Phylogenomics of the lepidopteran endoparasitoid wasp subfamily Rogadinae (Hymenoptera: Braconidae) and related subfamilies

Rogadinae are a cosmopolitan, species‐rich braconid wasp subfamily whose species are endoparasitoids that attack larvae of a number of lepidopteran families. Members of this subfamily are characterized by pupating within the mummified host larval skin. The subfamily contains six tribes whose relationships have only been partially clarified: Aleiodini, Betylobraconini, Clinocentrini, Rogadini, Stiropiini and Yeliconini. The limits and composition of the closely related subfamilies to the Rogadinae, Hormiinae and Lysiterminae, also remain unclear. Here, we generated ultraconserved element data to reconstruct an almost fully resolved phylogeny for the members of Rogadinae and related subfamilies. Based on our best estimate of phylogeny, we confirm the monophyly of Rogadinae including Betylobraconini, synonymize Xenolobus Fahringer and Bequartia Cameron within the species‐rich genus Aleiodes Wesmael ( syn.n. ) based on DNA, and synonymize Promesocentrus van Achterberg with Pilichremylus Belokobylskij ( syn.n. ) based on morphology. We also consistently recovered Hormiinae and Lysiterminae as not reciprocally monophyletic, and thus propose to unite their members under Hormiinae. The ancestral host preference for Rogadinae was probably attacking concealed lepidopteran larvae, with the occurrence of at least two main subsequent transitions to attack both concealed and exposed hosts, one within Rogadini and a second within Aleiodini. We highlight the importance of natural history collections as a source for conducting genomic‐based studies using techniques that allow to obtain a substantial amount of data from considerably old preserved insect specimens.     

Molecular phylogeny of the paper wasp subgenus Polistes (Polistella) Ashmead, 1904 (Hymenoptera: Vespidae: Polistinae) from Vietnam

This study provides the first molecular phylogeny of the social wasp subgenus Polistella (Hymenoptera: Vespidae: Polistes) from Vietnam. Fragments of the mitochondrial COI and 16S rRNA genes were used to reconstruct the phylogenetic trees among 38 Polistes species plus two out-group species (Vespa soror du Buysson and Ropalidia fasciata (Fabricius)). Our results support the existence of several species-groups, including two that are congruent with the previous stigma and Stenopolistes groups defined on the basis of morphology. Moreover, we recovered a clade including the stigma group and the two species P. humilis and P. variabilis that was sister to all other species of Polistella. However, the results also challenged the definition of other groups of Polistella based on morphological data, as well as the definition of two species: P. brunus and P. affinis. This first study calls for further analyses including morphological characters to clarify the taxonomy and the classification of the group.     

A new rod-shaped virus from parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

A new rod-shaped nucleocapsids (NCs) was found inadvertently in Diachasmimorpha longicaudata accessory gland filaments (AGFs). The NCs were 30 nm in diameter and nearly 900 nm in length. They replicated in a small cell type of the AGFs in D. longicaudata, and following oviposition, invaded and proliferated in the hemocytes of a parasitized host Bactrocera dorsalis Hendel. This finding of a completely new virus in the AGF indicate that different geographical populations (subspecies) of D. longicaudata may carry different sybionts. This is the first report showing that the same wasp species, but from a different geographical populations, can carry an entirely different virus.     

Identification and characterization of antimicrobial peptides from the skin of the endangered frog Odorrana ishikawae

The endangered anuran species, Odorrana ishikawae, is endemic to only two small Japanese Islands, Amami and Okinawa. To assess the innate immune system in this frog, we investigated antimicrobial peptides in the skin using artificially bred animals. Nine novel antimicrobial peptides containing the C-terminal cyclic heptapeptide domain were isolated on the basis of antimicrobial activity against Escherichia coli. The peptides were members of the esculentin-1 (two peptides), esculentin-2 (one peptide), palustrin-2 (one peptide), brevinin-2 (three peptides) and nigrocin-2 (two peptides) antimicrobial peptide families. They were named esculentin-1ISa, esculentin-1ISb, esculentin-2ISa, palustrin-2ISa, brevinin-2ISa, brevinin-2ISb, brevinin-2ISc, nigrocin-2ISa and nigrocin-2ISb. Peptide primary structures suggest a close relationship with the Asian odorous frogs, Odorrana grahami and Odorrana hosii. These antimicrobial peptides possessed a broad-spectrum of growth inhibition against five microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis and Candida albicans). Nine different cDNAs encoding the precursor proteins were also cloned and showed that the precursor proteins exhibited a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and an antimicrobial peptide at the C-terminus.     

In vivo and in vitro activity of venom from the endoparasitic wasp Pimpla turionellae (L.) (Hymenoptera: Ichneumonidae)

The biological activity of venom from Pimpla turionellae L. (Hymenoptera: Ichneumonidae) was examined in vivo toward larvae and pupae of Galleriae mellonella L. (Lepidoptera: Pyralidae), and in vitro toward bacterial and fungal cultures, as well as cultured insect cells. Pupae of G. mellonella were far more susceptible to the venom than larvae. At low doses of venom [0.1 venom reservoir equivalents (VRE)], pupal abdominal mobility was inhibited within 30 min, and by 24 h, all pupae injected with venom concentrations >0.5 VRE were completely paralyzed. These same doses of venom resulted in an inhibition of adult emergence. Host larvae were far less sensitive to wasp venom as evidenced by all venom injected larvae remaining responsive to mechanical stimulation by 1 h post injection, even at concentrations equivalent to 1 venom reservoir. Eventually (>2 h at 25 degrees C), venom-injected larvae became immobile, then flaccid, and all died within 24 h post-injection. At lower concentrations of wasp venom, the onset of paralysis was delayed by comparison to that evoked by 1 VRE, and few host larvae were able to pupate. Development of host larvae to adult emergence was also reduced in a dose-dependent manner, with eclosion completely prevented at high concentrations (>0.5 VRE) of venom. Venom doses <0.5 VRE did not appear to induce paralysis or alter larval development. When venom was incubated with bacterial or fungal cultures, no antimicrobial activity was detected. However, wasp venom was found to be cytotoxic and cytolytic to cultured cells derived from the cabbage looper Trichoplusia ni Hubner (Lepidoptera: Noctuidae) and the yellow fever mosquito, Aedes aegypti (L.) (Diptera: Culcidae). Though both cell types displayed similar susceptibility in terms of LC50s, the lepidopteran cells responded much more rapidly with regard to the onset of morphological changes and the timing of cell death. A possible mode of action for the venom is discussed.     

Dispersal behavior of the parasitic wasp Cotesia urabae (Hymenoptera: Braconidae): A recently introduced biocontrol agent for the control of Uraba lugens (Lepidoptera: Nolidae) in New Zealand

Estimates of the dispersal range of a recently introduced biocontrol agent in its new environment are vital to understanding its relative searching capacity, and to foresee the maximum area that could be covered in a release event. In New Zealand, the solitary endoparasitoid Cotesia urabae Austin and Allen (Hymenoptera: Braconidae) was first released in January 2011 as a biological control agent for the gum leaf skeletoniser, Uraba lugens Walker (Lepidoptera: Nolidae). The objective of this study was to utilize an experimental approach to quantify the dispersal behavior of one generation of C. urabae. In our experiment, which used sentinel larvae as target hosts, parasitoids dispersed up to 20 m away from the release point but parasitism was highest within 5 m of the release site. A high level of parasitism was observed at the release tree (87.6%) which suggests that most of the females released may have stayed there. According to the dispersal model developed from the data collected, Cotesia would be able to disperse up to 53 m in one release event. In addition, significant differences were found between the different directions tested for dispersal, showing a clear downwind effect on dispersal suggesting that wind has a direct effect on the dispersal behavior of C. urabae in the field.     

Emergence and dispersal relative to natal nest in the digger wasp Stizus continuus (Hymenoptera: Crabronidae)

The position of the emerging point has rarely been investigated as a factor possibly affecting the future nest settlement behaviour in Hymenoptera, in particular within nest aggregations. We studied the emergence and dispersion patterns of the digger wasp Stizus continuus. Individuals emerged daily in clumped patterns, possibly revealing a certain synchrony of emergence from the same nests, and protandry appeared both at seasonal and daily level. Differences between the number of females that nested relatively close or far from their emergence holes (EH) were either significant or not, depending on the year, and observed dispersal distances from the natal nests did not differ from those obtained by random simulations. By contrast, females nested close to the nearest conspecific nest. Size did not affect the dispersion patterns. EH are thus not important cues for nest establishment, and conspecific nests are probably the key cue for nest-founding females. In addition, males did not prefer to establish territories close to their natal nest.     

Biogenic amine biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

Biogenic amines (BAs), such as octopamine, tyramine, dopamine, serotonin, and acetylcholine regulate various behaviors and physiological functions in insects. Here, we identified seven genes encoding BA biosynthetic enzymes and 16 genes encoding BA G protein-coupled receptors in the genome of the endoparasitoid wasp, Pteromalus puparum. We compared the genes with their orthologs in its host Pieris rapae and the related ectoparasitic wasp Nasonia vitripennis. All the genes show high (>90%) identity to orthologs in N. vitripennis. P. puparum and N. vitripennis have the smallest number of BA receptor genes among the insect species we investigated. We then analyzed the expression profiles of the genes, finding those acting in BA biosynthesis were highly expressed in adults and larvae and those encoding BA receptors are highly expressed in adults than immatures. Octα1R and 5-HT₇ genes were highly expressed in salivary glands, and a high messenger RNA level of 5-HT_1A was found in venom apparatuses. We infer that BA signaling is a fundamental component of the organismal organization, homeostasis and operation in parasitoids, some of the smallest insects.     

Sexual size dimorphism in the ontogeny of the solitary predatory wasp Symmorphus allobrogus (Hymenoptera: Vespidae)

Sex-specific patterns of individual growth, resulting in sexual size dimorphism (SSD), are a little studied aspect of the ontogeny related to the evolutionary history and affected by the ecology of a species. We used empirical data on the development of the predatory wasp Symmorphus allobrogus (Hymenoptera, Vespidae) to test the hypotheses that sexual differences of growth resulting in the female-biased SSD embrace the difference in (1) the egg size and the starting size of larva, (2) the larval development duration, and (3) the larval growth rate. We found that eggs developing into males and females have significant differences in size. There was no significant difference between the sexes in the duration of larval development. The relative growth rate and the food assimilation efficiency of female larvae were significantly higher than compared to those of male larvae. Thus, the SSD of S. allobrogus is mediated mainly by sexual differences in egg size and larval growth rate.     

Interactions between Phaedrotoma scabriventris Nixon (Hymenoptera: Braconidae) and Diglyphus isaea Walker (Hymenoptera: Eulophidae), parasitoids of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae)

Liriomyza leafminer flies represent a serious threat to horticultural production in East Africa. Total field parasitism rates recorded in Kenya are below 5%, with the indigenous ectoparasitoid Diglyphus isaea Walker being one of the key parasitoid species. The International Centre of Insect Physiology and Ecology (icipe), in collaboration with the International Potato Centre (CIP), imported into Kenya the endoparasitoid Phaedrotoma scabriventris Nixon to improve natural control of leafminers. The objective of this study was to investigate the interactions between D. isaea and P. scabriventris when used together for the biological control of Liriomyza species. These interactions were studied under laboratory conditions, using treatments that involved single, simultaneous and sequential releases of the different parasitoid species onto plants infested by L. huidobrensis larvae. While used separately, parasitism rates of D. isaea and P. scabriventris were 30.4 ± 10.9% and 63.6 ± 7.7% respectively. However, when used simultaneously, the total parasitism rate increased to 77.0 ± 5.3%. Although P. scabriventris had no effect on D. isaea, the presence of D. isaea reduced the specific parasitism rate of P. scabriventris. In addition, both parasitoids induced leafminer mortality through larval-feeding and stinging. However, feeding and stinging mortality induced by D. isaea (41.9 ± 9.1%) was significantly higher compared to P. scabriventris (11.9 ± 8.7). Similarly, pupal mortality due to feeding and stinging activity was 49.1 ± 6.5% and 21.6 ± 1.9% when exposed to D. isaea and P. scabriventris respectively. The implication for simultaneous use of both parasitoids in East Africa is discussed.     

Characterization of microsatellite loci isolated from the wasp, Microstigmus nigrophthalmus (Hymenoptera)

Fifty‐two microsatellite loci were characterized in 22–31 unrelated females of the wasp (Microstigmus nigrophthalmus) collected from the Mata do Paraiso, Viçosa, M.G., Brazil. Fifty‐one of these loci were developed from a microsatellite‐enriched genomic library derived from M. nigrophthalmus and one was derived from the wasp, Ormyrus nitidulus. The genus Microstigmus represents an independent origin of social behaviour in the Hymenoptera and is thus of great potential in the study of social evolution.     

First report of chromosomes of the parasitoid wasp Trissolcus basalis (Wollaston) (Hymenoptera: Platygastridae: Telenominae)

Here we present the first data on the chromosome complement of Trissolcus basalis gathered in conjunction with the ongoing whole genome sequencing efforts for this species. The cytogenetic investigation revealed a diploid karyotype with 20 chromosomes, comprising one pair of large metacentrics and nine pairs of acrocentrics that gradually decrease in length.     

Exogenous JH and ecdysteroid applications alter initiation of polydnaviral replication in an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera)

Polydnaviruses are a group of double-stranded DNA viruses and are symbiotically associated with some ichneumonoid wasps. As proviruses, the replication of polydnaviruses occurs in the female reproductive organ at the pupal stage. This study analyzed the effects of two developmental hormones, juvenile hormone (JH) and ecdysteroid, on the viral replication of Cotesia plutellae bracovirus (CpBV). All 23 CpBV segments identified contained a conserved excision/rejoining site ('AGCTTT') from their proviral segments. Using quantitative real-time PCR based on this excision/rejoining site marker, initiation of CpBV replication was determined to have occurred on day 4 on the pupal stage. Pyriproxyfen, a JH agonist, significantly inhibited adult emergence of C. plutellae, whereas RH5992, an ecdysteroid agonist, had no inhibitory effect. Although RH5992 had no effect dose on adult development, it significantly accelerated viral replication. The results of immunoblotting assays against viral coat proteins support the effects of the hormone agonists on viral replication.     

Host regulation and the embryonic development of the endoparasitoid Toxoneuron nigriceps (Hymenoptera: Braconidae)

Insect endoparasitoids modulate the host physiology through the injection of maternal-derived substances into the host, inducing physiological and hormonal changes in the host's internal environment to benefit parasitoid development. These changes are direct to control host development and regulate nutrient availability to the developing parasitoid, and they are synchronized with parasitoid development. Eggs of some of these parasitoids have low yolk content and require nutrients from the host hemolymph to initiate and complete embryogenesis. We report changes in the amino acid composition and protein profile of the host hemolymph of the endoparasitoid Toxoneuron nigriceps, and improved the in vitro culture of pre-germ band stage eggs. The protein profile of parasitized larvae was similar to controls throughout the embryonic development, but total amino acid concentration decreased in the first 2 h after parasitization, significantly increasing in the following hours up to 8 h. Amino acid levels were higher in parasitized larvae from 16 to 28 h after parasitization. Comparison of single amino acids indicated amino acids involved in energy metabolism (Krebs cycle) followed a trend during parasitoid embryogenesis, and their changes were correlated with embryonic development. Improvement in the in vitro development of 6 h-old eggs of T. nigriceps was obtained by adding factors released by the host fat body to the artificial medium, while a cell lysate stimulated embryogenesis and allowed the full development of newly laid eggs in vitro.     

Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.     

Antimicrobial peptides are present in immune and host defense cells of the human respiratory and gastroinstestinal tracts

Previous studies have implicated antimicrobial peptides in the host defense of the mammalian intestinal and respiratory tract. The aim of the present study has been to characterize further the expression of these molecules in non-epithelial cells of the human pulmonary and digestive systems by detailed immunohistochemical analysis of the small and large bowel and of the large airways and lung parenchyma. Additionally, cells obtained from bronchoalveolar lavage were analyzed by fluorescent activated cell sorting and immunostaining of cytospin preparations. hBD-1, hBD-2, and LL-37 were detected in lymphocytes and macrophages in the large airways, lung parenchyma, duodenum, and colon. Lymphocytes positive for the peptides revealed a staining pattern and distribution that largely matched that of CD3-positive and CD8-positive T-cells. Macrophages with positive staining for the antimicrobial peptides also stained positively for CD68 and CD74. In view of the morphology of the LL-37-positive and hBD-2-positive mucosal lymphocytes, they are probably also B-cells. Thus, antimicrobial peptides of the defensin and cathelicidin families are present in a variety of non-epithelial cells of mucosal organs. These findings confirm that antimicrobial peptides have multiple functions in the biology of the mucosa of these organs. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ba 1641/5–1 and Ba 1641/6–1)     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.