Julien Varaldi

EVIDENCE FOR FEMALE MORTALITY IN WOLBACHIA‐MEDIATED CYTOPLASMIC INCOMPATIBILITY IN HAPLODIPLOID INSECTS: EPIDEMIOLOGIC AND EVOLUTIONARY CONSEQUENCES

Abstract.— Until now, only two Wolbachia‐mediated cytoplasmic incompatibility (CI) types have been described in haplodiploid species, the first in Nasonia (Insect) and the second in Tetranychus (Acari). They both induce a malebiased sex ratio in the incompatible cross. In Nasonia, CI does not reduce fertility since incompatible eggs develop as haploid males, whereas in Tetranychus CI leads to a partial mortality of incompatible eggs, thus reducing the fertility of females. Here, we study Wolbachia infection in a Drosophila parasitoid, Leptopilina heterotoma (Hymenoptera: Figitidae). A survey of Wolbachia infection shows that all natural populations tested are totally infected. Crosses between infected males and cured females show complete incompatibility: almost no females are produced. Moreover, incompatible eggs die early during their development, unlike Nasonia. This early death allows the parasitized Drosophila larva to achieve its development and to emerge. Thus, uninfected females crossed with infected males have reduced offspring production consisting only of males. Evidence of this CI type in insects demonstrates that the difference in CI types of Nasonia and Tetranychus is not due to specific factors of insects or acari. Using theoretical models, we compare the invasion processes of different strategies of Wolbachia: CI in diploid species, the two CI types in haplodiploid species, and parthenogenesis (the classical effect in haplodiploid species). Models show that CI in haplodiploid species is less efficient than in diploid ones. However, the Leptopilina type is advantageous compared to the Nasonia type. Parthenogenesis may be more or less advantageous, depending on the infection cost and on the proportion of fertilized eggs. Finally, we can propose different processes of Wolbachia strategy evolution in haplodiploid species from Nasonia CI type to Leptopilina CI type or parthenogenesis.

Superparasitism Evolution: Adaptation or Manipulation?

Superparasitism refers to the oviposition behavior of parasitoid females who lay their eggs in an already parasitized host. This often yields intense competition among larvae that are sharing the same host. Why would a female oviposit in such hostile habitat instead of looking for a better quality, unparasitized host? Here we present a continuous‐time model of host‐parasitoid interaction and discuss alternative scenarios. This model is first used to analyze the evolution of the superparasitism behavior of a solitary proovigenic parasitoid under both time and egg limitation. Then, following the recent discovery by Varaldi et al., we allow the parasitoid to be infected by a virus that alters the superparasitism behavior of its host to enhance its own horizontal transmission. The analysis of the coevolution of this manipulative behavior with the oviposition behavior of uninfected females clarifies and quantifies the conflict that emerges between the parasitoid and its virus. The model also yields new testable predictions. For example, we expect that uninfected parasitoids should superparasite less after coevolving with the manipulative virus. More generally, this model provides a theoretical framework for analyzing the evolution of the manipulation of parasitoid life‐history traits by microparasites.

Infection polymorphism and cytoplasmic incompatibility in Hymenoptera-Wolbachia associations

Most cases of Wolbachia infection so far documented in haplodiploid Hymenoptera are associated with parthenogenesis induction. Only three examples of Wolbachia-mediated cytoplasmic incompatibility (CI) have been reported, resulting either in haploidisation of fertilised eggs, which develop into viable males, or in their death. To better document this variability, we studied two new Wolbachia-wasp associations involving Drosophila parasitoids. In Trichopria cf. drosophilae, individuals are infected by two different Wolbachia variants, populations are nearly totally infected, and Wolbachia induces incomplete CI resulting in death of the fertilised eggs. On the other hand, Pachycrepoideus dubiusharbours only one bacterial variant, populations are polymorphic for infection, and Wolbachia has no detectable effect. These two cases show that the range of variation in Wolbachias effects in Hymenoptera is as wide as in diploids, extending from complete CI to an undetectable effect. Cases so far studied show some parallel between the strength of incompatibility, the number of Wolbachia variants infecting each wasp, and the natural infection frequency. These empirical data support theoretical models predicting evolution of CI towards lower levels, resulting in the decline and ultimate loss of infection, and place multiple infections as being an important factor in the evolution of host-Wolbachia associations.

Influence of the virus LbFV and of Wolbachia in a host-parasitoid interaction.

Symbionts are widespread and might have a substantial effect on the outcome of interactions between species, such as in host-parasitoid systems. Here, we studied the effects of symbionts on the outcome of host-parasitoid interactions in a four-partner system, consisting of the parasitoid wasp Leptopilina boulardi, its two hosts Drosophila melanogaster and D. simulans, the wasp virus LbFV, and the endosymbiotic bacterium Wolbachia. The virus is known to manipulate the superparasitism behavior of the parasitoid whereas some Wolbachia strains can reproductively manipulate and/or confer pathogen protection to Drosophila hosts. We used two nuclear backgrounds for both Drosophila species, infected with or cured of their respective Wolbachia strains, and offered them to L. boulardi of one nuclear background, either infected or uninfected by the virus. The main defence mechanism against parasitoids, i.e. encapsulation, and other important traits of the interaction were measured. The results showed that virus-infected parasitoids are less frequently encapsulated than uninfected ones. Further experiments showed that this viral effect involved both a direct protective effect against encapsulation and an indirect effect of superparasitism. Additionally, the Wolbachia strain wAu affected the encapsulation ability of its Drosophila host but the direction of this effect was strongly dependent on the presence/absence of LbFV. Our results confirmed the importance of heritable symbionts in the outcome of antagonistic interactions.

Divergent strategies in low temperature environment for the sibling species Drosophila melanogaster and D. simulans: overwintering in extension border areas of France and comparison with African populations

The two sibling species D. /melanogaster and D. simulans adopt different overwintering strategies in northern border areas situated in France. If the winter is mild, both species reappear in early spring to refound the population. If the winter is cold, with several weeks of temperatures below 0 °C, D. melanogaster leave their shelters in April whereas D. simulans, which do not use shelters, reappear in late June, probably after returning from further south. Here, we tried to identify life-history characteristics responsible for this difference. For this, we studied developmental duration, viability, fecundity, fertility and longevity, and compared the abilities of French and African populations to survive when food supplies were inadequate, at different temperatures (14, 11, 7 °C). These temperatures are lower than those commonly used in the laboratory but closer to real conditions encountered in the wild. When the temperature was mild (14 or 11 °C) and the food supply was adequate, D. simulans performed better than D. melanogaster: it had a higher fecundity, a longer life expectancy and the males remained fertile, allowing outdoor reproduction late in winter. However, D. simulans was less resistant in more extreme conditions. At 7 °C D. simulans survived shorter on normal medium and its ability to survive when food supplies were inadequate was insufficient to allow outdoor overwintering. In contrast, D. melanogaster could not reproduce during winter: its fecundity was low and males were sterile at 11 °C. Nevertheless, if only protein-deficient resources were available, temperate D. melanogaster could survive for longer than D. simulans at all the temperatures tested. This greater resistance to underfeeding allows the species to survive until spring, in shelters for several months. A comparison of French and African population performances showed differences in the evolution of the two species during the colonization of more northern areas. African D. simulans, which are efficient at mild temperatures, underwent few modifications. In contrast, the viability of D. melanogaster improved at low developmental temperatures. This species also displayed higher fecundity, longer survival and higher underfeeding resistance at low temperatures. The relationship between the long retention genotype and underfeeding resistance or survival ability observed in French D. melanogaster populations may not exist in African populations.

Prevalence of a virus inducing behavioural manipulation near species range border

The densities of conspecific individuals may vary through space, especially at the edge of species range. This variation in density is predicted to influence the diffusion of species‐specific horizontally transmitted symbionts. However, to date there is very little data on how parasite prevalence varies around the border of a host species. Using a molecular epidemiology approach, we studied the prevalence of a vertically and horizontally transmitted virus at the edge of the geographic range of its insect host, the Drosophila parasitoid wasp Leptopilina boulardi. L. boulardi is a Mediterranean parasitoid species showing a recent range expansion to the north (in France). The LbFV virus manipulates the behaviour of females, increasing their tendency to lay additional eggs in already parasitized Drosophila larvae (superparasitism). This is beneficial for the virus because it allows the virus to be horizontally transferred during superparasitism. We show that LbFV prevalence is very high in central populations, intermediate in marginal populations and almost absent from newly established peripheral populations of L. boulardi. We failed to detect any influence of temperature and diapause on viral transmission efficiency but we observed a clear relationship between prevalence and parasitoid density, and between parasitoid density and the occurrence of superparasitism, as predicted by our epidemiological model. Viral strains were all efficient at inducing the behavioural manipulation and viral gene sequencing revealed very low sequence variation. We conclude that the prevalence reached by the virus critically depends on density‐dependent factors, i.e. superparasitism, underlying the selective pressures acting on the virus to manipulate the behaviour of the parasitoid.

Cost induced by viral particles manipulating superparasitism behaviour in the parasitoid Leptopilina boulardi.

Vertically transmitted symbionts can be maintained in a host population only if they do not reduce host fitness, unless they compensate by manipulation of their hosts reproduction or have alternative mode of transmission. In Leptopilina boulardi, a parasitoid of Drosophila larvae, some females are infected by viral particles showing both maternal and horizontal transmission. Horizontal transmission occurs when larvae from infected and uninfected individuals of L. boulardi compete in the same host. This situation is facilitated by the increasing tendency to accept already parasitized hosts that viral infection induces in L. boulardi females. Estimation of the adaptive significance of this behavioural modification requires measuring the effect of viral presence on other parasitoid physiological features. Here, we show that viral infection in females imposes no cost on adult survival, a low cost on developmental rate and tibia length, and leads to a strong reduction of locomotor activity. Surprisingly, infected females show higher egg load which could be accounted for by a redirection of energy allocation to egg production. The high intensity of superparasitism in infected females induced a dramatic decrease in pre-imaginal survival of the parasitoids offspring, representing a potential indirect cost of infection. Low overall pathogeny induced by viral particles appears to be well suited to both transmission modes, both of them requiring females ability to locate and (super)parasitize hosts.

Molecular detection, penetrance, and transmission of an inherited virus responsible for behavioral manipulation of an insect parasitoid.

ABSTRACT For insects, the prevalence of numerous vertically transmitted viruses can be high in their host populations. These viruses often have few, if any, pathological effects on their hosts, and consequently, many of them can remain unnoticed for long periods, despite their potential role in the evolution of the host phenotype. Some females of Leptopilina boulardi, a solitary parasitoid of Drosophila larvae, are infected by an inherited virus (LbFV) that manipulates the behavior of the wasp by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). This behavioral alteration allows horizontal transmission of the virus within superparasitized Drosophila larvae. Using suppressive subtractive hybridization with infected and uninfected lines, we identified one putative viral sequence. Based on this sequence, we developed a simple PCR test. We tested the correlation between the superparasitism phenotype and PCR amplification of the putative viral marker using several experimental conditions (including horizontal transfers) and several parasitoid genotypes. All of the results revealed that there was a perfect match between the superparasitism phenotype and the amplification profile, which validated use of the molecular marker as a tool to track the presence of the virus and provided the first genomic data for this fascinating virus. The results also show that there was very efficient horizontal and vertical transmission of LbFV, which probably explains its high prevalence in the French populations that we sampled (67 and 70% of infected females). This manipulative virus is likely to play a major role in the ecology and evolution of its parasitoid host.

A virus-shaping reproductive strategy in a Drosophila parasitoid.

Insect parasitoids are often infected with heritable viruses. Some of them, such as polydnaviruses, have evolved toward an obligatory relationship with the parasitoid because they are necessary to protect the parasitoid egg from the host immune reaction. However, recent and past discoveries have revealed the presence of facultative inherited viruses in parasitoids for which no clear phenotypic effect was observed. In this chapter, we present how such an inherited virus was recently discovered in the Drosophila parasitoid, Leptopilina boulardi. We show that this virus is responsible for an increase in the superparasitism tendency of the infected females. This alteration is beneficial for the virus, since superparasitism conditions permit the horizontal transmission of the virus. We review theoretical developments suggesting that this leads to a conflict of interest between the parasitoid and the virus. The direct and indirect influence of the virus on several other fitness traits has also been studied both empirically and theoretically, in particular the egg load. Finally, because the frequency of horizontal transmission is a crucial parameter for the evolution of the superparasitism manipulation, we present an attempt to select the virus for high or low manipulation intensity.

EVOLUTION AND MANIPULATION OF PARASITOID EGG LOAD

In proovigenic parasitoids such as Leptopilina boulardi, the female emerges with a limited egg load and no further eggs are produced during its adult life. A female thus runs the risk of exhausting this limited supply of eggs before the end of her life. Given that the production of an egg is costly, what is the evolutionarily stable egg load at emergence? This question has attracted a lot of attention in the last decade. Here, we analyze a model that allows us to track both the evolution and the population dynamics of a solitary, proovigenic parasitoid. First, we show how host-parasitoid dynamics feedbacks on the evolution of parasitoid egg load. Second, we use this model to consider the situation in which the parasitoid can be infected by a virus that manipulates the oviposition behavior of the females. In particular, we model the effect of the LbFV virus in L. boulardi, a virus that is known to enhance its horizontal transmission by increasing superparasitism (i.e., the laying of eggs in a host already parasitized). Specifically, we model (1) the effect of the virus on parasitoid egg load strategies, and (2) the evolution of egg load manipulation by the virus. This analysis yields two alternative, yet not mutually exclusive, adaptive explanations for the observation that females infected by the virus harbor higher egg loads than uninfected females. Infected females could either respond plastically to the infection status, or be manipulated by the virus. Further experimental work is required to distinguish between these two hypotheses. In a broader context, we present a general theoretical framework that allows us to study the epidemiology, the evolution, the coevolution, and the evolution of manipulation of various reproductive strategies of parasitoids.