Mosquitoes (Diptera: Culicidae) of the Amazonian savannas of French Guiana with a description of two new species.

Amazonian savannas are among the most noteworthy landscape components of the coastal plain of French Guiana. Although they cover only 0.22% of the territory, they bring together a large part of the animal and plant diversity of this overseas region of France. This article outlines the results of the first study dedicated to mosquitoes (Diptera: Culicidae) of Amazonian savannas. Samplings were conducted in eight independent savannas evenly distributed along a transect of 170 km on the coastal plain of French Guiana. A total of 50 mosquito species were recorded, which is about 20% of the culicid fauna currently known in French Guiana. Among them, Culex (Melanoconion) organaboensis sp. nov. and Cx. (Mel.) zabanicus sp. nov. are newly described based on both morphological features of the male genitalia and a DNA barcode obtained from type specimens. Diagnostic characters to assist their identification are provided and their placement within the infrasubgeneric classification of the subgenus Melanoconion is discussed.

Review on Main Arboviruses Circulating on French Guiana, An Ultra-Peripheric European Region in South America.

French Guiana (FG), a French overseas territory in South America, is susceptible to tropical diseases, including arboviruses. The tropical climate supports the proliferation and establishment of vectors, making it difficult to control transmission. In the last ten years, FG has experienced large outbreaks of imported arboviruses such as Chikungunya and Zika, as well as endemic arboviruses such as dengue, Yellow fever, and Oropouche virus. Epidemiological surveillance is challenging due to the differing distributions and behaviors of vectors. This article aims to summarize the current knowledge of these arboviruses in FG and discuss the challenges of arbovirus emergence and reemergence. Effective control measures are hampered by the nonspecific clinical presentation of these diseases, as well as the Aedes aegypti mosquito's resistance to insecticides. Despite the high seroprevalence of certain viruses, the possibility of new epidemics cannot be ruled out. Therefore, active epidemiological surveillance is needed to identify potential outbreaks, and an adequate sentinel surveillance system and broad virological diagnostic panel are being developed in FG to improve disease management.

Identification of Neotropical Culex Mosquitoes by MALDI-TOF MS Profiling.

The mosquito (Diptera: Culicidae) fauna of French Guiana encompasses 242 species, of which nearly half of them belong to the genus Culex. Whereas several species of Culex are important vectors of arboviruses, only a limited number of studies focus on them due to the difficulties to morphologically identify field-caught females. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of mosquitoes. Culex females collected in French Guiana were morphologically identified and dissected. Abdomens were used for molecular identification using the COI (cytochrome oxidase 1) gene. Legs and thorax of 169 specimens belonging to 13 Culex species, (i.e., Cx. declarator, Cx. nigripalpus, Cx. quinquefasciatus, Cx. usquatus, Cx. adamesi, Cx. dunni, Cx. eastor, Cx. idottus, Cx. pedroi, Cx. phlogistus, Cx. portesi, Cx. rabanicolus and Cx. spissipes) were then submitted to MALDI-TOF MS analysis. A high intra-species reproducibility and inter-species specificity of MS spectra for each mosquito body part tested were obtained. A corroboration of the specimen identification was revealed between MALDI-TOF MS, morphological and molecular results. MALDI-TOF MS protein profiling proves to be a suitable tool for identification of neotropical Culex species and will permit the enhancement of knowledge on this highly diverse genus.

Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification.

Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens-the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics.

Outbreak of Oropouche Virus in French Guiana.

Oropouche fever is a zoonotic dengue-like syndrome caused by Oropouche virus. In August-September 2020, dengue-like syndrome developed in 41 patients in a remote rainforest village in French Guiana. By PCR or microneutralization, 23 (82.1%) of 28 tested patients were positive for Oropouche virus, documenting its emergence in French Guiana.

Illustrated Morphological Keys for Fleas (Siphonaptera) in Madagascar.

Madagascar has an important diversity of fleas (Siphonaptera), which almost species do not exist elsewhere. Scientists have provided huge efforts to improve knowledge on Malagasy fleas since the middle of 1900s particularly by investigating topics such as taxonomy, systematics, biogeography, and flea vector role. Since then, new species discovery has increased and currently, 48 species are recorded which the majority is endemic. Therefore, it becomes necessary to have updated keys to identify species membership. This paper presents morphological-based keys to identify at genus and species levels adult fleas reported as occurring in Madagascar. Illustrations are proposed to make easier the observation of morphological criteria, which may be tricky for sibling species.

Experimental bluetongue virus infection of Culicoides austropalpalis, collected from a farm environment in Victoria, Australia.

Following the emerging bluetongue virus transmission in European temperate regions, we question the vector competence of the abundant Culicoides austropalpalis Lee and Reye in South-East temperate Australia. Field collected Culicoides midges were membrane fed with a bluetongue virus serotype 1 (BTV-1). The average feeding rate was 50%. After 13 days, survival rate was 25% and virus RNA presence was checked by quantitative PCR targeting viral genome segment 10. Virus RNA was found in 7.4% of individually tested females with relative viral RNA load values lower than freshly fed females, indicating that viral replication was low or null. A second qPCR targeting viral genome segment 1 confirmed the presence of virus RNA in only four out of 29 previously positive specimens. After 10 days culture on Culicoides cells, none of these four confimed positive samples did show subsequent cytopathogenic effect on Vero cells or BTV antigen detection by ELISA. As control for this virus activity detection, 12 days after microinjection of BTV-1, Culex annulirostris mosquitoes showed, after culture on Kc cells, cytopathogenic effect on Vero cells, with ELISA-confirmed infection. Despite its abundance in farm environment of the temperate Australian regions, the results of this study make C. austropalpalis of unlikely epidemiological importance in the transmission of BTV in Australia.

The Culex Mosquitoes (Diptera: Culicidae) of French Guiana: A Comprehensive Review With the Description of Three New Species.

The taxonomically intricate genus Culex Linnaeus includes numerous known vector species of parasites and viruses to humans. The aim of this article is to comprehensively review the Culex species which occur in French Guiana to provide a stronger taxonomic foundation for future studies on this genus in South America. The occurrence of Culex species was investigated in light of current taxonomic knowledge through an extensive examination of voucher specimens deposited in the entomological collections of four French depositories and additional specimens recently collected at various localities in French Guiana. Based on this review, 104 Culex species classified in eight subgenera are confirmed to occur in French Guiana. Compared to the most recent checklist, 18 species are added, and 10 species excluded, resulting in a total number of 242 valid mosquito species known to occur in French Guiana. Three nominal species are synonymized, three others are newly described, and a last one is transferred to another informal infrasubgeneric group. Overall, this review also highlights the limits of the use of only bibliographic data when dealing with taxonomically complex groups of insects.

Correction: Broad dengue neutralization in mosquitoes expressing an engineered antibody.

[This corrects the article DOI: 10.1371/journal.ppat.1008103.].

Broad dengue neutralization in mosquitoes expressing an engineered antibody.

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.

Whole Transcriptome Analysis of Aedes albopictus Mosquito Head and Thorax Post-Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton's tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV.

RNASeq Analysis of Aedes albopictus Mosquito Midguts after Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is an emerging pathogen around the world and causes significant morbidity in patients. A single amino acid mutation in the envelope protein of CHIKV has led to a shift in vector preference towards Aedesalbopictus. While mosquitoes are known to mount an antiviral immune response post-infection, molecular interactions during the course of infection at the tissue level remain largely uncharacterised. We performed whole transcriptome analysis on dissected midguts of Aedes albopictus infected with CHIKV to identify differentially expressed genes. For this, RNA was extracted at two days post-infection (2-dpi) from pooled midguts. We initially identified 25 differentially expressed genes (p-value < 0.05) when mapped to a reference transcriptome. Further, multiple differentially expressed genes were identified from a custom de novo transcriptome, which was assembled using the reads that did not align with the reference genome. Thirteen of the identified transcripts, possibly involved in immunity, were validated by qRT-PCR. Homologues of seven of these genes were also found to be significantly upregulated in Aedes aegypti midguts 2 dpi, indicating a conserved mechanism at play. These results will help us to characterise the molecular interaction between Aedes albopictus and CHIKV and can be utilised to reduce the impact of this viral infection.

Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs.

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells.

Wolbachia-infected mosquitoes are refractory to super-infection with arthropod-borne pathogens, but the role of host cell signaling proteins in pathogen-blocking mechanisms remains to be elucidated. Here, we use an antibody microarray approach to provide a comprehensive picture of the signaling response of Aedes aegypti-derived cells to Wolbachia. This approach identifies the host cell insulin receptor as being downregulated by the bacterium. Furthermore, siRNA-mediated knockdown and treatment with a small-molecule inhibitor of the insulin receptor kinase concur to assign a crucial role for this enzyme in the replication of dengue and Zika viruses in cultured mosquito cells. Finally, we show that the production of Zika virus in Wolbachia-free live mosquitoes is impaired by treatment with the selective inhibitor mimicking Wolbachia infection. This study identifies Wolbachia-mediated downregulation of insulin receptor kinase activity as a mechanism contributing to the blocking of super-infection by arboviruses.

Dengue virus infection changes Aedes aegypti oviposition olfactory preferences.

Aedes aegypti mosquitoes, main vectors for numerous flaviviruses, have olfactory preferences and are capable of olfactory learning especially when seeking their required environmental conditions to lay their eggs. In this study, we showed that semiochemical conditions during Aedes aegypti larval rearing affected future female choice for oviposition: water-reared mosquitoes preferred to lay eggs in water or p-cresol containers, while skatole reared mosquitoes preferred skatole sites. Using two independent behavioural assays, we showed that this skatole preference was lost in mosquitoes infected with dengue virus. Viral RNA was extracted from infected female mosquito heads, and an increase of virus load was detected from 3 to 10 days post infection, indicating replication in the insect head and possibly in the central nervous system. Expression of selected genes, potentially implied in olfactory learning processes, were also altered during dengue infection. Based on these results, we hypothesise that dengue virus infection alters gene expression in the mosquito's head and is associated with a loss of olfactory preferences, possibly modifying oviposition site choice of female mosquitoes.

Electrophysiological evidence of RML12 mosquito cell line towards neuronal differentiation by 20-hydroxyecdysdone.

Continuous cell lines from insect larval tissues are widely used in different research domains, such as virology, insect immunity, gene expression, and bio pharmacology. Previous study showed that introduction of 20-hydroxyecdysone to Spodoptera cell line induced a neuron-like morphology with neurite extensions. Despite some results suggesting potential presence of neuro-receptors, no study so far has shown that these neuron-induced cells were functional. Here, using microelectrode arrays, we showed that the mosquito cell line, RML12, differentiated with 20-hydroxyecdysone, displays spontaneous electrophysiological activity. Results showed that these cells can be stimulated by GABAergic antagonist as well as nicotinic agonist. These results provide new evidence of neuron-like functionality of 20-hydroxyecdysone induced differentiated mosquito cell line. Finally, we used this new model to test the effects of two insecticides, temephos and permethrin. Our analysis revealed significant changes in the spiking activity after the introduction of these insecticides with prolonged effect on the neuronal activity. We believe that this differentiated mosquito neuronal cell model can be used for high-throughput screening of new pesticides on insect nervous system instead of primary neurons or in vivo studies.

Species diversity and phylogeny of fleas of small terrestrial mammals in the forests of the Central Highlands of Madagascar.

Fleas are holometabolous insects forming the order of Siphonaptera. Some studies have been carried out on biology and systematic of Malagasy fleas, but little is known about their phylogenetic relationships. In this study, we focused on flea species occurring in the forests of the Central Highlands and also, on the determination of their phylogenetic relationships. Three families, five genera and thirteen species were identified. The family Pulicidae includes four species (Centetipsylla madagascariensis Rothschild, Synopsyllus fonquerniei Wagner Roubaud, S. estradei Klein and S. robici Klein); Leptopsyllidae has eight species (Paractenopsyllus vauceli Klein, P. petiti Klein, P. viettei Klein, P. grandidieri Klein, P. goodmani Duchemin, P. rouxi Duchemin, P. raxworthyi Duchemin Ratovonjato and Tsaractenus rodhaini Duchemin), and Ctenophtalmidae one species (Dinopsyllus brachypecten Smit). All are endemic to Madagascar and each differs geographically. Flea phylogenetic relationships were inferred using four molecular markers (ITS2, mtCOII, 16SrRNA and 12S rRNA) and using Neighbor-Joining, Maximum Parsimony and Bayesian methods with addition of Genbank sequences of exotic species. The Family Pulicidae was monophyletic while the families Leptopsyllidae and Ctenophtalmidae were paraphyletic. Malagasy fleas are homogeneous and all species adhere to current classification schemes.

Neurotropism and behavioral changes associated with Zika infection in the vector Aedes aegypti.

Understanding Zika virus infection dynamics is essential, as its recent emergence revealed possible devastating neuropathologies in humans, thus causing a major threat to public health worldwide. Recent research allowed breakthrough in our understanding of the virus and host pathogenesis; however, little is known on its impact on its main vector, Aedes aegypti. Here we show how Zika virus targets Aedes aegypti's neurons and induces changes in its behavior. Results are compared to dengue virus, another flavivirus, which triggers a different pattern of behavioral changes. We used microelectrode array technology to record electrical spiking activity of mosquito primary neurons post infections and discovered that only Zika virus causes an increase in spiking activity of the neuronal network. Confocal microscopy also revealed an increase in synapse connections for Zika virus-infected neuronal networks. Interestingly, the results also showed that mosquito responds to infection by overexpressing glutamate regulatory genes while maintaining virus levels. This neuro-excitation, possibly via glutamate, could contribute to the observed behavioral changes in Zika virus-infected Aedes aegypti females. This study reveals the importance of virus-vector interaction in arbovirus neurotropism, in humans and vector. However, it appears that the consequences differ in the two hosts, with neuropathology in human host, while behavioral changes in the mosquito vector that may be advantageous to the virus.

Zika virus-induced hyper excitation precedes death of mouse primary neuron.

BACKGROUND: Zika virus infection in new born is linked to congenital syndromes, especially microcephaly. Studies have shown that these neuropathies are the result of significant death of neuronal progenitor cells in the central nervous system of the embryo, targeted by the virus. Although cell death via apoptosis is well acknowledged, little is known about possible pathogenic cellular mechanisms triggering cell death in neurons. METHODS: We used in vitro embryonic mouse primary neuron cultures to study possible upstream cellular mechanisms of cell death. Neuronal networks were grown on microelectrode array and electrical activity was recorded at different times post Zika virus infection. In addition to this method, we used confocal microscopy and Q-PCR techniques to observe morphological and molecular changes after infection. RESULTS: Zika virus infection of mouse primary neurons triggers an early spiking excitation of neuron cultures, followed by dramatic loss of this activity. Using NMDA receptor antagonist, we show that this excitotoxicity mechanism, likely via glutamate, could also contribute to the observed nervous system defects in human embryos and could open new perspective regarding the causes of adult neuropathies. CONCLUSIONS: This model of excitotoxicity, in the context of neurotropic virus infection, highlights the significance of neuronal activity recording with microelectrode array and possibility of more than one lethal mechanism after Zika virus infection in the nervous system.

Detection of Elizabethkingia spp. in Culicoides Biting Midges, Australia.

The bacterial pathogen Elizabethkingia is known to exist in certain species of mosquito but was unknown in other arthropods. We report the detection and identification of Elizabethkingia in species of Culicoides biting midge in Australia, raising the possibility of bacterial transmission via this species.