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.     

Swarming behaviour of the mosquito Culex pipiens quinquefasciatus: a quantitative analysis

ABSTRACT. Analysis of video recordings of Culex pipiens quinquefasciatus Say swarms showed that the overall swarming patterns of males and virgin females are similar, even though the short-term characteristics of their flight, such as speed and turning angle, differ. This suggests they have the same response to the visual cue of the swarm 'marker'. The swarming pattern of an individual consists of elliptical loops which, in the short-term, define an area smaller than the swarm as a whole. The foci of these ellipses drift gradually with respect to the marker; individuals do not seem to have preferred positions within the swarm, but drift at random. Male mosquitoes identify and locate females by the sound of their wing-beats. Diffuse sound at the wing-beat frequency of female C.p. quinquefasciatus (500–600 Hz) caused males within the swarm to slow down significantly. Their turning behaviour remained unchanged, so the ellipses decreased in size. The swarm as a whole collapsed into a smaller volume, centred over the marker. Apparently, the response of males to sound consists of at least two parts: initially they alter their flight speed, and then alter their turning behaviour once they have located the source of the sound.     

Cytoplasmic incompatibilities in the mosquito Culex pipiens: How to explain a cytotype polymorphism?*

Although cytoplasmic incompatibilities have been used as a means of eradicating the mosquito Culex pipiens, the population dynamics of these sterilities in relation to the coexistence of multiple incompatible cytotypes in a single area has not been investigated, except in the case of two unidirectionally incompatible cytotypes. An analytical model of the evolution of n cytotypes in an infinite panmictic population has been developed in order to investigate polymorphic equilibrium. A necessary criterion for the stability of such an equilibrium is established; it is shown that a stable polymorphism cannot exist between incompatible cytotypes. This result is discussed in the light of population dynamics and genetics of Culex pipiens, and of our present knowledge on incompatibilities. The consequences of a geographic structuring and of homogamy are considered. A careful reconsideration of previous experimental results disclosed probable nuclear effects and a serious experimental weakness: with the common procedure of backcrossing hybrid females to males of constant genotype it is not possible to rule out probable nuclear effects with paternal expression. It is concluded that incompatibilities in Culex pipiens may have a nuclear-cytoplasmic determinism.     

Olfactory memory in the mosquito Culex quinquefasciatus

The cosmotropical urban mosquito Culex quinquefasciatus Say (Diptera: Culicidae) uses chemical cues to locate suitable water pools for oviposition. Although gravid females are innately attracted to or repelled by certain compounds, this study found that an individual mosquito's preferences for these odours could be altered greatly by prior experience. Mosquitoes reared in water containing skatole, at a level normally repellent to ovipositing females, preferred to oviposit in water containing that compound rather than in water with an otherwise attractive odour compound (P-cresol). This behaviour occurred regardless of whether mosquitoes were tested individually or in groups of up to 50 per cage. The F1 progeny of conditioned mosquitoes did not exhibit the parental preference, but were as susceptible to conditioning as their parents. Moreover, rearing mosquitoes in infusions of hay or animal (guinea-pig) faeces produced a similar although less dramatic change, such that the innate propensity for hay infusion could be cancelled by rearing in guinea-pig faeces infusion. The results demonstrated a change in odour preference by Cx. quinquefasciatus following exposure to the odour during development or pupal eclosion, suggesting that some form of larval conditioning or early adult imprinting occurred. Precisely when that conditioning occurred remains to be determined.     

Crossing type variability associated with cytoplasmic incompatibility in Australian populations of the mosquito Culex quinquefasciatus Say

An analysis of cytoplasmic crossing type variation in Australian populations of Culex quinquefasciatus, a member of the Culex pipiens complex of mosquitoes, revealed high levels of variability causing partial incompatibility between natural populations. Segregating crossing types were commonly found together within sampled sites. No correlation was evident between similarity of crossing type and environmental parameters of the sites, nor distance between sites. The nature of the observed variation did not support the hypothesis of paternally expressed nuclear 'restorer' genes. Such high levels of crossing type variation would be likely to impede attempts to control populations of the Culex pipiens complex using cytoplasmic incompatibility.     

Modification of female circadian flight-activity by a male accessory gland pheromone in the mosquito, Culex pipiens quinquefasciatus

ABSTRACT. The pattern of circadian flight activity in female Culex pipiens quinquefasciatus was modified after insemination or injection with an extract of male accessory glands. The changes were linked with an apparent alteration in the timing of one of the major components of the rhythm. In LD 12:12, virgin females were highly active at 'dusk' and 'dawn' and in the latter part of the dark phase; inseminated females were less active at 'dusk' and 'dawn' and were active in the first half of the dark phase. In DD, there were two peaks in each cycle, the initial (evening) peak occurring at the same time after all treatments; the peaks were approximately 12 h apart in virgin females, but only 6–7 h apart in females which had been inseminated or injected with accessory gland extract. The accessory gland substance (pheromone) also appeared to cause a progressive increase in activity. These induced changes are consistent with a switch to host-seeking behaviour, which, under natural conditions, leads to a peak of biting in the middle of the night.     

Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens.

Reproductive incompatibilities called cytoplasmic incompatibilities are known to affect a large number of arthropod species and are mediated by Wolbachia, a maternally transmitted microorganism. The crossing relationships between strains of potential hosts define their incompatibility types and it is generally assumed that differences between strains of Wolbachia induce different crossing types. Among all the described host species, the mosquito, Culex pipiens, displays the greatest variability of cytoplasmic incompatibility crossing types. We analysed mitochondrial and bacterial DNA variability in Culex pipiens in order to investigate some possible causes of incompatibility crossing type variability. We sequenced fragments of the ftsZ gene, and the A + T-rich control region of the mtDNA. We also sequenced the second subunit of the mitochondrial cytochrome oxidase (COII) gene, in Culex pipiens and a closely related species, C. torrentium, in order to verify the usefulness of the A + T-rich region for the present purposes. No variability was found in the Wolbachia ftsZ gene fragment, and very limited variation of the mitochondrial marker whatever the compatibility type or the origin of the host. A low variability was found in the A + T-rich region and comparison of divergence of the A + T-rich region and COII gene between C. pipiens and C. torrentium did not reveal any special constraints affecting this region. In contrast to observations in other host species, variability of incompatibility crossing types is not due to multiple infections by distantly related Wolbachia strains.     

Salivary gland proteins of the mosquito Culex quinquefasciatus

Several properties of the salivary glands of Culex quinquefasciatus mosquitoes were analysed. The amount of protein in female salivary glands increased from 0.26 microg on day one after emergence to about 1.4 microg on day seven. The major polypeptides found in the female salivary glands had molecular weights of 35.7, 28.3, and 20.5 kDa. Antibodies produced by mice immunized by bites of Culex quinquefasciatus female mosquitoes reacted with the 35.7 and 28.3 kDa polypeptides, showing that these molecules were secreted by mosquitoes during blood feeding. The salivary glands of C. Quinquefasciatus females displayed the same morphological and biochemical organization as that of Aedes aegypti mosquitoes, accumulating apyrase in the distal portions and alpha-glucosidase in the proximal portions of the gland. Arch.     

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.     

Composition of fatty acids in Culex pipiens quinquefasciatus: a developmental study

In anticipation of antioxidants affecting the lipid content, uptake, or metabolic biosynthesis within the mosquito Culex pipiens quinquefasciatus, a preliminary study was conducted to determine the background quantities of total fatty acids found in the 4th instar larvae, 1-day and 2-day ‘pupae’ (pharate adults) reared on two different diets. It was found that the preponderant fatty acid detected was palmitoleic acid (16:1), which accounted for 40% of all fatty acids present. The greatest amount of saturated fatty acid was palmitic acid (16:0) which constituted 82% of the saturated fatty acids and 21% of the total fatty acids. About 16% of the total percentage of fatty acids was myristoleic acid (14:1). It was 21% of the unsaturated fatty acids. The approximate ratio in all categories of unsaturated-saturated fatty acids was 3:1. There were no abrupt fluctuations in percentage composition of fatty acids over the developmental period studied, nor did the ratio of unsaturated-saturated change appreciably.     

The etiological agent of cytoplasmic incompatibility in Culex pipiens

All individuals of Culex pipiens (wide sense) which have been examined were infected with Wolbachia pipientis. Larvae reared in tetracycline were freed of these symbiotes and remained free (aposymbiotic) in future generations. When males were freed of their symbiotes, they no longer displayed incompatibility. Aposymbiotic males were compatible with all females, whether infected or not. Aposymbiotic females, on the other hand, laid fertile eggs after mating with aposymbiotic males, but not after mating with normal males. Most eggs laid by aposymbiotic females after mating with normal males showed no development at all, even though the females had been inseminated.     

Cytoplasmic factors in the gonads of Culex pipiens complex mosquitoes

Electron microscopic examination of the gonads of six species of the $\text{Culex pipiens}$ complex revealed rickettsia-like, intracellular symbionts in $\text{C.pipiens}$, $\text{C.molestus}$, $\text{C.pallens}$ and $\text{C.fatigans}$.¹ In the two endemic Australian members, $\text{C.a}$$\text{C.globocoxitus}$, no rickettsia-like symbionts were found, but virus-like particles were seen in the cytoplasm of the ovaries and certain associated tissues.     

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.     

Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum

The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus by the fungus Lagenidium giganteum has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters investigated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that the rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.     

Autogeny induced by a nonsteroidal ecdysone agonist tebufenozide in the mosquito Culex quinquefasciatus

The effects of tebufenozide, a nonsteroidal agonist of ecdysone, on egg development and reproduction in the anautogenic mosquito Culex quinquefasciatus Say (Culicidae, Diptera) are examined. The study population originates from Hyderabad, India. A dose‐dependent toxicity is observed when varying concentrations (10 µg L^?1 to 10 pg L^?1 ) of a flowable formulation of tebufenozide, known as Mimic 2F, are applied to first‐instar larvae in water. The median lethal dose (LC₅₀) against larvae is very low at 1.29 ng L^?1 H₂O. Doses below the LC₅₀ induce vitellogenesis in the females that subsequently emerge, and these females lay eggs autogenically (without blood feeding). Autogenous egg clutches are laid through three ovarian cycles. The number of eggs laid in each clutch is lower compared with an anautogenous clutch. An autogenic population has been maintained for over 15 generations after the primary induction, during which time the females have not been allowed to feed on blood. By comparison, doses higher than the LC₅₀ inhibit ovarian development. There is a gradual degeneration of the ovaries, and the females die approximately 20 days after imaginal ecdysis without commencing vitellogenesis. The results are discussed in relation to the endocrine and nutritional regulation of vitellogenesis in C. quinquefasciatus, and the possibility of using tebufenozide to regulate populations of some species of 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.