Male Cx Research Articles

Clock genes regulate mating activity rhythms in the vector mosquitoes, Aedes albopictus and Culex quinquefasciatus.

Endogenous circadian rhythms result from genetically-encoded molecular clocks, whose components and downstream output factors cooperate to generate cyclic changes in activity. Mating is an important activity of mosquitoes, however, the key aspects of mating rhythm patterns and their regulatory mechanisms in two vector mosquito species, Aedes albopictus and Culex quinquefasciatus, remain unclear. We determined and compared the diel mating activity rhythms of these two mosquito species and discovered that Ae. albopictus had mating peaks in the light/dark transition periods (ZT0-3 and ZT9-12), while Cx. quinquefasciatus only had a mating peak at ZT12-15. Knockouts of the clock (clk) orthologous genes (Aalclk and Cxqclk) resulted in phase delay or phase reversal of the mating peaks in Ae. albopictus and Cx. quinquefasciatus, respectively. In addition, the temporal expression pattern of the desaturase orthologous genes, desat1, in both mosquito species was also different in respective wild-type strains and showed phase changes similar to the mating rhythms in clk mutant strains. Inhibition of desat1 expression resulted in decreased mating activity in male mosquitoes of both species but not females. In addition, desat1 regulated cuticular hydrocarbons' synthesis in both species. Silencing desat1 in male Ae. albopictus resulted in decreases of nonadecane and tricosane, which promoted mating, with concomitant increases of heptacosane, which inhibited mating. Silencing desat1 in male Cx. quinquefasciatus also resulted in decreases of tricosane, which promoted mating. These results suggest that Aalclk and Cxqclk have significant roles in the mating activity rhythms in both Ae. albopictus and Cx. quinquefasciatus by regulating the temporal expression of the desat1 gene under LD cycles, which affects sex pheromone synthesis and mating. This work provides insights into the molecular regulatory mechanism of distinct mating rhythm of Ae. albopictus and Cx. quinquefasciatus and may provide a basis for the control of these two important vector mosquitoes.

Arboviruses and Blood Meal Sources in Zoophilic Mosquitoes at Human-Wildlife Interfaces in Kenya.

Background: Zoophilic mosquitoes play an important role in the transmission of arboviruses of medical importance at human-wildlife interfaces, yet arbovirus surveillance efforts have been focused mostly on anthropophilic mosquitoes. Understanding the diversity of zoophilic mosquitoes and their associated feeding patterns and arboviruses can inform better vector control strategies. Materials and Methods: We morphologically identified mosquitoes collected from two game reserves in Kenya, the Maasai Mara National Reserve (MMNR) and locations near the Shimba Hills National Reserve (SHNR). Representative mosquitoes were also identified by cytochrome c oxidase subunit 1 (COI) barcode sequencing. In addition, we identified the vertebrate hosts of mosquito blood meals from the contents of each mosquito's abdomen by high-resolution melting (HRM) analysis and sequencing of COI, 16S ribosomal RNA, and cytochrome b gene PCR products. Similarly, mosquito arbovirus infections were identified by HRM analysis and sequencing of Alphavirus- and Flavivirus-specific RT-PCR products. Results: Of 2858 mosquitoes collected, 51 were engorged with blood meals from seven different vertebrate hosts, including humans, birds, domestic, and peridomestic animals and wildlife. Culex was the most abundant mosquito genus, with Culex pipiens being the most abundant species in both study regions. Among MMNR samples, we detected dengue serotype-2 virus (DENV-2) for the first time in Aedes tarsalis and Aedes tricholabis, as well as Sindbis virus in male Cx. pipiens. We also detected DENV-2 in Aedes aegypti sampled from locations near the SHNR. Human and diverse wildlife blood meals were identified, including bushbuck blood in the dengue-infected Ae. tarsalis and both human and hippopotamus blood in a single Eretmapodites chrysogaster mosquito. Conclusions: Our findings highlight the potential risk of sylvatic dengue and Sindbis transmission to humans by zoophilic mosquitoes at human-wildlife interfaces in Africa. Of specific importance, we provide evidence of sylvatic DENV-2 in Ae. tarsalis and Ae. tricholabis, representing potential new dengue vectors.

Vertical transmission of Zika virus in Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes

Several mosquito species have been described as vectors for the Zika virus (ZIKV), such as those in the Aedes, Anopheles, Mansonia and Culex genera. Our previous survey studies were found the ZIKV RNA positive in both male, female and larvae of Culex quinquefasciatus Say and Aedes aegypti (L.) mosquitoes collected from active ZIKV infected patients’ homes in Thailand. Therefore, the aims of this study were to investigate whether ZIKV could be vertically transmitted in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. Laboratory and field colonies of these mosquito species were maintained and artificially fed with ZIKV in human blood. Fully engorged mosquitoes (F0) were selected and reared for the vertical transmission study. The subsequent mosquito generations were fed with human blood without the virus. ZIKV in the mosquitoes was detected by hemi-nested RT-PCR and sequencing. C6/36 cells were used to isolate ZIKV from samples that tested positive by hemi-nested RT-PCR. Moreover, ZIKV was identified by immunocytochemical staining 7 days after infection in several organs of infected F0 females, including the salivary glands, midguts, yoke granules and facet cells of the eye. The localization of the ZIKV antigen was identified by the presence of the specific antibody in the salivary glands, midguts, yoke granules and facet cells. ZIKV was detected in female and male Cx. quinquefasciatus until the F6 and F2 generations, respectively. The isolated virus showed cytopathic effects in C6/36 cells by 5 days postinfection. The results suggested that the vertical transmission of ZIKV occurs in Cx. quinquefasciatus in the laboratory. However, we were able to detect the presence of ZIKV in Ae. aegypti in only the F1 generation in both male and female mosquitoes, and Ae. albopictus mosquitoes were not able to vertically transmit the virus at all. Data obtained from this study could be valuable for developing a better understanding of the role of Cx. quinquefasciatus as a potential vector for ZIKV transmission in Thailand and may be useful in creating more effective mosquito vector control strategies in the future.

Arboviruses isolated from the Barkedji mosquito-based surveillance system, 2012-2013

BackgroundA mosquito-based arbovirus surveillance system was set up at Barkedji, Senegal after the first outbreak of Rift valley fever in West Africa in 1988. This system was recently updated using more sampling methods and collecting in greater number of ponds and villages sites.MethodsFor the current study, mosquitoes were sampled biweekly between July and December 2012 and 2013 using CDC+CO2 light traps set at ground and canopy level, mosquito nets baited with goat, sheep, human or chicken, light traps baited with goat, sheep and chicken; bird-baited traps using pigeons or chickens placed either at the ground or canopy level. Collected mosquitoes were identified, pooled and screened for arboviruses.ResultsA total of 42,969 mosquitoes in 4,429 pools were processed for virus isolation. Ten virus species were identified among 103 virus isolates. West Nile virus (WNV; 31 isolates), Barkedji virus (BARV; 18), Sindbis virus (SINV; 13), Usutu virus (USUV; 12), Acado virus (ACAV; 8), Ndumu virus (NDUV; 9), Sanar virus (SANV; 7), Bagaza virus (BAGV; 3), Rift valley fever virus (RVFV; 1), and Yaounde virus (YAOV; 1) were isolated from 9 ponds (91 strains) and 7 villages (12 strains). Only 3 virus species (WNV, NDU and SINV) were isolated from villages. The largest numbers of isolates were collected in October (29.1% of total isolates) and November (50.5%). Viruses were isolated from 14 mosquito species including Cx. neavei (69.9% of the strains), Cx. antennatus (9.7%), and Ma. uniformis (4.8%). NDUV, ACAV, and SINV are herein reported for the first time in the Barkedji area. Isolation of ACAV and SANV from a pool of male Ma. uniformis and USUV and BARV from a pool of male Cx. neavei, are reported for the first time to our knowledge.ConclusionOur data indicate that the Barkedji area is characterized by a high diversity of viruses of medical, veterinary and unknown importance. Arboviruses were first detected in July at the beginning of the rainy season and peaked in abundance in October and November. The Barkedji area, an enzootic focus of several potentially emerging arboviruses, should be surveilled annually to be prepared to deal with future disease emergence events.

Laboratory evaluation of differential attraction of Culex pipiens pallens to the volatiles of flowers, fruits, and seed pods

The attraction of Culex pipiens pallens Coquillett to various potential sugar sources in their habitats remains unknown. In the present study, we evaluated the attraction of Cx. pipiens pallens to the volatiles of different kinds of flowers, fruits, and seed pods by multiple choice tests under laboratory conditions. Nine of the 21 tested fresh tissues of flowers were attractive to females and six of them were attractive to males. The volatiles of six flowers (Ligustrum quihoui, Abelia chinensis, Ophiopogon japonicus, Phaseolus vulgaris, Osmanthus fragrans, and Nerium indicum) were attractive to both female and male Cx. pipiens pallens, and the flowers of Torenia fournieri and Lavandula angustifolia were potentially repellent to female and male mosquitoes, respectively. Besides, the volatiles of three fruits and seed pods (Broussonetia papyrifera, Amygdalus persica, and Cucumis melo) were attractive to both female and male Cx. pipiens pallens while the seed pods of Ficus carica and Pyrus bretschneideri were only attractive to females and males, respectively. This study provides valuable information on developing novel control methods against Cx. pipiens pallens in China.

Susceptibility of adult mosquitoes to insecticides in aqueous sucrose baits

Mosquitoes characteristically feed on plant-derived carbohydrates and honeydew just after emergence and intermittently during their lives. Development of toxic baits focusing on this carbohydrate-seeking behavior may potentially contribute to localized control. In the present study, ten insecticides were fed to female Culex quinquefasciatus, Anopheles quadrimaculatus, and Aedes taeniorhynchus in a 10% sucrose solution. Active ingredients representative of five classes of insecticides (pyrethroids, phenylpyroles, pyrroles, neonicotinoids, and macrocyclic lactones) were selected for comparison with commercial formulations used to facilitate incorporation of active ingredients into aqueous sucrose solutions. Sucrose as a phagostimulant significantly enhanced mortality to toxicants. In general, the most effective active ingredients were fipronil, deltamethrin and imidacloprid, followed by spinosad, thiamethoxam, bifenthrin, permethrin, and cyfluthrin. The least effective ingredients were chlorfenapyr and ivermectin. For some of the ingredients tested, Cx. quinquefasciatus was the least susceptible species. One-day-old male Cx. quinquefasciatus were more susceptible than females; however, no differences existed between one- and seven-day-old mosquitoes. There were no differences in susceptibility between unfed and gravid ten-day-old female Cx. quinquefasciatus to bifenthrin. In conclusion, several pesticides from different classes of compounds have potential for use in development of toxic baits for mosquitoes.

Population genetics of the mosquito Culex pipiens pallens reveals sex-linked asymmetric introgression by Culex quinquefasciatus

The Culex pipiens complex in Asia includes a temperate subspecies, Culex pipiens pallens, of uncertain taxonomic status. The shape of the male genitalia suggests it is a hybrid between Cx. pipiens and Cx. quinquefasciatus. We studied populations of Cx. p. pallens in Japan, Korea, and China and compared them to local populations of Cx. quinquefasciatus and Cx. p. pipiens. We examined variation in a nuclear intron in the acetylcholinesterase-2 gene [ACE] and eight microsatellite loci. We found a distinct microsatellite signature for Cx. p. pallens indicating restricted gene flow between Eastern and Western populations of Cx. pipiens, supporting the existence of two subspecies. Furthermore, a multilocus genotype analysis revealed current hybridization between Cx. p. pallens and Cx. quinquefasciatus in southern Japan, Republic of Korea, and China but not in Hokkaido, in northern Japan. Surprisingly, however, we found that the sex-linked ACE locus in chromosome I has introgressed asymmetrically through the males such that all male Cx. p. pallens have a copy of the Cx. quinquefasciatus ACE locus. This result highlights some of the potential consequences of hybridization between local and introduced species to disease transmission worldwide.

West Nile Virus from Female and Male Mosquitoes (Diptera: Culicidae) in Subterranean, Ground, and Canopy Habitats in Connecticut

In total, 93,532 female mosquitoes (Diptera: Culicidae) were captured in traps placed in subterranean (catch basin), ground (approximately 1.5 m above ground), and canopy (approximately 7.0 m above ground) habitats in Stamford and Stratford, CT, during 2003-2005. Culex pipiens L. was the most abundant (64.8%) of the 31 species identified. Significantly greater numbers of Cx. pipiens were captured in canopy-placed mosquito magnet experimental traps, and significantly greater numbers were collected in catch basin-placed (Centers for Disease Control) CDC traps than in CDC traps placed elsewhere. Culex restuans Theobald was captured in significantly greater numbers in traps placed in catch basins. Aedes vexans (Meigen), Aedes cinereus Meigen, and Aedes cantator (Coquillett) were significantly more abundant in ground traps. In total, 429 isolations of West Nile virus (WNV) were made from seven species of mosquitoes from late June through the end of October during 2003 through 2005. Three hundred ninety-eight (92.8%) isolates were from Cx. pipiens. Others were from Cx. restuans (n = 16), Culex salinarius Coquillett (n = 5), Ae. vexans (n = 4), Ae. cantator (n = 3), Aedes triseriatus (Say) (n = 2), and Ae. cinereus (n = 1). Multiple isolates from Cx. pipiens were made each week, primarily during the later part of July through the end of September. Weekly minimum infection rates (MIRs) were lower in 2004 (highest weekly MIR = 7.1) when no human cases were reported in Connecticut in comparison with 2003 and 2005 (highest weekly MIR = 83.9) when human cases were documented. Frequencies of infected pools were significantly higher in Cx. pipiens captured in traps in the canopy and significantly higher in catch basin placed traps than in traps at ground level. The physiological age structure of Cx. pipiens captured in the canopy was significantly different from that of Cx. pipiens collected in catch basins. Invariably, Cx. pipiens captured in the canopy were nulliparous or parous with ovaries in Christophers' stage 2, whereas 58.7% of the females captured in catch basins possessed ovaries filled with mature oocytes in Christophers' stage 5. Our results suggest that females in the canopy are seeking hosts, and after digestion of the bloodmeal and development of mature oocytes, they descend to catch basins for shelter and deposition of eggs. WNV was isolated from three, one, and two pools of male Cx. pipiens captured in catch basin-, ground-, and canopy-placed traps, respectively, and from six nulliparous Cx. pipiens females collected in the canopy. Weekly MIR ranged from 1.2 to 31.1 per 1,000 male specimens. These data show that mosquitoes become infected by means other than by blood feeding, possibly by transovarial transmission. The placement of traps in tree canopies and in catch basins can be used to augment current practices of placement of traps near the ground for surveillance of mosquitoes infected with WNV and for studies of the ecology of WNV.

Distribution and hybridization between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina

To better undesrtand the distribution of Culex pipiens and Cx. quinquefasciatus in Argentina, samples were collected from six localities situated in a North-South line from Castelli (Chaco Province) to Puerto Madryn (Chubut Province). Identification was based on the morphology of male genitalia. Only Cx. quinquefasciatus was found in Castelli and Esperanza, while in Rosario, 95.3% belonged to this species and 4.7% represented hybrid forms. Southern samples included only Cx. pipiens. With the purpose of verfying if Cx. pipiens and Cx. quinquefasciatus hybridize, different crosses between the two species were perfomed. All crosses produced viable egg rafts. Hatching ranged from 70 to 100%, except in one cross, female Cx. pipiens x male Cx. quinquefasciatus, where a high incompatibility was observed (11.1%hatch). The F1 hybrids obtained all crosses were fertile. The finding of hybrid forms in nature can be interpreted as evidence for subspecific status of Cx. pipiens and Cx. quinquefasciatus in Argentina.

Persistence of Mosquito-Borne Viruses in Kern County, California, 1983–1988

The persistence of arboviruses was studied from 1983 to 1988 in mixed agriculture, marsh, riparian, and foothill habitats in Kern County, CA. Western equine encephalomyelitis (WEE) virus was isolated frequently during 1983 from Culex tarsalis and Aedes melanimon and was detected by the seroconversion of sentinel chickens. WEE virus then disappeared, even though vector competence studies during 1984-1986 showed that Cx. tarsalis was able to transmit WEE virus. St. Louis encephalitis (SLE) virus was detected sporadically in 3 of the 6 years of the study by isolation from Cx. tarsalis and/or by sentinel chicken seroconversion. When mosquito pools were screened for virus in suckling mice, Turlock (TUR) and Hart Park (HP) viruses were isolated from Cx. tarsalis during each summer. Vertical transmission of HP was indicated by the isolation of virus from a pool of male Cx. tarsalis. California encephalitis (CE) virus was isolated repeatedly from host-seeking Ae. melanimon females, males, and adults reared from field-collected immatures, verifying vertical transmission in nature. Horizontal transmission of CE virus among both jackrabbits (Lepus californicus) and desert cottontails (Sylvilagus auduboni) appeared to amplify Ae. melanimon infection rates during the summer of 1985, but elevated herd immunity depressed infection rates during 1986. Thus, CE, HP, and TUR viruses persisted in Kern County, while WEE virus appeared to become extinct and required reintroduction. The sporadic occurrence of SLE virus activity remains unexplained, but its persistence may require both vertical transmission and reintroduction.