Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens . The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 × 10⁻⁴ M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 × 10⁻⁴ M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10⁻⁴ M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.     

Differentiation between the urban mosquito Culex pipiens pipiens F. molestus and Culex torrentium (Diptera: Culicidae) by the molecular genetic methods

The molecular characters suitable for the differentiation between the autogenous form of Culex pipiens pipiens (the urban mosquito) and the species Culex torrentium, which was previously identified erroneously as an anautogenous form of C. pipiens pipiens (Shaikevich, Vinogradova, 2004), are given. These mosquitoes differ in the infection by the endosymbiotic bacteria Wolbachia pipientis, 6 transitions among 247 nucleotide sequences of 3' part of the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA), in the mtDNA fragments resulted from the restriction analysis, and in the lengths of second internal transcribed spacer (ITS2) sequences in the ribosomal DNA.     

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.     

Population of the mosquitoes Culex pipiens molestus (Diptera: Culicidae) in dwellings of Lensk city

Culex pipiens molestus has been distributed in Siberia and the Russian Far East since 80s of XX century. In early 90s, C. p. molestus reached South Yakutia (Nerungri city), and nowadays it occurs in South-West Yakutia (Lensk city). The number of breeding places with larvae and pupae of mosquitoes was not numerous in Lensk; among 56 examined houses, the places with larvae and pupae mosquitoes were registered in seven houses, while the adult mosquitoes occurred in the majority of the houses. The occurrence of larvae varied from 12.5 to 33.3%, occurrence of adults-from 12.5 to 100%. The coefficient of variation of larvae in the samples was equal to 0.92, and adults had the following coefficients: 0.84 in the houses without basement; 0.92 in the houses with dry basements; 1.05 in the houses with pools in basements. This data are referred to the casual type, it means that the distribution of mosquitoes in space is dispersed. Imago spread actively in stone house of Lensk that provides mosquitoes with new places of breeding, and supports the stability of population in new areas.     

Genetic differentiation of Culex pipiens (Diptera: Culicidae) in China

The population genetic structures of Culex pipiens Linnaeus were evaluated in China over a 2000 km transect that encompasses the two subspecies, C. p. pallens and C. p. quinquefasciatus. Four polymorphic allozyme loci were investigated in 1376 mosquitoes sampled from 20 populations across four provinces. These loci were not statistically dependent with no apparent heterozygote deficit or excess. On a regional scale (intra-province), a low (Fst=0.007-0.016) and significant genetic differentiation was found, with no clear geographical pattern. On a wider scale (inter-province), the genetic differentiation was higher (Fst=0.059), and an isolation by distance emerged. The results are compared with previous population genetic surveys of this mosquito species in different geographic areas over the world. The overall pattern suggests that Culex pipiens requires considerable distance (500-1000 km) to show isolation by distance, irrespective of the subspecies (C. p. pipiens, C. p. quinquefasciatus and C. p. pallens) or the geographic location.     

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.     

一个致倦库蚊杀虫剂敏感品系的筛选

化学防治是控制蚊虫传播疾病的主要方法, 抗性监测表明我国蚊虫已对有机磷、 有机氯、 氨基甲酸酯和拟除虫菊酯类杀虫剂产生了不同程度的抗性。蚊虫抗药性的分子机制主要包括靶标抗性和三大解毒酶家族带来的代谢抗性。筛选对杀虫剂敏感的品系是抗性监测和抗性机理研究必不可少的材料。本研究通过从一个致倦库蚊Culex pipiens quinquefasciatus野生种群筛选无乙酰胆碱酯酶G119S突变且具有低活性羧酸酯酶、 P450单加氧酶和谷胱甘肽-S-转移酶的单雌系, 建立了一个对杀虫剂敏感的致倦库蚊品系。该品系的羧酸酯酶活性是敏感品系S-lab的2.5倍, P450单加氧酶和谷胱甘肽-S-转移酶的活性与S-lab相当。生物测定表明, 与S-lab相比, 该品系对有机磷杀虫剂有低于2倍的抗性, 对氨基甲酸酯和拟除虫菊酯类杀虫剂没有抗性, 可以作为相对敏感品系用于抗性监测。     

The taxonomy and biology of two sympatric sibling species of Culex, C. pipiens and C. torrentium (Diptera, Culicidae)

The mosquito Culex torrentium Martini is a sibling species of C. pipiens L. and was first discovered in Britain in 1951. However, it is now evident that it was present in this country from at least 1900. Collections from a few southern localities in England indicate that the size of its population is about a third that of C. pipiens.
A comparative taxonomic study showed that neither the eggs nor larvae can be separated from those of C. pipiens , and that only in certain instances can the pupae of the two species be distinguished. Males are readily separated on the structure of the terminalia. Previously it was considered that C. torrentium could be distinguished by the presence of a pre-alar patch of scales, but it is shown that in a few specimens they are absent and in others they may become easily detached.
Larvae of both species frequently occur together in the same habitats, and no differences were observed in the biology of their immature stages. Serological tests showed that adults of both species feed on birds. Adult females of both species hibernate during the autumn, but whereas those of C. pipiens are common in man-made shelters, C. torrentium is absent.     

Larvicidal potency of selected xerophytic plant extracts on Culex pipiens (Diptera: Culicidae)

Chemical insecticides released into the environment may have adverse biological effects. Therefore, there is a need for ecofriendly insecticides for mosquito control. Xerophytic plant extracts that may provide more ecofriendly active component were evaluated against Culex pipiens 4th instars. Plant extracts prepared using different solvents with a Soxhlet apparatus and different concentrations were tested against Culex pipiens larvae. The effects were observed at 24 h and 72 h intervals and LD₅₀ and LD₉₀ values determined. Chloroform (CHCl₃) and ethyl acetate (EtOAc) extracts of Althaea ludwigii were the most effective against Cx. pipiens 4^th instars, but were highly dependent on extract concentrations and exposure time. Results suggest that A. ludwigii extracts contain bioactive compounds, such as phenols and saponins, that may provide effective Cx. pipiens larval control. However, the extract was found to be toxic to zebrafish larvae, and may be toxic to other aquatic fauna. Further studies to determine the active components and toxicity to other fauna are needed.     

41种植物精油对淡色库蚊的熏蒸活性

为发现具有较高杀蚊活性的精油, 推动植物精油类卫生杀虫剂的创制, 本研究采用三角瓶熏蒸法测定了17科41种植物精油对淡色库蚊Culex pipiens pallens雌成蚊的熏蒸活性, 并进一步采用密闭圆筒法验证其杀虫毒力大小。三角瓶熏蒸法测定表明, 在12 μL/L浓度下, 留兰香油（spearmint oil）、 薰衣草油（lavender oil）、 山苍子油（Litsea cubeba oil）等26种精油均有较强的熏蒸作用, KT₅₀小于15 min。该26种精油用密闭圆筒熏蒸法进一步测定表明, 在浓度为10 μL/L时, 冬青油（wintergreen oil）、 艾叶油（blumea oil）、 薄荷油（peppermint oil）、 桉叶油（Eucalyptus robusta oil）和蓝桉油（E. globulus oil）等植物精油熏蒸活性较强, 其KT₅₀分别为16.91, 21.20, 22.57, 18.43和19.48 min。结果证明冬青油、 艾叶油、 薄荷油、 桉叶油和蓝桉油等5种精油对淡色库蚊具有较强的熏蒸活性, 具备作为淡色库蚊防控剂开发的潜力, 值得进一步研究。     

Cultivation of the mosquito Culex pipiens pipiens f. molestus (Diptera, Culicidae) without blood feeding

Optimal photoperiod and temperature conditions for rearing of the mosquito Culex pipiens pipiens f. molestus without blood feeding (through autogenous eggs) were experimentally substantiated. This method may be used in order to obtain relatively small quantities of mosquitoes or for preservation of a laboratory colony. Optimal conditions of rearing are the short-day photoperiod (12 h of light per day) at 20 degrees C, when 87 % of the females produce autogenous eggs rafts consisting of 52 eggs on the average. It should be emphasized that larval food must be adequate and abundant, because the quality and quantity of it has a pronounced effect on the autogeny rate.     

Seasonal dynamics of oviposition in non-autogenous population of Culex pipiens pipiens (Diptera: Culicidae) in the Leningrad province

Seasonal patterns of oviposition in a synanthropic unautogenous population of Culex pipiens pipiens mosquitoes from 69 suburban settlements (60 degrees 30' N, 30 degrees E') of the Leningrad province were studied during the period 1998-2002 years. The numbers of egg rafts laid in six artificial pools (barrels) were counted every day; altogether 692 rafts were collected. The general patterns of oviposition activity were similar for all studied years, in spite of their differences in the summer temperature regimes. The first peak of oviposition was observed during the second decade of July, the second peak--during the third decade of July--the first decade of August. The first and second summer generations of mosquitoes were responsible for these oviposition peaks, whereas the third generation completely entered the reproductive diapause. Thus, the oviposition activity was successfully used for populational monitoring of C. p. pipiens, that was for the first time recommended by Madder and co-authors (1980), taking into consideration the simplicity and economy of this technique. The differences in the attractiveness of distinct reservoirs for oviposition were recorded. The females preferred the barrels located on open sun-lit space and waters with organic pollution settled by mosquito larvae. Windly and rainy weather and also low (below 10 degrees C) night temperatures suppressed mosquito oviposition.     

Monitoring of Insecticide Resistance in Field-Collected Populations of Culex pipiens pallens (Diptera: Culicidae)

A susceptible strain and six field populations of the northern house mosquito, Culex pipiens pallens Coquillett, were collected in Seoul, Paju, Daejeon, Heongseong, Jinhae, and Buan, and tested for their susceptibility to seven insecticides by topical application. In tests with female Cx. pipiens pallens, marked regional variations of insecticide susceptibility were observed. Five field-collected populations of Cx. pipiens pallens, except for the Seoul colony, exhibitedhightoveryhighlevelsofresistanceto cyfluthrin [resistance ratio (RR), 96–554] and deltamethrin (RR, 192–959). Low to moderate levels of resistance were observed in bendiocarb (RR, 14–46), chlorpyrifos [RR, 7–38; exceptions: Buan (RR, 63) and Jinhae (RR, 80) colonies], λ-cyhalothrin (RR, 5–23), S-bioallethrin (RR, 1–47), and permethrin [RR, 2–27; exception: Jinhae colony (RR, 247)]. These results indicate that careful selection and rotational use of these insecticides should result in continued satisfactory control against field populations of Cx. pipiens pallens.     

Effects of blood meal source on the reproduction of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Culex pipiens quinquefasciatus were fed blood meals from a live chicken (LC), chicken blood in Alsever's (AC) solution, defibrinated bovine blood (DB), or bovine blood in citrate (CB) and incubated at 28° C. The effects of different blood meal sources were evaluated with respect to rates of blood feeding and reproduction (i.e., fecundity and fertility) over two gonotrophic cycles. Mosquitoes that fed on the first blood meal were subjected to a second blood meal as follows (first blood meal / second blood meal): LC/LC, LC/DB, DB/DB, CB/CB, AC/AC. Fecundity and fertility of Cx. p. quinquefasciatus were significantly (P < 0.05) greater in mosquitoes fed LC blood; however, fecundity and fertility in different treatment groups varied by gonotrophic cycle. These results contribute to our understanding of the impact of blood meal source on feeding and reproduction in Cx. p. quinquefasciatus. The potential impacts of blood meal source on virus transmission experiments are discussed.     

Bioassay of some Egyptian isolates of Bacillus thuringiensis against Culex pipiens (Diptera: Culicidae)

Some Egyptian isolates of Bacillus thuringiensis (BT) were grown on economic media contain 4% of fodder yeast in tap water and incubated under shaking conditions for four days. The biological activities of these isolates against Culex pipiens (Diptera: Culicidae) were carried out to determine their effectiveness against field and laboratory strains of 3rd larval instar. All isolates of BT were more pathogenic to laboratory strain. causing up to 84% larval mortality. The insecticidal activities of these isolates were extended to the pupal stage causing a significant effect on pupal mortality in both strains tested. A pronounced effect on adult emergence was noticed with remarkable adult malformations especially in the case of the isolate No. 2. The reproductivety of females was affected significantly by all isolates applied.     

Mosquito Distribution in Northwestern Russia: Species of the Genera Anopheles Meigen,Coquillettidia Dyar,Culex L., and Culiseta Felt (Diptera,Culicidae)

Entomological Review - Records of 16 species of the mosquito genera Anopheles, Coquillettidia, Culex, and Culiseta (Diptera: Culicidae) in Northwestern Russia are mapped. The values of the sum of...