Molecular genetic methods for the identification of the urban mosquito Culex pipiens pipiens F. molestus (Diptera, Culicidae)

Molecular genetic methods for the identification of two ecotypes, or forms (pipiens and molestus) of the Culex pipiens pipiens mosquitoes, which are known as active bloodsuckers and vectors of various agents of diseases, are proposed. For the DNA analysis, two populations of the urban mosquitoes (the molestus ecotype) from St. Petersburg and Moscow and two populations from the overground reservoirs in the Leningrad Province and neighboring areas of Moscow (the pipiens ecotype) have been studied. These ecotypes differ by six transitions among 247 nucleotide sequences of 3' part of the cytochrome oxidase subunit I (COI) gene of mitochondrial DNA (mtDNA), by the mtDNA fragments resulted from the restriction analysis and by the lengths of second internal transcribed spacer (ITS2) sequences in the ribosomal DNA.     

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.     

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.     

致倦库蚊主要组织结构的石蜡切片观察

【目的】蚊虫是传播人类多种疾病的重要媒介害虫, 对其组织形态学的认识是开展众多领域研究的基础。本文通过研究致倦库蚊Culex pipiens quinquefasciatus成虫组织结构及形态, 为媒介蚊虫的抗药性研究及有效防治提供基础材料。【方法】采用改进的石蜡切片法和HE染色, 结合活体内脏器官解剖及光学显微镜观察, 从形态学和组织学水平对致倦库蚊组织结构做详尽展示。【结果】获得结构完整、 染色清晰、 定位准确的消化排泄系统、 生殖系统、 神经系统、 呼吸系统等HE染色石蜡切片。【结论】探讨了改进制片和染色过程中一些步骤及注意事项。研究结果为利用原位杂交、 免疫组化等方法研究蚊虫体内抗药性基因的准确定位及基因功能分析提供了可靠的基础。     

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.     

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.     

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae)

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.     

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.     

Spacing behaviour in resting Culex pipiens (Diptera, Culicidae): a computer modelling approach

Abstract. We compared two models based on Monte Carlo simulations with the spacing patterns of resting adults of the mosquito Cx pipiens L. (form molestus ) (Diptera, Culicidae). The comparison with the first model which assumed a distribution of complete spatial randomness showed that there was a tendency for mosquitoes to repel other mosquitoes which were too close, resulting in high local regularity. The second model took this repulsion into account. A final test of global patchiness using Model 2 indicated that the spacing behaviour of Cx pipiens was not sufficiently described by local regularity only, but also required a gregarious element. This supports the hypothesis that the molestus form of Cx pipiens shows spaced-out gregariousness in the sense of Kennedy & Crawley (1967).     

ٍSome biologically active microorganisms have the potential to suppress mosquito larvae (Culex pipiens,Diptera: Culicidae)

Malaria is a disease caused by protozoan species of the genus Plasmodium. It is widespread and becoming a challenge in several African countries in the tropical and subtropical regions. In 2010, a report was published showing that over 1.2 million death cases were occurred globally due to malaria in just one year. The transmission of the disease from one person to another occurs via the bite of the Anopheles female. It is known that Plasmodium ovale, P. vivax, P. malariae, P. falciparum, and P. knowlesi are the highly infective malaria species. The problem of this disease is the absence of any effective medical treatment or vaccine, making the mosquito control is the only feasible way for disease prevention. Pesticides are currently the most widely used method for mosquito control, despite its well-known negative effects, including health hazards on human, the increasing insecticidal resistance, and the negative impact on the environment and beneficial organisms. Biological control (also called: biocontrol) of insects has been a promising method to overcome the negative effects of using chemical insecticides, as it depends on just using the natural enemies of pests to either minimize their populations or eradicate them. This article provides an overview of the recent and effective biological means to control malaria, such as bacteria, fungi, viruses, larvivorous fish, toxorhynchites larva and nematodes. In addition, the importance, advantages, and disadvantages of the biocontrol methods will be discussed in comparison with the traditionally used chemical methods of malaria control with special reference to nanotechnology as a novel method for insects’ control.     

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus (Diptera: Culicidae) in Petrozavodsk City

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus in Petrozavodsk city was studied for the first time. Immature mosquito stages were found in 21 of 68 basements flooded with polluted waters, the occurrence of the mosquitoes varied in different districts of the sity. The mean siphonal index of the mosquito larvae from 6 underground populations varied from 3.8 up to 4.2, i. e. within the range typical for C. p. p. f. molestus.     

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。     

Evidence of natural Wolbachia infections and molecular identification of field populations of Culex pipiens complex (Diptera: Culicidae) mosquitoes in western Turkey

Establishing reliable risk projection information about the distribution pattern of members of the Culex pipiens complex is of particular interest, as these mosquitoes are competent vectors for certain disease‐causing pathogens. Wolbachia, a maternally inherited bacterial symbiont, are distributed in various arthropod species and can induce cytoplasmic incompatibility, i.e., reduced egg hatch, in certain crosses. It is being considered as a tool for population control of mosquito disease vectors. The Aegean region is characterized by highly populated, rural, and agricultural areas and is also on the route of the migratory birds. In this study, a fragment of the 658 bp of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which includes the barcode region, was employed to differentiate Cx. pipiens complex species found in this region. Moreover, for the first time, the prevalence of Wolbachia endobacteria in these natural populations was examined using PCR amplification of a specific wsp gene. Our results revealed a widespread (more than 90%, n=121) presence of the highly efficient West Nile virus vector Cx. quinquefasciatus in the region. We also found that Wolbachia infection is widespread; the average prevalence was 62% in populations throughout the region. This study provided valuable information about the composition of Cx. pipiens complex mosquitoes and the prevalence of Wolbachia infection in these populations in the Aegean region. This information will be helpful in tracking mosquito‐borne diseases and designing and implementing Wolbachia‐based control strategies in the region.     

Characterization of Culex pipiens Complex (Diptera: Culicidae) Populations in Colorado,USA Using Microsatellites

Mosquitoes such as those in the Culex pipiens complex are important vectors of disease. This study was conducted to genetically characterize Cx. pipiens complex populations in the state of Colorado, USA, and to determine the number of genetic clusters represented by the data. Thirteen populations located among four major river basins were sampled (n = 597 individuals) using a panel of 14 microsatellites. The lowest-elevation sites had the highest Expected Heterozygosity (H_E) values (range 0.54–0.65). AMOVA results indicated the presence of statistically significant amounts of variation within each level when populations were analyzed as one group or when they were grouped either by river basin or by their position on the east or west side of the Rocky Mountains. Most pairwise F_ST values were significant via permutation test (range 0–0.10), with the highest values from comparisons with Lamar, in southeast CO. A neighbor joining tree based on Cavalli–Sforza and Edwards’s chord distances was consistent with the geographic locations of populations, as well as with the AMOVA results. There was a significant isolation by distance effect, and the cluster analysis resolved five groups. Individuals were also assayed with an additional microsatellite marker, Cxpq78, proposed to be monomorphic in Cx. pipiens but polymorphic in the closely related but biologically distinct species Cx. quinquefasciatus. Low frequencies (≤3%) of Cx. quinquefasciatus alleles for this marker were noted, and mostly confined to populations along the Interstate 25 corridor. Pueblo was distinct in that it had 10% Cx. quinquefasciatus alleles, mostly of one allele size. The degree of population genetic structure observed in this study is in contrast with that of Cx. tarsalis, the other major vector of WNV in the western U.S., and likely reflects the two species’ different dispersal strategies.     

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal

Background  

Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group.     

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.