Ecdysteroid titer and oocyte growth in the northern house mosquito, Culex pipiens L

In an anautogenous strain of the northern house mosquito, Culex pipiens, the ovaries reached the resting stage (follicle length = 90 microns) three days after adult emergence. Follicle length increased from 90 to 550 microns between 0 and 60 hr after a blood meal. Total ecdysteroids reached a peak at 400 fmol/insect at 36 hr after a blood meal then declined rapidly. The ratio of 20-hydroxyecdysone to ecdysone increased in conjunction with the total ecdysteroid level. Oocyte growth beyond the resting stage and initiation of vitellogenesis was dependent on a head factor which was released within 4-8 min of the start of the blood meal.     

Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.     

Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization

In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including six genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and two upregulated throughout diapause. In addition, two genes are diapause downregulated and five remain unchanged during diapause. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions.     

Energy metabolism during diapause in Culex pipiens mosquitoes

Diapause in overwintering adult female Culex pipiens mosquitoes plays an important role in the transmission of West Nile and other encephalitis-inducing flaviviruses. To investigate the dynamic metabolic processes that control Cx. pipiens diapause, we used radioactive tracer techniques with [¹⁴C]-glucose to investigate the metabolic fate and flux of glucose in adult mosquitoes reared under diapause (18 °C, short day) and non-diapause (27 °C, long day) conditions. We found that by 72 h post-¹⁴C-labeling of 1-day-old mosquitoes, the diapause-destined mosquitoes had converted 46% more ¹⁴C-labled glucose into ¹⁴C-labled lipid than mosquitoes reared under non-diapausing conditions. When 5-day-old mosquitoes were fed [¹⁴C]-glucose, and then switched to water only, the non-diapausing mosquitoes oxidized nearly three times more ¹⁴C-labled glycogen and lipid by day 7 than diapausing-mosquitoes. This increased energy expenditure in non-diapausing mosquitoes is most likely due to temperature- and light-dependent increases in the basal metabolic rate. Amongst the diapausing-mosquitoes we analyzed over a subsequent 7-week period, we found that the amount of ¹⁴C-labeled glycogen decreased steadily for the first month of diapause, whereas, ¹⁴C-labeled-lipid levels were not significantly decreased until after day 35 of diapause, indicating that flux through glycogenolysis is higher than lipolysis during the first month of diapause. Lastly, our analysis revealed that 38% of the initial ¹⁴C-labled lipid that was synthesized during the adult pre-diapause phase was still present following the first gonotrophic cycle. About 33% of this remaining ¹⁴C-labeled lipid was localized to the newly developed eggs, suggesting that lipid sparing processes during a minimal 7-week long diapause may enhance egg production.     

Microsatellite loci from the northern house mosquito (Culex pipiens), a principal vector of West Nile virus in North America

Microsatellites were isolated and characterized in the northern house mosquito, Culex pipiens, a widespread pest species and important vector of diseases such as West Nile virus. An enrichment protocol yielded 150 positive clones. We designed primers to amplify 17 unique (GT)_n microsatellites, eight of which amplified cleanly and were polymorphic. A survey of 29 individuals showed that these loci are highly variable with the number of alleles ranging from seven to 19 and expected heterozygosity ranging from 0.66 to 0.93. These markers will be useful for studies of population structure and intraspecific variation in epidemiological characteristics of Cx. pipiens.     

Variability and expression of ankyrin domain genes in Wolbachia variants infecting the mosquito Culex pipiens

Wolbachia strains are maternally inherited endosymbiotic bacteria that infect many arthropod species and have evolved several different ways of manipulating their hosts, the most frequent way being cytoplasmic incompatibility (CI). CI leads to embryo death in crosses between infected males and uninfected females as well as in crosses between individuals infected by incompatible Wolbachia strains. The mosquito Culex pipiens exhibits the highest crossing type variability reported so far. Our crossing data support the notion that CI might be driven by at least two distinct genetic units that control the CI functions independently in males and females. Although the molecular basis of CI remains unknown, proteins with ankyrin (ANK) domains represent promising candidates since they might interact with a wide range of host proteins. Here we searched for sequence variability in the 58 ANK genes carried in the genomes of Wolbachia variants infecting Culex pipiens. Only five ANK genes were polymorphic in the genomes of incompatible Wolbachia variants, and none correlated with the CI pattern obtained with 15 mosquito strains (representing 14 Wolbachia variants). Further analysis of ANK gene expression evidenced host- and sex-dependent variations, which did not improve the correlation. Taken together, these data do not support the direct implication of ANK genes in CI determinism.     

Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito

The α-proteobacteria Wolbachia are among the most common intracellular bacteria and have recently emerged as important drivers of arthropod biology. Wolbachia commonly act as reproductive parasites in arthropods by inducing cytoplasmic incompatibility (CI), a type of conditional sterility between hosts harboring incompatible infections. In this study, we examined the evolutionary histories of Wolbachia infections, known as wPip, in the common house mosquito Culex pipiens, which exhibits the greatest variation in CI crossing patterns observed in any insect. We first investigated a panel of 20 wPip strains for their genetic diversity through a multilocus scheme combining 13 Wolbachia genes. Because Wolbachia depend primarily on maternal transmission for spreading within arthropod populations, we also studied the variability in the coinherited Cx. pipiens mitochondria. In total, we identified 14 wPip haplotypes, which all share a monophyletic origin and clearly cluster into five distinct wPip groups. The diversity of Cx. pipiens mitochondria was extremely reduced, which is likely a consequence of cytoplasmic hitchhiking driven by a unique and recent Wolbachia invasion. Phylogenetic evidence indicates that wPip infections and mitochondrial DNA have codiverged through stable cotransmission within the cytoplasm and shows that a rapid diversification of wPip has occurred. The observed pattern demonstrates that a considerable degree of Wolbachia diversity can evolve within a single host species over short evolutionary periods. In addition, multiple signatures of recombination were found in most wPip genomic regions, leading us to conclude that the mosaic nature of wPip genomes may play a key role in their evolution.     

Meiosis in translocation heterozygotes in the mosquito Culex pipiens

Adult Culex pipiens males irradiated with both X-rays and neutrons were crossed to untreated females and F₁-egg rafts were checked for dominant lethality. F₁-progenies were outcrossed with normal individuals in order to obtain lines with inherited semisterility. From a total of 120 lines that showed a certain amount of sterility 12 lines were studied cytologically. 10 lines showed reciprocal chromosome exchanges.—At late pachytene and diplotene cross configurations with large asynaptic regions at the center of the cross are obligatory. Bivalents, chains of three, chains of four, and ring configurations are present at metaphase and anaphase I. The different frequencies of the occurrence of such multiples are dependent on the chromosomes involved in the exchange, the length of the pairing segments and the chiasma frequencies in these segments. Chiasma frequency in the interstitial segments is reduced by means of chiasma interference over the centromere and by asynapsis near the breakage points. — Alternate, adjacent-1- and adjacent-2-distributions are present to a different extent. Alternate distribution is most, adjacent-2-distribution least frequent. — The role of translocations and the probability of their becoming effective in pest eradication programs is discussed.     

Population genetics of insecticide resistance in the mosquito Culex pipiens

Thirty years of control of the mosquito Culex pipiens using organophosphate insecticides (OP) has selected for OP-resistance alleles on a world-wide scale. As reviewed here, studies at the levels of gene and population allow identification of the main forces driving this process of adaptation. Three loci are involved in OP-resistance in C. pipiens. For two of these, adaptive mutations were found to be rare events, such that the ubiquitous distribution of certain resistance alleles could only be explained as deriving from a single origin by mutation followed by extensive migration. Population structure analyses confirmed that long-distance migration is frequent. Thus, different resistance alleles could accumulate and compete within populations soon after their origin by mutation. The different selection pressures acting on these alleles, i.e. their selective advantage in the presence of OP and their disadvantage (resistance cost) in absence of OP, were also analysed. Substantial differences in resistance cost among alleles present within the Mediterranean area were discovered. Long-term surveys of Mediterranean populations confirmed the pivotal importance of resistance cost in shaping the evolution of this adaptive polymorphism. Some hypotheses on the functional links between the nature of the initial mutation events and the subsequent evolution of polymorphism are discussed.     

A potential role for ribosomal protein S2 in the gene network regulating reproductive diapause in the mosquito Culex pipiens

We propose that a shut-down in expression of ribosomal protein S2 (rpS2) contributes to regulation of diapause in adult females of Culex pipiens. While this gene is expressed continuously in nondiapausing females reared under long-day conditions, it is strongly down-regulated 5–18 days after adult eclosion in females reared under the short-day conditions that induce diapause. The possibility that this shut-down in expression of rpS2 contributes to the arrest in ovarian development characteristic of diapause is bolstered by the diapause-like arrest in follicle growth observed when nondiapausing females are injected with dsrpS2. A control gene encoding another ribosomal protein, L19, is expressed equally in nondiapausing and diapausing females, and RNA interference directed against rpL19 did not arrest follicle growth, thus indicating that the response we observed in knocking down expression of rpS2 is not common to all ribosomal proteins. Diapause in C. pipiens is readily terminated with juvenile hormone (JH), and in this study we demonstrated that an exogenous application of JHIII can rescue the arrest in follicle growth caused by dsrpS2. Together, these results suggest that rpS2 plays a critical role in arresting the ovarian development associated with diapause in this mosquito.     

Tracking factors modulating cytoplasmic incompatibilities in the mosquito Culex pipiens

Wolbachia are maternally inherited endosymbiotic bacteria that infect many arthropod species and may induce cytoplasmic incompatibility (CI), resulting in abortive embryonic development. One Wolbachia host, Culex pipiens complex mosquitoes, displays high levels of variability in both CI crossing types (cytotypes) and DNA markers. We report here an analysis of 14 mosquito strains, containing 13 Wolbachia variants, and with 13 different cytotypes. Cytotypes were Wolbachia-dependent, as antibiotic treatment rendered all strains tested compatible. Cytotype distributions were independent of geographical distance between sampling sites and host subspecies, suggesting that Wolbachia does not promote a reproductive isolation depending on these parameters. Backcross analysis demonstrated a mild restoring effect of the nuclear genome, indicating that CI is mostly cytoplasmically determined for some crosses. No correlation was found between the phenotypic and genotypic variability of 16 WO prophage and transposon markers, except for the WO prophage Gp15 gene, which encodes a protein similar to a bacterial virulence factor. However, Gp15 is partially correlated with CI expression, suggesting that it could be just linked to a CI gene.     

Coupled oscillators controlling circadian flight activity in the mosquito, Culex pipiens quinquefasciatus

ABSTRACT. In both virgin and inseminated female Culex pipiens quinquefasciatus Say, circadian flight-activity has two major components: evening (E) and morning (M) in virgins, and evening (E) and night (N) in inseminated females. These components probably represent the activity of constituent oscillators of the underlying pacemaker system. In DD (constant dark) the E and M peaks are approximately 12 h apart in virgins, the E and N peaks 6–7 h apart in inseminated females. Entrainment to regimes between LD 6:18 and 18:6 appears to have only small effects on the relative position of these components, and after a change to DD they quickly relax towards a common phase-relation. Entrainment to LD 12:12 followed by release into DD or constant dim light (intensities 0.005-5 be) showed that light has a differential effect on the components, initially increasing the period of the E component more than that of the apparently more stable M and N components. Thus with increasing light intensity a bimodal cycle fuses into a unimodal cycle. Light also affected the level of activity, causing big increases in the activity of both virgin and inseminated females at 0.05 lx, but depressing activity, at least initially, at 5 lx. These results indicate that, under natural conditions, moonlight may have big effects, both on the level of activity and on the underlying circadian pacemaker.