A nucleopolyhedrovirus from Uranotaenia sapphirina (Diptera: Culicidae)

In this report we present data on biology, gross pathology, ultrastructure, and host range studies of a naturally occurring nucleopolyhedrovirus from the mosquito Uranotaenia sapphirina (UrsaNPV). Development of this virus was restricted to nuclei of epithelial cells in posterior midgut and distal gastric caecum. Occlusion bodies contained numerous singly enveloped rod-shaped virions. Early occlusion bodies were irregularly shaped and seemed to subsequently coalesce to form larger polyhedra. Mature occlusion bodies had a unique dumbbell shape, and lacked a polyhedron envelope and crystalline structure. Developmental and structural features of UrsaNPV were generally similar to other mosquito NPVs, with major differences in occlusion body shape and size. Transmission tests showed that only members of Uranotaenia (Ur. sapphirina and Ur. lowii) were susceptible to this virus. Transmission was facilitated by magnesium. Field collected Ur. sapphirina larvae had a relatively high rate of dual infections with UrsaNPV and UrsaCPV (cypovirus).     

蓝带蚊属一新种（双翅目：蚊科）

记述采白云南省西双版纳景洪县竹筒积水内蓝带蚊属伪费蚊亚属1新种．讨论了新种与相似种的鉴别特征。模式标本保存于云南省寄生虫病防治研究所。     

Establishment and characterization of a cell line from the mosquito Culex tritaeniorhynchus (Diptera: Culicidae)

We established a continuous cell line from the embryo of the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae), a known major vector of the Japanese encephalitis virus (family Flaviviridae, genus Flavivirus) in Asia. The cell line, designated NIID-CTR, was serially subcultured in VP-12 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS). It continued to grow for more than 60 passages over a 750-d period. The NIID-CTR cell line mainly comprised two morphologically distinct types of cells with adhesive properties: spindle-shaped and round cells. Most of the NIID-CTR cells at the 45th passage were diploid (2n = 6). The growth kinetics of the NIID-CTR cells was significantly affected by the FBS concentration in the medium. The population doubling time of the NIID-CTR cells was 20 h in the presence of 10 % FBS and 76 h in its absence. The DNA sequence of the mitochondrial cytochrome oxidase I gene confirmed that the NIID-CTR cell line was derived from C. tritaeniorhynchus. The cells were highly susceptible to Japanese encephalitis and Dengue viruses, thus providing a valuable tool for the study of mosquito-borne flaviviruses.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

Molecular and biological characterization of a Cypovirus from the mosquito Culex restuans

A cypovirus from the mosquito Culex restuans (named CrCPV) was isolated and its biology, morphology, and molecular characteristics were investigated. CrCPV is characterized by small (0.1-1.0 microm), irregularly shaped inclusion bodies that are multiply embedded. Laboratory studies demonstrated that divalent cations influenced transmission of CrCPV to Culex quinquefasciatus larvae; magnesium enhanced CrCPV transmission by approximately 30% while calcium inhibited transmission. CrCPV is the second cypovirus from a mosquito that has been confirmed by using molecular analysis. CrCPV has a genome composed of 10 dsRNA segments with an electropherotype similar to the recently discovered UsCPV-17 from the mosquito Uranotaenia sapphirina, but distinct from the lepidopteran cypoviruses BmCPV-1 (Bombyx mori) and TnCPV-15 (Trichoplusia ni). Nucleotide and deduced amino acid sequence analysis of CrCPV segment 10 (polyhedrin) suggests that CrCPV is closely related (83% nucleotide sequence identity and 87% amino acid sequence identity) to the newly characterized UsCPV-17 but is unrelated to the 16 remaining CPV species from lepidopteran hosts. A comparison of the terminal segment regions of CrCPV and UsCPV-17, an additional method for differentiating various Cypovirus species, revealed a high level of conservation. Therefore, we propose that CrCPV is a member of the Cypovirus-17 group and designate this species as CrCPV-17.     

Isolation and characterization of microsatellite loci in the mosquito Anopheles stephensi Liston (Diptera: Culicidae)

The mosquito Anopheles stephensi is an important malaria vector in India, Pakistan, Iran and Afghanistan. Differences in egg morphology and chromosomal characters have been described between urban and rural forms of this mosquito but the population genetic structure remains unclear. In India this species is mainly urban, rural populations are largely zoophilic and not thought to transmit malaria. In eastern Afghanistan and the Punjab and Northwest Frontier Province, Pakistan, it is the major malaria vector. We have developed primers for 16 microsatellite loci to assist in defining the population structure and epidemiological importance of this mosquito.     

Reduced productivity in adult yellowfever mosquito (Diptera: Culicidae) populations

Male and female Aedes aegypti (L.) mosquitoes of the laboratory strain ROCK were irradiated with 130 mw of argon 514.5 nm laser microbeams for 0.04, 0.25, 0.4, and 0.5 s, respectively. Egg production, percentage hatch, and productivity (average number of adults surviving after 3 wk) were used to assess mutagenic effects. Mortality was high for males in all laser radiation groups and increased with time of exposure. Except for the group treated for 0.25 s, significant reductions in total F1 progeny also were demonstrated for all other experimentals when male parents were exposed to laser radiation. Females showed a high mortality when subjected to 0.4- and 0.5-s laser radiation. No F1 progeny were produced when parental females were exposed for 0.25, 0.4, and 0.5 s. Numbers of F1 progeny from females exposed to 0.04 s of laser radiation were significantly reduced. A comparison of weekly mean number of progeny showed that the important differences in productivity occurred during the first and second week, respectively, when either male or female adult parents were subjected to laser radiation.     

piggyBac-mediated germline transformation of the malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Dear Editor, Anopheles sinensis is the primary vector of vivax malaria in China.Given the risks posed by An.sinen-sis,many methods have been used to control thi...     

Characterization of microsatellite markers in the mosquito Ochlerotatus caspius (Diptera: Culicidae)

Seven polymorphic microsatellite loci were developed for the mosquito species Ochlerotatus caspius, using an enriched genomic library. The number of alleles per locus varied between two and 11; the expected heterozygosity (H_E) ranged from 0.18 to 0.77. These microsatellite primers should prove useful for population genetic studies of this mosquito species.     

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well‐supported clusters. Intraspecific Kimura 2‐parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra‐ and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.     

Genetic lineages in the yellow fever mosquito Aedes (Stegomyia) aegypti (Diptera: Culicidae) from Peru

The yellow fever mosquito Aedes aegypti was introduced in Peru in 1852 and was considered to be eradicated in 1958. In 2001, Ae. aegypti had been recorded in 15 out of 24 Peruvian Departments. Peru has great ecological differences between the east and west sides of Andes. Because of this, we consider that Ae. aegypti populations of both east and west sides can have a genetically distinct population structure. In this study we examined genetic variability and genealogical relationships among three Ae. aegypti Peruvian populations: Lima, Piura (west Andes), and Iquitos (east Andes) using a fragment of the ND4 gene of the mitochondrial genome. Three haplotypes were detected among 55 samples. Lima and Iquitos showed the same haplotype (Haplotype I), whereas Piura has two haplotypes (Haplotype II and III). Haplotype II is four mutational steps apart from Haplotype I, while Haplotype III is 13 mutational steps apart from Haplotype I in the network. The analysis of molecular variation showed that mostly of the detected genetic variation occurs at interpopulational level. The significant value Phi(st) suggests that Piura population is structured in relation to Lima and Iquitos populations and the gene flow of the ND4 is restricted in Piura when compared to Lima and Iquitos. Genetic relationship between haplotype I and haplotype II suggests introduction of the same mtDNA lineage into those localities. However the existence of a genetically distant haplotype III also suggests introduction of at least two Ae. aegypti lineages in Peru.     

Ultrastructure of the spermatozoon of the mosquito Toxorhynchites (Diptera, Culicidae)

The spermatozoon of Toxorhynchites brevipalpis conradti Gruenberg is long and filiform. It contains an anterior nucleus, an axoneme of the 9 + 9 +"1" pattern and two mitochondrial derivatives. Comparison of our observations with bibliographical data shows that spermatozoon ultrastructure is homogeneous in the family Culicidae (Culicinae, Anophelinae and Toxorhyn-chitinae). The 9 + 9 +"1" pattern of the sperm axoneme may be considered a synapomorphy of the Culicidae.     

Contributions to the mosquito fauna (Diptera, Culicidae) of Belgorod Province

The results of long-term studies of mosquitoes in Belgorod Province are reported. The material was collected at 54 sites situated in 15 districts of the province. Different biotopes were investigated during the spring-autumn period. Larvae and adult mosquitoes were used for identification. A total of 27 mosquito species were found in the region examined. The following species were recorded for the first time: Ae. (Och.) communis. Ae. (Och.) diantaeus. Ae. (Och.) pulchritarsis. Ae. (Och.) sticticus, and Cx. (Bar.) modestus; and also the form Cx. (Cux.) pipiens pipiens biotype molestus. The frequency of occurrence of each species in different biotopes and different localities is given. The most widespread and common species in Belgorod Province include Ae. (Och.) cantans (Mg.), Ae. (Och.) cataphylla Dyar, Ae. (Fin.) geniculatus (Ol.), Ae. (Adm.) vexans (Mg.), Cx. (Cux.) pipiens, and Cx. (Cux.) pipiens pipiens biotype molestus.     

The Anopheles maculipennis complex (Diptera: Culicidae) in Iran: molecular characterization and recognition of a new species

Mosquitoes of the Anopheles maculipennis complex were collected in nine provinces of Iran (Esfahan, Fars, Gilan, Golestan, Kohkiluyeh va Boyerahmad, Mazandaran, Tehran, Azarbaijan-e Gharbi and Zanjan) between June 1983 and September 2002. The nuclear rDNA ITS2 sequences of 86 specimens were compared with those of seven species of the complex available in GenBank. Three genetically distinct species of the complex were distinguished: A. maculipennis Meigen, A. sacharovi Favre and a previously unrecognized species. The last species is most similar to, but clearly distinct from, A. martinius Shingarev and A. sacharovi. The taxonomy of A. martinius and A. sacharovi is critically reviewed, and justification is provided for formally recognizing the third species as Anopheles persiensis sp.n. The new species is the first culicid to be characterized and named principally on the basis of DNA evidence. Anopheles persiensis was collected only in the northern Caspian Sea littoral provinces of Gilan and Mazandaran, and it seems likely that this species could be responsible for malaria transmission in this region that was previously attributed to A. maculipennis. A species-specific RFLP-PCR assay based on ITS2 sequences was developed to facilitate further studies of the three species in Iran.     

Phylogeography of the medically important mosquito Aedes (Ochlerotatus) vigilax (Diptera: Culicidae) in Australasia

Aim Within Australia the Carpentaria Barrier has been identified as an important biogeographical barrier. Here we test this long‐standing hypothesis using a medically important mosquito, Aedes (Ochlerotatus) vigilax. Location We obtained samples of Ae. vigilax from throughout its Australian distribution and from New Caledonia. Methods We constructed a distributional database from 602,417 specimens and obtained sequence data from 66 female specimens from 16 localities. The distributional database of Ae. vigilax comprised historical data from 13 organizations and was used to develop our molecular sampling strategy. Genetic structure within Ae. vigilax was examined via haplotype networks, F_ST values, analysis of molecular variance and neutrality test statistics based on one mitochondrial (cytochrome c oxidase subunit I, COI) and two nuclear (alpha amalyse and zinc finger) loci. The historical demography of Ae. vigilax was investigated using extended Bayesian skyline plot (EBSP) methods, with past migration rates estimated using migrate . Results We identified three distinct lineages within Ae. vigilax; however, two of the three lineages show a large distributional overlap across the Carpentaria Barrier. The mitochondrial locus suggested a pattern of significant genetic differentiation, with high F_ST values, significant genetic differentiation within the COI locus, and significantly more variation between the lineages than within. A higher number of migrants per generation were estimated for the overlapping lineages and both the neutrality test statistics and EBSP suggested the occurrence of post‐population expansion in these lineages. Main conclusions Significant deviation from genetic neutrality, in combination with estimates of migration and the demographic history of Ae. vigilax lineages, suggests that the incongruence of the Ae. vigilax phylogeny with the hypothesized Carpentaria Barrier could be attributed to the separation of eastern and western populations of Ae. vigilax around 770 ka and subsequent secondary contact within the last 100 kyr. Sea‐level and precipitation fluctuations within the Carpentaria area during the late Quaternary could have facilitated the current biogeographical patterns of Ae. vigilax. Mosquitoes represent one of the most medically important insect groups; however, understanding the factors that influence past and present distributions of mosquitoes is critical in the face of a range of emerging arboviruses.