Culex Pipiens Quinquefasciatus Research Articles

Localization of nitric oxide–producing hemocytes in Aedes and Culex mosquitoes infected with bacteria

Mosquitoes are significant vectors of various pathogens. Unlike vertebrates, insects rely solely on innate immunity. Hemocytes play a crucial role in the cellular part of the innate immune system. The gaseous radical nitric oxide (NO) produced by hemocytes acts against pathogens and also functions as a versatile transmitter in both the immune and nervous systems, utilizing cyclic guanosine monophosphate (cGMP) as a second messenger. This study conducted a parallel comparison of NO synthase (NOS) expression and NO production in hemocytes during Escherichia coli K12 infection in four vector species: Aedes aegypti, Aedes albopictus, Culex pipiens molestus, and Culex pipiens quinquefasciatus. Increased NOS expression by NADPH diaphorase (NADPHd) staining and NO production by immunofluorescence against the by-product L-citrulline were observed in infected mosquito hemocytes distributed throughout the abdomens. NADPHd activity and citrulline labeling were particularly found in periostial hemocytes near the heart, but also on the ventral nerve chord (VNC). Pericardial cells of Ae. aegypti and Cx. p. molestus showed increased citrulline immunofluorescence, suggesting their involvement in the immune response. Oenocytes displayed strong NADPHd and citrulline labeling independent of infection status. This comparative study, consistent with findings in other species, suggests a widespread phenomenon of NO’s role in hemocyte responses during E. coli infection. Found differences within and between genera highlight the importance of species-specific investigations.

Novel strains of Culex flavivirus and Hubei chryso-like virus 1 from the Anopheles mosquito in western Kenya

Surveillance of mosquito vectors is critical for early detection, prevention and control of vector borne diseases. In this study we used advanced molecular tools, such as DNA barcoding in combination with novel sequencing technologies to discover new and already known viruses in genetically identified mosquito species. Mosquitoes were captured using BG sentinel traps in Western Kenya during May and July 2019, and homogenized individually before pooled into groups of ten mosquitoes. The pools and individual samples were then used for molecular analysis and to infect cell cultures. Of a total of fifty-four (54) 10-pools, thirteen (13) showed cytopathic effect (CPE) on VeroB4 cells, eighteen (18) showed CPE on C6/36 cells. Eight (8) 10-pools out of the 31 CPE positive pools showed CPE on both VeroB4 and C6/36 cells. When using reverse transcriptase polymerase chain reaction (RT-PCR), Sanger sequencing and Twist Comprehensive Viral Research Panel (CVRP) (Twist Biosciences), all pools were found negative by RT-PCR when using genus specific primers targeting alphaviruses, orthobunyaviruses and virus specific primers towards o'nyong-nyong virus, chikungunya virus and Sindbis virus (previously reported to circulate in the region). Interestingly, five pools were RT-PCR positive for flavivirus. Two of the RT-PCR positive pools showed CPE on both VeroB4 and C6/36 cells, two pools showed CPE on C6/36 cells alone and one pool on VeroB4 cells only. Fifty individual mosquito homogenates from the five RT-PCR positive 10-pools were analyzed further for flavivirus RNA. Of these, 19 out of the 50 individual mosquito homogenates indicated the presence of flavivirus RNA. Barcoding of the flavivirus positive mosquitoes revealed the mosquito species as Aedes aegypti (1), Mansonia uniformis (6), Anopheles spp (3), Culex pipiens (5), Culex spp (1), Coquilletidia metallica (2) and Culex quinquefasciatus (1). Of the 19 flavivirus positive individual mosquitoes, five (5) virus positive homogenates were sequenced. Genome sequences of two viruses were completed. One was identified as the single-stranded RNA Culex flavivirus and the other as the double-stranded RNA Hubei chryso-like virus 1. Both viruses were found in the same Anopheles spp. homogenate extracted from a sample that showed CPE on both VeroB4 and C6/36 cells. The detection of both viruses in a single mosquito homogenate indicated coinfection. Phylogenetic analyses suggested that the Culex flavivirus sequence detected was closely related to a Culex flavivirus isolated from Uganda in 2008. All four Hubei chryso-like virus 1 segments clusters closely to Hubei chryso-like virus 1 strains isolated in Australia, China and USA. Two novel strains of insect-specific viruses in Anopheles mosquitoes were detected and characterized.

Talaromyces sp. Ethyl Acetate Crude Extract as Potential Mosquitocide to Control Culex pipiens quinquefasciatus.

Vector control is considered an effective approach to controlling diseases spread by mosquito bites. Entomopathogenic fungi are widely used in agriculture to control insect pests, and fungal metabolites can potentially be developed as effective mosquitocides. In this study, a high-throughput screening method was used to search for potential mosquitocides in the Global Fungal Extract Library (GFEL). We tested the larvicidal activity of 264 fungal ethyl acetate crude extracts against Culex pipiens quinquefasciatus. Nine fungal extracts caused moderate to high mortality rates (>50%), with two fungal extracts (58A7 and 101H12) causing a 100% mortality rate. The lethal concentrations for 50% of the population (LC50) were 44.27 mg/L and 31.90 mg/L, respectively. Fraction 14 had a high mortality rate, with an LC50 value of 12.13 mg/L, and was isolated from 58A7 (Fractions 1-11) and 101H12 (Fractions 12-15). Further analyses showed that Fraction 14 was made up of vermistatin and dihydrovermistatin. In a Cx. p. quinquefasciatus larvicidal bioassay, vermistatin (LC50 = 28.13 mg/L) was more toxic than dihydrovermistatin (LC50 = 83.87 mg/L). Our findings suggested that the active fungal extract 101H12 from Talaromyces sp. and its compound vermistatin could be developed as mosquitocides.

Trans-Generational Symbiont Transmission Reduced at High Temperatures in a West Nile Virus Vector Mosquito Culex quinquefasciatus

The influence of environmental factors on the efficacy of the endosymbiont Wolbachia used in mosquito and pathogen control are poorly characterized and may be critical for disease control. We studied the vector mosquito Culex pipiens quinquefasciatus (Say) to determine the effect of temperature on the composition of the relative abundance of Wolbachia spp. and the microbiome, as well as key immune genes of interest in the Toll and IMD pathways. 16S barcode sequencing was used to determine the microbiome composition and qPCR was used to determine the relative abundance of Wolbachia spp. based on the highly utilized marker Wolbachia surface protein (wsp) gene. We found no effect of temperature within a single generation on the relative abundance of Wolbachia or immune gene expression, nor on the alpha or beta diversity of the microbiome. However, there was a significant difference in the abundance of Wolbachia between generations at high temperatures (≥ 28°C), but not at lower temperatures (≤ 23°C). These results support the idea that Wolbachia are reduced at higher temperatures between generations, which has an influence on the establishment of pathogens including West Nile Virus (WNV). Modulation of the Toll or IMD mosquito immune pathways was not indicated. Wolbachia endosymbiosis and trans-generation transmission appears especially sensitive to high temperatures, which may have implications for Wolbachia-based vector control strategies under climate change scenarios.

Mapping the Distributions of Mosquitoes and Mosquito-Borne Arboviruses in China.

The geographic expansion of mosquitos is associated with a rising frequency of outbreaks of mosquito-borne diseases (MBD) worldwide. We collected occurrence locations and times of mosquito species, mosquito-borne arboviruses, and MBDs in the mainland of China in 1954−2020. We mapped the spatial distributions of mosquitoes and arboviruses at the county level, and we used machine learning algorithms to assess contributions of ecoclimatic, socioenvironmental, and biological factors to the spatial distributions of 26 predominant mosquito species and two MBDs associated with high disease burden. Altogether, 339 mosquito species and 35 arboviruses were mapped at the county level. Culex tritaeniorhynchus is found to harbor the highest variety of arboviruses (19 species), followed by Anopheles sinensis (11) and Culex pipiens quinquefasciatus (9). Temperature seasonality, annual precipitation, and mammalian richness were the three most important contributors to the spatial distributions of most of the 26 predominant mosquito species. The model-predicted suitable habitats are 60–664% larger in size than what have been observed, indicating the possibility of severe under-detection. The spatial distribution of major mosquito species in China is likely to be under-estimated by current field observations. More active surveillance is needed to investigate the mosquito species in specific areas where investigation is missing but model-predicted probability is high.

Screening of olfactory genes related to blood-feeding behaviors in Culex pipiens quinquefasciatus and Culex pipiens molestus by transcriptome analysis.

BackgroundCulex pipiens quinquefasciatus Say (Cx. quinquefasciatus) and Culex pipiens form molestus Forskal (Cx. molestus) in the Culex pipiens complex group show considerable differences in host seeking, blood feeding, mating behavior and in vector competence. Blood-feeding mosquito behaviors are closely related to their olfactory gene expression and olfactory gene repertoire composition. Comparing olfactory genes between these two subspecies with significantly different blood-feeding behaviors can support further research on the molecular mechanism of the Culex pipiens complex olfactory sensory system, providing a new approach for determining candidate attractant or repellent compounds.MethodsNon-blood-feeding (NBF) and post-blood-feeding (PBF) olfactory system transcriptomes of the two subspecies were sequenced, and the biological functions of their differentially expressed genes were described by bioinformatics analysis. A quantitative polymerase chain reaction (qPCR) was applied to validate the RNA-seq data. The roles of particular olfactory receptors in Cx. quinquefasciatus blood-feeding behaviors were evaluated by RNAi.ResultsFive, 7, 24, and 3 Cx. quinquefasciatus-specific OBPs, Cx. molestus-specific OBPs, Cx. quinquefasciatus-specific ORs and Cx. molestus-specific ORs were identified, respectively. The majority of selected ORs were consistent with the predicted transcriptome sequencing results after qRT-PCR validation. OR5 was expressed only in Cx. quinquefasciatus, and OR65 was the only gene upregulated after blood feeding in Cx. molestus. The blood-feeding rates of the OR5 and OR78 dsRNA groups were significantly lower (4.3%±3.1% and 13.3%±11.5%) than those of the enhanced green fluorescence protein (EGFP) group (64.5%±8.7%).ConclusionMost OBPs and ORs were expressed in both subspecies but showed divergence in expression level. OR5 and OR65 might be species-specific expressed genes that regulate the olfactory behaviors of Cx. quinquefasciatus and Cx. molestus, respectively. The RNA interference of OR5 and OR78 could inhibit the blood-feeding behavior of Cx. quinquefasciatus, providing new targets for screening effective repellent compounds to control mosquito-borne diseases effectively and efficiently.

Random Forest Analysis of Impact of Abiotic Factors on Culex pipiens and Culex quinquefasciatus Occurrence

The Culex pipiens complex of mosquitoes are significant vectors of several pathogens resulting in infectious human diseases in North America, including but not limited to West Nile encephalitis, Rift Valley Fever, and Lymphatic filariasis. Among this complex are C. pipiens form pipiens and Culex quinquefasciatus. While morphologically similar, the mosquitoes exhibit unique life histories that suit them uniquely to divergent niches, wherein C. pipiens can thrive despite the cold winters of the northern United States and C. quinquefasciatus is able to survive periods of drought typical in the southern states. Here, Random Forests machine-learning algorithms were employed to model and explore which environmental parameters best explain mosquito occurrence in historical trapping data across the continental United States of America, and test correlation with abundance data. The models explained between 71 and 97% of the presence or absence of the two mosquitoes based on historical climatic data. The results of this study will improve vector management programs by explaining which environmental variables will provide the most accurate predictions of mosquito presence at a given site.

A Conserved Female-Specific Requirement for the GGT Gene in Mosquito Larvae Facilitates RNAi-Mediated Sex Separation in Multiple Species of Disease Vector Mosquitoes.

Although several emerging mosquito control technologies are dependent on mass releases of adult males, methods of sex-sorting that can be implemented globally have not yet been established. RNAi screens led to the discovery of siRNA, which targets gamma-glutamyl transpeptidase (GGT), a gene which is well conserved in multiple species of mosquitoes and located at the sex-determining M locus region in Aedes aegypti. Silencing the A. aegypti, Aedes albopictus, Anopheles gambiae, Culex pipiens, and Culex quinquefasciatus GGT genes resulted in female larval death, with no significant impact on male survival. Generation of yeast strains that permitted affordable expression and oral delivery of shRNA corresponding to mosquito GGT genes facilitated larval target gene silencing and generated significantly increased 5 males:1 female adult ratios in each species. Yeast targeting a conserved sequence in Culex GGT genes was incorporated into a larval mass-rearing diet, permitting the generation of fit adult male C. pipiens and C. quinquefasciatus, two species for which labor-intensive manual sex separation had previously been utilized. The results of this study indicate that female-specific yeast-based RNAi larvicides may facilitate global implementation of population-based control strategies that require releases of sterile or genetically modified adult males, and that yeast RNAi strategies can be utilized in various species of mosquitoes that have progressed to different stages of sex chromosome evolution.

Trypanosomes of the Trypanosoma theileri Group: Phylogeny and New Potential Vectors

Trypanosomes belonging to Trypanosoma theileri group are mammalian blood parasites with keds and horse fly vectors. Our aim is to study to vector specificity of T. theileri trypanosomes. During our bloodsucking Diptera survey, we found a surprisingly high prevalence of T. theileri trypanosomes in mosquitoes (154/4051). Using PCR and gut dissections, we detected trypanosomes of T. theileri group mainly in Aedes mosquitoes, with the highest prevalence in Ae. excrucians (22%), Ae. punctor (21%), and Ae. cantans/annulipes (10%). Moreover, T. theileri group were found in keds and blackflies, which were reported as potential vectors for the first time. The vectorial capacity was confirmed by experimental infections of Ae. aegypti using our isolates from mosquitoes; sand fly Phlebotomus perniciosus supported the development of trypanosomes as well. Infection rates were high in both vectors (47–91% in mosquitoes, 65% in sandflies). Furthermore, metacyclic stages of T. theileri trypanosomes were observed in the gut of infected vectors; these putative infectious forms were found in the urine of Ae. aegypti after a second bloodmeal. On the contrary, Culex pipiens quinquefasciatus was refractory to experimental infections. According to a phylogenetic analysis of the 18S rRNA gene, our trypanosomes belong into three lineages, TthI, ThII, and a lineage referred to as here a putative lineage TthIII. The TthI lineage is transmitted by Brachycera, while TthII and ThIII include trypanosomes from Nematocera. In conclusion, we show that T. theileri trypanosomes have a wide range of potential dipteran vectors, and mosquitoes and, possibly, sandflies serve as important vectors.

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Molecular detection of zoonotic filarioids in Culex spp. from Portugal.

To investigate the role of dipterans in the transmission of Onchocerca lupi and other zoonotic filarioids, samples were collected from different sites in Algarve, southern Portugal, morphologically identified and molecularly tested for filarioids. Culex sp. (72.8%) represented the predominant genus followed by Culicoides sp. (11.8%), Ochlerotatus sp. (9.7%), Culiseta sp. (4.5%), Aedes sp. (0.9%) and Anopheles sp. (0.3%). Nineteen (2.8%) specimens scored positive for filarioids, with Culex pipiens quinquefasciatus (2%) positive for Dirofilaria immitis (1.4%), Dirofilaria repens, Acanthocheilonema reconditum, Onchocerca lupi, unidentified species of Filarioidea (0.2%, each) and Onchocercidae (0.6%). Additionally, Culiseta longiareolata (6.5%), Ochlerotatus caspius (3%) and Culex laticinctus (0.2%) scored positive for unidentified Onchocercidae, A. reconditum and for O. lupi, respectively. This is the first report of the occurrence of DNA of O. lupi, D. repens and A. reconditum in Culex spp. in Portugal. Information regarding the vectors and the pathogens they transmit may help to adopt proper prophylactic and control measures.

Dose dependence of susceptibility and transmission for an Argentinean West Nile virus strain in local Culex pipiens quinquefasciatus (Diptera: Culicidae).

West Nile virus (WNV) activity has been documented in the central region of Argentina since 2005, but its maintenance network has not been elucidated yet. We evaluated the susceptibility to WNV oral infection and transmission in a Culex pipiens quinquefasciatus mosquito population by means of a dose-dependent vector competence assay. Mosquitoes were orally infected with five different viral loads and evaluated for viral infection, dissemination and transmission. The evaluated population was susceptible to WNV oral infection. Disseminated infections were detected in all the viral doses but transmission was only detected in the higher doses (6 and 7 log10 plaque-forming units/ml). A linear relationship between doses and transmission rates could be established. A minimum transmission threshold suggests that the Cu. p. quinquefasciatus from Argentina could acquire WNV infection from local urban birds and transmit the virus.

Investigation of bovine ephemeral fever virus transmission by putative dipteran vectors under experimental conditions

BackgroundBovine ephemeral fever virus (Rhabdoviridae: Ephemerovirus) (BEFV) causes bovine ephemeral fever (BEF), an economically important disease of cattle and water buffalo. Outbreaks of BEF in Africa, Australia, Asia and the Middle East are characterized by high rates of morbidity and highly efficient transmission between cattle hosts. Despite this, the vectors of BEFV remain poorly defined.MethodsColony lines of biting midges (Culicoides sonorensis) and mosquitoes (Aedes aegypti, Culex pipiens and Culex quinquefasciatus) were infected with a strain of BEFV originating from Israel by feeding on blood–virus suspensions and by intrathoracic inoculation. In addition, in vivo transmission of BEFV was also assessed by allowing C. sonorensis inoculated by the intrathoracic route to feed on male 6 month-old Holstein-Friesian calves.ResultsThere was no evidence of BEFV replication within mosquitoes fed on blood/virus suspensions for mosquitoes of any species tested for each of the three colony lines. In 170 C. sonorensis fed on the blood/virus suspension, BEFV RNA was detected in the bodies of 13 individuals and in the heads of two individuals, indicative of fully disseminated infections and an oral susceptibility rate of 1.2%. BEFV RNA replication was further demonstrated in all C. sonorensis that were inoculated by the intrathoracic route with virus after 5, 6 or 7 days post-infection. Despite this, transmission of BEFV could not be demonstrated when infected C. sonorensis were allowed to feed on calves.ConclusionsNo evidence for infection or dissemination of BEFV (bovine/Israel/2005-6) in mosquitoes of three different species was found. Evidence was found for infection of C. sonorensis by the oral route. However, attempts to transmit BEFV to calves from infected C. sonorensis failed. These results highlight the challenge of defining the natural vector of BEFV and of establishing an in vivo transmission model. The results are discussed with reference to the translation of laboratory-based studies to inference of vector competence in the field.Graphical abstract

Mosquito Diversity and Population Genetic Structure of Six Mosquito Species From Hainan Island.

Hainan is a tropical island in southern China with abundant mosquito species, putting Hainan at risk of mosquito-borne virus disease outbreaks. The population genetic diversity of most mosquito species on Hainan Island remains elusive. In this study, we report the diversity of mosquito species and the genetic diversity of the predominant species on Hainan. Field populations of adults or larvae were collected from 12 regions of Hainan Island in 2018 and 2019. A fragment of the mitochondrial cytochrome c oxidase subunit I (coxI) gene was sequenced from 1,228 mosquito samples and used for species identification and genetic diversity analysis. Twenty-three known mosquito species from the genera Aedes, Armigeres, Culex, Mansonia, and Anopheles and nine unconfirmed mosquito species were identified. Aedes albopictus, Armigeres subalbatus, and Culex pipiens quinquefasciatus were the most prevalent mosquito species on Hainan. The regions north of Danzhou, Tunchang, and Qionghai exhibited high mosquito diversity (26 species). The order of the total haplotype diversity and nucleotide diversity of the populations from high to low was as follows: Culex tritaeniorhynchus, Ar. subalbatus, Culex pallidothorax, Culex gelidus, Ae. albopictus, and C. p. quinquefasciatus. Tajima’s D and Fu’s Fs tests showed that Ae. albopictus, C. p. quinquefasciatus, C. tritaeniorhynchus, and C. gelidus had experienced population expansion, while the Ar. subalbatus and C. pallidothorax populations were in genetic equilibrium. Significant genetic differentiation existed in the overall populations of Ae. albopictus, Ar. subalbatus, C. p. quinquefasciatus, and C. pallidothorax. The Ae. albopictus populations on Hainan were characterized by frequent gene exchange with populations from Guangdong and four other tropical countries, raising the risk of viral disease outbreaks in these regions. Two subgroups were reported in the Ar. subalbatus populations for the first time. Our findings may have important implications for vector control on Hainan Island.

Culex interfor and Culex saltanensis (Diptera: Culicidae) are susceptible and competent to transmit St. Louis encephalitis virus (Flavivirus: Flaviviridae) in central Argentina.

St. Louis encephalitis virus (SLEV) is endemic and autochthonous on the American continent. Culex pipiens quinquefasciatus is a vector of SLEV; however, Culex interfor and Culex saltanensis have also been found to be naturally infected with SLEV. The aim of this study was to determine the vector competence of C. interfor and C. saltanensis for SLEV from Argentina compared with C. p. quinquefasciatus. Female of the Culex species were orally infected by feeding on viraemic chicks that had been inoculated with SLEV. Abdomens, legs and saliva blood-fed mosquitoes were analysed by viral plaque assay. Mosquitoes were susceptible to orally acquired infection, dissemination and transmission of SLEV in the saliva. Our results demonstrate that C. saltanensis and C. interfor are susceptible to SLEV and competent for its transmission.

Potential mosquito (Diptera: Culicidae) vectors of Dirofilaria immitis from residential entryways in Northeast Arkansas

The nematode Dirofilaria immitis causes serious disease of canines in the United States. Transmitted by a variety of mosquito species, several studies have examined the prevalence of D. immitis in mosquitoes. However, no prevalence studies have focused on diurnal mosquitoes that could potentially enter homes. Mosquitoes were collected under doorway awnings at four residences to identify the diurnal blood-feeding mosquito species and prevalence of potential vectors harboring D. immitis in Jonesboro, Arkansas. Mosquitoes were collected from May to December 2016 using pheromone traps and aerial nets. Each mosquito was individually dissected and observed for larvae in the head, thorax, and abdomen to identify infective and non-infective larvae. Ten mosquito species from five genera were identified. Larvae, presumed to be D. immitis, were observed in five mosquito species, including Aedes albopictus, Anopheles quadrimaculatus, Culex pipiens quinquefasciatus, Culiseta inornata, and Psorophora columbiae and four genera, including Anopheles spp., Culex spp., Culiseta spp., and Psorophora spp. The diurnal mosquito, Ae. albopictus composed 3.4% of the total mosquitoes collected. An. quadrimaculatus and Anopheles spp. were the most abundant mosquitoes, composing 59.7 and 19.6% of the total mosquitoes collected, respectively. Of the 536 mosquitoes dissected, 60 (11.2%) were observed with D. immitis, and 33 (55.0%) of the heartworm-positive mosquitoes harbored infective (L3) larvae in the head or proboscis upon dissection. The high proportion of infective D. immitis larvae found in the mosquitoes aggregating under doorway awnings may increase the exposure of indoor dogs and cats to heartworm disease in northeast Arkansas.

Potential Vector Competence of Mosquitoes to Transmit Baiyangdian Virus, a New Tembusu-Related Virus in China.

A new duck Tembusu-related flavivirus, Baiyangdian virus (BYDV), caused duck egg-drop syndrome in China. The rapid spread, unknown transmission routes, and zoonotic nature, raise serious concern about BYDV as a potential threat to human health. The study provides the first evaluation on the vector competence of Culex and Aedes mosquitoes to transmit BYDV in China. The results show that Culex tritaeniorhynchus, Culex pipiens pallens, Culex pipiens quinquefasciatus, and Aedes albopictus can become infected with BYD-1 virus (BYDV-1) on different days after oral infection. Although the viral copies in Ae. albopictus was higher than that in Cx. p. quinquefasciatus at 13 days postinfection (χ2 = 10.385, p = 0.016), there was no significant differences between infection rates of four mosquito species (χ2 = 3.98, p = 0.137). In transmission experiment, healthy ducks were infected after being bitten by virus-positive mosquitoes and BYDV-1 disseminated to and replicated in the duck brains. These findings verified the potential role of Cx. p. quinquefasciatus and Cx. tritaeniorhynchus as vectors of BYDV-1. BYDV-1 was also detected in salivary gland of Cx. p. pallens, which indicated that this virus could be transmitted by mosquitoes. These results provide evidence for the role of Culex mosquitoes in the transmission cycles involving BYDV-1 and avian hosts in China.

Repellent, Insecticidal and Antimicrobial Activities of Leaf Essential Oils from Three Eucalyptus Species.

Developing effective and eco-friendly antimicrobials and pesticides has become a highly important issue. The repellent, insecticidal and antimicrobial activity of essential oils (EOs) isolated by hydrodistillation from dried leaves of the three Eucalyptus species (E. cloeziana, E. umbellata and E. benthamii) were investigated. During GC/MS analysis, α-pinene (47.36 %), 1,8-cineol (38.53 %) and α-pinene (35.31 %) were identified as major components of E. cloeziana, E. umbellata and E. benthamii, respectively. The EOs from E. cloeziana exhibited the longest effective protection time (465 min, at 50.0 % w/w) for humans among the EOs studied. The effective protection time was 30 min and 300 min at concentrations of 12.5 % (w/w) and 25.0 % (w/w), respectively. Fumigating insecticidal activity of EOs from three Eucalyptus species was tested by airtight fumigation in conical flask, which indicated that essential oils had a highly and rapidly insecticidal activity on Culex pipiens quinquefasciatus. The antimicrobial activity of EOs was evaluated by using disc diffusion and agar dilution methods. There was no significant difference in the antibacterial activity of EOs from E. cloeziana and E. umbellate and they had the same MICs (20 mL/L) on Staphylococcus aureus, Salmonella typhi, Bacillus subtilis and Escherichia coli. E. benthamii had the worst microbial inhibitory effect among the three Eucalyptus essential oils and the MIC value for the test species is 40 mL/L except for Rhodotorula Harrison (10 mL/L).

Ecology and Contribution of Anopheles hancocki Edwards, 1929 in Malaria Transmission in a Village of South Cameroon Forest Block

Anopheles hancocki is one of the secondary vectors of malaria whose larval ecology in the South Cameroonian forest block is still unknown. This information is however fundamental for developing efficient and sustainable control strategies against this mosquito in localities where it acts as a disease vector. The present study describes the larval ecology of A. hancocki and its contribution in malaria transmission in a riverbank village of the Ayos Health District. Mosquito sampling took place in 2018 on a quarterly rate, combining the dipping method for larval collection with adult mosquito capture on volunteers. For each of the breeding sites, physicochemical characteristics were measured and larvae were collected and reared to adult. Molecular alongside morpho-taxonomic techniques were used for the identification of mosquito species. Physiological age was determined based on the appearance of their ovarian tracheoles and CSP Elisa test was used to assess infectivity. In total, 3,618 adult mosquitoes belonging to seven species were collected in the study area, namely A. hancocki, Anopheles gambiae, Anopheles moucheti, Mansonia africana, Culex pipiens quinquefasciatus, Aedes albopictus and Aedes aegypti. Breeding sites of A. hancocki were similar to those of A. gambiae. However, the total dissolved solids and conductivity values were significantly higher in A. gambiae breeding sites than in those of A. hancocki. A. hancocki was the most aggressive mosquito species and represented 45.6% of the local aggressive culicidofauna. Male species' nocturnal cycle of aggression showed maximum activity between 8 pm and 10 pm. Females of this species were significantly older than those of A. gambiae and contributed to 40% of malaria transmission in the locality with an average annual Entomological Inoculation Rate (EIR) of 2.92 ib/p/year lower than that of A. gambiae (3.65 ib/p/year). Except few differences, ecological requirements for the development of A. hancocki larvae are similar to that of A. gambiae. A. hancocki contributes to the perennial transmission of malaria in the Ayos area; implementation of vector control strategies is therefore needed.

Molecular identification and phylogenetic analysis of acetylcholinesterase-2 gene from <i>Culex pipiens</i> and <i>Culex quinquefasciatus</i> (Diptera: Culicine) in Nigeria

West Nile virus (WNV) is maintained in an enzootic cycle between ornithophilic mosquitoes and birds. There is paucity of information on population density of these vectors and the molecular details in Nigeria. We determined relative abundance and employed molecular methods to detect Culex species. Mosquitoes were caught from April 2013 to February 2014. Genomic DNA extraction was carried out and acetylcholinesterase 2 gene amplified by Polymerase Chain Reaction in different Culex species. Culex pipiens quinquefasciatus and Cx. p. pipiens were identified in different locations, while some other Culex species could not be identified. Culex p. quinquefasciatus were sequenced by Sanger's method. Vector density was 100 Culex species per man hour. Identification of Culex pipiens and Cx. quinquefasciatus from different locations in Ibadan, Nigeria reaffirms the presence of vectors and highlights possibility of enzootic transmission of WNV. Surveillance should be sustained in equine population and bird reservoirs.