Chromosome-level assembly of Culex pipiens molestus and improved reference genome of Culex pipiens pallens (Culicidae,Diptera)

Culex pipiens molestus and Culex pipiens pallens are two distinct bioforms in the Culex pipiens complex that are important vectors of several pathogens and are widely distributed around the world. In the current study, we present a high-quality chromosome-level genome of Cx. pipiens f. molestus and describe the genetic characteristics of this genome. The assembly genome was 559.749 Mb with contig and scaffold N50 values of 200.952 Mb and 0.370 Mb, and more than 94.78% of the assembled bases were located on 3 chromosomes. A total of 19,399 protein-coding genes were predicted. Many gene families were expanded in the genome of Cx. pipiens f. molestus, particularly those of the chemosensory protein (CSP) and gustatory receptor (GR) gene families. In addition, utilizing Hi-C data, we improved the previously assembled draft genome of Cx. pipiens f. pallens, with scaffold N50 of 186.195 Mb and contig N50 of 0.749 Mb, and more than 97.02% of the assembled bases were located on three chromosomes. This reference genome provides a foundation for genome-based investigations of the unique ecological and evolutionary characteristics of Cx. pipiens f. molestus, and the findings in this study will help to elucidate the mechanisms involved in species divergence in the Culex pipiens complex.     

An Odorant Receptor from the Southern House Mosquito Culex pipiens quinquefasciatus Sensitive to Oviposition Attractants

Background

Insect odorant receptors (ORs) are heteromers comprised of highly variable odorant-binding subunits associated with one conserved co-receptor. They are potential molecular targets for the development of novel mosquito attractants and repellents. ORs have been identified in the malaria mosquito, Anopheles gambiae, and in the yellow fever mosquito, Aedes aegypti. However, they are still unknown in the Southern house mosquito, Culex quinquefasciatus, which transmits pathogens that cause human diseases throughout the world, including West Nile Virus in the United States.

Methodology

We have employed a combination of bioinformatics, molecular cloning and electrophysiology approaches to identify and characterize the response profile of an OR in Cx. quinquefasciatus. First, we have unveiled a large multigenic family of one-hundred-fifty-eight putative ORs in this species, including a subgroup of conserved ORs in three mosquito species. Using the Xenopus oocytes expression system, we have determined the response profile of CquiOR2, an antennae-specific OR, which shares high identity with putative orthologs in Anopheles gambiae (AgamOR2) and Aedes aegypti (AaegOR2).

Conclusion

We show that CquiOR2 is highly sensitive to indole, an oviposition attractant for Cx. quinquefasciatus. The response profile of CquiOR2 expressed in Xenopus oocytes resembles that of an olfactory receptor neuron housed in the antennal short blunt-tipped sensilla (A2) of Cx. quinquefasciatus, which are natural detectors for oviposition attractants. This first Culex OR de-orphanized is, therefore, a potential molecular target for screening oviposition attractants.     

Behavioural response of the house mosquitoes Culex quinquefasciatus and Culex pipiens molestus to avian odours and its reliance on carbon dioxide

How Culex (Diptera: Culicidae) mosquitoes select and discriminate between potential avian hosts is critical for understanding the epidemiology of West Nile virus. Therefore, the present authors studied the behavioural responses of Culex quinquefasciatus (Say) and Culex pipiens molestus (Forsskål) to headspace volatiles of three avian species [chicken and pigeon (sexes analysed separately), and magpie], presented either alone or in combination with 600 p.p.m. carbon dioxide (CO₂). The attraction of Cx. quinquefasciatus to the headspace volatiles of both sexes of chicken, and of female pigeon, in combination with CO₂ was significantly higher than that achieved by the CO₂ and solvent control. Although Cx. p. molestus was attracted to headspace volatiles of chickens and magpies, it was repelled by those of female pigeons when combined with CO₂. An increased effect between the avian volatiles and CO₂ was observed for Cx. quinquefasciatus, whereas the addition of CO₂ had no effect on the attraction of Cx. p. molestus females. The results of this study demonstrate that Cx. quinquefasciatus and Cx. p. molestus are attracted to the odour of potential avian hosts. Future studies aimed at identifying the bioactive volatile compounds in the headspace of chickens may contribute to the potential development of effective surveillance and control tools against Culex species.     

Ecological Distribution and CQ11 Genetic Structure of Culex pipiens Complex (Diptera: Culicidae) in Italy

Mosquitoes in the Culex pipiens complex are considered to be involved in the transmission of a range of pathogens, including West Nile virus (WNV). Although its taxonomic status is still debated, the complex includes species, both globally distributed or with a more limited distribution, morphologically similar and characterised by different physiological and behavioural traits, which affect their ability as vectors. In many European countries, Cx. pipiens and its sibling species Culex torrentium occur in sympatry, exhibiting similar bionomic and morphological characters, but only Cx. pipiens appears to play a vector role in WNV transmission. This species consists of two biotypes, pipiens and molestus, which can interbreed when in sympatry, and their hybrids can act as WNV-bridge vectors, due to intermediate ecological features. Considering the yearly WNV outbreaks since 2008 and given the morphological difficulties in recognising species and biotypes, our aim was to molecularly identify and characterised Cx. pipiens and Cx. torrentium in Italy, using recently developed molecular assays. Culex torrentium was not detected; as in other European countries, the pipiens and molestus biotypes were widely found in sympatry with hybrids in most environments. The UPGMA cluster analysis applied to CQ11 genotypic frequencies mainly revealed two groups of Cx. pipiens populations that differed in ecological features. The high propensity of the molestus biotype to exist in hypogean environments, where the habitat’s physical characteristics hinder and preclude the gene flow, was shown. These results confirmed the CQ11 assay as a reliable diagnostic method, consistent with the ecological and physiological aspects of the populations analysed. Since the assessment of the actual role of three biotypes in the WNV circulation remains a crucial point to be elucidated, this extensive molecular screening of Cx. pipiens populations can provide new insights into the ecology of the species and may give useful indications to plan and implement WNV surveillance activities in Italy.     

我国尖音库蚊复合组蚊虫的杂交及其与Wolbachia感染的关系

为了了解我国尖音库蚊复合组蚊虫间杂交卵的不孵化现象和明确该现象与共生微生物Wolbachia感染的关系,对该复合组实验室种群4个亚种进行了笼内杂交和抗生素处理后的杂交。试验表明: 在复合组蚊虫中骚扰库蚊Culex pipiens molestus与淡色库蚊Cx. Pipiens pallens、致倦库蚊Cx. Ipiens quinquefasciatus与尖音库蚊Cx. Pipiens pipiens之间存在有单向胞质不融合现象,骚扰库蚊的雄虫与尖音库蚊、致倦库蚊和淡色库蚊的雌虫杂交卵的孵化率分别为0.06%、0.46%和0.19%;该胞质不融合现象可以通过抗生素处理而消除,处理后骚扰库蚊雄虫与其余3个亚种雌虫F₃杂交卵的孵化率均有提高,分别为89.49%（t=3.90×10^-28＜t_0.01=2.704）、23.39%（t=9.15×10^-7＜t_0.01=2.660_）和22.27%（t=5.08×10^-4＜t_0.01=2.750）,并可因抗生素处理而产生新的不融合类型。     

Development and evaluation of an efficient and real-time monitoring system for the vector mosquitoes,Aedes albopictus and Culex quinquefasciatus

BackgroundThe surveillance of vector mosquitoes is essential for prevention and control of mosquito-borne diseases. In this study, we developed an internet-based vector mosquito monitor, MS-300, and evaluated its efficiency for the capture of the important vector mosquitoes, Aedes albopictus and Culex quinquefasciatus, in laboratory and field trials.Methodology/Principal findingsThe linear sizes of adult Ae. albopictus and Cx. quinquefasciatus were measured and an infrared window was designed based on these data. A device to specifically attract these two species and automatically transmit the number of captured mosquitoes to the internet was developed. The efficiency of the device in capturing the two species was tested in laboratory, semi-field and open field trials. The efficiency results for MS-300 for catching and identifying Ae. albopictus in laboratory mosquito-net cages were 98.5% and 99.3%, and 95.8% and 98.6%, respectively, for Cx. quinquefasciatus. In a wire-gauze screened house in semi-field trials, the efficiencies of MS-300 baited with a lure in catching Ae. albopictus and Cx. quinquefasciatus were 54.2% and 51.3%, respectively, which were significantly higher than 4% and 4.2% without the lure. The real-time monitoring data revealed two daily activity peaks for Ae. albopictus (8:00–10:00 and 17:00–19:00), and one peak for Cx. quinquefasciatus (20:00–24:00). During a 98-day surveillance trial in the field, totals of 1,118 Ae. albopictus and 2,302 Cx. quinquefasciatus were captured by MS-300. There is a close correlation between the number of captured mosquitoes and the temperature in the field, and a positive correlation in the species composition of the captured samples among the mosquitoes using MS-300, BioGents Sentinel traps and human landing catches.Conclusions/SignificanceThe data support the conclusion that MS-300 can specifically and efficiently capture Ae. albopictus and Cx. quinquefasciatus, and monitor their density automatically in real-time. Therefore, MS-300 has potential for use as a surveillance tool for prevention and control of vector mosquitoes.     

Vertebrate-Aedes aegypti and Culex quinquefasciatus (Diptera)-arbovirus transmission networks: Non-human feeding revealed by meta-barcoding and next-generation sequencing

BackgroundAedes aegypti mosquito-borne viruses including Zika (ZIKV), dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) have emerged and re-emerged globally, resulting in an elevated burden of human disease. Aedes aegypti is found worldwide in tropical, sub-tropical, and temperate areas. The characterization of mosquito blood meals is essential to understand the transmission dynamics of mosquito-vectored pathogens.Methodology/principal findingsHere, we report Ae. aegypti and Culex quinquefasciatus host feeding patterns and arbovirus transmission in Northern Mexico using a metabarcoding-like approach with next-generation deep sequencing technology. A total of 145 Ae. aegypti yielded a blood meal analysis result with 107 (73.8%) for a single vertebrate species and 38 (26.2%) for two or more. Among the single host blood meals for Ae. aegypti, 28.0% were from humans, 54.2% from dogs, 16.8% from cats, and 1.0% from tortoises. Among those with more than one species present, 65.9% were from humans and dogs. For Cx. quinquefasciatus, 388 individuals yielded information with 326 (84%) being from a single host and 63 (16.2%) being from two or more hosts. Of the single species blood meals, 77.9% were from dogs, 6.1% from chickens, 3.1% from house sparrows, 2.4% from humans, while the remaining 10.5% derived from other 12 host species. Among those which had fed on more than one species, 11% were from dogs and humans, and 89% of other host species combinations. Forage ratio analysis revealed dog as the most over-utilized host by Ae. aegypti (= 4.3) and Cx. quinquefasciatus (= 5.6) and the human blood index at 39% and 4%, respectively. A total of 2,941 host-seeking female Ae. aegypti and 3,536 Cx. quinquefasciatus mosquitoes were collected in the surveyed area. Of these, 118 Ae. aegypti pools and 37 Cx. quinquefasciatus pools were screened for seven arboviruses (ZIKV, DENV 1–4, CHIKV, and West Nile virus (WNV)) using qRT-PCR and none were positive (point prevalence = 0%). The 95%-exact upper limit confidence interval was 0.07% and 0.17% for Ae. aegypti and Cx. quinquefasciatus, respectivelyConclusions/significanceThe low human blood feeding rate in Ae. aegypti, high rate of feeding on mammals by Cx. quinquefasciatus, and the potential risk to transmission dynamics of arboviruses in highly urbanized areas of Northern Mexico is discussed.     

Identification of QTLs Conferring Resistance to Deltamethrin in Culex pipiens pallens

Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus. Deltamethrin is an insecticide that is widely used for mosquito control, however resistance to this and other insecticides has become a major challenge in the control of vector-borne diseases that appear to be inherited quantitatively. Furthermore, the genetic basis of insecticide resistance remains poorly understood. In this study, quantitative trait loci (QTL) mapping of resistance to deltamethrin was conducted in F₂ intercross segregation populations using bulked segregation analysis (BSA) and amplified fragment length polymorphism markers (AFLP) in Culex pipiens pallens. A genetic linkage map covering 381 cM was constructed and a total of seven QTL responsible for resistance to deltamethrin were detected by composite interval mapping (CIM), which explained 95% of the phenotypic variance. The major QTL in linkage group 2 accounted for 62% of the variance and is worthy of further study. 12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the Basic Local Alignment Search Tool (BLAST) tool to the genome sequence of the closely related Culex quinquefasciatus. Our results suggest that resistance to deltamethrin is a quantitative trait under the control of a major QTL in Culex pipiens pallens. Cloning of related AFLP markers confirm the potential utility for anchoring the genetic map to the physical map. The results provide insight into the genetic architecture of the trait.     

The haemagglutination activity in the different developmental stages and changes of this activity caused by analogues of insect hormones in two members of the Culex pipiens complex of mosquitoes

Abstract. Haemagglutination activity was studied in last larval instars, pupae and adults of both sexes of the Culex pipiens mosquito complex. In females of Culex pipiens quinquefasciatus Say, an anautogenous member of C. pipiens complex, activity was detected in the homogenate of the head‐thorax complex, gut and remaining abdomen (after removing the gut), before and after sucking water, glucose solution or blood. Similar investigations were made in females of autogenous Culex pipiens molestus Forskal, a member of the same mosquito complex. The results were compared with data obtained for males of these subspecies, plus the larvae and pupae. The results show that haemagglutination activity in adult females depends not on the sucking of blood, but on the age of the females. In newly emerged females, the level of activity was very low, but after 24 h or 48 h of activity was highly enhanced, even in females that had no possibility to suck water or other liquids including blood. Furthermore, in C. p. molestus, the haemagglutination activity does not change in hungry or engorged females, and a high level of activity was found in the gut of last larval instars, a developmental stage that never takes blood. Treatment with methoprene reduces haemagglutination activity in both sexes, but 20‐hydroxycdysone application decreased activity in the gut of females. It is suggested that haemagglutination activity may be regulated indirectly by the endocrine system. The possibility that haemagglutination activity may depend on some aspect of the digestive system that has no direct connection to blood uptake is discussed. In addition, the activity may be important not only for the elimination of infections, but also for the processing of food and the utilization and transport of nutrients.     

Sympatric occurrence of Culex pipiens (Diptera,Culicidae) biotypes pipiens,molestus and their hybrids in Portugal,Western Europe: feeding patterns and habitat determinants

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host‐feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri‐urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri‐urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.     

Occurrence of L1014F and L1014S mutations in insecticide resistant Culex quinquefasciatus from filariasis endemic districts of West Bengal,India

IntroductionLymphatic filariasis causes long term morbidity and hampers the socio-economic status. Apart from the available treatments and medication, control of vector population Culex quinquefasciatus Say through the use of chemical insecticides is a widely applied strategy. However, the unrestrained application of these insecticides over many decades has led to resistance development in the vectors.MethodsIn order to determine the insecticide susceptibility/resistance status of Cx. quinquefasciatus from two filariasis endemic districts of West Bengal, India, wild mosquito populations were collected and assayed against six different insecticides and presence of L1014F; L1014S kdr mutations in the voltage-gated sodium channel gene was also screened along with the use of synergists to evaluate the role of major detoxifying enzymes in resistance development.ResultsThe collected mosquito populations showed severe resistance to insecticides and the two synergists used–PBO (piperonyl butoxide) and TPP (triphenyl phosphate), were unable to restore the susceptibility status of the vector thereupon pointing towards a minor role of metabolic enzymes. kdr mutations were present in the studied populations in varying percent with higher L1014F frequency indicating its association with the observed resistance to pyrethroids and DDT. This study reports L1014S mutation in Cx. quinquefasciatus for the first time.     

Comparative proteomics reveals mechanisms that underlie insecticide resistance in Culex pipiens pallens Coquillett

Mosquito control based on chemical insecticides is considered as an important element of the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. In contrast to target site resistance, other mechanisms are far from being fully understood. Global protein profiles among cypermethrin-resistant, propoxur-resistant, dimethyl-dichloro-vinyl-phosphate-resistant and susceptible strain of Culex pipiens pallens were obtained and proteomic differences were evaluated by using isobaric tags for relative and absolute quantification labeling coupled with liquid chromatography/tandem mass spectrometric analysis. A susceptible strain of Culex pipiens pallens showed elevated resistance levels after 25 generations of insecticide selection, through iTRAQ data analysis detected 2,502 proteins, of which 1,513 were differentially expressed in insecticide-selected strains compared to the susceptible strain. Finally, midgut differential protein expression profiles were analyzed, and 62 proteins were selected for verification of differential expression using iTRAQ and parallel reaction monitoring strategy, respectively. iTRAQ profiles of adaptation selection to three insecticide strains combined with midgut profiles revealed that multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Culex pipiens pallens. Significant molecular resources were developed for Culex pipiens pallens, potential candidates were involved in metabolic resistance and reducing penetration or sequestering insecticide. Future research that is targeted towards RNA interference of the identified metabolic targets, such as cuticular proteins, cytochrome P450s, glutathione S-transferases and ribosomal proteins proteins and biological pathways (drug metabolism—cytochrome P450, metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, ribosome) could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Culex pipiens pallens.     

Development of Resistance to Pyrethroid in Culex pipiens pallens Population under Different Insecticide Selection Pressures

Current vector control programs are largely dependent on pyrethroids, which are the most commonly used and only insecticides recommended by the World Health Organization for insecticide-treated nets (ITNs). However, the rapid spread of pyrethroid resistance worldwide compromises the effectiveness of control programs and threatens public health. Since few new insecticide classes for vector control are anticipated, limiting the development of resistance is crucial for prolonging efficacy of pyrethroids. In this study, we exposed a field-collected population of Culex pipiens pallens to different insecticide selection intensities to dynamically monitor the development of resistance. Moreover, we detected kdr mutations and three detoxification enzyme activities in order to explore the evolutionary mechanism of pyrethroid resistance. Our results revealed that the level of pyrethroid resistance was proportional to the insecticide selection pressure. The kdr and metabolic resistance both contributed to pyrethroid resistance in the Cx. pipiens pallens populations, but they had different roles under different selection pressures. We have provided important evidence for better understanding of the development and mechanisms of pyrethroid resistance which may guide future insecticide use and vector management in order to avoid or delay resistance.     

ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016

A field investigation of arboviruses was conducted in Dejiang, Guizhou Province in the summer of 2016. A total of 8,795 mosquitoes, belonging to four species of three genera, and 1,300 midges were collected. The mosquito samples were identified on site according to their morphology, and the pooled samples were ground and centrifuged in the laboratory. The supernatant was incubated with mosquito tissue culture cells (C6/36) and mammalian cells (BHK-21) for virus isolation. The results indicated that 40% (3,540/8,795) were Anopheles sinensis, 30% (2,700/8,795) were Culex pipiens quinquefasciatus, and 29% (2,530/8,795) were Armigeres subbalbeatus. Furthermore, a total of eight virus isolates were obtained, and genome sequencing revealed two Zika viruses (ZIKVs) isolated from Culex pipiens quinquefasciatus and Armigeres subbalbeatus, respectively; three Japanese encephalitis viruses (JEVs) isolated from Culex pipiens quinquefasciatus; two Banna viruses (BAVs) isolated from Culex pipiens quinquefasciatus and Anopheles sinensis, respectively; and one densovirus (DNV) isolated from Culex pipiens quinquefasciatus. The ZIKVs isolated from the Culex pipiens quinquefasciatus and Armigeres subbalbeatus mosquitoes represent the first ZIKV isolates in mainland China. This discovery presents new challenges for the prevention and control of ZIKV in China, and prompts international cooperation on this global issue.     

A rural–urban latitudinal study of the distributions of Culex quinquefasciatus and Culex pipiens bioforms in their southernmost sympatric fringe

Mosquitoes grouped in the complex Culex pipiens L. (Diptera: Culicidae) are important vectors of medical and veterinary diseases. In the South American sympatric region, Cx. pipiens and Culex quinquefasciatus Say coexist and potentially hybridize. To identify key drivers of their geographical distribution, mosquito immatures were collected from flower vases of eight urban/rural cemetery pairs within a 5° latitudinal transect along Buenos Aires Province, Argentina. The specimens were identified by molecular methods and their relative proportion modelled as a function of environmental variables. At the beginning of the warm season, northern and southern cemeteries presented exclusively Cx. quinquefasciatus and Cx. pipiens, respectively, with different proportions of both at mid latitudes. By the end of the summer, Cx. quinquefasciatus was present throughout the study area, exclusively in 11 of the 16 cemeteries both rural and urban, whereas Cx. pipiens was predominant only in the southernmost pair. Mean annual temperature, photoperiod variability and time of the season were key drivers of their distributions. All specimens of Cx. pipiens were identified as form molestus and no hybrids were recognized. The reported distribution patterns and the potential absence of Cx. pipiens f. pipiens and hybrids are discussed, along with their implications in disease transmission.