Combining transinfected Wolbachia and a genetic sexing strain to control Aedes albopictus in laboratory-controlled conditions.

The global expansion of Aedes albopictus has stimulated the development of environmentally friendly methods aiming to control disease transmission through the suppression of natural vector populations. Sterile male release programmes are currently being deployed worldwide, and are challenged by the availability of an efficient sex separation which can be achieved mechanically at the pupal stage and/or by artificial intelligence at the adult stage, or through genetic sexing, which allows separating males and females at an early development stage. In this study, we combined the genetic sexing strain previously established based on the linkage of dieldrin resistance to the male locus with a Wolbachia transinfected line. For this, we introduced either the wPip-I or the wPip-IV strain from Culex pipiens in an asymbiotic Wolbachia-free Ae. albopictus line. We then measured the penetrance of cytoplasmic incompatibility and life-history traits of both transinfected lines, selected the wPip-IV line and combined it with the genetic sexing strain. Population suppression experiments demonstrated a 90% reduction in population size and a 50% decrease in hatching rate. Presented results showed that such a combination has a high potential in terms of vector control but also highlighted associated fitness costs, which should be reduced before large-scale field assay.

Synchronicity of viral shedding in molossid bat maternity colonies.

Infection dynamics in vertebrates are driven by biological and ecological processes. For bats, population structure and reproductive cycles have major effects on RNA virus transmission. On Reunion Island, previous studies have shown that parturition of pregnant females and aggregation of juvenile Reunion free-tailed bats (Mormopterus francoismoutoui) are associated with major increase in the prevalence of bats shedding RNA viruses. The synchronicity of such shedding pulses, however, is yet to be assessed between viruses but also maternity colonies. Based on 3422 fresh faeces collected every 2-5 weeks during four consecutive birthing seasons, we report the prevalence of bats shedding astroviruses (AstVs), coronaviruses (CoVs) and paramyxoviruses (PMVs) in two maternity colonies on Reunion Island. We found that the proportion of bats shedding viruses is highly influenced by sampling collection periods, and therefore by the evolution of the population age structure. We highlight that virus shedding patterns are consistent among years and colonies for CoVs and to a lesser extent for PMVs, but not for AstVs. We also report that 1% of bats harbour co-infections, with two but not three of the viruses, and most co-infections were due to CoVs and PMVs.

Genetic characterization of dengue virus serotype 1 circulating in Reunion Island, 2019-2021, and the Seychelles, 2015-2016.

BACKGROUND: An unprecedent increase in the number of cases and deaths reported from dengue virus (DENV) infection has occurred in the southwestern Indian ocean in recent years. From 2017 to mid-2021 more than 70,000 confirmed dengue cases were reported in Reunion Island, and 1967 cases were recorded in the Seychelles from 2015 to 2016. Both these outbreaks displayed similar trends, with the initial circulation of DENV-2 which was replaced by DENV-1. Here, we aim to determine the origin of the DENV-1 epidemic strains and to explore their genetic characteristics along the uninterrupted circulation, particularly in Reunion. METHODS: Nucleic acids were extracted from blood samples collected from dengue positive patients; DENV-1 was identified by RT-qPCR. Positive samples were used to infect VERO cells. Genome sequences were obtained from either blood samples or infected-cell supernatants through a combination of both Illumina or MinION technologies. RESULTS: Phylogenetic analyses of partial or whole genome sequences revealed that all DENV-1 sequences from Reunion formed a monophyletic cluster that belonged to genotype I and were closely related to one isolate from Sri Lanka (OL752439.1, 2020). Sequences from the Seychelles belonged to the same major phylogenetic branch of genotype V, but fell into two paraphyletic clusters, with greatest similarity for one cluster to 2016-2017 isolate from Bangladesh, Singapore and China, and for the other cluster to ancestral isolates from Singapore, dating back to 2012. Compared to publicly available DENV-1 genotype I sequences, fifteen non-synonymous mutations were identified in the Reunion strains, including one in the capsid and the others in nonstructural proteins (NS) (three in NS1, two in NS2B, one in NS3, one in NS4B, and seven in NS5). CONCLUSION: In contrast to what was seen in previous outbreaks, recent DENV-1 outbreaks in Reunion and the Seychelles were caused by distinct genotypes, all likely originating from Asia where dengue is (hyper)endemic in many countries. Epidemic DENV-1 strains from Reunion harbored specific non-synonymous mutations whose biological significance needs to be further investigated.

Daphnanes diterpenes from the latex of Hura crepitans L. and their PKCζ-dependent anti-proliferative activity on colorectal cancer cells.

Hura crepitans L. (Euphorbiaceae) is a thorn-covered tree widespread in South America, Africa and Asia which produces an irritating milky latex containing numerous secondary metabolites, notably daphnane-type diterpenes known as Protein Kinase C activators. Fractionation of a dichloromethane extract of the latex led to the isolation of five new daphnane diterpenes (1-5), along with two known analogs (6-7) including huratoxin. Huratoxin (6) and 4',5'-epoxyhuratoxin (4) were found to exhibit significant and selective cell growth inhibition against colorectal cancer cell line Caco-2 and primary colorectal cancer cells cultured as colonoids. The underlying mechanism of 4 and 6 was further investigated revealing the involvement of PKCζ in the cytostatic activity.

New Phenolic Lipids from the Leaves of Clausena harmandiana Inhibit SARS-CoV-2 Entry into Host Cells.

Induced by the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic underlined the clear need for antivirals against coronaviruses. In an effort to identify new inhibitors of SARS-CoV-2, a screening of 824 extracts prepared from various parts of 400 plant species belonging to the Rutaceae and Annonaceae families was conducted using a cell-based HCoV-229E inhibition assay. Due to its significant activity, the ethyl acetate extract of the leaves of Clausena harmandiana was selected for further chemical and biological investigations. Mass spectrometry-guided fractionation afforded three undescribed phenolic lipids (1-3), whose structures were determined via spectroscopic analysis. The absolute configurations of 1 and 2 were determined by analyzing Mosher ester derivatives. The antiviral activity against SARS-CoV-2 was subsequently shown, with IC50 values of 0.20 and 0.05 µM for 2 and 3, respectively. The mechanism of action was further assessed, showing that both 2 and 3 are inhibitors of coronavirus entry by acting directly on the viral particle. Phenolic lipids from Clausena harmandiana might be a source of new antiviral agents against human coronaviruses.

Spread of SARS-CoV-2 Variants on Réunion Island, France, 2021.

In January 2021, after detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, genomic surveillance was established on Réunion Island to track the introduction and spread of SARS-CoV-2 lineages and variants of concern. This system identified 22 SARS-CoV-2 lineages, 71% of which were attributed to the Beta variant.

Bombali Ebolavirus in Mops condylurus Bats (Molossidae), Mozambique.

We detected Bombali ebolavirus RNA in 3 free-tailed bats (Mops condylurus, Molossidae) in Mozambique. Sequencing of the large protein gene revealed 98% identity with viruses previously detected in Sierra Leone, Kenya, and Guinea. Our findings further support the suspected role of Mops condylurus bats in maintaining Bombali ebolavirus.

One Health compartmental analysis of ESBL-producing Escherichia coli on Reunion Island reveals partitioning between humans and livestock.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-Ec) is a major cause of infections worldwide. An understanding of the reservoirs and modes of transmission of these pathogens is essential, to tackle their increasing frequency. OBJECTIVES: We investigated the contributions of various compartments (humans, animals, environment), to human colonization or infection with ESBL-Ec over a 3 year period, on an island. METHODS: The study was performed on Reunion Island (Southwest Indian Ocean). We collected ESBL-Ec isolates prospectively from humans, wastewater and livestock between April 2015 and December 2018. Human specimens were recovered from a regional surveillance system representative of the island's health facilities. These isolates were compared with those from livestock and urban/rural wastewater, by whole-genome sequencing. RESULTS: We collected 410 ESBL-Ec isolates: 161 from humans, 161 from wastewater and 88 from animals. Phylogenomic analysis demonstrated high diversity (100 STs), with different STs predominating among isolates from humans (ST131, ST38, ST10) and animals (ST57, ST156). The large majority (90%) of the STs, including ST131, were principally associated with a single compartment. The CTX-M-15, CTX-M-27 and CTX-M-14 enzymes were most common in humans/human wastewater, whereas CTX-M-1 predominated in animals. Isolates of human and animal origin had different plasmids carrying blaCTX-M genes, with the exception of a conserved IncI1-ST3 blaCTX-M-1 plasmid. CONCLUSIONS: These molecular data suggest that, despite their high level of contamination, animals are not a major source of the ESBL-Ec found in humans living on this densely populated high-income island. Public health policies should therefore focus primarily on human-to-human transmission, to prevent human infections with ESBL-Ec.

Fitness costs associated with a GABA receptor mutation conferring dieldrin resistance in Aedes albopictus.

Understanding the dynamics of insecticide resistance genes in mosquito populations is pivotal for a sustainable use of insecticides. Dieldrin resistance in Aedes albopictus is conferred by the alanine to serine substitution (A302S or RdlR allele) in the γ-aminobutyric acid (GABA) receptor encoded by the Rdl gene. On Reunion Island, dieldrin resistance was initially reported in natural Ae. albopictus populations sampled in 2008 despite the ban of dieldrin since 1994. To monitor insecticide resistance in Ae. albopictus on the island and to identify its drivers, we measured (i) the frequency of resistance alleles in 19 distinct natural populations collected between 2016 and 2017, (ii) fitness costs associated with dieldrin resistance in laboratory-controlled experiments, and (iii) the resistance conferred by RdlR to fipronil, an insecticide widely used on the island and reported to cross-react with RdlR. The results show a persistence of RdlR in Ae. albopictus natural populations at low frequencies. Among the measured life history traits, mortality in pre-imaginal stages, adults' survival as well as the proportion of egg-laying females were significantly affected in resistant mosquitoes. Finally, bioassays revealed resistance of RdlR mosquitoes to fipronil, suggesting that the use of fipronil in natura could select for the RdlR allele. This study shows that dieldrin resistance is persistent in natural mosquito populations likely as a result of combined effects between fitness costs associated with RdlR and selection exerted by cross-reacting environmental insecticides such as fipronil.

Investigation of astrovirus, coronavirus and paramyxovirus co-infections in bats in the western Indian Ocean.

Co-infections have a key role in virus transmission in wild reservoir hosts. We investigated the simultaneous presence of astroviruses, coronaviruses, and paramyxoviruses in bats from Madagascar, Mayotte, Mozambique, and Reunion Island. A total of 871 samples from 28 bat species representing 8 families were tested by polymerase chain reactions (PCRs) targeting the RNA-dependent RNA-polymerase genes. Overall, 2.4% of bats tested positive for the presence of at least two viruses, only on Madagascar and in Mozambique. Significant variation in the proportion of co-infections was detected among bat species, and some combinations of co-infection were more common than others. Our findings support that co-infections of the three targeted viruses occur in bats in the western Indian Ocean region, although further studies are needed to assess their epidemiological consequences.

Changes in Bacterial Diversity, Composition and Interactions During the Development of the Seabird Tick Ornithodoros maritimus (Argasidae).

Characterising within-host microbial interactions is essential to understand the drivers that shape these interactions and their consequences for host ecology and evolution. Here, we examined the bacterial microbiota hosted by the seabird soft tick Ornithodoros maritimus (Argasidae) in order to uncover bacterial interactions within ticks and how these interactions change over tick development. Bacterial communities were characterised through next-generation sequencing of the V3-V4 hypervariable region of the bacterial 16S ribosomal RNA gene. Bacterial co-occurrence and co-exclusion were determined by analysing networks generated from the metagenomic data obtained at each life stage. Overall, the microbiota of O. maritimus was dominated by four bacterial genera, namely Coxiella, Rickettsia, Brevibacterium and Arsenophonus, representing almost 60% of the reads. Bacterial diversity increased over tick development, and adult male ticks showed higher diversity than did adult female ticks. Bacterial networks showed that co-occurrence was more frequent than co-exclusion and highlighted substantial shifts across tick life stages; interaction networks changed from one stage to the next with a steady increase in the number of interactions through development. Although many bacterial interactions appeared unstable across life stages, some were maintained throughout development and were found in both sexes, such as Coxiella and Arsenophonus. Our data support the existence of a few stable interactions in O. maritimus ticks, on top of which bacterial taxa accumulate from hosts and/or the environment during development. We propose that stable associations delineate core microbial interactions, which are likely to be responsible for key biological functions.

Development of Sustainable Chemistry in Madagascar: Example of the Valuation of CNSL and the Use of Chromones as an Attractant for Mosquitoes.

This article describes a part of the results obtained from the cooperation between the University of Lyon1 (France) and the University of Antananarivo (Madagascar). It shows (among others) that useful research can be carried out in developing countries of the tropics if their social, technical, and economic conditions are taken into account. The concepts and methods associated with so-called "green chemistry" are particularly appropriated for this purpose. To illustrate this approach, two examples are shown. The first deals with industrial ecology and concerns waste transformation from the production of cashew nut into an amphiphilic product, oxyacetic derivatives. This product was obtained with a high yield and in a single step reaction. It exhibited an important surfactant property similar to those of the main fossil-based ones but with a much lower ecological impact. The second talks about chemical ecology as an alternative to insecticides and used to control dangerous mosquito populations. New substituted chromones were synthesized and showed biological activities toward Aedes albopictus mosquito species. Strong repellent properties were recorded for some alkoxylated products if others had a significant attractant effect (Kairomone) depending on their stereochemistry and the length of the alkyl chain.

Analysis of partial sequences of the RNA-dependent RNA polymerase gene as a tool for genus and subgenus classification of coronaviruses.

The recent reclassification of the Riboviria, and the introduction of multiple new taxonomic categories including both subfamilies and subgenera for coronaviruses (family Coronaviridae, subfamily Orthocoronavirinae), represents a major shift in how official classifications are used to designate specific viral lineages. While the newly defined subgenera provide much-needed standardization for commonly cited viruses of public health importance, no method has been proposed for the assignment of subgenus based on partial sequence data, or for sequences that are divergent from the designated holotype reference genomes. Here, we describe the genetic variation of a 387 nt region of the coronavirus RNA-dependent RNA polymerase (RdRp), which is one of the most used partial sequence loci for both detection and classification of coronaviruses in molecular epidemiology. We infer Bayesian phylogenies from more than 7000 publicly available coronavirus sequences and examine clade groupings relative to all subgenus holotype sequences. Our phylogenetic analyses are largely coherent with whole-genome analyses based on designated holotype members for each subgenus. Distance measures between sequences form discrete clusters between taxa, offering logical threshold boundaries that can attribute subgenus or indicate sequences that are likely to belong to unclassified subgenera both accurately and robustly. We thus propose that partial RdRp sequence data of coronaviruses are sufficient for the attribution of subgenus-level taxonomic classifications and we supply the R package, MyCoV, which provides a method for attributing subgenus and assessing the reliability of the attribution.

Bat Astrovirus in Mozambique.

Astroviruses (AstVs) are responsible for infection of a large diversity of mammalian and avian species, including bats, aquatic birds, livestock and humans. We investigated AstVs circulation in bats in Mozambique and Mayotte, a small island in the Comoros Archipelago located between east Africa and Madagascar. Biological material was collected from 338 bats and tested for the presence of the AstV RNA-dependent RNA-polymerase gene with a pan-AstV semi-nested polymerase chain reaction assay. None of the 79 samples obtained from Mayotte bats (Pteropus seychellensis comorensis and Chaerephon pusillus) tested positive; however, 20.1% of bats sampled in Mozambique shed AstVs at the time of sampling and significant interspecific variation in the proportion of positive bats was detected. Many AstVs sequences obtained from a given bat species clustered in different phylogenetic lineages, while others seem to reflect some level of host-virus association, but also with AstVs previously reported from Malagasy bats. Our findings support active circulation of a large diversity of AstVs in bats in the western Indian Ocean islands, including the southeastern African coast, and highlight the need for more detailed assessment of its risk of zoonotic transmission to human populations.

Synthesis, Bioassays and Field Evaluation of Hydroxycoumarins and their Alkyl Derivatives as Repellents or Kairomones for Aedes albopictus Skuse (Diptera: Culicidae).

In recent years, a significant increase in mosquito-borne diseases has been recorded worldwide. Faced with the limitations of existing methods for controlling the vector mosquito population, the development of attractants to bait traps and repellents to limit host-vector contacts could be promising and environmentally-friendly control strategies. The purpose of this study was to evaluate the effect of hydroxycoumarins and their alkyls derivatives against Aedes albopictus, the main vector of several arboviruses. Synthesis, bioassays and field trials were carried out in Madagascar. The results showed that 3, 4 and 6-hydroxycoumarins are attractive to this mosquito, 4-hydroxycoumarin being the most effective both in the laboratory and under field conditions. In addition, a good synergistic effect was found with octenol to attract mosquitoes and especially Ae. albopictus in comparison to other mosquito species living in sympatry. On the contrary, the 4-s-butoxycoumarin and 4-s-pentoxycoumarin derivatives had a repellent effect with the former showing the most significant effect. Further optimization of the dose and structure of these products will be carried out in order to maximize their utility for the control of Ae. albopictus and other mosquitoes.

Comprehensive proteome profiling in Aedes albopictus to decipher Wolbachia-arbovirus interference phenomenon.

BACKGROUND: Aedes albopictus is a vector of arboviruses that cause severe diseases in humans such as Chikungunya, Dengue and Zika fevers. The vector competence of Ae. albopictus varies depending on the mosquito population involved and the virus transmitted. Wolbachia infection status in believed to be among key elements that determine viral transmission efficiency. Little is known about the cellular functions mobilized in Ae. albopictus during co-infection by Wolbachia and a given arbovirus. To decipher this tripartite interaction at the molecular level, we performed a proteome analysis in Ae. albopictus C6/36 cells mono-infected by Wolbachia wAlbB strain or Chikungunya virus (CHIKV), and bi-infected. RESULTS: We first confirmed significant inhibition of CHIKV by Wolbachia. Using two-dimensional gel electrophoresis followed by nano liquid chromatography coupled with tandem mass spectrometry, we identified 600 unique differentially expressed proteins mostly related to glycolysis, translation and protein metabolism. Wolbachia infection had greater impact on cellular functions than CHIKV infection, inducing either up or down-regulation of proteins associated with metabolic processes such as glycolysis and ATP metabolism, or structural glycoproteins and capsid proteins in the case of bi-infection with CHIKV. CHIKV infection inhibited expression of proteins linked with the processes of transcription, translation, lipid storage and miRNA pathways. CONCLUSIONS: The results of our proteome profiling have provided new insights into the molecular pathways involved in tripartite Ae. albopictus-Wolbachia-CHIKV interaction and may help defining targets for the better implementation of Wolbachia-based strategies for disease transmission control.

High-throughput sequencing of transposable element insertions suggests adaptive evolution of the invasive Asian tiger mosquito towards temperate environments.

Invasive species represent unique opportunities to evaluate the role of local adaptation during colonization of new environments. Among these species, the Asian tiger mosquito, Aedes albopictus, is a threatening vector of several human viral diseases, including dengue and chikungunya, and raises concerns about the Zika fever. Its broad presence in both temperate and tropical environments has been considered the reflection of great "ecological plasticity." However, no study has been conducted to assess the role of adaptive evolution in the ecological success of Ae. albopictus at the molecular level. In the present study, we performed a genomic scan to search for potential signatures of selection leading to local adaptation in one-hundred-forty field-collected mosquitoes from native populations of Vietnam and temperate invasive populations of Europe. High-throughput genotyping of transposable element insertions led to the discovery of more than 120,000 polymorphic loci, which, in their great majority, revealed a virtual absence of structure between the biogeographic areas. Nevertheless, 92 outlier loci showed a high level of differentiation between temperate and tropical populations. The majority of these loci segregate at high insertion frequencies among European populations, indicating that this pattern could have been caused by recent adaptive evolution events in temperate areas. An analysis of the overlapping and neighbouring genes highlighted several candidates, including diapause, lipid and juvenile hormone pathways.

Bacterial diversity of cosmopolitan Culex pipiens and invasive Aedes japonicus from Germany.

Symbiotic bacteria have gained significant attention in recent years. For example, microbiota of some mosquito species seems to influence the development and transmission of pathogens. Furthermore, several attempts using bacteria as a paratransgenetic tool have been made in order to assist the control of mosquito-borne diseases. In this study, we examined the bacterial diversity of wild-caught adult Culex (Cx.) pipiens and laboratory-reared adult Aedes japonicus (Ae. japonicus) in Germany using a culture-independent method. Genomic DNA was extracted from each specimen and submitted to PCR amplification of eubacterial 16S rDNA. After the cloning reaction, 28 bacterial transformants per sample containing the 16S rDNA inserts were selected per each sample for sequencing. The analysed specimens of Cx. pipiens as well as of Ae. japonicus showed a diverse bacterial community including some common bacterial genera. Blast analysis allowed to identify 21 bacterial genera belonging to 2 phyla among the 23 specimens of Cx. pipiens. The 14 analysed Ae. japonicus revealed 11 bacterial genera belonging to 3 phyla. In both mosquito species, identified isolates were mainly Proteobacteria. Only 4 of the bacterial genera were found in both mosquito species, with the most prevalent genera Sphingomonas and Rahnella in Cx. pipiens and in Ae. japonicus respectively. Most of the bacterial genera found in our study have been identified in other mosquito species before. Due to the currently scarce data situation, ongoing examinations on the very abundant bacterial genera or species are strongly required to determine their relevance for the biology and adaptiveness of mosquitoes including pathogen-host relationship.

The Bacteriome of Bat Flies (Nycteribiidae) from the Malagasy Region: a Community Shaped by Host Ecology, Bacterial Transmission Mode, and Host-Vector Specificity.

The Nycteribiidae are obligate blood-sucking Diptera (Hippoboscoidea) flies that parasitize bats. Depending on species, these wingless flies exhibit either high specialism or generalism toward their hosts, which may in turn have important consequences in terms of their associated microbial community structure. Bats have been hypothesized to be reservoirs of numerous infectious agents, some of which have recently emerged in human populations. Thus, bat flies may be important in the epidemiology and transmission of some of these bat-borne infectious diseases, acting either directly as arthropod vectors or indirectly by shaping pathogen communities among bat populations. In addition, bat flies commonly have associations with heritable bacterial endosymbionts that inhabit insect cells and depend on maternal transmission through egg cytoplasm to ensure their transmission. Some of these heritable bacteria are likely obligate mutualists required to support bat fly development, but others are facultative symbionts with unknown effects. Here, we present bacterial community profiles that were obtained from seven bat fly species, representing five genera, parasitizing bats from the Malagasy region. The observed bacterial diversity includes Rickettsia, Wolbachia, and several Arsenophonus-like organisms, as well as other members of the Enterobacteriales and a widespread association of Bartonella bacteria from bat flies of all five genera. Using the well-described host specificity of these flies and data on community structure from selected bacterial taxa with either vertical or horizontal transmission, we show that host/vector specificity and transmission mode are important drivers of bacterial community structure.