Effects of daily fluctuating temperatures on the Drosophila-Leptopilina boulardi parasitoid association.

Koinobiont parasitoid insects, which maintain intimate and long-term relationships with their arthropod hosts, constitute an association of ectothermic organisms that is particularly sensitive to temperature variations. Because temperature shows pronounced natural daily fluctuations, we examined if experiments based on a constant temperature range can mask the real effects of the thermal regime on host-parasitoid interactions. The effects of two fluctuating thermal regimes on several developmental parameters of the Drosophila larval parasitoid Leptopilina boulardi were analyzed in this study. Regime 1 included a range of 16-23-16°C and regime 2 included a range of 16-21-26-21-16°C (mean temperature 20.1°C) compared to a 20.1°C constant temperature. Under an average temperature of 20.1°C, which corresponds to a cold condition of L. boulardi development, we showed that the success of parasitism is significantly higher under a fluctuating temperature regime than at constant temperature. A fluctuating regime also correlated with a reduced development time of the parasitoids. In contrast, the thermal regime did not affect the ability of Drosophila to resist parasitoid infestation. Finally, we demonstrated that daily temperature fluctuation prevented the entry into diapause for this species, which is normally observed at a constant temperature of 21°C. Overall, the results reveal that constant temperature experiments can produce misleading results, highlighting the need to study the thermal biology of organisms under fluctuating regimes that reflect natural conditions as closely as possible. This is particularly a major issue in host-parasitoid associations, which constitute a good model to understand the effect of climate warming on interacting species.

Additional heritable virus in the parasitic wasp Leptopilina boulardi: prevalence, transmission and phenotypic effects.

Parasitoid wasps can be found in association with heritable viruses. Although some viruses have been shown to profoundly affect the biology and evolution of parasitoid wasps, the genetic and phenotypic diversity of parasitoid-associated viruses remains largely unexplored. We previously discovered a behaviour-manipulating DNA virus in the parasitoid wasp Leptopilina boulardi. In this species, which lays its eggs inside Drosophila larvae, Leptopilina boulardi filamentous virus (LbFV) forces the females to lay their eggs in already parasitized Drosophila larvae. This behavioural manipulation increases the chances for the horizontal transmission of the virus. Here, we describe in the same parasitoid species another virus, which we propose to call Leptopilina boulardi toti-like virus (LbTV). This double-stranded RNA virus is highly prevalent in insect laboratory lines as well as in parasitoids caught in the field. In some cases, LbTV was found in coinfection with LbFV, but did not affect the behaviour of the wasp. Instead we found that the presence of LbTV correlates with an increase in the number of offspring, mostly due to increased survival of parasitoid larvae. LbTV is vertically transmitted mostly through the maternal lineage even if frequent paternal transmission also occurs. Unlike LbFV, LbTV is not horizontally transmitted. Its genome encodes a putative RNA-dependent RNA polymerase (RdRp) showing similarities with RdRps of Totiviridae. These results underline the high incidence and diversity of inherited viruses in parasitoids as well as their potential impact on the phenotype of their hosts.

Competitive outcome of multiple infections in a behavior-manipulating virus/wasp interaction.

Infections by multiple parasites are common in nature and may impact the evolution of host-parasite interactions. We investigated the existence of multiple infections involving the DNA virus LbFV and the Drosophila parasitoid Leptopilina boulardi. This vertically transmitted virus forces infected females to lay their eggs in already parasitized Drosophila larvae (a behavior called superparasitism), thus favoring its spread through horizontal transmission. Previous theoretical work indicated that the evolution of the level of the manipulation strongly depends on whether infected parasitoids can be re-infected or not. Here, we describe a strain of LbFV that differs from the reference strain by showing a deletion within the locus used for PCR detection. We used this polymorphism to test for the existence of multiple infections in this system. Viral strains did not differ on their vertical or horizontal transmission rates nor on the way they affect the parasitoid's phenotype, including their ability to manipulate behavior. Although already infected parasitoids were much less susceptible to new infection than uninfected ones, frequent coinfection was detected. However, following coinfection, competition between viral strains led to the rapid elimination of one strain or the other after a few generations of vertical transmission. We discuss the implications of these results for the evolution of the behavioral manipulation.

Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: new insights into the systematics of this worldwide pest.

BACKGROUND: The taxonomy of the species complex Bemisia tabaci, a serious agricultural pest worldwide, is not well resolved yet, even though species delimitation is critical for designing effective control strategies. Based on a threshold of 3.5% mitochondrial (mtCOI) sequence divergence, recent studies have identified 28 putative species. Among them, mitochondrial variability associated with particular symbiotic compositions (=cytotypes) can be observed, as in MED, which raises the question of whether it is a single or a complex of biological species. RESULTS: Using microsatellites, an investigation was made of the genetic relatedness of Q1 and ASL cytotypes that belong to MED. Samples of the two cytotypes were collected in West Africa where they live in sympatry on the same hosts. Genotyping revealed a high level of differentiation, without evidence of gene flow. Moreover, they differed highly in frequencies of resistance alleles to insecticides, which were much higher in Q1 than in ASL. CONCLUSION: Q1 and ASL are sufficiently reproductively isolated for the introgression of neutral alleles to be prevented, suggesting that they are actually different species. This indicates that nuclear genetic differentiation must be investigated within groups with less than 3.5% mtCOI divergence in order to elucidate the taxonomy of B. tabaci at a finer level. Overall, these data provide important information for pest management.

Nuclear and cytoplasmic differentiation among Mediterranean populations of Bemisia tabaci: testing the biological relevance of cytotypes.

BACKGROUND: The taxonomy of the species complex Bemisia tabaci is still an unresolved issue. Recently, phylogenetic analysis based on mtCOI identified 31 cryptic species. However, mitochondrial diversity is observed within these species, associated with distinct symbiotic bacterial communities forming associations, which here are called cytotypes. The authors investigated the biological significance of two cytotypes (Q1 and Q2) belonging to the Mediterranean species, which have only been found in allopatry in the Western Mediterranean to date. Sampling was done over a few years in Western Europe, and sympatric situations were found that allowed their reproductive compatibility to be tested in the field with the use of microsatellites. RESULTS: The field survey indicated that, in spite of its recent introduction, Q2 is well established in France and Spain, where it coexists with Q1. Microsatellite data showed that, in allopatry, Q1 and Q2 are highly differentiated, while there is little or no genetic differentiation when they coexist in sympatry, suggesting a high rate of hybridisation. Crossing experiments in the lab confirmed their interfertility. CONCLUSION: Q1 and Q2 hybridise, which confirms that they belong to the same species, in spite of the high degree of genetic differentiation at both the cytoplasmic and nuclear levels, and also suggests that their symbiotic bacteria do not prevent hybridisation.

An inherited virus influences the coexistence of parasitoid species through behaviour manipulation.

The potential role of pathogens or parasites in maintaining species coexistence is well documented. However, the impact of vertically transmitted symbionts, that can markedly modify their host's biology, is largely unknown. Some females of the Drosophila parasitoid Leptopilina boulardi are infected with an inherited virus (LbFV). The virus forces females to lay supernumerary eggs in already parasitised hosts, thus allowing its horizontal transmission. Using two independent experimental procedures, we found that LbFV impacts inter-specific competition between L. boulardi and the related L. heterotoma. While L. boulardi rapidly outcompetes L. heterotoma in the absence of the virus, L. heterotoma was able to maintain or even to eliminate L. boulardi in the presence of LbFV. By forcing females to superparasitise, LbFV induced egg wastage in L. boulardi thus explaining its impact on the competition outcome. We conclude that this symbiont whose transmission is L. boulardi-density-dependant may affect the coexistence of Leptopilina species.

Evidence of diversity and recombination in Arsenophonus symbionts of the Bemisia tabaci species complex.

BACKGROUND: Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology. RESULTS: Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers. CONCLUSIONS: This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.

Endosymbiont metacommunities, mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex.

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA-based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont-host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.

The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia tabaci is correlated with the presence of a specific symbiotic bacterium species.

Tomato yellow leaf curl virus (TYLCV) (Geminiviridae: Begomovirus) is exclusively vectored by the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). TYLCV transmission depends upon a 63-kDa GroEL protein produced by the vector's endosymbiotic bacteria. B. tabaci is a species complex comprising several genetically distinct biotypes that show different secondary-symbiont fauna. In Israel, the B biotype harbors Hamiltonella, and the Q biotype harbors Wolbachia and Arsenophonus. Both biotypes harbor Rickettsia and Portiera (the obligatory primary symbionts). The aim of this study was to determine which B. tabaci symbionts are involved in TYLCV transmission using B. tabaci populations collected in Israel. Virus transmission assays by B. tabaci showed that the B biotype efficiently transmits the virus, while the Q biotype scarcely transmits it. Yeast two-hybrid and protein pulldown assays showed that while the GroEL protein produced by Hamiltonella interacts with TYLCV coat protein, GroEL produced by Rickettsia and Portiera does not. To assess the role of Wolbachia and Arsenophonus GroEL proteins (GroELs), we used an immune capture PCR (IC-PCR) assay, employing in vivo- and in vitro-synthesized GroEL proteins from all symbionts and whitefly artificial feeding through membranes. Interaction between GroEL and TYLCV was found to occur in the B biotype, but not in the Q biotype. This assay further showed that release of virions protected by GroEL occurs adjacent to the primary salivary glands. Taken together, the GroEL protein produced by Hamiltonella (present in the B biotype, but absent in the Q biotype) facilitates TYLCV transmission. The other symbionts from both biotypes do not seem to be involved in transmission of this virus.

Ecology and life history evolution of frugivorous Drosophila parasitoids.

Parasitoids and their hosts are linked by intimate and harmful interactions that make them well suited to analyze fundamental ecological and evolutionary processes with regard to life histories evolution of parasitic association. Drosophila aspects of what parasitoid Hymenoptera have become model organisms to study aspects that cannot be investigated with other associations. These include the genetic bases of fitness traits variations, physiology and genetics of resistance/virulence, and coevolutionary dynamics leading to local adaptation. Recent research on evolutionary ecology of Drosophila parasitoids were performed mainly on species that thrive in fermenting fruits (genera Leptopilina and Asobara). Here, we review information and add original data regarding community ecology of these parasitoids, including species distribution, pattern of abundance and diversity, host range and the nature and intensity of species interactions. Biology and the evolution of life histories in response to habitat heterogeneity and possible local adaptations leading to specialization of these wasps are reported with special emphasis on species living in southern Europe. We expose the diversity and intensity of selective constraints acting on parasitoid life history traits, which vary geographically and highlight the importance of considering both biotic and abiotic factors with their interactions to understand ecological and evolutionary dynamics of host-parasitoid associations.

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.

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

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.

Inherited intracellular ecosystem: symbiotic bacteria share bacteriocytes in whiteflies.

Symbiotic relationships with bacteria are common within the Arthropoda, with interactions that substantially influence the biology of both partners. The symbionts' spatial distribution is essential for understanding key aspects of this relationship, such as bacterial transmission, phenotype, and dynamics. In this study, fluorescence in situ hybridization was used to localize five secondary symbionts from various populations and biotypes of the sweet potato whitefly Bemisia tabaci: Hamiltonella, Arsenophonus, Cardinium, Wolbachia, and Rickettsia. All five symbionts were found to be located with the primary symbiont Portiera inside the bacteriocytes--cells specifically modified to house bacteria--but within these cells, they occupied various niches. The intrabacteriocyte distribution pattern of Rickettsia differed from what has been described previously. Cardinium and Wolbachia were found in other host tissues as well. Because all symbionts share the same cell, bacteriocytes in B. tabaci represent a unique intracellular ecosystem. This phenomenon may be a result of the direct enclosure of the bacteriocyte in the egg during oogenesis, providing a useful mechanism for efficient vertical transmission by "hitching a ride" with Portiera. On the other hand, cohabitation in the same cell provides ample opportunities for interactions among symbionts that can either facilitate (cooperation) or limit (warfare) symbiotic existence.

Artifical transfer and morphological description of virus particles associated with superparasitism behaviour in a parasitoid wasp.

In parasitoids, the adaptive significance of superparasitism (laying of egg(s) in already parasitized hosts) has been the subject of strong controversy. The current view is to interpret this behaviour as an adaptation to increased competition for hosts, because the supernumerary egg still has a chance to win possession for the host. However, we recently discovered that in the solitary parasitoid Leptopilina boulardi, superparasitism is rather caused by an unknown infectious element: stable non superparasitizing lineages (NS) are transformed into stable superparasitizing lineages (S) after eggs from both lineages have competed inside the same host (superparasitism). In this report, we investigate the nature and location of the causative agent. Involvement of bacteria is unlikely because antibiotic treatments do not affect wasp phenotype and because bacterial 16S ribosomal DNA was not detected using PCR. We report successful injection experiments showing that the causative agents are located in wasp poison gland and ovaries and are stably inherited. Electron microscopic studies demonstrate that long filamentous virus particles located in wasp oviducts are strongly associated with superparasitism behaviour, leading to reconsider the adaptive significance of this behaviour in parasitoids. Interestingly, parasitoids are often infected with similar viruses for which no phenotypic effect has been documented. This raises the possibility that they could induce the same behavioural manipulation.

Between- and within-host species selection on cytoplasmic incompatibility-inducing Wolbachia in haplodiploids.

The most common effect of the endosymbiont Wolbachia is cytoplasmic incompatibility (CI), a form of postzygotic reproductive isolation that occurs in crosses where the male is infected by at least one Wolbachia strain that the female lacks. We revisited two puzzling features of Wolbachia biology: how Wolbachia can invade a new species and spread among populations, and how the association, once established in a host species, can evolve, with emphasis on the possible process of infection loss. These questions are particularly relevant in haplodiploid species, where males develop from unfertilized eggs, and females from fertilized eggs. When CI occurs in such species, fertilized eggs either die (female mortality type: FM), or develop into males (male development type: MD), raising one more question: how transition among CI types is possible. We reached the following conclusions: (1) the FM type is a better invader and should be retained preferentially after a new host is captured; (2) given the assumptions of the models, FM and MD types are selected on neither the bacterial side nor the host side; (3) selective pressures acting on both partners are more or less congruent in the FM type, but divergent in the MD type; (4) host and symbiont evolution can drive infection to extinction for all CI types, but the MD type is more susceptible to the phenomenon; and (5) under realistic conditions, transition from MD to FM type is possible. Finally, all these results suggest that the FM type should be more frequent than the MD type, which is consistent with the results obtained so far in haplodiploids.