Gene expression profiles of the Southern house mosquito Culex quinquefasciatus during exposure to permethrin

Insecticide resistance is a major obstacle to the management of disease‐vectoring mosquitoes worldwide. The genetic changes and detoxification genes involved in insecticide resistance have been extensively studied in populations of insecticide‐resistant mosquitoes, however few studies have focused on the resistance genes upregulated upon insecticide exposure and the possible regulation pathways involved in insecticide resistance. To characterize the changes in gene expression during insecticide exposure, and to investigate the possible connection of known regulation pathways with insecticide resistance, we conducted RNA‐Seq analysis of a highly permethrin‐resistant strain of Culex quinquefasciatus following permethrin exposure. Gene expression profiles revealed a total of 224 upregulated and 146 downregulated genes when compared to a blank acetone carrier treated control, respectively, suggesting that there were multiple, but specific genes involved in permethrin resistance. Functional enrichment analysis showed that the upregulated genes contained multiple detoxification genes including a glutathione S‐transferase and multiple cytochrome P450 genes, as well as several immune‐related genes, while the downregulated genes consisted primarily of proteases and carbohydrate metabolism and transport. Further analysis showed that permethrin exposure resulted in a decrease in the expression of serum storage proteins and likely represented a delay in the development of the fourth instar possibly due to a decrease in feeding. This effect was more pronounced in an insecticide‐resistant strain than in an insecticide‐susceptible strain and may represent a behavioral mechanism of insecticide resistance in Culex mosquitoes.     

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.     

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.     

Assessing natural infection with Zika virus in the southern house mosquito,Culex quinquefasciatus,during 2016 in Puerto Rico

The epidemic of Zika in the Western hemisphere has led to intense investigations of all species important in the transmission of Zika virus (ZikV), including putative mosquito vectors. Although evidence points to Stegomyia (= Aedes) (Diptera: Culicidae) mosquitoes as the primary vectors in nature among humans, there remains the possibility that other common mosquito species may be implicated in the rapid spread of the virus. Herein, field‐caught Culex quinquefasciatus (Diptera: Culicidae) collected during June 2016 in different neighbourhoods in San Juan, Puerto Rico were examined for the presence of natural infection with ZikV. Stegomyia aegypti (= Aedes aegypti) from the same locations were also analysed. None of the Cx. quinquefasciatus tested showed natural infection for ZikV, whereas S. aegypti tested positive at seven sites. The present results suggest that Cx. quinquefasciatus was not involved in the transmission of ZikV in San Juan, Puerto Rico in 2016.     

Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus

Haematophagy, the utilization of blood as food, has evolved independently among insects such as mosquitoes, bedbugs, fleas, and others. Accordingly, several distinct biological adaptations have occurred in order to facilitate the finding, ingestion and digestion of blood from vertebrate sources. Although blood meals are essential for survival and reproduction of these insects, mechanical and chemical stresses are caused by the ingestion of a sizable meal (frequently twice or more times the weight of the insect) containing large amounts of cytotoxic molecules such as haem. Here we present data showing that the stresses caused by a blood meal induce cell death in the midgut epithelium of Culex quinquefasciatus mosquitoes. The process involves apoptosis, ejection of dead cells to the midgut lumen and differentiation of basal regenerative cells to replace the lost digestive cells. The basal cell differentiation in blood-fed mosquito midguts represents an additional mechanism by which insects cope with the stresses caused by blood meals. C. quinquefasciatus adult females are unable to replace lost cells following a third or fourth blood meal, which may have a significant impact on mosquito longevity, reproduction and vectorial capacity.     

Epoxide hydrolase activities and epoxy fatty acids in the mosquito Culex quinquefasciatus

Culex mosquitoes have emerged as important model organisms for mosquito biology, and are disease vectors for multiple mosquito-borne pathogens, including West Nile virus. We characterized epoxide hydrolase activities in the mosquito Culex quinquefasciatus, which suggested multiple forms of epoxide hydrolases were present. We found EH activities on epoxy eicosatrienoic acids (EETs). EETs and other eicosanoids are well-established lipid signaling molecules in vertebrates. We showed EETs can be synthesized in vitro from arachidonic acids by mosquito lysate, and EETs were also detected in vivo both in larvae and adult mosquitoes by LC-MS/MS. The EH activities on EETs can be induced by blood feeding, and the highest activity was observed in the midgut of female mosquitoes. The enzyme activities on EETs can be inhibited by urea-based inhibitors designed for mammalian soluble epoxide hydrolases (sEH). The sEH inhibitors have been shown to play diverse biological roles in mammalian systems, and they can be useful tools to study the function of EETs in mosquitoes. Besides juvenile hormone metabolism and detoxification, insect epoxide hydrolases may also play a role in regulating lipid signaling molecules, such as EETs and other epoxy fatty acids, synthesized in vivo or obtained from blood feeding by female mosquitoes.     

Variations in life tables of geographically isolated strains of the mosquito Culex quinquefasciatus

Variations in the life tables and other biological attributes of four strains of Culex quinquefasciatus Say (Diptera: Culicidae) from geographically isolated regions of India that had been reared to the fifth generation in the laboratory were assessed under a standardized rearing regime under constant laboratory conditions. Two strains from arid habitats [Jodhpur (JD) and Bikaner (BKN)], one from a semi-arid inland habitat [Bathinda (BTH)], one from a semi-arid coastal habitat [Jamnagar (JMN)] and a standard laboratory strain (LAB) were compared. Horizontal life-table parameters were measured for each strain. Egg mortality ranged from 4.4% (JD and BTH) to 19.5% (BKN). The lowest rate of adult emergence and highest female : male ratio were found in BKN, and the highest rate of adult emergence and lowest female : male ratio were recorded in BTH. The egg-hatching period was longest in BTH and shortest in LAB. The duration from oviposition to adult emergence was longest in JD and shortest in LAB. Females lived longer than males in all strains. The net reproductive rates (R(0) ) of all field-derived strains (122.9-162.2) differed significantly between strains and were significantly greater than that of LAB (107.6). Similarly, both the intrinsic rate of increase (r(m) ) and finite rate of increase (λ) were found to be lower in LAB than in the field strains, but the mean generation time (T) and doubling time (DT) were longest in LAB. For several life-table attributes, JD and BTH clustered together and were more similar to JMN than to BKN and LAB. The results indicate that BTH, BKN and JD can be characterized as r-strategists, more so than JMN. Overall fecundity increased with age. Differences in annual temperature ranges and mean annual rainfall between locations were positively correlated (r = 0.46-0.97) with egg production, female life expectancy, R(0) , r(m) , λ and T. The results suggest that strains of Cx. quinquefasciatus from different geographical areas with contrasting habitats vary in their survival and reproductive strategies accordingly.     

Synergism between permethrin and propoxur against Culex quinquefasciatus mosquito larvae

To see if synergism occurs between carbamate and pyrethroid insecticides, we tested permethrin and propoxur as representatives of these two classes of compounds used for mosquito control. Larvicidal activity of both insecticides was assessed separately and together on a susceptible strain of the mosquito Culex quinquefasciatus (Diptera: Culicidae) by two methods. When mixed at a constant ratio (permethrin : propoxur 1 : 60 based on LC50) and tested at serial concentrations to plot dose/mortality regression, significant synergy occurred between them (co-toxicity coefficient = 2.2), not just an additive effect. For example, when the mixture gave 50% mortality, the same concentrations of permethrin and propoxur alone would have given merely 2 x 1% mortality. When a sublethal dose (LC0) of permethrin or propoxur was added to the other (range LC10-LC95), synergism occurred up to the LC80 level. Synergistic effects were attributed to the complementary modes of action by these two insecticide classes acting on different components of nerve impulse transmission. Apart from raising new possibilities for Culex control, it seems appropriate to consider using such mixtures or combinations for insecticide-treated mosquito nets in situations with insecticide-resistant Anopheles malaria vectors.     

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.     

光活化多炔类化合物对蚊幼虫的毒力

采用自制的光活化实验装置,测定了11个合成的多炔类化合物对致倦库蚊Culex quinquefasciatus 4龄幼虫的光活化毒力,发现部分化合物在近紫外光照射条件下,能明显地提高光活化毒杀效应,测得化合物5(1-苯基-4-(3,4-亚甲基二氧)苯基-丁二炔)光照与未光照处理LC₅₀分别是0.35 μg/mL和8.89 μg/mL。实验中发现蚊虫先接触药后,再进行光照处理,才能较好地发挥毒效,而且毒杀效应与光照时间呈正相关。模拟田间试验表明,太阳光能显著提高化合物5毒杀蚊幼虫的药效。利用抗氧化剂进行猝灭作用试验,间接地证明化合物5的光活毒杀机理是与过氧化作用有关。分析结构与活性关系,发现二苯基-丁二炔衍生物比二烷基取代丁二炔活性高,苯基上不同取代基也影响光活毒杀效果,它们的活性顺序是：亚甲基二氧基>甲氧基>邻硝基>间硝基>甲基酯。     

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 .     

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.     

Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus

Francisella tularensis is an environmental bacterium capable of infecting a wide spectrum of species from mammals and birds to reptiles. It has been demonstrated that F. tularensis can invade and survive within protozoa, but an association with aquatic insects has not been thoroughly investigated. We examined the interaction of F. tularensis LVS biofilms and Culex quinquefasciatus larvae to determine the effects on larvae and adults. Our results demonstrate that F. tularensis LVS can form and persist as biofilms in natural water and that the mosquito larvae of C. quinquefasciatus readily feed on biofilm and planktonic forms of F. tularensis LVS. Larvae raised in both bacteria-only cultures suffered significant delays in pupation. Adults resulting from larvae continuously exposed to the bacteria had significantly reduced wing lengths in males and fecundity of both sexes. The bacteria may be exerting these effects through localization and persistence within the midgut and Malpighian tubule cells of the larvae. The study of oral acquisition of pathogens by insect larvae can significantly contribute to the study of environmental persistence of pathogens. We show that oral uptake of F. tularensis LVS by C. quinquefasciatus larvae results in not only larval effects but also has effects on adult mosquitoes. These effects are important in understanding both the ecology of tularemia as well as bacterial interactions with aquatic invertebrates.     

Protein hydrolysates and associated bacterial contaminants as oviposition attractants for the mosquito Culex quinquefasciatus

Abstract. Six protein or protein hydrolysate solutions were tested for activity as attractants for ovipositing Culex quinquefasciatus mosquitoes in the laboratory. Four of these solutions (egg albumin, lactalbumin hydrolysate, casein hydrolysate and yeast hydrolysate) were attractive to ovipositing females at varying concentrations, when compared to distilled water controls. Soy hydrolysate was repellent at 1%, but not significantly attractive or repellent at lower concentrations. 'Nulure', a tephritid fly bait containing protein hydrolysate, also had no significant effect on oviposition behaviour. Gravid females mostly oviposited within the first 4 h of the scotophase, regardless of the presence or absence of an oviposition attractant.
Lactalbumin hydrolysate 1% solution, with or without 0.1% neomycin antibiotic, was attractive to Cx quinquefasciatus . This effect was reduced by the presence of neomycin which, alone, had no effect on oviposition. Hence both lactalbumin hydrolysate and bacterial contaminants were shown to be attractive to gravid Cx quinquefasciatus .     

Survival of West Nile virus‐challenged Southern house mosquitoes,Culex pipiens quinquefasciatus,in relation to environmental temperatures

We investigated the effect of West Nile virus (WNV) infection on survival in two colonies of Culex pipiens quinquefasciatus Say (Diptera: Culicidae) originating from Vero Beach and Gainesville, FL. Mosquitoes were fed West Nile virus‐infected blood and checked daily for survival. Exposure to WNV decreased survival among Cx. p. quinquefasciatus from Gainesville relative to unexposed individuals at 31° C. In contrast, exposure to WNV enhanced survival among Cx. p. quinquefasciatus from Vero Beach relative to unexposed individuals at 27° C. These results may suggest that exposure to WNV and associated infection could increase or decrease components of fitness, dependent on environmental temperature and intraspecific variation in Cx. p. quinquefasciatus. The relationship between lifespan (time of death in days) and WNV titer differed in the colonies at 31° C and 27° C, suggesting that intraspecific species variation in response to temperature impacts interactions with WNV. While further work is needed to determine if similar effects occur under field conditions, this suggests intraspecific variation in vector competence for WNV and adult survival of Cx. p. quinquefasciatus, both aspects of vectorial capacity.     

A rapid knockdown effect of Penicillium citrinum for control of the mosquito Culex quinquefasciatus in Thailand

Twenty local isolates of entomopathogenic fungi were determined for control of the larvae and adults of Culex quinquefasciatus. In a laboratory experiment, a Penicillium sp. CM-010 caused 100 % mortality of third-instar larvae within 2 h using a conidial suspension of 1 × 10⁶ conidia ml^?1. Its LC₅₀ was 3 × 10⁵ conidia ml^?1, and the lethal time (LT₅₀) was 1.06 h. Cloning and sequencing of its internal transcribed spacer region indicated that this Penicillium species is Penicillium citrinum (100 % identity in 434 bp). Mortality of the adult was highest with Aspergillus flavus CM-011 followed with Metarhizium anisopliae CKM-048 from 1 × 10⁹ conidia ml^?1. P. citrinum CM-010 at 1 × 10⁶ conidia ml^?1 killed 100 % larvae within 2 h while Bacillus thuringiensis var. israelensis at 5 ITU ml^?1 required 24 h. This P. citrinum CM-010 also greatly reduced survival of C. quinquefasciatus larvae in an unreplicated field test. Light and transmission electron micrographs showed that the fungal conidia were ingested by the larvae and deposited in the gut. The metabolite patulin was produced by P. citrinum CM-010 instead of citrinin.     

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.