Isolation, characterization and PCR multiplexing of microsatellite loci for two sub-species of terrestrial isopod Porcellio dilatatus (Crustacea, Oniscidea).

Several microsatellite markers have already been developed for different terrestrial isopod species such as Armadillidium vulgare, A. nasatum and Porcellionides pruinosus. In all these species, the endosymbiont Wolbachia has a feminizing effect that generates a female bias in sex ratio and reduces the number of reproductive males. Thus this can potentially decrease the genetic diversity of host populations. However, in some other isopod species, Wolbachia induces cytoplasmic incompatibility (CI); the most commonly described effect of Wolbachia in arthropods. The CI by rendering some crossings incompatible can reduce the gene flow and strengthen genetic differentiation between isopod populations. To date, the influence of Wolbachia inducing CI on population structure of terrestrial isopods has never been investigated. In this study, we developed 10 polymorphic microsatellite markers shared by two sub-species of Porcellio dilatatus. Crossings between the two sub-species are partially incompatible due to two CI-inducing Wolbachia strains. These new microsatellite markers will allow us to investigate the effect of CI on host genetic differentiation in this species complex.

Bidirectional cytoplasmic incompatibility caused by Wolbachia in the terrestrial isopod Porcellio dilatatus.

In the terrestrial isopod species Porcellio dilatatus, unidirectional Cytoplasmic Incompatibility (CI) between two morphs (P. d. dilatatus and P. d. petiti) caused by a Wolbachia strain (wPet) infecting the morph P. d. petiti has been previously described by experiments initiated four decades ago. Here, we studied another Wolbachia that has been recently detected in a population of the morph P. d. dilatatus. The MLST markers reveal that this Wolbachia is a new strain called wDil distinct from wPet also belonging to the isopod clade of Wolbachia. Quantifications of both Wolbachia strains in the gonads of the two P. dilatatus morphs revealed that all males exhibit similar Wolbachia titers while the titers in females depend on the Wolbachia strain they host. Crossing experiments showed that both wDil and wPet induced partial unidirectional CI with different intensities. Moreover, these two strains induced bidirectional CI when individuals were both infected with one of the two different Wolbachia strains. This way, we demonstrated that P. dilatatus can be infected by two closely related Wolbachia strains (wDil and wPet), that seem to have different modification-rescue systems.

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.

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies.

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus, the Wolbachia wPet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus. To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus. We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results.

Complete genomic sequences of Tomato yellow leaf curl Mali virus isolates infecting tomato and pepper from the North Province of Cameroon.

Complete genomic sequences of Tomato yellow leaf curl Mali virus isolates infecting tomato and pepper from the North Province of Cameroon.

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Ecology and spatiotemporal dynamics of sandflies in the Mediterranean Languedoc region (Roquedur area, Gard, France).

BACKGROUND: Phlebotomine sandflies are hematophagous insects widely present in Western Mediterranean countries and known for their role as Leishmania vectors. During the last ten years, the risk of leishmaniasis re-emergence has increased in France. However, sandfly biology and ecology in the South of France remain poorly known because the last detailed study on their spatiotemporal dynamics was performed over 30 years ago. The aim of the present study was to update our knowledge on sandfly ecology by determining their spatiotemporal dynamics and by investigating the relationship between environmental/climatic factors and the presence and abundance of sandflies in the South of France. METHODS: An entomological survey was carried out during three years (2011-2013) along a 14 kilometer-long transect. The findings were compared with the data collected along the same transect in 1977. Data loggers were placed in each station and programmed to record temperature and relative humidity every six hours between April 2011 and November 2014. Several environmental factors (such as altitude, slope and wall orientation (North, East, West and South)) were characterized at each station. RESULTS: Four sandfly species were collected: Phlebotomus ariasi and Sergentomyia minuta, which were predominant, Ph. perniciosus and Ph. mascittii. Sandfly activity within the studied area started in May and ended in October with peaks in July-August at the optimum average temperature. We found a positive effect of altitude and temperature and a negative effect of relative humidity on Ph. ariasi and Se. minuta presence. We detected interspecific differences and non-linear effects of these climatic variables on sandfly abundance. Although the environment has considerably changed in 30 years, no significant difference in sandfly dynamics and species diversity was found by comparing the 1977 and 2011-2013 data. CONCLUSION: Our study shows that this area maintains a rich sandfly fauna with high Ph. ariasi population density during the active season. This represents a risk for Leishmania transmission. The analysis revealed that the presence and abundance of Ph. ariasi and Se. minuta were differently correlated with the environmental and climatic factors. Comparison with the data collected in 1977 highlighted the sandfly population stability, suggesting that they can adapt, in the short and long term, to changing ecosystems.

Biological invasions of geminiviruses: case study of TYLCV and Bemisia tabaci in Reunion Island.

In the last 20 years, molecular ecology approaches have proven to be extremely useful to identify and assess factors associated with viral emerging diseases, particularly in economically and socially important tropical crops such as maize (maize streak disease) and cassava (cassava mosaic disease). Molecular ecology approaches were applied in Reunion Island to analyze the epidemic of tomato yellow leaf curl disease, which has been affecting the island since the end of the 1990s. Before the invasive biotype B (currently known as Middle East-Asia Minor 1 cryptic species) of Bemisia tabaci spread across the world, Reunion Island (South West Indian Ocean) only hosted an indigenous biotype of B. tabaci, Ms (currently known as Indian Ocean cryptic species). Wild hybrids between invasive and indigenous species were subsequently characterized over multiple generations. Endosymbiont analysis of the hybrid population indicated that matings were non-random. Similarly, while no indigenous begomoviruses have ever been reported on Reunion Island, the two main strains of one of the most damaging and emerging plant viruses in the world, the Mild and Israel strains of the Tomato yellow leaf curl virus (TYLCV-Mld and TYLCV-IL), were introduced in 1997 and 2004 respectively. While these introductions extensively modified the agricultural landscape of Reunion Island, they also provided an invaluable opportunity to study the ecological and genetic mechanisms involved in biological invasion and competition.