Experimental infection of Artibeus lituratus bats and no detection of Zika virus in neotropical bats from French Guiana, Peru, and Costa Rica suggests a limited role of bats in Zika transmission.

Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics.

Adaptive duplication and genetic diversification of protein kinase R contribute to the specificity of bat-virus interactions.

Several bat species act as asymptomatic reservoirs for many viruses that are highly pathogenic in other mammals. Here, we have characterized the functional diversification of the protein kinase R (PKR), a major antiviral innate defense system. Our data indicate that PKR has evolved under positive selection and has undergone repeated genomic duplications in bats in contrast to all studied mammals that have a single copy of the gene. Functional testing of the relationship between PKR and poxvirus antagonists revealed how an evolutionary conflict with ancient pathogenic poxviruses has shaped a specific bat host-virus interface. We determined that duplicated PKRs of the Myotis species have undergone genetic diversification, allowing them to collectively escape from and enhance the control of DNA and RNA viruses. These findings suggest that viral-driven adaptations in PKR contribute to modern virus-bat interactions and may account for bat-specific immunity.

Venezuelan Equine Encephalitis Complex Alphavirus in Bats, French Guiana.

Although essential for control strategies, knowledge about transmission cycles is limited for Venezuelan equine encephalitis complex alphaviruses (VEEVs). After testing 1,398 bats from French Guiana for alphaviruses, we identified and isolated a new strain of the encephalitogenic VEEV species Tonate virus (TONV). Bats may contribute to TONV spread in Latin America.

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses.

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo-mock community (33 arthropod taxa from 16 orders), and guano-based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano-based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one-third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.

Integrating population genetics to define conservation units from the core to the edge of Rhinolophus ferrumequinum western range.

The greater horseshoe bat (Rhinolophus ferrumequinum) is among the most widespread bat species in Europe but it has experienced severe declines, especially in Northern Europe. This species is listed Near Threatened in the European IUCN Red List of Threatened Animals, and it is considered to be highly sensitive to human activities and particularly to habitat fragmentation. Therefore, understanding the population boundaries and demographic history of populations of this species is of primary importance to assess relevant conservation strategies. In this study, we used 17 microsatellite markers to assess the genetic diversity, the genetic structure, and the demographic history of R. ferrumequinum colonies in the western part of its distribution. We identified one large population showing high levels of genetic diversity and large population size. Lower estimates were found in England and northern France. Analyses of clustering and isolation by distance suggested that the Channel and the Mediterranean seas could impede R. ferrumequinum gene flow. These results provide important information to improve the delineation of R. ferrumequinum management units. We suggest that a large management unit corresponding to the population ranging from Spanish Basque Country to northern France must be considered. Particular attention should be given to mating territories as they seem to play a key role in maintaining high levels of genetic mixing between colonies. Smaller management units corresponding to English and northern France colonies must also be implemented. These insular or peripheral colonies could be at higher risk of extinction in the near future.

Coexistence of two sympatric cryptic bat species in French Guiana: insights from genetic, acoustic and ecological data.

BACKGROUND: The distinction between lineages of neotropical bats from the Pteronotus parnellii species complex has been previously made according to mitochondrial DNA, and especially morphology and acoustics, in order to separate them into two species. In these studies, either sample sizes were too low when genetic and acoustic or morphological data were gathered on the same individuals, or genetic and other data were collected on different individuals. In this study, we intensively sampled bats in 4 caves and combined all approaches in order to analyse genetic, morphologic, and acoustic divergence between these lineages that live in the same caves in French Guiana. RESULTS: A multiplex of 20 polymorphic microsatellite markers was developed using the 454-pyrosequencing technique to investigate for the first time the extent of reproductive isolation between the two lineages and the population genetic structure within lineages. We genotyped 748 individuals sampled between 2010 and 2015 at the 20 nuclear microsatellite loci and sequenced a portion of the cytochrome c oxydase I gene in a subset of these. Two distinct, non-overlapping haplogroups corresponding to cryptic species P. alitonus and P. rubiginosus were revealed, in accordance with previous findings. No spatial genetic structure between caves was detected for both species. Hybridization appeared to be quite limited (0.1-4%) using microsatellite markers whereas introgression was more common (7.5%) and asymmetric for mitochondrial DNA (mtDNA). CONCLUSIONS: The extremely low rate of hybridization could be explained by differences in life cycle phenology between species as well as morphological and acoustical distinction between sexes in one or the other species. Taken together, these results add to our growing understanding of the nature of species boundaries in Pteronotus parnelli, but deserve more in-depth studies to understand the evolutionary processes underlying asymmetric mtDNA introgression in this group of cryptic species.