Mosquito Specimens Research Articles

Prediction of species composition ratios in pooled specimens of the Anopheles Hyrcanus group using quantitative sequencing

BackgroundPlasmodium vivax is transmitted by members of the Anopheles Hyrcanus Group that includes six species in the Republic of Korea: Anopheles sinensis sensu stricto (s.s.), Anopheles pullus, Anopheles kleini, Anopheles belenrae, Anopheles lesteri, and Anopheles sineroides. Individual Anopheles species within the Hyrcanus Group demonstrate differences in their geographical distributions, vector competence and insecticide resistance, making it crucial for accurate species identification. Conventional species identification conducted using individual genotyping (or barcoding) based on species-specific molecular markers requires extensive time commitment and financial resources.ResultsA population-based quantitative sequencing (QS) protocol developed in this study provided a rapid estimate of species composition ratios among pooled mosquitoes as a cost-effective alternative to individual genotyping. This can be accomplished by using species- or group-specific nucleotide sequences of the mitochondrial cytochrome C oxidase subunit I (COI) and the ribosomal RNA internal transcribed spacer 2 (ITS2) region as species identification alleles in a two-step prediction protocol. Standard genomic DNA fragments of COI and ITS2 genes were amplified from each Anopheles species using group-specific universal primer sets. Following sequencing of the COI or ITS2 amplicons generated from sets of standard DNA mixtures, equations were generated via linear regression to predict species-specific nucleotide sequence frequencies at different positions. Species composition ratios between An. sineroides, An. pullus and An. lesteri were estimated from QS of the COI amplicons based on the mC.260A, mC.122C and mC.525C alleles at the first step, followed by the prediction of species composition ratios between An. sinensis, An. kleini and An. belenrae based on QS of the ITS2 amplicons using the rI.370G and rI.389T alleles. The COI copy number was not significantly different between species, suggesting the reliability of COI-based prediction. In contrast, ITS2 showed a slightly but significantly higher copy number in An. belenrae, requiring an adjustment of its predicted composition ratio. A blind test proved that predicted species composition ratios either from pooled DNA specimens or pooled mosquito specimens were not statistically different from the actual values, demonstrating that the QS-based prediction is accurate and reliable.ConclusionsThis two-step prediction protocol will facilitate rapid estimation of the species composition ratios in field-collected Anopheles Hyrcanus Group populations and is particularly useful for studying the vector ecology of Anopheles population and epidemiology of malaria.

Sindbis virus infection of mosquito species in the wetlands of northwestern Iran and modeling the probable ecological niches of SINV vectors in the country

Sindbis virus (SINV) and Chikungunya virus (CHIKV) are among the most widely spread mosquito-borne viruses worldwide. Due to the key role of mosquitoes in the transmission cycle of vector-borne diseases, models such as Maximum Entropy (MaxEnt) have been used in recent years to predict the environmental suitability and ecological niches of mosquito vectors.Infection of three mosquito species (Anopheles maculipennis s.l., Culex tritaeniorhynchus, and Culiseta longiareolata) with CHIKV has recently been reported in Iran. However, given the importance of vector-borne diseases in the country, there is a need for extensive studies on the infection of mosquitoes with CHIKV and SINV in different areas of the country. Accordingly, the current research was conducted to investigate the infection of mosquitoes with the two aforementioned viruses in the northwestern part of Iran and also to model the ecological niches of the vectors of these mosquito-borne viruses in the country.In the current study, 4639 mosquito specimens, consisting of 2515 adults and 2124 larvae, were collected from the wetlands of West Azerbaijan Province and identified. Ten species belonging to four genera were identified in this study. The specimens were allocated to 149 pools for the determination of infection with CHIKV and SINV. The amplification pattern of five pools comprising two mosquito species (Culex pipiens complex and Cx. Theileri) corresponded to the reference strain of SINV, and the isolates were sequenced to confirm the presence of SINV genome. No cases of CHIKV infection were found among the 149 examined mosquito pools. Data on the distribution of Cx. Pipiens complex and Cx. Theileri were mapped using ArcMap 10.5. Prediction maps of the presence probability for these species revealed that they are most likely to be found in and spread to the north, northwest, south, and southeastern areas of the country and in areas with abundant water resources.For the first time in Iran, our study investigated the presence probability of SINV vectors using ecological niche modeling. Ecological niche profiling showed that the most suitable habitats for Cx. pipiens are mainly concentrated in the north and northwestern parts of the country, whereas Cx. theileri is mostly located in the northwest and western regions. However, there were some other areas of low suitability for these two species in the country. Further studies in a broader geographical area with more species of mosquitos and the determination of infection with other mosquito-borne viruses can provide a clear understanding of the potential spread of mosquito-borne diseases in various regions of Iran.

Epidemiological and entomological studies of malaria transmission in Tibati, Adamawa region of Cameroon 6\xa0years following the introduction of long-lasting insecticide nets

BackgroundMalaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets.MethodsCross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing.ResultsOverall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis.ConclusionOur study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon.Graphical

Polymerase Chain Reaction and GyrA Nucleotide Sequence Analysis of Wolbachia Endosymbionts (Rickettsiales: Anaplasmataceae) in Various Species of Culicidae, Cimex lectularius (Hemiptera: Cimicidae) and Dirofilaria immitis (Rhabditida: Onchocercidae)

Wolbachia Hertig and Wolbach (Rickettsiales: Anaplasmataceae) are non-culturable, bacterial endosymbionts that have been found in a broad range of arthropods and other invertebrate species. They have been implicated in human and veterinary pathologies, and may play a major role in embryonic development and evolution of host species. Given the apparent ubiquity of Wolbachia in certain animal taxa suggested by previous studies, there are still many unanswered questions about its biology. Like other obligate intracellular bacteria, they are difficult to cultivate outside of their host and often are analyzed using molecular methods. Polymerase chain reaction (PCR) assays have been developed previously for Wolbachia detection within host species, and several genes have been explored for strain typing and phylogenetic reconstruction. However, given the expansive host range and biological complexity of symbiotic relationships between Wolbachia and its host species, new methods could help accelerate the pace of Wolbachia research. As part of an overarching goal to study the distribution of Wolbachia in local mosquitoes and in the heartworm, Dirofilaria immitis (Leidy) (Rhabditida: Onchocercidae), we aimed to develop cost-effective methods that can be used in strain identification and analysis. We developed a novel PCR assay targeting the gyrA gene of Wolbachia and explored various methods of sample preparation. Presumptive Wolbachia were detected in mosquito specimens from several genera, as well as from D. immitis samples obtained from canine necropsy. DNA sequence analysis of the PCR products confirmed the identity of Wolbachia and revealed variability within some regions of the gyrA gene that correspond to host species. Consequently, this gene could be useful for future phylogenetic and population studies.

Autochthonous Transmission of West Nile Virus by a New Vector in Iran, Vector-Host Interaction Modeling and Virulence Gene Determinants.

Using molecular techniques and bioinformatics tools, we studied the vector-host interactions and the molecular epidemiology of West Nile virus (WNV) in western Iran. Mosquitoes were collected during 2017 and 2018. DNA typing assays were used to study vector-host interactions. Mosquitoes were screened by RT-PCR for the genomes of five virus families. WNV-positive samples were fully sequenced and evolutionary tree and molecular architecture were constructed by Geneious software and SWISS-MODEL workspace, respectively. A total of 5028 mosquito specimens were collected and identified. The most prevalent species was Culex (Cx.) pipiens complex (57.3%). Analysis of the blood-feeding preferences of blood-fed mosquitoes revealed six mammalian and one bird species as hosts. One mosquito pool containing non-blood-fed Cx. theileri and one blood-fed Culex pipiens pipiens (Cpp.) biotype pipiens were positive for WNV. A phylogram indicated that the obtained WNV sequences belonged to lineage 2, subclade 2 g. Several amino acid substitutions suspected as virulence markers were observed in the Iranian WNV strains. The three-dimensional structural homology model of the E-protein identified hot spot domains known to facilitate virus invasion and neurotropism. The recent detection of WNV lineage 2 in mosquitoes from several regions of Iran in consecutive years suggests that the virus is established in the country.

Modeling host-feeding preference and molecular systematics of mosquitoes in different ecological niches in Canada

Several mosquito-borne viruses (mobovirus) cause infections in Canada. Ecological data on mosquito species and host range in Canada remains elusive. The main aim of the current study is to determine the host range and molecular systematics of mosquito species in Canada. Mosquitoes were collected using BG-Sentinel traps and aspirators at 10 trapping sites in Canada during 2018 and 2019. Mosquitoes collected were identified via morphology and molecular techniques. Mosquito sequences were aligned by MUSCLE algorithm and evolutionary systematics were drawn using MEGA and SDT software. Moreover, the source of blood meals was identified using a DNA barcoding technique. A total of 5,708 female mosquitoes over 34 different taxa were collected. DNA barcodes and evolutionary tree analysis confirmed the identification of mosquito species in Canada. Of the total collected samples, 201 specimens were blood-fed female mosquitoes in 20 different taxa. Four mosquito species represented about half (51.47%) of all collected blood-fed specimens: Aede cinereus (39 specimens, 19.11%), Aedes triseriatus (23, 11.27%), Culex pipiens (22, 10.78%), and Anopheles punctipennis (21, 10.29%). The most common blood meal sources were humans (49 mosquito specimens, 24% of all blood-fed mosquito specimen), pigs (44, 21.5%), American red squirrels (28, 13.7%), white-tailed deers (28, 13.7%), and American crows (16, 7.8%). Here, we present the first analysis of the host-feeding preference of different mosquito species in Canada via molecular techniques. Our results on mosquito distribution and behavior will aid in the development of effective mitigation and control strategies to prevent or reduce human/animal health issues in regards to moboviruses.

New records of mosquito species (Diptera: Culicidae) in La Comarca Lagunera, Durango, Mexico

Introduction: Many mosquitoes are of great medical and veterinary importance because they transmit numerous pathogens which cause diseases in animals and humans; thus, knowing their taxonomy and distribution is pivotal for implementing the correct control strategies. The aim of this study was to determine the occurrence of mosquito species and their distribution in La Comarca Lagunera in the state of Durango, Mexico.Method: Adult mosquitoes were collected at different resting sites in four municipalities between August and November 2018 using Insectzookas. Aquatic habitats were also sampled for immature stages. Adult specimens were killed using lethal chambers with triethylamine vapors; while larvae and pupae were stored in individual tubes to obtain the adult stages and associated exuviae. All material was transported to the Molecular Biology Laboratory of the Parasitology Department of the Universidad Autónoma Agraria Antonio Narro, Unidad Laguna (UAAAN-UL) for mounting and taxonomical identification. Species were identified using taxonomic keys for the region.Results: In total, 689 mosquito specimens (286 males and 403 females) were collected belonging to 15 species, among them Anopheles franciscanus McCracken, Culex erythrothorax Dyar, and Toxorhynchites moctezuma (Dyar and Knab) are new records for Durango State. The most abundant species was Cx. quinquefasciatus Say with 364 (56.2%) specimens, followed by Aedes aegypti (Linnaeus) (21.3%) and Ae. vexans (Meigen) (7.7%).Conclusion: 13 out of 15 species collected are associated with the transmission of an arbovirus of relevant medical or veterinary importance in Mexico. There are now 38 species recorded for Durango. The information in this study is directly relevant for the Health Ministry in Durango State for the control of vector borne diseases in the region.

Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

First record of West Nile Virus detection inside wild mosquitoes in Khartoum capital of Sudan using PCR.

This study aimed to explore the presence of West Nile Virus (WNV) inside four species of mosquitoes: Culex univittatus (Theobald), Culex quinquefasciatus (Say) Aedes vittatus (Bigot) and Aedes vexans (Meigen). Adult wild mosquitoes were collected from different sites: Soba West, Hellat Kuku, Shambat, and Khartoum North Central Live Stock Market (KCLM). Surveys were carried out at Khartoum State during two phases: pre to the rainy season and post to the rainy season. Mosquito specimens were identified using classical keys then preserved at −80 °C freezer for two weeks till the virus examination using polymerase chain reaction (PCR) were carried out. WNV has been detected inside the three species of mosquitoes: A. vexans, C. univittatus, and C. quinquefasciatus. The species were collected from Hellat Kuku, (Shambat and Hellat Kuku), and (Shambat and KCLM) respectively. Two species of mosquitoes were positive for the virus: C. quinquefasciatus and C. univittatus. Positive results for the virus during the first phase of the study; males of C. quinquefasciatus and C. univittatus collected during the second phase of the study were also tested for the existence of the virus and they were positive. For our knowledge this study represents first record of WNV inside wild mosquitoes in Sudan. PCR technique provided reliable information because specific primer-probe sets were used for the detection of the virus. Extra studies are required to incriminate these species of mosquitoes as potential vectors of WNV.

Study of Abiotic and Biotic Parameters Affecting the Abundance of Mosquito Larvae (Diptera: Culicidae) in the Region of Fez (Morocco)

Mosquitoes cause significant human health issues. However, very few studies have attempted to examine the question of how abiotic and biotic factors affect the abundance of Culicidae in the larval habitat. The objective of this study was to analyze the influence of biotic and abiotic factors on the increase of the most common mosquito species in the Fez region (Central Morocco). Larvae mosquitoes were sampled by standard dipping technique in four different types of macrohabitats, between November 2015 and November 2016. Each mosquito specimen was morphologically identified by the Moroccan Culicidae key and the Brunhes key. The analysis was done using R analysis software. We collected a total of 772 mosquito larvae belonging to nine different species, five of which are considered of medical interest. Culex pipiens (Linnaeus, 1758), known as the major vector in the transmission of West Nile virus fever (WNV), was the most common species of all mosquito larvae collected. The results of Poisson regression analysis showed that factors such as the presence of green filamentous algae, vegetation cover, and debris were found to be positively significant in the distribution of the genus Culex. However, there was insufficient evidence to determine the parameters that are capable of estimating the abundance of Anopheles. The findings have also estimated that biotic and abiotic factors can lead to significant variation in the abundance of Culex perexiguus (Theobald, 1903), Culex theileri (Theobald, 1903), and Culex pipiens (Linnaeus, 1758). Identifying the priority parameters governing the proliferation of mosquitoes in the region of Fez can be one of the key elements for better vector control.

Nine years of mosquito monitoring in Germany, 2011\u20132019, with an updated inventory of German culicid species

Before the background of increasingly frequent outbreaks and cases of mosquito-borne diseases in various European countries, Germany recently realised the necessity of updating decade-old data on the occurrence and spatiotemporal distribution of culicid species. Starting in 2011, a mosquito monitoring programme was therefore launched with adult and immature mosquito stages being collected at numerous sites all over Germany both actively by trapping, netting, aspirating and dipping, and passively by the citizen science project ‘Mueckenatlas’. Until the end of 2019, about 516,000 mosquito specimens were analysed, with 52 (probably 53) species belonging to seven genera found, including several species not reported for decades due to being extremely rare (Aedes refiki, Anopheles algeriensis, Culex martinii) or local (Culiseta alaskaensis, Cs. glaphyroptera, Cs. ochroptera). In addition to 43 (probably 44 including Cs. subochrea) out of 46 species previously described for Germany, nine species were collected that had never been documented before. These consisted of five species recently established (Ae. albopictus, Ae. japonicus, Ae. koreicus, An. petragnani, Cs. longiareolata), three species probably introduced on one single occasion only and not established (Ae. aegypti, Ae. berlandi, Ae. pulcritarsis), and a newly described cryptic species of the Anopheles maculipennis complex (An. daciae) that had probably always been present but not been differentiated from its siblings. Two species formerly listed for Germany could not be documented (Ae. cyprius, Ae. nigrinus), while presence is likely for another species (Cs. subochrea), which could not be demonstrated in the monitoring programme as it can neither morphologically nor genetically be reliably distinguished from a closely related species (Cs. annulata) in the female sex. While Cs. annulata males were collected in the present programme, this was not the case with Cs. subochrea. In summary, although some species regarded endemic could not be found during the last 9 years, the number of culicid species that must be considered firmly established in Germany has increased to 51 (assuming Cs. subochrea and Ae. nigrinus are still present) due to several newly emerged ones but also to one species (Ae. cyprius) that must be considered extinct after almost a century without documentation. Most likely, introduction and establishment of the new species are a consequence of globalisation and climate warming, as three of them are native to Asia (Ae. albopictus, Ae. japonicus, Ae. koreicus) and three (Ae. albopictus, An. petragnani, Cs. longiareolata) are relatively thermophilic. Another thermophilic species, Uranotaenia unguiculata, which had been described for southwestern Germany in 1994 and had since been found only at the very site of its first detection, was recently documented at additional localities in the northeastern part of the country. As several mosquito species found in Germany are serious pests or potential vectors of disease agents and should be kept under permanent observation or even be controlled immediately on emergence, the German mosquito monitoring programme has recently been institutionalised and perpetuated.

Emergence of Zika Virus in Culex tritaeniorhynchus and Anopheles sinensis Mosquitoes in China.

Zika virus (ZIKV) has been isolated from mosquitoes such as Aedes, Mansonia uniformis, and Culex perfuscus; However, the isolation of ZIKV from Anopheles sinensis and Culex tritaeniorhynchus has not yet been reported. In June and July 2018, 22,985 mosquitoes and 57,500 midges were collected in Jiangxi Province in southeastern China. Among them, six strains of ZIKV were isolated from mosquitoes: four from An. sinensis and two from Cx. tritaeniorhynchus. Molecular genetic analysis showed that the ZIKV isolated from An. sinensis and Cx. tritaeniorhynchus belonged to genotype 2 in the Asian evolutionary branch of ZIKV. In addition, the ZIKV strains isolated from An. sinensis and Cx. tritaeniorhynchus had amino acid substitutions identical to ZIKV strains prevalent in South America since 2015. This study is the first to isolate ZIKV from mosquito specimens collected in the wild of Jiangxi Province, China; This is also the first time that ZIKV has been isolated from An. sinensis and Cx. tritaeniorhynchus. Given that An. sinensis and Cx. tritaeniorhynchus have a very wide geographical distribution in China and even in eastern and southern Asia, the isolation of several strains of ZIKV from these two mosquitoes poses new challenges for the prevention and control of ZIKV infection in the mainland of China and countries and regions with the same distribution of mosquitoes.

Bartonella species in medically important mosquitoes, Central Europe.

Here, we provide the first mass molecular screening of medically important mosquitoes for Bartonella species using multiple genetic markers. We examined a total of 72,115 mosquito specimens, morphologically attributed to Aedes vexans (61,050 individuals), Culex pipiens (10,484 individuals) and species of the Anopheles maculipennis complex (581 individuals) for Bartonella spp. The initial screening yielded 63 Bartonella-positive A. vexans mosquitoes (mean prevalence 0.1%), 34 Bartonella-positive C. pipiens mosquitoes (mean prevalence 0.3%) and 158 Bartonella-positive A. maculipennis group mosquitoes (mean prevalence 27.2%). Several different Bartonella ITS sequences were recovered. This study highlights the need for molecular screening of mosquitoes, the most important vectors of arthropod-borne pathogens, for potential bacterial agents.

Nocturnal Mosquitoes of Pará State in the Brazilian Amazon: Species Composition, Habitat Segregation, and Seasonal Variation.

Mosquitoes (Diptera: Culicidae) are one of the most important disease vector species in the world. Many species have a high degree of anthropophilia and are often found in human habitations. In the present study, we have inventoried the nocturnal mosquito assemblage in intra-, peri-, and extradomicile environments in four municipalities in Pará, Brazil. At each municipality, a residence was selected and the mosquitoes were sampled using the protected human attraction capture and Shannon trap methods in April (rainy season) and August 2018 (dry season). We have collected a total of 696 mosquito specimens belonging to 8 genera and 17 species. The most abundant species were Mansonia (Mansonoides) titillans (Walker) (366/696, 52.6%), Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga (97/696, 13.9%), and Culex (Culex) quinquefasciatus Say (93/696, 13.4%). Mosquito richness, abundance, and composition did not differ between intra-, peri-, and extradomicile environments suggesting limited habitat segregation among the different species. However, mosquito species richness and mosquito species abundance were significantly higher during the rainy season than during the dry season, suggesting increased mosquito activity during the rainy season. We detected several important vector species of human diseases including Aedes (Stegomyia) aegypti (Linnaeus), Anopheles (Nyssorhynchus) darlingi Root, Haemagogus (Conopostegus) leucocelaenus (Dyar and Shannon), Coquillettidia (Coquillettidia) venezuelensis (Theobald), and Culex (Culex) quinquefasciatus which are the main transmitters of dengue, malaria, yellow fever, mayaro, and oropouche fever, respectively. As inventories of disease-carrying mosquitoes in the region are very scarce, mainly in residential environments, our results suggest high potential for mosquito-borne disease transmission in Pará State.

Identification of Aedes mosquitoes by MALDI-TOF MS biotyping using protein signatures from larval and pupal exuviae

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) biotyping is an innovative strategy, applied successfully for the identification of numerous arthropod families including mosquitoes. The effective mosquito identification using this emerging tool was demonstrated possible at different steps of their life-cycle, including eggs, immature and adult stages. Unfortunately, for species identification by MS, the euthanasia of the mosquito specimen is required.MethodsTo avoid mosquito euthanasia, the present study assessed whether aedine mosquitoes could be identified by MALDI-TOF MS biotyping, using their respective exuviae. In this way, exuviae from the fourth-instar and pupal stages of Aedes albopictus and Aedes aegypti were submitted to MALDI-TOF MS analysis.ResultsReproducible and specific MS spectra according to aedine species and stage of exuviae were observed which were objectified by cluster analyses, composite correlation index (CCI) tool and principal components analysis (PCA). The query of our reference MS spectra database (DB) upgraded with MS spectra of exuviae from fourth-instar larvae and pupae of both Aedes species revealed that 100% of the samples were correctly classified at the species and stage levels. Among them, 93.8% (135/144) of the MS profiles reached the threshold log score value (LSV > 1.8) for reliable identification.ConclusionsThe extension of reference MS spectra DB to exuviae from fourth-instar and pupal stages made now possible the identification of mosquitoes throughout their life-cycle at aquatic and aerial stages. The exuviae presenting the advantage to avoid specimen euthanasia, allowing to perform complementary analysis on alive mosquitoes.

Detection and characterization of Ilheus and Iguape virus genomes in historical mosquito samples from Southern Brazil

In Brazil, flaviviruses have caused massive outbreaks. Surveillance programs designed to monitor virus activity in vectors provides a system for mapping disease distribution and for identifying specific vector species for targeted control. The present study aimed to describe the detection, whole genome characterization and phylogenetic analysis of Ilheus virus (ILHV) and Iguape virus (IGUV) strains obtained from historical mosquito's samples. Twelve isolates of pooled mosquito specimens (inoculated in neonate mouse brain) collected in the state of São Paulo, Brazil, in 1993, 1994 and 1997 were investigated. Viral RNA was extracted and analyzed by qRT-PCR using Flavivirus genus-specific primers. Positive samples were sequenced and underwent phylogenetic analyses. Flavivirus was detected in 50% of the specimens. Positive samples were successfully Sanger sequenced. Three Anopholes cruzii pools collected in 1994 were positive for IGUV. One Culex sp. pool, one Anopheles triannulatus pool, and one Coquillettidia juxtamansonia pool, collected in 1994, were positive for ILHV. Metagenomic sequencing successfully characterize one ILHV and four IGUV full genomes, and revealed a high degree of homology between the Brazilian ILHV and IGUV strains and isolates available in GenBank. Phylogenetic analysis of partial ILHV NS5 gene revealed three distinct lineages (clades), an indication of genetic heterogeneity in strains circulating in Brazil. Nucleotide insertions and a high-level of nucleotide diversity were observed in the NS1 protein and capsid region of IGUV strains, respectively. Detection of ILHV and IGUV in mosquitoes from Southeastern Brazil confirms the historical circulation of these viruses in this area. Furthermore, this first evidence of ILHV in Anopheles triannulatus suggests the potential importance of Anopheles mosquitoes in the IGUV transmission cycle. Genomic and phylogenetic analysis of these viruses provided insights into their diversity and evolution, which are important for the emergence patterns of flaviviruses and their evolutionary trends in Brazil, an endemic country for several arbovirus. in In-depth studies of ILHV and IGUV including vector competence and molecular studies are needed to shed light on their epidemiology and potential risk of future emergence.

Isolation and identification of Arbovirus in Hainan province, 2017-2018

Objective: To understand the types and distribution of Arboviruses in Hainan province. Methods: Blood-sucking insects were collected in Hainan province from 2017 to 2018. After laboratory treatment, BHK-21 cells and C6/36 cells were inoculated with grinding supernatant of all blood-sucking insects to isolate all of involving virus. Arbovirus genes in blood-sucking insects were detected in parallel by RT-PCR method. Results: A total of 15 062 mosquitoes were classified into four genera (Culex, Armigeres, Aedes, Anopheles) and 11 360 midges were collected. Culex tritaeniorhynchus was in the majority and accounted for 92.88% (13 990/15 062) of all the mosquitoes collected. Four strains of virus isolates were notified by tissue culture method. Three strains of viruses belonged to Japanese encephalitis virus (JEV), with the other one as Getah virus (GETV). Five pools of JEV gene amplification were positive, from Culex tritaeniorhynchus. Results from the phylogenetic analysis showed that they belonged to genotype JEV-Ⅰ. The minimum infection rate of JEV was 0.57‰ (8/13 990). A total of 5 pools of Akabane virus (AKV) gene amplification were positive. The minimum infection rate of AKV was 0.44‰ (5/11 360). Based on the S gene and M gene sequences of the virus, data from the phylogenetic analysis showed that the five AKV strains carried by midges in Hainan province were in a separate evolutionary branch and with formed unique geographical distribution. Conclusions: JEV and GETV had been isolated again from the mosquito specimens in this survey, since the 1980s. AKV was detected from the midge specimens in Hainan province. These results showed the needs of strengthening the programs on detection and monitor of JEV, GETV and AKV that were related to animal and human diseases in order to reduce the risks of related diseases in this area.

The mosquitoes of Armenia: review of knowledge and results of a field survey with first report of Aedes albopictus.

Background: In 2016, a field study was implemented in all Armenian provinces in order to update knowledge on the presence and distribution of both native and invasive mosquito species. Larvae and adult mosquitoes were sampled and identified on the basis of their morphology. Supplementary field surveys were performed in 2017–2018. Results: Between June 20 and July 12, 2016, 117 localities were visited. A total number of 197 sampling units were checked, of which 143 (73%) were positive for mosquitoes (with 1–6 species per sampling unit). A total number of 4157 mosquito specimens were identified to species or species complex level. Ten species represent first records for Armenia: Aedes albopictus, Ae. annulipes, Ae. cataphylla, Ae. cinereus/geminus (probably Ae. cinereus), Ae. flavescens, Anopheles plumbeus, Coquillettidia richiardii, Culex martinii, Cx. torrentium and Culiseta subochrea. The invasive species Ae. albopictus was recorded in a single locality (Bagratashen) at the border point with Georgia, along the main road Tbilisi-Yerevan. This species was further recorded in 2017 and 2018, demonstrating its establishment and spread in north Armenia. These surveys confirm the presence of vectors of malaria parasites (in particular An. sacharovi) and West Nile virus (Cx. pipiens). Conclusion: The knowledge of the Armenian mosquito fauna is extended to a list of 28 species. The record of Aedes albopictus, an important potential vector of many arboviruses, has important implications for public health.

Variety of Anopheles mosquito in Salamwates Village, Dongko Subdistrict Trenggalek District, East Java Province

Malaria is an infectious disease caused by parasites from the genus Plasmodium and transmitted through the bites of Anopheles mosquitoes. This study was an observational analytic research by using adult mosquito specimens. The mosquitoes were caught using indoor net trapping and outdoor cow baited net trapping started at 18.45 to 24.00 for six catching times. Outdoor cow baited net trapping caught 61 mosquitoes from seven different species namely An. aconitus, An. annularis, An. barbirostris, An. indefinitus, An. kochi, and An. vagus. Human bait trap only trapped one species, An. indefinites. The most often caught mosquito was a zoophilic An. barbirostris, while An. indefinitus was the only spesies of anthropozoophilic mosquito and suspected as the malaria vector because they were caught using indoor human bait trap.

The mosquitoes (Diptera: Culicidae) of Nuevo León, Mexico, with descriptions of two new species.

To document the diversity and distribution of mosquitoes inhabiting the Mexican state of Nuevo León, collection trips were conducted to all physiographic regions (Grand Northamerican Plains, Coastal Plain of North Gulf, and Sierra Madre Oriental) and subregions across the state. A total of 3,176 specimens were collected. Additionally, we re-examined mosquito specimens in two Mexican entomological collections: The Collection of Insects and Mites of Medical Importance and the Collection of Arthropods of Medical Importance. These represent the two culicid subfamilies Anophelinae and Culicinae, 8 tribes, 12 genera, 25 subgenera, and 64 named species. Of these, 1 tribe, 2 genera, 5 subgenera, and 14 species are new records for the mosquito fauna of Nuevo León. Three undescribed species were collected. Two are described in this study: Aedes (Ochlerotatus) amateuri Ortega & Zavortink n. sp., and Aedes (Protomacleaya) lewnielseni Ortega & Zavortink n. sp. The third belongs to the genus Wyeomyia. Twelve species previously recorded from Nuevo León were not collected during this study. Taxonomic notes, new distribution limits, and comments about the medical importance of some species are reported.