to the multi-functional use of iridomyrmecin.

Sperm morphology of Muscidifurax uniraptor (Hymenoptera: Chalcidoidea: Pteromalidae).

Sperm morphology of the parasitoid Muscidifurax uniraptor was investigated under light and transmission electron microscopy. M. uniraptor sperm are filiform, spiraled, approximately 150 µm in length, with a distinctive head, hooded by an extracellular sheath and a flagellum. This extracellular layer, from which many filaments radiate, measures approximately 90 nm in thickness and covers a small acrosome and the anterior nuclear region. The acrosome is composed of an acrosomal vesicle and a perforatorium with its base inserted in the nuclear tip. The nucleus is filled with homogeneously compacted chromatin. The centriolar adjunct extends towards the anterior portion in a spiral around the nucleus for 3.5 µm in length. The two mitochondrial derivatives begin exactly at the centriole adjunct base and, in cross-section, have a circular shape with equal areas that are smaller than the axoneme diameter. It is coiled, with 9 + 9 + 2 microtubules and begins from the centriole, just below the nuclear base. The axoneme is connected to the mitochondrial derivatives by two small irregularly shaped masses. Between the derivatives and the axoneme, the 'center-flagellar material' is observed. Overall, these characteristics are recognized in other Chalcidoidea, especially in the eurytomids, but together they form a set of species-specific data.

First instar larvae morphology of Opiinae (Hymenoptera: Braconidae) parasitoids of Anastrepha (Diptera: Tephritidae) fruit flies. Implications for interspecific competition.

The morphology of the first instars of the Opiinae braconids Diachasmimorpha longicaudata, Doryctobracon areolatus, Doryctobracon crawfordi, Utetes anastrephae and Opius hirtus (the first is exotic, and the others are natives to Mexico), parasitoids of Anastrepha fruit flies, are described and compared. The possible implications on interspecific competition among these species are discussed. The most significant adaptations found were: (1) the mouth apparatus, where the large mandibles and fang-shaped maxillary lobes present in D. longicaudata and U. anastrephae larvae were absent in O. hirtus, D. areolatus and D. crawfordi larvae, and (2) the degree of mobility for exploration and escape, such as the lateral and caudal appendages that were only present in D. longicaudata (ventrolateral appendages in the base of the head capsule), U. anastrephae (caudal lobe with two appendages) and D. areolatus (caudal lobe with a round apex with a globular shape). The first instar larvae of the species D. longicaudata show morphological adaptations that apparently confer competitive advantages against the larvae of D. areolatus, D. crawfordi and O. hirtus. However, the first instar larvae of U. anastrephae show larger mandibles, an adaptation that could enable this species to resist competition from D. longicaudata.

Redescription of Campoletis sonorensis (Cameron, 1886) (Hymenoptera, Ichneumonidae, Campopleginae), parasitoid of Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera, Noctuidae) in Brazil.

The fall armyworm Spodoptera frugiperda (Lepidoptera; Noctuidae) is a voracious pest of numerous crops of economic importance throughout the New World. In Brazil, its larvae are attacked by several species of parasitoid wasps, making them potential candidate as biological control agents against this pest. A survey of the parasitoid fauna on S. frugiperda in maize crops throughout Brazil reveals two species of Campoletis, which are morphologicaly very similar species. In this paper we combine these data with pictures from the type material of C. sonorensis and C. flavicincta, as well as their descriptions to provide a redescription to Campoletis sonorensis (Cameron, 1886) using for this both morphological characters and DNA Barcoding (Hebert et al., 2003) information, in an attempt to help with the correct identification of the taxa to improve biological control studies.

Redescription of Campoletis sonorensis (Cameron, 1886) (Hymenoptera, Ichneumonidae, Campopleginae), parasitoid of Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera, Noctuidae) in Brazil / Redescrição de Campoletis sonorensis (Cameron, 1886) (Hymenoptera, Ichneumonidae, Campopleginae), parasitoide de Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera, Noctuidae) no Brasil

Abstract The fall armyworm Spodoptera frugiperda (Lepidoptera; Noctuidae) is a voracious pest of numerous crops of economic importance throughout the New World. In Brazil, its larvae are attacked by several species of parasitoid wasps, making them potential candidate as biological control agents against this pest. A survey of the parasitoid fauna on S. frugiperda in maize crops throughout Brazil reveals two species of Campoletis, which are morphologicaly very similar species. In this paper we combine these data with pictures from the type material of C. sonorensis and C. flavicincta, as well as their descriptions to provide a redescription to Campoletis sonorensis (Cameron, 1886) using for this both morphological characters and DNA Barcoding (Hebert et al., 2003) information, in an attempt to help with the correct identification of the taxa to improve biological control studies.

Resumo Spodoptera frugiperda é uma praga voraz de diversas culturas de importância econômica no Novo Mundo. No Brasil, suas larvas podem ser atacadas por diversas espécies de vespas parasitóides que são candidatos a agentes de controle biológico contra essa praga. Pesquisando os parasitóides da fauna de Spodoptera frugiperda em cultivos de milho no Brasil foram encontradas duas espécies do gênero Campoletis, muito similares morfologicamente. Este trabalho apresenta uma redescrição para Campoletis sonorensis usando caracteres morfológicos e DNA Barcoding (Hebert et al., 2003) com o objetivo de evitar erros de identificação desse grupo, aprimorando estudos de controle biológico.

Exocrine glands in the legs of the social wasp Vespula vulgaris.

This study brings a survey of the exocrine glands in the legs of Vespula vulgaris wasps. We studied workers, males, virgin queens as well as mated queens. A variety of 17 glands is found in the different leg segments. Among these, five glands are novel exocrine structures for social insects (trochanter-femur gland, ventrodistal tibial gland, distal tibial sac gland, ventral tibial gland, and ventral tarsomere gland). Most leg glands are present in the three leg pairs of all castes. This may indicate a mechanical function. This is likely for the numerous glands that occur near the articulation between the various leg segments, where lubricant production may be expected. Other possible functions include antenna cleaning, acting as a hydraulic system, or pheromonal. Further research including leg-related behavioural observations and chemical analyses may help to clarify the functions of these glandular structures in the legs.