Establishment and characterization of a cell line from the mosquito Culex tritaeniorhynchus (Diptera: Culicidae)

We established a continuous cell line from the embryo of the mosquito Culex tritaeniorhynchus Giles (Diptera: Culicidae), a known major vector of the Japanese encephalitis virus (family Flaviviridae, genus Flavivirus) in Asia. The cell line, designated NIID-CTR, was serially subcultured in VP-12 medium supplemented with 10 % heat-inactivated fetal bovine serum (FBS). It continued to grow for more than 60 passages over a 750-d period. The NIID-CTR cell line mainly comprised two morphologically distinct types of cells with adhesive properties: spindle-shaped and round cells. Most of the NIID-CTR cells at the 45th passage were diploid (2n = 6). The growth kinetics of the NIID-CTR cells was significantly affected by the FBS concentration in the medium. The population doubling time of the NIID-CTR cells was 20 h in the presence of 10 % FBS and 76 h in its absence. The DNA sequence of the mitochondrial cytochrome oxidase I gene confirmed that the NIID-CTR cell line was derived from C. tritaeniorhynchus. The cells were highly susceptible to Japanese encephalitis and Dengue viruses, thus providing a valuable tool for the study of mosquito-borne flaviviruses.     

Identification and classification of detoxification enzymes from Culex quinquefasciatus (Diptera: Culicidae)

Molecular characterization of the insecticide resistance has become a hot research topic ever since the first disease transmitting arthropod (Anopheles gambiae) genome sequence has unveiled in 2002. A recent publication of the Culex quinquefasciatus genome sequence has opened up new opportunities for molecular and comparative genomic analysis of multiple mosquito genomes to characterize the insecticide resistance. Here, we utilized a whole genome sequence of Cx. quinquefasciatus to identify putatively active members of the detoxification supergene families, namely cytochrome P450s (P450s), glutathione-S-transferases (GSTs), and choline/carboxylesterases (CCEs). The Culex genome analysis revealed 166 P450s, 40 GSTs, and 62 CCEs. Further, the comparative genomic analysis shows that these numbers are considerably higher than the other dipteran mosquitoes. These observed speciesspecific expansions of the detoxification super gene family members endorse the popular understanding of the involvement of these gene families in protecting the organism against multitudinous classes of toxic substances during its complex (aquatic and terrestrial) life cycle. Thus, the generated data set may provide an initial point to start with to characterize the insecticide resistance at a molecular level which could then lead the development of an easy to use molecular marker to monitor the incipient insecticide resistance in field environs.     

Life cycle of Culex quinquefasciatus Say (Diptera: Culicidae) under uncontrolled conditions

Aspects of the growth and development were described for immature stages of Culex quinquefasciatus Say, 1826 (Diptera: Culicidae), an antropophilic mosquito species found frequently in Bogotá, Colombia. Two experiments were carried out during January-February and September-October of 2001 under ambient environmental conditions. Oviposition occured 5-8 days after blood ingestion. Females laid eggs in plastic containers filled with pooled water with high organic material content. The number of eggs per raft varied between 152 and 203. For the 2 trials, the hatch rate was 50% and 75%. The asynchronous egg hatch, the short duration of the pupal stage (11% of the total development time, and the high efficiency of adult emergence from the pupal stage (98.6%) were noted. In general, these development times were shorter compared to those reported by other authors. Moreover, the high percentages of hatch (83.6%), pupation (86.6%) and emergence (98.6%) under the average temperature conditions of 14.8 degrees C and 15.1 degrees C, and average relative humidity of 72.5% and 74.1%, respectively) demonstrated adaptation of C. quinquefasciatus to Bogotá's cool, high altitude environment. These characteristics, together with its vectorial capacity and the resistance to chemical control methods, could make this species become a risk for the health of human populations.     

Attraction of female Culex quinquefasciatus Say (Diptera: Culicidae) to odors from chicken feces

Odors from fresh chicken feces in water elicited upwind flight of host-seeking female Culex quinquefasciatus mosquitoes in a dual-choice olfactometer. Acidification of the slurry of chicken feces and water resulted in increased attraction, whereas alkaline slurries of chicken feces and water controls did not attract female mosquitoes. This is the first reported example of avian fecal odor eliciting upwind flight of female mosquitoes. Headspace odors from acidified slurries were sampled using solid phase micro-extraction (SPME) coated fibers. Eight volatile aldehydes [(E)-2-decenal, undecanal, dodecanal, tetradecanal, pentadecanal, hexadecanal, heptadecanal, and octadecanal] identified in the headspace of acidified chicken feces elicited electroantennogram responses from antennae of C. quinquefasciatus females. An improved electroantennogram technique in which four antennae were used in parallel for monitoring the GC effluent is described.     

Association of esterases with insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae)

The southern house mosquito, Culex quinquefasciatus Say, is a competent vector of human disease and an important target of mosquito abatement programs. However, these management programs have been compromised by development of insecticide resistance. In the current study, susceptibilities to naled and resmethrin, two adulticides used in mosquito abatement, were monitored using a topical and contact bioassay, respectively, in five field- collected populations of C. quinquefasciatus (MARC, HOOD1, HOOD2, MINLOVE, and THIB). Frequencies of resistance, measured as survival after treatment with discriminating concentrations (i.e., sufficient to kill > 90% of a reference susceptible strain) were high (88.0-96.8%) in all field collections treated with naled, but were variable (3.3-94.2%) with resmethrin. In addition, esterase activities in mosquitoes from these collections were quantified using alpha-naphthyl acetate and ranged from 1.08 to 3.39 micromol alpha-naphthol produced min(-1) mg prot(-1). Heightened activities were associated with decreased insecticide susceptibility in HOOD1, THIB, and MINLOVE but not HOOD2. Esterases were visualized using native polyacrylamide gel electrophoresis, and intra- and interstrain differences in banding patterns were detected. In addition, esterases from MINLOVE mosquitoes were more numerous and intensely staining when compared with those from a laboratory-susceptible strain. Finally, naled synergized the toxicity of resmethrin in populations with decreased insecticide susceptibility and increased esterase activity by 2.5-(MINLOVE) to three-fold (THIB). Results from this study will allow management strategies for populations of C. quinquefasciatus to be optimized, and provide a foundation for further studies exploring use of esterase inhibitors as synergists of pyrethroid toxicity.     

Immune defense mechanisms of Culex quinquefasciatus (Diptera: Culicidae) against Candida albicans infection

Mosquitoes have an efficient defense system against infection. The cellular immune defense mechanism initiated by the mosquito Culex quinquefasciatus infected with the fungus Candida albicans was investigated in this study. Differences in the hemocyte counts in hemolymph perfused from uninoculated, saline-inoculated, and C. albicans-infected mosquitoes were compared using a light microscope. Phagocytosis was also investigated using electron microscopy. Four types of hemocytes were identified in control mosquitoes: prohemocytes (9.8%), plasmatocytes (38.8%), granular cells (44.2%), and oenocytoids (7.3%). Between 3 and 18 h postinoculation the total hemocyte count was significantly higher in infected, compared to uninfected, mosquitoes. Differential hemocyte counts from infected mosquitoes at 3, 6, and 18 h after inoculation showed that the relative proportion of plasmatocytes (48.6, 50.7, 45%) was higher and, concomitantly, the proportion of granular cells was lower (38, 36.8, 35%, respectively). Yeast cells were phagocytosed and limited growth was observed within the plasmatocytes. Melanized nodules were found attached to different insect tissues at 24 to 72 h following infection. These results suggest that phagocytosis, followed by nodule formation, was capable of clearing the hemolymph of yeast cells.     

Laboratory and field evaluation of an oviposition trap for Culex quinquefasciatus (Diptera: Culicidae)

An ovitrap (BR-OVT) based on physical and chemical stimuli for attracting gravid Culex quinquefasciatus (Diptera: Culicidae) females was developed and evaluated under laboratory and field conditions. Attractants were assayed using alternative chamber bioassays prior to being used in the BR-OVT oviposition trap. A significant preference of gravid females for sites containing conspecific egg rafts was observed, as a response to the natural oviposition pheromone, as well as for sites treated with the synthetic pheromone erythro-6-acetoxy-5-hexadecanolide. Five- to 20-day old grass infusion was strongly attractive to gravid females for laying eggs. On the other hand, entomopathogenic Bacillus sphaericus (Bs) did not influence the choice of an oviposition site when used in combination with grass infusion and can therefore be used as a larvicide in ovitraps. Results from field trials showed that the BR-OVT with grass infusion and with or without Bs works as a preferred oviposition site for Cx. quinquefasciatus. The BR-OVT was more effective for egg collection when placed indoors and comparison with the number of egg rafts laid in cesspits over 40 days indicates that this very simple ovitrap may be a useful tool for monitoring populations of the most important of the vectors of bancroftian filariasis.     

Potential for integrated control of Culex quinquefasciatus (Diptera: Culicidae) using larvicides and guppies

Acute toxicity tests were carried out to determine the effect of three larvicides, spinosad, pirimiphos methyl, and chlorpyrifos, on Culex quinquefasciatus Say, Anopheles gambiae s.s. Giles (Diptera: Culicidae), and guppies, Poecilia reticulata Peters (Pisces: Poeciliidae). Thereafter, larvae of Cx. quinquefasciatus were introduced to P. reticulata in containers of different volumes with low concentrations of each larvicide at established predator to prey densities of 1–35; 5–70 and 10–350 (fish to larvae) respectively. The experiment was replicated six times, and the larval consumption was counted after 24 h. Spinosad and pirimiphos methyl were significantly toxic to Cx. quinquefasciatus, the less susceptible mosquito species. Guppy consumption of Culex larvae was highest at a predator to prey density 5–70. Feeding activity of guppies increased in the spinosad treatment at 49 μg L⁻¹ compared to control and the synthetic larvicides. The synthetic larvicides generally reduced consumption of larvae except for the lowest concentration of pirimiphos methyl which increased it at the highest predator to prey density in a 3 L container. The highest percentage of Culex larvae was consumed by fish in the presence of spinosad at 49 μg L⁻¹ and a predator to prey density of 5–70 in 0.5 L plastic containers. Thus, predator to prey density, container size, type and concentration of larvicide are all important factors to be considered in integrated management of mosquito larvae.     

Reproductive aspects of the mosquito Culex quinquefasciatus (Diptera:Culicidae) infected with Wuchereria bancrofti (Spirurida: Onchocercidae)

This study reports on the relationship between Wuchereria bancrofti infection and female body size, intake of blood and fecundity in the mosquito Culex quinquefasciatus, vector of this filarial parasite in Recife (Brazil). Adults from field collected larvae were infected via a membrane feeding procedure, using blood with parasitaemia ranging from 724-6,000 mf/ml. A positive correlation was observed between mosquito size (measured by wing length) and egg production in uninfected females. However, this relationship did not exist in W. bancrofti infected mosquitoes. This change is unlikely to be the result of changes in blood ingestion as no significant difference was found when infected and uninfected females were compared. Variation in egg production observed between trials could not be associated with parasite density in the blood. These results suggest infection with W. bancrofti may disrupt the relationship between mosquito size and egg production during the first gonotrophic cycle of C. quinquefasciatus such that fecundity is sometimes reduced. However, this overall affect is variable and many groups of mosquitoes do not respond in this way.     

Flight muscle-specific expression of act88F: GFP in transgenic Culex quinquefasciatus Say (Diptera: Culicidae)

A strategy to engineer a strain of Culex mosquitoes refractory to filarial transmission is described. A requirement for success of the strategy is identification of a flight muscle-specific promoter that functions in the mosquito. A GFP marker gene under the control of the promoter region of the Drosophila melanogaster act88F gene was inserted into the genome of Culex quinquefasciatus. Transformation was confirmed by Mendelian genetics. Hybridization of a genomic Southern blot to a radiolabeled probe verified that the entire donor plasmid integrated into the mosquito genome. GFP expression in the transgenic mosquitoes was restricted to the flight muscles.     

The Effect of Sucrose Concentration on Olfactory-Based Associative Learning in Culex quinquefasciatus Say (Diptera: Culicidae)

A classical conditioning approach was used to examine the ability of Culex quinquefasciatus Say (Diptera: Culicidae) to associate odor with a sugar-meal of varying quality. The objectives of this study were to investigate the impact of different sucrose concentrations (5 %, 10 %, or 50 %) on positive response to conditioning and to examine sucrose concentration preference following exposure to a 10 % solution. Mosquitoes conditioned in conjunction with all three sucrose concentrations showed evidence of learning; including the concentration of the conditioning stimulus, and the sex of the mosquito. Using colored solutions to determine feeding patterns of experienced mosquitoes indicated male mosquitoes showed no preference but females showed a preference for 10 % over 5 % sucrose but not between 10 % and 50 % sucrose solutions.     

Age modifies the effect of body size on fecundity in Culex quinquefasciatus Say (Diptera: Culicidae)

Fecundity of mosquitoes can vary with many factors and can have a strong effect on population growth. This study reports the effects of body size, blood meal size, and age on the reproductive output of nulliparous Culex quinquefasciatus, a vector of arboviruses and other pathogens. Mated adult female mosquitoes from a colony were reared under standard conditions and fed on chickens at different ages post‐eclosion. Blood meal size and wing length were recorded, as well as the number of eggs in the first‐cycle egg raft. Each of these factors had a significant influence on fecundity considered in a simple regression context. Multiple regression analysis revealed a significant interaction effect between age and body size on fecundity. Up to 13 days of age, fecundity was positively correlated with body size, but in mosquitoes older than 13 days, this relationship was not significantly different from zero. These results are discussed in terms of the known physiology of this and other species.     

The salivary gland chromosomes of Culex (Culex) vishnui (Culicidae: Diptera)

Culex vishnui is a member of the vishnui group comprising six closely resembling species. The salivary gland chromosomes are longer than those found in Anopheles, more fragile and spread with difficulty. The banding pattern is compared with that of Culex pipiens pipiens and Culex pipiens fatigans. Homologies are closest between vishnui and fatigans. The incidence of asynapsis between the homologues is very high.     

Enzymatic Characterization of Insecticide Resistance Mechanisms in Field Populations of Malaysian Culex quinquefasciatus Say (Diptera: Culicidae)

Background

There has been no comprehensive study on biochemical characterization of insecticide resistance mechanisms in field populations of Malaysian Culex quinquefasciatus. To fill this void in the literature, a nationwide investigation was performed to quantify the enzyme activities, thereby attempting to characterize the potential resistance mechanisms in Cx. quinquefasciatus in residential areas in Malaysia.

Methodology/Principal Findings

Culex quinquefasciatus from 14 residential areas across 13 states and one federal territory were subjected to esterases, mixed function oxidases, glutathione-S-transferase and insensitive acetylcholinesterase assays. Enzyme assays revealed that α-esterases and β-esterases were elevated in 13 populations and 12 populations, respectively. Nine populations demonstrated elevated levels of mixed function oxidases and glutathione-S-transferase. Acetylcholinesterase was insensitive to propoxur in all 14 populations. Activity of α-esterases associated with malathion resistance was found in the present study. In addition, an association between the activity of α-esterases and β-esterases was also demonstrated.

Conclusions/Significance

The present study has characterized the potential biochemical mechanisms in contributing towards insecticide resistance in Cx. quinquefasciatus field populations in Malaysia. Identification of mechanisms underlying the insecticide resistance will be beneficial in developing effective mosquito control programs in Malaysia.     

Point Mutations Associated with Organophosphate and Carbamate Resistance in Chinese Strains of Culex pipiens quinquefasciatus (Diptera: Culicidae)

Acetylcholinesterase resistance has been well documented in many insects, including several mosquito species. We tested the resistance of five wild, Chinese strains of the mosquito Culex pipiens quinquefasciatus to two kinds of pesticides, dichlorvos and propoxur. An acetylcholinesterase gene (ace1) was cloned and sequenced from a pooled sample of mosquitoes from these five strains and the amino acids of five positions were found to vary (V185M, G247S, A328S, A391T, and T682A). Analysis of the correlation between mutation frequencies and resistance levels (LC₅₀) suggests that two point mutations, G247S (r² = 0.732, P = 0.065) and A328S (r² = 0.891, P = 0.016), are associated with resistance to propoxur but not to dichlorvos. Although the V185M mutation was not associated with either dichlorvos or propoxur resistance, its RS genotype frequency was correlated with propoxur resistance (r² = 0.815, P = 0.036). And the HWE test showed the A328S mutation is linked with V185M, also with G247S mutation. This suggested that these three mutations may contribute synergistically to propoxur resistance. The T682A mutation was negatively correlated with propoxur (r² = 0.788, P = 0.045) resistance. Knowledge of these mutations may help design strategies for managing pesticide resistance in wild mosquito populations.     

Establishment and characterization of a new human cell line (EJ) derived from endometrial carcinoma

We present a new cell line, EJ established from an invasive endometrioid adenocarcinoma of the uterine corpus in a 56-year-old patient. The cells show rapid growth in culture with a doubling time of 16 h and high migration activity. Monolayer-cultured cells were polygonal in shape showing a tendency to pile up without contact inhibition. Subcutaneous transplantation of the EJ cells into nude mice formed solid tumors that were histologically diagnosed as adenocarcinoma, whereas no metastasis was observed. Cultured EJ cells produced tissue polypeptide antigen (IPA). Genetic and molecular analyses revealed high telomerase activity but not estrogen receptor alpha expression. Using the DNA sequencing technique, we have screened EJ cells for p53 mutation in exon 5 to 8 but no mutation of p53 was observed. This cell line appears to represent the development of a more malignant clone with divergent receptor function and growth behavior, and provides us with an interesting new tool for the study of tumorigenesis in the human endometrium.     

Influence of light and habitat on predation of Culex quinquefasciatus (Diptera: Culicidae) larvae by the waterbugs (Hemiptera: Heteroptera)

The influence of light and habitat structure on the predation of Culex quinquefasciatus larvae by the common heteropteran water bug, Diplonychus (=Sphaerodema) annulatus, D. rusticus and Anisops bouvieri was assessed in the laboratory. It was revealed that water bugs predate more in presence of light than in dark conditions. While A. bouvieri consumed more prey in structured conditions, D. annulatus and D. rusticus consumed more prey in open conditions. The selection of prey size as well as the respective numbers varied between predators and treatments significantly. Prey vulnerability (PV), an indicator of predatory efficiency, was highest for D. annulatus, moderate for D. rusticus and low for A. bouvieri. Prey consumption and PV values under different treatment conditions indicate that for belostomatid water bugs D. annulatus and D. rusticus, the order of prey consumption under different habitat and light/dark combinations is light open > dark open > light vegetated > dark vegetated. In the case of the backswimmer, A. bouvieri, the order of prey consumption appeared to be light structured > dark structured > light open > dark open. These findings were consistent with resource‐partitioning by water bug species sharing the same guild. If the observed results are extended to natural settings the efficiency of these predators in controlling mosquito populations will vary with the structural complexity of habitats and the intensity of light.