Genetic manipulation allows in vivo tracking of the life cycle of the son-killer symbiont,Arsenophonus nasoniae,and reveals patterns of host invasion,tropism and pathology

Maternally heritable symbionts are common in arthropods and represent important partners and antagonists. A major impediment to understanding the mechanistic basis of these symbioses has been lack of genetic manipulation tools, for instance, those enabling transgenic GFP expression systems for in vivo visualization. Here, we transform the ‘son-killer’ reproductive parasite Arsenophonus nasoniae that infects the parasitic wasp Nasonia vitripennis with the plasmid pOM1-gfp, re-introduce this strain to N. vitripennis and then used this system to track symbiont life history in vivo. These data revealed transfer of the symbiont into the fly pupa by N. vitripennis during oviposition and N. vitripennis larvae developing infection over time through feeding. A strong tropism of A. nasoniae to the N. vitripennis ovipositor developed during wasp pupation, which aids onward transmission. The symbiont was also visualized in diapause larvae. Occasional necrotic diapause larvae were observed which displayed intense systemic infection alongside widespread melanotic nodules indicative of an active but failed immune response. Our results provide the foundation for the study of this symbiosis through in vivo tracking of the fate of symbionts through host development, which is rarely achieved in heritable microbe/insect interactions.     

Reproductive strategies under multiparasitism in natural populations of the parasitoid wasp Nasonia (Hymenoptera)

Parasitoid Nasonia wasps adjust their progeny sex ratio to the presence of conspecifics to optimize their fitness. Another trait under female control is the induction of offspring diapause. We analysed progeny sex ratios and the proportion of diapausing offspring of individual Nasonia females in host patches parasitized by two species, Nasonia vitripennis and Nasonia giraulti, in North American field populations using microsatellite fingerprinting. Both Nasonia species produced similar sex ratios on hosts that were co‐parasitized by their own species as by the other species, indicating that females do not distinguish between con‐ and heterospecific clutches. The sex ratios of the diapause and adult fractions of mixed broods from single females were not correlated. We found further indications that N. vitripennis females take the emergence time of the offspring into account in their sex allocation. The reproductive strategies of Nasonia under multiparasitism are largely adaptive, but also partially constrained by information.     

GENETIC AND DEVELOPMENTAL BASIS OF F2 HYBRID BREAKDOWN IN NASONIA PARASITOID WASPS

Speciation is responsible for the vast diversity of life, and hybrid inviability, by reducing gene flow between populations, is a major contributor to this process. In the parasitoid wasp genus Nasonia, F₂ hybrid males of Nasonia vitripennis and Nasonia giraulti experience an increased larval mortality rate relative to the parental species. Previous studies indicated that this increase of mortality is a consequence of incompatibilities between multiple nuclear loci and cytoplasmic factors of the parental species, but could only explain ～40% of the mortality rate in hybrids with N. giraulti cytoplasm. Here we report a locus on chromosome 5 that can explain the remaining mortality in this cross. We show that hybrid larvae that carry the incompatible allele on chromosome 5 halt growth early in their development and that ～98% die before they reach adulthood. On the basis of these new findings, we identified a nuclear‐encoded OXPHOS gene as a strong candidate for being causally involved in the observed hybrid breakdown, suggesting that the incompatible mitochondrial locus is one of the six mitochondrial‐encoded NADH genes. By identifying both genetic and physiological mechanisms that reduce gene flow between species, our results provide valuable and novel insights into the evolutionary dynamics of speciation.     

Nasonia vitripennis venom causes targeted gene expression changes in its fly host

Parasitoid wasps are diverse and ecologically important insects that use venom to modify their host's metabolism for the benefit of the parasitoid's offspring. Thus, the effects of venom can be considered an ‘extended phenotype’ of the wasp. The model parasitoid wasp Nasonia vitripennis has approximately 100 venom proteins, 23 of which do not have sequence similarity to known proteins. Envenomation by N. vitripennis has previously been shown to induce developmental arrest, selective apoptosis and alterations in lipid metabolism in flesh fly hosts. However, the full effects of Nasonia venom are still largely unknown. In this study, we used high throughput RNA sequencing (RNA‐Seq) to characterize global changes in Sarcophaga bullata (Diptera) gene expression in response to envenomation by N. vitripennis. Surprisingly, we show that Nasonia venom targets a small subset of S. bullata loci, with ~2% genes being differentially expressed in response to envenomation. Strong upregulation of enhancer of split complex genes provides a potential molecular mechanism that could explain the observed neural cell death and developmental arrest in envenomated hosts. Significant increases in antimicrobial peptides and their corresponding regulatory genes provide evidence that venom could be selectively activating certain immune responses of the hosts. Further, we found differential expression of genes in several metabolic pathways, including glycolysis and gluconeogenesis that may be responsible for the decrease in pyruvate levels found in envenomated hosts. The targeting of Nasonia venom effects to a specific and limited set of genes provides insight into the interaction between the ectoparasitoid wasp and its host.     

Two-sex life table of Nasonia vitripennis (Hymenoptera: Pteromalidae) and the effects of Wolbachia on its reproduction and parasitism of Musca domestica (Diptera: Muscidae)

Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) is now emerging as genetic model organism for development and genetics research. As a parasitic insect, the egg, larval, pupal, and early adult developmental stages of N. vitripennis occur within the enclosed fly pupae, which differ a lot from the life cycle of other insects that undergo complete metamorphosis. Previous report on the life table of N. vitripennis was based on females only. In this study, the two-sex life table approach was used to examine the parasitic efficiency of N. vitripennis within the pupae of Musca domestica L. (Diptera: Muscidae), and the influence of low temperature and the endosymbiotic bacteria Wolbachia on wasp population growth were investigated. Wolbachia could improve the fecundity of N. vitripennis and prolong the host life history. We propose the preservation of M. domestica pupae at 4 °C for 15 days is suitable in practical use. Age-stage, two-sex life table analysis revealed the stage structure and variability of N. vitripennis population growth, and also provide useful information about the effects of Wolbachia on its reproduction and parasitism of M. domestica.     

Hybrid origin of a B chromosome (PSR) in the parasitic wasp Nasonia vitripennis

Little is known about the origin and evolution of supernumerary (B) chromosomes. This study utilizes molecular markers to examine the evolutionary history and microstructural organization of the supernumerary paternal-sex-ratio (PSR) chromosome of the parasitic wasp Nasonia vitripennis. Copies of the retrotransposon NATE were previously isolated from PSR and the genomes of N. vitripennis and related wasp species. A phylogenetic analysis of sequences representing 29 elements from PSR and seven wasp species, coupled with a hybridization analysis of elements in genomic DNA provides evidence that PSR was recently transferred into N. vitripennis from a species in the genus Trichomalopsis. A linear region of the PSR chromosome was compared by Southern blot analysis with genomic DNA from N. vitripennis, Nasonia longicornis, Trichomalopsis americanus, and Trichomalopsis dubius. A region organized similarly to the region on PSR was not evident in any of the species, thus a progenitor region was not identified. However, the hybridizations revealed that this region of PSR is primarily composed of repetitive sequences that appear dispersed in these wasp genomes, and might represent additional mobile elements. At least three different dispersed repeats are present in the 18 kb region of PSR. The abundance of tandem and dispersed repetitive sequences in this relatively small region provides additional evidence for the degenerate structure of the PSR chromosome. Received: 19 December 1996; in revised form: 14 April 1997 / Accepted: 24 April 1997     

High‐throughput olfactory conditioning and memory retention test show variation in Nasonia parasitic wasps

Most of our knowledge on learning and memory formation results from extensive studies on a small number of animal species. Although features and cellular pathways of learning and memory are highly similar in this diverse group of species, there are also subtle differences. Closely related species of parasitic wasps display substantial variation in memory dynamics and can be instrumental to understanding both the adaptive benefit of and mechanisms underlying this variation. Parasitic wasps of the genus Nasonia offer excellent opportunities for multidisciplinary research on this topic. Genetic and genomic resources available for Nasonia are unrivaled among parasitic wasps, providing tools for genetic dissection of mechanisms that cause differences in learning. This study presents a robust, high‐throughput method for olfactory conditioning of Nasonia using a host encounter as reward. A T‐maze olfactometer facilitates high‐throughput memory retention testing and employs standardized odors of equal detectability, as quantified by electroantennogram recordings. Using this setup, differences in memory retention between Nasonia species were shown. In both Nasonia vitripennis and Nasonia longicornis, memory was observed up to at least 5 days after a single conditioning trial, whereas Nasonia giraulti lost its memory after 2 days. This difference in learning may be an adaptation to species‐specific differences in ecological factors, for example, host preference. The high‐throughput methods for conditioning and memory retention testing are essential tools to study both ultimate and proximate factors that cause variation in learning and memory formation in Nasonia and other parasitic wasp species.     

Genetics of sex determination in the haplodiploid wasp <Emphasis Type="Italic">Nasonia vitripennis</Emphasis> (Hymenoptera: Chalcidoidea)

The parasitoid wasp Nasonia vitripennis reproduces by haplodiploidy; males are haploid and females are diploid. Sex determination in Nasonia is not governed by complementary alleles at one or more sex loci. As in most other insects, the sex-determining pathway consists of the basal switch doublesex that is sex-specifically regulated by transformer. Analysis of a polyploid and a mutant gynandromorphic strain, suggested a parent-specific effect (imprinting) on sex determination in Nasonia. Zygotic activity of transformer is autoregulated and depends on a combination of maternal provision of tra mRNA and a paternal genome set. This constitutes a novel way of transformer control in insect sex determination implying maternal imprinting. The nature of the maternal imprint is not yet known and it remains to be determined how broadly the Nasonia sex-determining mechanism applies to other haplodiploids.     

Der Einfluss der Temperatur auf die Anstichaktivität beiNasonia vitripennis undSpalangia nigra [Chalcid.: Pteromalidae]

Zusammenfassung Bei den 2Pteromalidae Nasonia vitripennis (Walker) undSpalangia nigra latreille wird gezeigt, wie die Temperatur w?hrend des Anstichs die Zahl der abgelegten Eier beeinflusst. Die Anstichaktivit?t beginnt bei beiden Arten bei etwa 12,5°C, hat bei 25°C ihr Optimum und nimmt bis 40°C wieder ab. Bei dem Greg?rparasitenN. vitripennis ist bei 25°C in Folge gesteigerter lokomotorischer Aktivit?t die Zahl der parasitierten Puppen am h?chsten, die Zahl der Nachkommen pro Puppe jedoch niedriger als bei 20°C und 30°C. Die niedrigere Eizahl pro Puppe bei 25°C führt zu einer Verschiebung des Geschlechtsverh?ltnisses zugunsten der ♀♀. Beide Parasiten k?nnen in einem breiten Temperaturbereich anstechen. Die weite Potenz dieser plurivoltinen Arten wird als eine Anpassung an ein breites Wirtsspektrum angesehen.

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Summary Experiments with the 2 PteromalidsNasonia vitripennis (Walker) andSpalangia nigra Latreille show in which way the number of eggs depends on the temperature prevalent during the stinging. The stinging activity starts at about 12,5°C, has its optimum at 25°C, has its optimum at 25°C to 30°C and decreases from 30°C to 40°C. withN. vitripennis the number of parasitized pupae is largest at 25°C the number of offsprings, however, smaller than at 20°C and 30°C. Thus at 25°C the sex ratio is changed in favour of the ♀♀. Both parasites can sting within a broad scope of temperature. This great capacity of the two plurivoltine species is regarded as an adaptation to a borad spectrum of hosts.

     

Temperature‐dependant development of Nasonia vitripennis on five forensically important carrion fly species

The effects of temperature and host species on the development of Nasonia vitripennis Walker (Hymenoptera: Pteromalidae), a forensically important parasitoid of carrion flies, were studied under laboratory conditions. Development time of N. vitripennis on five species of Calliphoridae (Diptera), Calliphora albifrontalis Malloch, Calliphora dubia Macquart, Lucilia sericata Meigen, Chrysomya rufifacies Macquart, and Chrysomya megacephala Fabricius, were determined under eight constant temperatures (15, 18, 21, 24, 27, 30, 33, and 36 °C). Thermal requirements for development (developmental thresholds, thermal constant, and optimum temperature) of N. vitripennis in each host species were estimated using linear and nonlinear models. Upper and lower developmental thresholds ranged between 36.6–38.4 and 9.6–11.1 °C, respectively. The optimum temperature for development was estimated at between 30.6 and 31.8 °C. Statistical differences in the development time of N. vitripennis on the various calliphorid host species were evident within all temperature treatments, particularly at the upper and lower temperature range investigated. As such, it is recommended that insect‐based estimates of time since death in forensic investigations relying on parasitoid evidence should use host‐specific development data where available.     

Fecundity and development of the ectoparasitic waspNasonia vitripennis are dependent on host quality

The gregarious, ectoparasitoidNasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) was offered pupae representing seven fly species, but only members of two families (Sarcophagidae and Muscidae) were parasitized. Host acceptance as an oviposition site did not imply host suitability for parasitoid growth:N. vitripennis produced fewer progeny, a higher proportion of males, required a longer development time, and produced smaller adult wasps onMusca domestica L. (Diptera: Muscidae) than on the three sarcophagid species tested [Sarcophaga bullata Parker,S. crassipalpis Macquart, andPeckia abnormis (Enderlein) (Diptera: Sarcophagidae)]. The physiological and nutritional status of a preferred host,S. bullata, influenced oviposition behavior and development ofN. vitripennis. Progeny allocation and sex ratio, which were regulated by the female parasitoid during oviposition, differed on living and dead nondiapausing hosts and on diapausing pupae. Differences in the host's nutritional condition was reflected in changes of the wasp's development time and adult body size. Envenomation was essential for successful development of the parasitoid on nondiapausing hosts, but venom injection byN. vitripennis did not increase the suitability of diapausing or dead pupae. The results suggest that wasp development is enhanced by changes induced in the host by parasitism.     

Regulation and function of tailless in the long germ wasp Nasonia vitripennis

In the long germ insect Drosophila, the gene tailless acts to pattern the terminal regions of the embryo. Loss of function of this gene results in the deletion of the anterior and posterior terminal structures and the eighth abdominal segment. Drosophila tailless is activated by the maternal terminal system through Torso signaling at both poles of the embryo, with additional activation by Bicoid at the anterior. Here, we describe the expression and function of tailless in a long germ Hymenoptera, the wasp Nasonia vitripennis. Despite the morphological similarities in the mode of development of these two insects, we find major differences in the regulation and function of tailless between Nasonia and Drosophila. In contrast to the fly, Nasonia tll appears to rely on otd for its activation at both poles. In addition, the anterior domain of Nasonia tll appears to have little or no segmental patterning function, while the posterior tll domain has a much more extensive patterning role than its Drosophila counterpart.     

Adaptive latitudinal cline of photoperiodic diapause induction in the parasitoid Nasonia vitripennis in Europe

Living in seasonally changing environments requires adaptation to seasonal cycles. Many insects use the change in day length as a reliable cue for upcoming winter and respond to shortened photoperiod through diapause. In this study, we report the clinal variation in photoperiodic diapause induction in populations of the parasitoid wasp Nasonia vitripennis collected along a latitudinal gradient in Europe. In this species, diapause occurs in the larval stage and is maternally induced. Adult Nasonia females were exposed to different photoperiodic cycles and lifetime production of diapausing offspring was scored. Females switched to the production of diapausing offspring after exposure to a threshold number of photoperiodic cycles. A latitudinal cline was found in the proportion of diapausing offspring, the switch point for diapause induction measured as the maternal age at which the female starts to produce diapausing larvae, and the critical photoperiod for diapause induction. Populations at northern latitudes show an earlier switch point, higher proportions of diapausing individuals and longer critical photoperiods. Since the photoperiodic response was measured under the same laboratory conditions, the observed differences between populations most likely reflect genetic differences in sensitivity to photoperiodic cues, resulting from local adaptation to environmental cycles. The observed variability in diapause response combined with the availability of genomic tools for N. vitripennis represent a good opportunity to further investigate the genetic basis of this adaptive trait.     

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.     

The effect of host spatial distribution on patterns of parasitism by Nasonia vitripennis

Patterns of parasitism of Calliphora sp. pupae by Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) are explored under various conditions of host aggregation. Increasing the hosts' spatial aggregation decreased the number of hosts actually parasitised but had no significant effect on the total number of hosts attacked. Increasing host aggregation also resulted in a smaller parasitoid population with a more male biased sex ratio.

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Résumé Nous avons examiné les effets de différents niveaux d'agrégation des hôtes (pupes de Calliphora) sur l'efficacité parasitaire de Nasonia vitripennis. Une augmentation de l'agrégation des pupes a diminué le nombre d'hôtes parasités bien que le nombre de pupes attaquées ait été peu modifié. Cette différence a été attribuée à un accroissement des interactions comportementales entre les parasitoïdes et à une tendance au superparasitisme plus élevée lors des distributions les plus agrégatives. Plus les hôtes étaient concentrés, moins l'effectif de parasitoïdes achevant leur développement et émergeant était important. La fréquence des males a augmenté avec la concentration des pupes. Nous avons estimé que les facteurs déterminants ont été les interactions comportementales entre femelles et leur aptitude à modifier le taux sexuel de leur descendance immédiate.Nous en avons conclu que N. vitripennis est peut-être mieux adaptée aux populations d'hôtes distribuées au hasard, type de distribution le plus vraisemblable dans les populations naturelles.

     

Inhibition of melanization by a Nasonia defensin-like peptide: Implications for host immune suppression

The parasitic wasp Nasonia vitripennis suppresses host immune mechanisms that include melanization reactions. Melanization is an important immune response of hosts induced by wasp infection and thus its inhibition represents a successful strategy for parasitism. However, the molecular basis associated with such inhibition is largely unknown in N. vitripennis. Here, we report recombinant expression, structural and functional characterization of a Nasonia-derived defensin-like peptide (called nasonin-3) whose recombinant product exerts inhibitory effect on host melanization. The possible role of nasonin-3 in immune suppression is also discussed.     

Influence of parasitism and soil compaction on pupation of the green bottle fly,Lucilia sericata

The influence of parasitoids and soil compaction on pupation behavior of blow flies was examined in a host–parasitoid system involving Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Larvae of L. sericata were introduced to containers with soil of different compaction levels, with or without parasitoids. Although females of N. vitripennis did not significantly affect the pupation depth of L. sericata, they increased the rate of pupal development by 15.0–23.7 h at 28.4 ± 1.2 °C, and increased the clumping of puparia. Pupation depth of L. sericata was negatively related to soil compaction; mean depth of pupation was 4.4 cm in uncompacted soil and 0.5 cm in high‐compaction soil. In high‐compaction soil, pupal development increased by 10.5–18.8 h at 25.2 ± 0.3 °C, and puparia were clumped. These results provide a framework for locating puparia in forensic investigations and releasing appropriate parasitoids for biological control of blow flies.     

Host acceptance and sex allocation of Nasonia wasps in response to conspecifics and heterospecifics

Species recognition is an important aspect of an organism''s biology. Here, we consider how parasitoid wasps vary their reproductive decisions when their offspring face intra- and interspecific competition for resources and mates. We use host acceptance and sex ratio behaviour to test whether female Nasonia vitripennis and Nasonia longicornis discriminate between conspecifics and heterospecifics when ovipositing. We tested pairs of conspecific or heterospecific females ovipositing either simultaneously or sequentially on a single host, using strains varying in their recent history of sympatry. Both N. vitripennis and N. longicornis rejected parasitized hosts more often than unparasitized hosts, although females were more likely to superparasitize their own species in the sequential treatment. However, sex ratio behaviour did not vary, suggesting similar responses towards conspecifics and heterospecifics. This contrasts with theory predicting that heterospecifics should not influence sex ratios as their offspring do not influence local mate competition, where conspecifics would. These non-adaptive sex ratios reinforce the lack of adaptive kin discrimination in N. vitripennis and suggest a behavioural constraint. Discrimination between closely related species is therefore context dependent in Nasonia. We suggest that isolating mechanisms associated with the speciation process have influenced behaviour to a greater extent than selection on sex ratios.