High degree of single nucleotide polymorphisms in California Culex pipiens (Diptera: Culicidae) sensu lato

Resolution of systematic relationships among members of the Culex pipiens (L.) complex has important implications for public health as well as for studies on the evolution of sibling species. Currently held views contend that in California considerable genetic introgression occurs between Cx. pipiens and Cx. quinquefasciatus Say, and as such, these taxa behave as if they are a single species. Development of high throughput SNP genotyping tools for the analysis of Cx. pipiens complex population structure is therefore desirable. As a first step toward this goal, we sequenced 12 gene fragments from specimens collected in Marin and Fresno counties. On average, we found a higher single nucleotide polymorphism (SNP) density than any other mosquito species reported thus far. Coding regions contained significantly higher GC content (median 54.7%) than noncoding regions (42.4%; Wilcoxon rank sum test, P = 5.29 x 10(-5)). Differences in SNP allele frequencies observed between mosquitoes from Marin and Fresno counties indicated significant genetic divergence and suggest that SNP markers will be useful for future detailed population genetic studies of this group. The high density of SNPs highlights the difficulty in identifying species within the complex and may be associated with the large degree of phenotypic variation observed in this group of mosquitoes.     

Establishment and characterization of a cell line from the mosquito Culex (Culex) theileri (Diptera: Culicidae) and its susceptibility to infection with arboviruses

The mosquito Culex (Culex) theileri Theobald is probably the major vector of Rift Valley fever (RVF) virus during epizootics on the inland plateau of southern Africa. A cell line was established from the tissues of this mosquito species to facilitate laboratory studies on RVF and other arboviruses. After temperature pretreatment, egg rafts from a laboratory colony were disinfected, mechanically disrupted, and grown in preconditioned Eagles minimum essential medium supplemented with 10% fetal calf serum. The cells were characterized by their protein content, karyology, and their susceptibility to three arboviruses. Although high infectivity titers were obtained, no cytopathic effects were produced by these viruses. The cell line was shown to be free from latent viruses. These results show that this cell line may be employed fruitfully in arbovirus studies.     

Natural infection of Culex theileri (Diptera: Culicidae) with Dirofilaria immitis (Nematoda: Filarioidea) on Madeira Island, Portugal

Field and laboratory studies were performed to verify whether Culex theileri Theobald functions as a natural vector of Dirofilaria immitis (Leidy) on Madeira Island, Portugal. CO2-baited light traps (EVS traps) were use to sample mosquitoes monthly basis between February 2002 and February 2003 in the area of Quebradas (Funchal). Three mosquito species were captured, including 58 Culex pipiens L., 790 Cx. theileri, and three Culiseta longiareolata (Macquart). Only C. theileri tested positive for D. immitis. The presence of this filarial worm was detected by direct observation, infectivity assay dissection technique, and polymerase chain reaction methods. Infected mosquitoes were recovered in October and December 2002 and January 2003. These data provide evidence that Cx. theileri could be the main vector of D. immitis in Funchal, Madeira.     

Crepuscular flight activity of Culex erraticus (Diptera: Culicidae)

Circadian patterns of flight activity in mosquitoes can influence pathogen transmission by regulating dispersal potential of vectors and contact rates between vectors and reservoir and/or dead-end hosts. We investigated circadian activity patterns of Culex erraticus (Dyar and Knab) at a wetland field site in central Alabama, by aspirating resting adults and questing females in the morning and evening hours, respectively. Mosquitoes were aspirated at regular time intervals to determine the time of day during which peak resting site-seeking and host-seeking activities occurred. Day-to-day variation in activity patterns due to wind, humidity, and temperature was examined using stepwise linear regression. We found a distinct peak in flight activity during the morning hours (2 h before and 2 h after sunrise) for females and males of Culex erraticus, the most commonly encountered species at the site. The exact time of the peak varied from day to day, and was largely a function of temperature. A less distinct peak in activity was observed for questing females in the evening, although flights generally commenced just after sunset and peaked 30-60 min after sunset. A significant amount of day-to-day variation in the number of questing females was attributable to relative humidity. Our study demonstrates predictable patterns of circadian activity for Cx. erraticus, a suspected bridge vector of eastern equine encephalitis virus. Moreover, these patterns are modulated by environmental conditions. This information may be used to develop vector control strategies and make predictions about factors that affect the spread of mosquito-vectored pathogens.     

Seasonal abundance of Culex tarsalis and Culex pipiens complex mosquitoes (Diptera: Culicidae) in California

Large-scale patterns in the seasonal abundance profiles of the arboviral mosquito vectors Culex tarsalis Coquillett and the species of the Culex pipiens complex were described based on a decade of counts from 868 New Jersey light traps located throughout the urban and agricultural areas of California. Mean seasonal abundance profiles varied markedly among hydrologic regions. For all species, abundance increased earlier and declined later in drier, warmer southern regions, but variation could not be explained fully by latitude. The observed patterns may be driven by temperature, availability of larval habitats, and for the Cx. pipiens complex, the taxonomic composition of local populations.     

Stable, germ-line transformation of Culex quinquefasciatus (Diptera: Culicidae)

A Hermes-based transposable element transformation system incorporating an enhanced green fluorescent protein (EGFP) marker was used to produce two transgenic lines of Culex quinquefasciatus (Say). The transformation frequency was approximately 12% and transformation of Culex was shown to be dependent on the presence of Hermes transposase. Injected Culex embryos were treated with four different heat shock regimes, two of which produced transformed individuals. These individuals were mated with wild-type mosquitoes and produced offspring which expressed the dominant EGFP gene in Mendelian ratios predicted for the stable integration of a gene at a single locus. The two transformed lines displayed distinct patterns of phenotypic expression, the expression of which has remained stable after fifteen generations. In these transgenic lines both the Hermes element and flanking plasmid DNA integrated into the Culex genome, as has been previously seen in Hermes-mediated transgenic strains of Aedes aegypti (L.). The high frequency of Culex transformation together with the dependence on the presence of Hermes transposase suggests that, as for Ae. aegypti, this mode of transposition into the germ-line genome occurs by an alternate mechanisms to the cut and paste type of transposition seen for this element in other insect species and in the somatic nuclei of mosquitoes. This is the first report of the genetic transformation of a species in the genus Culex and demonstrates that this medically important mosquito species can now, along with several other Culicine and Anopheline mosquito species, be genetically manipulated.     

Persistence of hepatitis B antigen in Culex quinquefasciatus (Diptera: Culicidae)

Groups of wild-caught Culex quinquefaciatus Say, previously tested for the presence of hepatitis B surface antigen (HBsAg), were tested for the presence of hepatitis B e antigen (HBeAg). This antigen was detected at low levels in blood-fed, half-gravid, and gravid groups. A colony of Cx. quinquefasciatus was established in the laboratory and tested for the persistence of HBsAg and HBeAg. Five days after feeding on blood infected with HBsAg and HBeAg, 9 of 20 (45%) pools of Cx. quinquefasciatus were HBeAg-positive and 5 of 20 (25%) pools were HBeAg-positive; low levels of HBsAg and HBeAg were still detectable 28 d after the infective meal in 2 of 20 (10%) and 1 of 20 (5%) pools, respectively. A crude protease extract was prepared from colony mosquitoes, and the effect of this extract on HBsAg and HBeAg present in human serum was tested in vitro. After 20 h, tests for both antigens were still strongly positive. Low levels of HBsAg were detected in ovaries 7 d after infection. Salivary glands were HBsAg- and HBeAg-negative.     

Environmental factors influencing oviposition by Culex (Culex) (Diptera: Culicidae) in two types of traps

Seasonal and habitat influences on the egg-laying activity of four species of Culex were compared in south Florida using jar- and vat-type oviposition traps. Egg rafts of Culex nigripalpus Theobald, Cx. quinquefasciatus Say, and Cx. salinarius Coquillett were collected in every month of the year, but rafts of Cx. restuans Theobald were absent during spring and summer. Cx. nigripalpus was the dominant species in the summer and fall, and Cx. quinquefasciatus and Cx. salinarius peaked during the winter and spring. Cx. restuans oviposited most rafts in the fall (98.4%). Most Cx. nigripalpus egg rafts were laid in vats (88.5%), and most Cx. quinquefasciatus egg rafts were deposited in jars (58.7%). Fewer Cx. quinquefasciatus rafts were found in a wooded area (32.4%) with a dense undergrowth than in a more open area (67.6%), but Cx. nigripalpus showed no ovipositional preference between these two habitats. The number of rafts oviposited by all species was reduced during periods of rainfall.     

Mark-release-recapture studies with Culex mosquitoes (Diptera: Culicidae) in Southern California

Cohorts of Culex adults were marked uniquely with date- and site-specific fluorescent dust colors and were released at centrally located residences and at peripheral breeding sources to study population dispersal, size, additions, and deletions. The recapture rate of Cx. quinquefasciatus females was higher and the mean distance dispersed was lower in residential than in agricultural or park habitats. Dispersal was associated with host-seeking activity and ranged from 0.6 to 1.0 km/d. Survivorship ranged from 0.65 to 0.84 per day, and population density ranged from 36,612 to 671,634 females per km2. The sampling efficiency of CO2-baited traps in residential habitats increased coincidentally with increasing population density. Gravid traps were most effective in residential habitats where there were few competitive oviposition sites. Teneral Cx. stigmatosoma were extremely dispersive, and few marked females were recaptured. Unmarked females were more abundant at CO2-baited traps in residential habitats than at traps near productive peripheral breeding sources. Few Cx. tarsalis were released, and the recapture rate in residential habitats was low when compared with rural sites.     

Overproduced esterases in Culex pipiens quinquefasciatus (Diptera: Culicidae) from Vietnam

The electrophoretic polymorphism of loci encoding for 10 enzymes was studied in Culex p. quinquefasciatus Say from six localities of Vietnam. The analysis of 11 "neutral genes" showed that differentiation among samples was low, but significant (Fst = 0.06), and significantly related to geographic distance between sample sites. These results are similar to those observed in other countries (Europe and west Africa). A single type of overproduced esterases (A2-B2) was observed, and its frequency was high (60-100%) in all samples. This situation is in sharp contrast with that observed in other countries of South East Asia (China, South Korea and Japan), where two or more types of overproduced esterases have been reported. A map summarizing the geographic distribution of Asian Cr. p. quinquefasciatus with overproduced esterases is provided.     

单核苷酸多态性及其数据库

随着人类基因组研究计划（human genome project,HGP)DNA序列测定工作的快速发展,研究人类基因组变异的重要性日益突出。将人类DNA变异的数据收集整理和建立起数据库。不仅可以用于研究人类的起源、进化以及现代人群遗传变异的发展机理。而且为检测与疾病,尤其是肿瘤和复杂性疾病如糖尿病,肥胖症,高血压和老年痴呆症等相关的基因提供了基础。基因组变异最常见的一种形式是单核苷酸多态性（single nucleotide polymorphism,SNP)。近两年来,美国、欧洲、日本和中国相继建立了SNP数据库,现就对SNP的研究近况和SNP数据库的建立与发展进行综述。对SNP数据库的应用价值和目前存在的问题加以讨论,并且在此基础上分析了研究中国人群基因组变异和发展具有中国特色的SNP数据库的必要性,中国是世界上人口最多的国家,建立中国人群特有的SNP资料库,不仅可以为研究中国人群基因组变异及其与复杂性疾病的相关性提供迅速可靠的情报,同时还将为发展新的医药产品提供科学的依据。并为全人类基因组变异的研究提供近1/5的资料。     

Evaluating sampling method bias in Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae) bloodmeal identification studies

Determining the bloodmeal hosts of the Culex vectors of encephalitis viruses such as West Nile virus is essential for understanding the role of these mosquitoes in enzootic and epidemic transmission. Although molecular techniques have increased our knowledge of blood feeding patterns by allowing host identification to the species level, few studies have focused on the role that sampling methods may play in determining these patterns. In the current study, we identified 644 bloodmeals from Culex tarsalis Coquillett and Culex quinquefasciatus Say females collected in CO2 traps (dry ice-baited Center for Disease Control traps), in gravid traps, and aspirated from resting sites. There was no significant difference in the bloodmeal host apportionment in sampling methods such as gravid traps and resting collections that collected fully engorged females. However, CO2 traps that collected partially fed females had a significantly different apportionment of hosts than either gravid or resting collections. Bloodfed females from CO2 traps had either fed on only a small subset of available host species or were biased toward more mammalian and fewer nonpasserine avian feeds than females from other collections. Because both full and partial bloodmeals can contribute to viral transmission, obtaining Culex bloodmeal collections from multiple sampling methods may be important to fully interpret the role of these mosquitoes as maintenance and/or bridge vectors.     

Catalog of 320 single nucleotide polymorphisms (SNPs) in 20 quinone oxidoreductase and sulfotransferase genes

Single nucleotide polymorphisms (SNPs) in genes encoding drug-metabolizing enzymes, transporters, receptors, and other drug targets have been widely implicated as contributors to differences among individuals as regards the efficacy and toxicity of many medications, as well as the susceptibility to complex diseases. By combining the polymerase chain reaction (PCR) technique with direct sequencing, we screened genomic DNAs from 48 Japa-nese volunteers for SNPs in genes encoding three quinone oxidoreductases (NQO1, NQO2, and PIG3) and 17 sulfotransferases (SULT1A1, SULT1A2, SULT1A3, SULT1C1, SULT1C2, SULT2A1, SULT2B1, ST1B2, TPST1, TPST2, SULTX3, STE, CST, HNK-1 ST, CHST2, CHST4, and CHST5). In all, we identified 320 SNPs from these 20 loci: 22 within coding elements, 21 in 5′ flanking regions, 10 in 5′ untranslated regions, 223 in introns, 19 in 3′ untranslated regions, and 25 in 3′ flanking regions. The ratio of transitions to transversions was approximately 2.3 to 1. Of the 22 coding SNPs, 6 were nonsynonymous substitutions that resulted in amino-acid substitutions. The high-density SNP maps we constructed from this data for each of the quinone oxidoreductases and sulfotransferases examined here should provide useful information for investigations designed to detect association(s) between genetic variations and common diseases or responsiveness to drug therapy. Received: January 12, 2001 / Accepted: January 19, 2001