Male Killing and Incomplete Inheritance of a Novel <Emphasis Type="Italic">Spiroplasma</Emphasis> in the Moth <Emphasis Type="Italic">Ostrinia zaguliaevi</Emphasis>

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.     

Cytoplasmic incompatibility involving <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Wolbachia</Emphasis> in the white-backed planthopper <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

Cytoplasmic incompatibility (CI) is a reproductive phenotype induced by bacterial endosymbionts in arthropods. Measured as a reduction in egg hatchability resulting from the crossing of uninfected females with bacteria-infected males, CI increases the frequency of bacteria-infected hosts by restricting the fertilization opportunities of uninfected hosts in populations. Wolbachia, a type of alpha-proteobacteria, is well known as a CI inducer in a wide range of arthropod species, while Cardinium, a member of the phylum Bacteroidetes, is known to cause CI in one wasp and three spider mite species. In this study, dual infection with Cardinium and Wolbachia induced strong CI in a single host, Sogatella furcifera (Horváth), a planthopper species that is naturally infected with both bacteria. Specifically, infection with Cardinium alone was found to cause a 76 % reduction in egg development, and dual infection with Cardinium and Wolbachia a 96 % reduction, indicating that Cardinium induces CI and the dual infection raises the CI level. This study was the first to document reproductive alteration by Cardinium in a diploid host species.     

<Emphasis Type="Italic">Wolbachia</Emphasis> infection increases recapture rate of field-released <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Wolbachia pipientis is a commonly occurring endosymbiont with well-characterised effects on host reproductive biology associated with its infection of the gonads. Wolbachia infections are also widespread in somatic tissues and consequently they have the potential to play a much broader role in host biology. Recently, Wolbachia was shown to alter the locomotion of Drosophila melanogaster in response to food cues in the laboratory. To determine whether this laboratory-based phenotype might translate to real differences for insects in the field, we performed a simple mark-release-recapture experiment with Wolbachia-infected D. melanogaster in a forested habitat. We demonstrate that infected flies are recaptured at twice the rate of uninfected flies, although infection does not affect the distance traveled by those flies recaptured. The differences in recapture could be explained by infection-induced changes in physiology or behavior. If generalizable, such changes may affect the interpretation of behavioral studies for Wolbachia-infected insects and have potential implications for the dynamics of Wolbachia infections in natural populations, including situations where Wolbachia-infected insects are being released for biological control.     

Transfer of a chromosomal <Emphasis Type="Italic">Maverick</Emphasis> to endogenous bracovirus in a parasitoid wasp

Bracoviruses are used by parasitoid wasps to allow development of their progeny within the body of lepidopteran hosts. In parasitoid wasps, the bracovirus exists as a provirus, integrated in a wasp chromosome. Viral replication occurs in wasp ovaries and leads to formation of particles containing dsDNA circles (segments) that are injected into the host body during wasp oviposition. We identified a large DNA transposon Maverick in a parasitoid wasp bracovirus. Closely related elements are present in parasitoid wasp genomes indicating that the element in CcBV corresponds to the insertion of an endogenous wasp Maverick in CcBV provirus. The presence of the Maverick in a bracovirus genome suggests the possibility of transposon transfers from parasitoids to lepidoptera via bracoviruses.     

Population dynamics of a maternally-transmitted <Emphasis Type="Italic">Spiroplasma</Emphasis> infection in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

A maternally-inherited spiroplasma endosymbiont of Drosophila hydei does not exert apparent phenotypes on both sexes of its host and is prevalent in natural populations of D. hydei. Our previous experiments using a laboratory stock of D. hydei revealed that low temperatures (such as 15°C and 18°C) dramatically lower the vertical transmission rates of this spiroplasma. Therefore, we hypothesized that, in temperate regions, the infection frequencies may decrease in cool seasons but increase in the summer season. To clarify the temporal population dynamics of the spiroplasma infection, D. hydei were collected from two Japanese populations in 2006–2008 from May to early August, representing the only period when a number of D. hydei are collectable in Japan, and examined for spiroplasma infection. Within each year, the frequency of spiroplasma infection fluctuated considerably in both populations. Consistent with our hypothesis, the infection frequency showed an increasing trend in both populations in 2007. However, the data in 2006 and 2008 did not show consistent patterns of increase. The population dynamics of spiroplasma infection may be affected but not critically determined by temperature. Moreover, despite the fluctuation within each year, the infection frequencies seemed to be stable across the years. The frequencies of spiroplasma infection in D. hydei populations may be stabilized by multiple factors. One of these factors may involve a context-dependent positive effect of spiroplasma on the fitness of D. hydei, as was recently observed in laboratory experiments.     

Oviposition Marking Behavior of <Emphasis Type="Italic">Diachasma alloeum</Emphasis>, (Hymenoptera: Braconidae), Parasitizing <Emphasis Type="Italic">Rhagoletis pomonella</Emphasis>, (Diptera: Tephritidae)

Diachasma alloeum (Muesebeck) (Hymenoptera: Braconidae) is a solitary larval endoparasitoid attacking Rhagoletis (Diptera: Tephritidae) species. Rhagoletis pomonella (Walsh) mark the surface of fruit after oviposition with an oviposition marking pheromone (OMP) which deters conspecific female flies. Herein we demonstrate that female D. alloeum wasps reared from either apple or hawthorn race R. pomenella larvae also deposit an OMP that reduces oviposition by conspecific female wasps. Significantly fewer wasps accepted fruit that had received prior wasp oviposition and OMP or OMP alone without oviposition compared with control fruit for a minimum of 7 days on both fruit types. Rinsing fruit with a 50% ethanol solution appeared to remove the OMP rendering fruit more acceptable for oviposition than marked fruit that was not rinsed. Wasps of each host race were able to detect and avoid the OMP of the sister race and fruit substrate type did not affect wasp response to the pheromone. The possibility of an internal marker deposited during oviposition is also discussed.     

Winter and spring ecology of <Emphasis Type="Italic">Anaphes nitens</Emphasis>, a solitary egg-parasitoid of the <Emphasis Type="Italic">Eucalyptus</Emphasis> snout-beetle <Emphasis Type="Italic">Gonipterus scutellatus</Emphasis>

We investigated the effects of temperature, photoperiod, food and host availability, and body size on the overwintering abilities of the egg parasitoid Anaphes nitens Girault (Hymenoptera, Mymaridae) under natural conditions. Seven groups of eighty females received one of four treatments (n = 20): (i) honey and hosts, (ii) water and hosts, (iii) honey, or (iv) water. Seven groups of forty males received only honey or water (n = 20). To test if short day-length is the main cue for larval dormancy, the experiment was replicated inside a climate chamber at 20°C and under a winter photoperiod. A. nitens overwinters because of quiescence or oligopause inside the hosts and increased adult longevity. Mean pre-emergence mortality was up to 26% indoors and 15.2% outdoors, males being more affected. Development time had a significant and positive effect on body size. Honey-fed females without hosts had the highest longevity (53 days). Mother’s diet and size affected development time, body size, longevity, and fecundity of the progeny. The results confirm the good adaptation of the parasitoid to the environmental conditions of NW Spain and its ability to synchronize its life cycle with the phenology of the host. Handling editor: Drik Babendreier.     

Strength in numbers: high parasite burdens increase transmission of a protozoan parasite of monarch butterflies (<Emphasis Type="Italic">Danaus plexippus</Emphasis>)

Parasites often produce large numbers of offspring within their hosts. High parasite burdens are thought to be important for parasite transmission, but can also lower host fitness. We studied the protozoan Ophryocystis elektroscirrha, a common parasite of monarch butterflies (Danaus plexippus), to quantify the benefits of high parasite burdens for parasite transmission. This parasite is transmitted vertically when females scatter spores onto eggs and host plant leaves during oviposition; spores can also be transmitted between mating adults. Monarch larvae were experimentally infected and emerging adult females were mated and monitored in individual outdoor field cages. We provided females with fresh host plant material daily and quantified their lifespan and lifetime fecundity. Parasite transmission was measured by counting the numbers of parasite spores transferred to eggs and host plant leaves. We also quantified spores transferred from infected females to their mating partners. Infected monarchs had shorter lifespans and lower lifetime fecundity than uninfected monarchs. Among infected females, those with higher parasite loads transmitted more parasite spores to their eggs and to host plant leaves. There was also a trend for females with greater parasite loads to transmit more spores to their mating partners. These results demonstrate that high parasite loads on infected butterflies confer a strong fitness advantage to the parasite by increasing between-host transmission.     

Resource-Dependent Clutch Size Decisions and Size-Fitness Relationships in a Gregarious Ectoparasitoid Wasp, <Emphasis Type="Italic">Bracon brevicornis</Emphasis>

Gregarious parasitic wasps, which lay more than one egg into or onto a host arthropod’s body, are usually assumed to lay an optimal number of eggs per host. If females would lay too few eggs, some resources may be wasted, but if females lay too many eggs, offspring may develop into substantially smaller-sized adults or may not develop successfully and die. The availability of hosts can further influence a female’s clutch size decision, as more eggs should be laid when hosts are scarce. Here, we analyzed clutch size decisions and the fitness consequences thereof in the ectoparasitic wasp Bracon brevicornis (Hymenoptera: Braconidae), a potential biocontrol agent against pest moth species. For experiments, larvae of the Mediterranean flower moth, Ephestia kuehniella (Lepidoptera: Pyralidae) were used. Using artificially created as well as naturally laid clutches of eggs, the effects of clutch size on fitness of first (F1) and second (F2) generation offspring were investigated. Our results revealed that the fitness consequences of large clutches included both increased mortality and smaller adult sizes of the emerging offspring (F1). Smaller F1 females matured fewer eggs during their lifetime and their offspring (F2) had reduced egg-to-adult survival probability. Naturally laid clutches varied with host size up to a maximum, which probably reflects egg limitation. Clutches remained smaller than the calculated optimal (Lack) clutch size and females responded to high host availability with a decreased number of eggs laid. We thus conclude that large clutches may result in significantly smaller offspring with reduced fitness, and that host size as well as host availability influence the clutch size decision made by B. brevicornis females.     

Host Age Preference Behavior in <Emphasis Type="Italic">Aphidius ervi</Emphasis> Haliday (Hymenoptera: Aphidiidae)

Many parasitoid species have preference for certain stages of hosts to parasitize but the underlying behavioral mechanisms of such preference are still poorly understood, making it difficult to evaluate host-parasitoid interactions and their effects on the success of biological control programs. Here, we report our work on a parasitoid Aphidius ervi Haliday on the pea aphid Acyrthosiphon pisum (Harris). We show that with the increase of host age, female parasitoids are more likely to encounter and to attack their hosts but the hosts develop increasingly greater defensive capabilities. Encounter almost always triggers attack attempt; however, increasing attack attempts do not proportionally lead to ovipositor probings and increasing ovipositor probings do not proportionally translate into ovipositions. These asymmetric responses may be interpreted as that A. ervi females prefer to parasitize older aphids for higher fitness return but those aphids can better defend themselves, and as a consequence, A. ervi females may achieve the highest gain by attacking aphids of intermediate ages. We suggest that A. ervi females forage in a manner consistent with the optimal foraging theory, trading off host handling time with fitness returns.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

Effect of <Emphasis Type="Italic">Wolbachia</Emphasis> infection and temperature variations on the fecundity of the Uzifly <Emphasis Type="Italic">Exorista sorbillans</Emphasis> (Diptera: Tachinidae)

Wolbachia are cytoplasmically inherited endosymbionts known to cause several reproductive alterations in insects which allow their spread in host populations. In the Uzifly Exorista sorbillans, endoparasites of silkworms, the prevalence of Wolbachia is high in the field. In the present study, we investigated Wolbachia’s effects on the Uzifly fitness traits by measuring fecundity and hatching rate in crosses involving infected and cured individuals. We found evidence for positive fitness effects associated with Wolbachia infection in females which could help promote the spread of Wolbachia in E. sorbillans populations. We tested two types of treatments for removing Wolbachia, antibiotic therapy and high temperature treatment and found an influence on the reproduction: females treated by antibiotics have a lower fecundity than females cured by high temperature which could indicate a negative effect of the antibiotherapy on females’ fitness. Furthermore, the monitoring of the Uzifly populations during 2 years revealed seasonal variations of the offspring production which may be linked to temperature.     

Artificial transfer of a thelytoky-inducing <Emphasis Type="Italic">Wolbachia</Emphasis> endosymbiont between strains of the endoparasitoid wasp <Emphasis Type="Italic">Asobara japonica</Emphasis> (Hymenoptera: Braconidae)

Infection with Wolbachia is known to induce diploidization of haploid eggs and enables the production of females from unfertilized eggs. Although there have been several attempts to achieve the artificial horizontal transfer of thelytoky-inducing Wolbachia in parasitoid wasps, the artificial induction of thelytoky has generally been unsuccessful. In this study, we used two strains of Asobara japonica as study materials—one infected with thelytoky-inducing Wolbachia and the other not. We investigated methods of artificially inducing thelytoky by transferring thelytoky-inducing Wolbachia from wasps of the infected strain (the donor wasps) to wasps that had been cured of Wolbachia and to wasps of the uninfected strain (the recipient wasps). To examine the efficiencies of various methods of transfection, we compared the survival and infection rates of recipient wasps that received microinjections at the pupal and adult stages and in different body parts. We also examined the infection rate of the recipients due to cannibalism of Wolbachia-infected pupae. Among those methods, only microinjection at the adult stage resulted in the successful artificial horizontal transfer of Wolbachia, and some of the Wolbachia-infected wasps showed incomplete thelytoky. A low Wolbachia titer in the artificially infected wasps may explain why the thelytoky was incomplete.     

Response of the wasp (<Emphasis Type="Italic">Cotesia glomerata</Emphasis>) to larvae of the large white butterfly (<Emphasis Type="Italic">Pieris brassicae</Emphasis>)

The large white butterfly (Pieris brassicae L) first invaded northernmost Japan from Siberia around 1994, and after a few years, began to expand its range. The wasp, Cotesia glomerata (L) parasitizes larvae of the small white butterfly (Pieris rapae crucivora Boisduval), a usual host in the same geographic area. Some Pieris brassicae larvae in Hokkaido have been parasitized by Cotesia glomerata, but the parasitism rate of Pieris brassicae larvae tends to be lower than that of Pieris rapae. To examine the process of parasitizing Pieris brassicae larvae, we observed how the parasitoid wasp responded to the host larvae on damaged leaves. Cotesia glomerata females tended to avoid Pieris brassicae larvae, and even when female wasps inserted their ovipositors into Pieris brassicae larvae, none laid eggs. The parasitoids obtained from Pieris rapae larvae failed to parasitize Pieris brassicae during the host-acceptance step.     

Differential energy allocation as an adaptation to different habitats in the parasitic wasp <Emphasis Type="Italic">Venturia canescens</Emphasis>

Environmental pressures are expected to favour organisms that optimally allocate metabolic resources to reproduction and survival. We studied the resource allocation strategies and the associated tradeoffs in the parasitoid wasp Venturia canescens, and their adaptation to the characteristics of the environment. In this species, individuals of two reproductive modes coexist in the same geographical locations, but they mainly occur in distinct habitats. Thelytokous (asexual) wasps are mostly found in anthropogenic habitats, where hosts tend to aggregate and food is absent. Arrhenotokous (sexual) wasps are exclusively found in natural habitats, where hosts are scattered and food is present. We analysed (1) the quantity of energy stored during ontogeny, (2) the tradeoff between reproduction and survival, by measuring egg load and longevity and (3) the host patch exploitation behaviour of the wasps at emergence. Arrhenotokous wasps emerged with more metabolic resources than thelytokous ones, especially glycogen, a nutrient that could be used for flying in search of hosts and/or food. Thelytokous wasps allocated more energy than arrhenotokous wasps to egg production: this would allow them to parasitize more hosts. The tradeoff between egg production and longevity was not revealed within reproductive modes, but when comparing them. At emergence, arrhenotokous wasps tended to exploit host patches less thoroughly than thelytokous wasps, suggesting that by leaving the host patch, they search for food. The results clearly showed adaptations to the characteristics of habitats preferentially inhabited by the two reproductive modes, and suggested a mechanism that facilitates their coexistence in natural conditions.     

Wasp Attacks and Spider Defence in the Orb Weaving Species <Emphasis Type="Italic">Zygiella x-notata</Emphasis>

Predator–prey relationships are generally based on arm-race. Wasps and spiders are both predators, which could be potential prey for each other. The orb weaver spider Zygiella x-notata is sometimes a prey for the wasp Vespula germanica. We observed the wasp hunting behaviour under natural conditions, and we tested the influence of the spider’s behaviour on the wasp attack success. Wasps were active predators during the reproductive period of the spider. Results showed that wasps located more easily male spiders than females particularly when they were engaged in mate guarding. Female location depended on the presence of a web, but also of prey or prey remains in the web. On the other hand, their location depend neither on the characteristics and the position of the retreat in the environment nor on the size of the web. After location, males were more often captured than females whatever their behaviour (mate guarding or not). Presence of prey remains or prey in the web did not increase the risk for the spider to be captured. There was also no influence of the retreat’s characteristics or of its position in the habitat on the risk for the spider to be captured; but wasp successful attacks were less numerous when silk was present around the entrance of the retreat or when the spider was completely inside. As prey and prey remains favoured location of spiders by the wasps, we tested spider web cleaning behaviour as a response to wasp predatory pressure. By throwing small polystyrene pellets in the webs, we observed that more 80% of the spiders rejected the pellets in less than one minute. Our data indicated that wasps were significant predators of Z. x-notata and wasp attack could have been a selective pressure that had favoured spider defensive behaviours such as web cleaning.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.     

Interactions between the Social Digger Wasp, <Emphasis Type="Italic">Cerceris rubida</Emphasis>, and Its Brood Parasitic Flies at a Mediterranean Nest Aggregation

The fitness consequence of many behaviours of the small digger wasp, Cerceris rubida (Hymenoptera: Crabronidae), the only European species of its genus in which females share nests, are still unknown. Here, I present novel data on the nesting patterns and nest parasites of an Italian population of this wasp, with emphasis on which behavioural strategies may have evolved to reduce brood parasitism. Nests were established mainly in horizontal surfaces with scarce vegetation and hard soil, resulting in spatially clumped nests; the extent of nest aggregation increased over a 6-year period. Wasp brood cells are attacked by the miltogrammine fly Pterella grisea (Diptera: Sarcophagidae), which waits for nest-returning wasps on perching sites and then follows them in flight (satellite flight), eventually landing on the nest entrance and larvipositing without entering further in the tunnel. This technique seems to be adaptive for the parasitic flies, which would be rejected from nests by the guarding wasps if attempting to enter. The daily activity of the flies closely matched the host wasp’s provisioning activity, but C. rubida females were able to partially confound the tracking flies by performing evasive manoeuvres while returning to the nest. Patches with higher nest density and nests with more resident females did suffer more fly landings on nest entrances (a prerequisite for larviposition). These trends, however, disappeared on a per nest basis and on a per wasp provisioning flight basis, respectively. Across two years, only 6% of brood cells were parasitized, a picture similar to what observed for miltogrammine flies at nest aggregations of other Cerceris spp., and new data are necessary to test if there is a benefit of either nest density or nest sharing against P. grisea parasitism.     

Relationship between <Emphasis Type="Italic">Psoroptes cuniculi</Emphasis> and the Internal Bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis>

The bacterium Serratia marcescens isolated from surface-sterilised Psoroptes cuniculi was found sensitive to the antibiotic Amikacin. Mites placed in this antibiotic for 48–72 h and then washed by centrifugation were found to be alive and S. marcescens-free. Two experimental infestations were undertaken in order to verify the ability of the S. marcescens-free mites to infect and to give ear skin lesions in healthy rabbits and to evaluate the differential ability of the S. marcescens-free and S. marcescens-infected mites to give ear skin lesions. All rabbits were found to be infested, but only rabbits infested with S. marcescens-free mites presented crusts in their ears, whereas mites and/or eggs were only detected in the ear cerumen of all rabbits infested with S. marcescens-infected mites. S. marcescens was isolated only from P. cuniculi mites taken from these latter rabbits. Results indicate that P. cuniculi mites do not need S. marcescens to live and to be able to infest a healthy rabbit. In addition, S. marcescens was not isolated from eggs and newly born larvae of S. marcescens-infected P. cuniculi, demonstrating that in a population of P. cuniculi this bacterium is not transmitted transovarially.