The effect of predatory fish exudates on the ovipostional behaviour of three mosquito species: Culex quinquefasciatus, Aedes aegypti and Culex tarsalis

Abstract.  Three mosquito species, Culex tarsalis Coquillett, Culex quinquefasciatus Say and Aedes aegypti L. (Diptera: Culicidae), were examined in laboratory binary choice experiments to investigate whether fish exudates from the mosquitofish, Gambusia affinis (Baird & Girard) (Cyprinodontiformes: Poecilliidae), deter oviposition and whether the responses of these mosquito species to fish exudates in oviposition sites are consistent with the risk of predation from fish experienced by each species in their respective natural breeding habitats. Culex tarsalis was deterred significantly from egg laying by the presence of fish exudates in oviposition cups, consistent with high levels of predation by fish in natural breeding sites. Egg laying by Cx quinquefasciatus was slightly reduced in water with fish exudates, but was not consistently deterred by water conditioned by mosquitofish, consistent with the species' relatively low risk of fish predation in natural habitats. Oviposition by container-breeding Ae. aegypti was not deterred by the presence of fish exudates in oviposition cups, consistent with a low risk of predation by fish in natural habitats.     

Comparative responses of ovipositing Anopheles gambiae and Culex quinquefasciatus females to the presence of Culex egg rafts and larvae

Field observations have demonstrated that gravid Anopheles gambiae Giles s.s. (Diptera: Culicidae) are selective in their choice of oviposition sites. For example, immature stages of An. gambiae s.s. are rarely found in water that contains Culex quinquefasciatus Say immatures. The possibility that this may, in part at least, reflect a response by ovipositing An. gambiae s.s. females to volatile signals associated with Culex juveniles was evaluated by testing the response of An. gambiae s.s. females to varying densities of Cx. quinquefasciatus egg rafts and/or larvae in oviposition choice assays. For comparison, the oviposition choices of Cx. quinquefasciatus to conspecific egg rafts and/or larvae were similarly assayed. At a low density of Cx. quinquefasciatus egg rafts (1-15 egg rafts/100 mL water), An. gambiae s.s. females laid more eggs in the treatment water than in the control, with a maximum of twice as many in the treatment water at 5 egg rafts/100 mL water. At higher egg raft densities and in all treatments that included Cx. quinquefasciatus larvae, oviposition decreased significantly in the treatment dishes in a density-dependent manner. As previous studies have indicated, ovipositing Cx. quinquefasciatus females were attracted to and laid egg rafts in dishes containing conspecific egg rafts and, interestingly, also in dishes containing larvae.     

Orientation of Culex mosquitoes to carbon dioxide-baited traps: flight manoeuvres and trapping efficiency

Females of Culex quinquefasciatus Say and Culex tarsalis Coquillet (Diptera: Culicidae) in the host-seeking stage were released and video recorded in three dimensions in a large field wind tunnel as they flew to four kinds of CO2-baited mosquito traps. The trapping efficiency (number of mosquitoes approaching compared to the number caught) was determined for each trap type. The Encephalitis Virus Surveillance (EVS), Mosquito Magnet Freedom (MMF) and Mosquito Magnet Liberty (MML) traps captured only 13-16% of approaching Cx. quinquefasciatus females, whereas the Mosquito Magnet-X (MMX) trap captured 58%. Similar results were obtained for Cx. tarsalis. Orientation behaviour and flight parameters of mosquitoes approaching the four traps were compared. Mosquitoes spent the most time orienting to the EVS trap. Flight speed decreased as mosquitoes entered the vicinity of each trap and a large portion of their time was spent within 30 cm downwind of the traps. Flights became highly tortuous downwind of the poorly performing traps and just upwind of the MMX trap. Differences between traps and possible explanations for the superior performance of the MMX trap are considered.     

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.     

Using fluorescently labelled M13-tailed primers to isolate 45 novel microsatellite loci from the arboviral vector Culex tarsalis

Culex tarsalis Coquillett (Diptera: Culicidae) is a highly efficient arbovirus vector. Spatial and temporal heterogeneity have been observed in Cx tarsalis for phenotypic traits including autogeny, virus susceptibility and host preference. Genetic differences between populations may in part explain these observations. Using the M13-tailed primer method, we identified 45 novel polymorphic microsatellite markers from microsatellite-enriched Cx tarsalis genomic libraries. The M13-tailed primer method was more efficient in identifying useful loci than traditional screening by acrylamide gel electrophoresis. These markers will be useful for investigating genetic questions in this important vector mosquito.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.     

Larvicidal activity of Pseudocalymma alliaceum and Allium sativum against Culex quinquefasciatus (Say)

The larvicidal activity of the plant extracts Pseudocalymma alliaceum and Allium sativum were determined against Culex quinquefasciatus. The hexane extract of P. alliaceum and the petroleum ether extract of A. sativum exhibited larvicidal efficacy against Cx. quinquefasciatus larvae. Extracts of P. alliaceum resulted in concentrations that produced 50% mortality LC₅₀ and LC₉₀ values of 2.49 and 15.06 ppm, respectively, after 24 h and 1.16 and 8.45 ppm after 48 h. Extracts of A. sativum resulted in LC₅₀ and LC₉₀ values of 8.38 and 29.15 ppm after 24 h and 7.28 and 44.19 ppm after 48 h of exposure, respectively. The results indicate that the plant extract component(s) present in the hexane extract of P. alliaceum leaves demonstrated greater potential as an efficient larvicide than A. sativum against Cx. quinquefasciatus.     

Laboratory evaluation of methanolic extract of Atlantia monophylla (Family: Rutaceae) against immature stages of mosquitoes and non-target organisms

Methanolic extracts of the leaves of Atlantia monophylla (Rutaceae) were evaluated for mosquitocidal activity against immature stages of three mosquito species, Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti in the laboratory.Larvae of Cx. quinquefasciatus and pupae of An. stephensi were found more susceptible, with LC50 values of 0.14 mg/l and 0.05 mg/l, respectively. Insect growth regulating activity of this extract was more pronounced against Ae. aegypti, with EI50 value 0.002 mg/l. The extract was found safe to aquatic mosquito predators Gambusia affinis, Poecilia reticulata, and Diplonychus indicus, with the respective LC50 values of 23.4, 21.3, and 5.7 mg/l. The results indicate that the mosquitocidal effects of the extract of this plant were comparable to neem extract and certain synthetic chemical larvicides like fenthion, methoprene, etc.     

Insecticide resistance in Culex quinquefasciatus mosquitoes after the introduction of insecticide-treated bed nets in Macha, Zambia

Culex quinquefasciatus, an arboviral and filarial vector, is present in high numbers throughout sub-Saharan Africa, and insecticide-resistant populations have been reported worldwide. In order to determine the insecticide resistance status of Cx. quinquefasciatus in Macha, Zambia, adult mosquitoes reared from eggs collected from oviposition traps were tested by bioassay. High levels of resistance to DDT, pyrethroids, malathion, and deltamethrin-treated net material were detected, and molecular assays revealed that the knockdown resistance (kdr) allele was frequent in the Cx. quinquefasciatus population, with 7.0% homozygous for the kdr L1014 allele and 38.5% heterozygous (0.263 kdr frequency). The kdr frequency was significantly higher in mosquitoes that had successfully fed on human hosts, and screening archived specimens revealed that kdr was present at lower frequency prior to the introduction of ITNs, indicating that ITNs might be a selective force in this population. Additionally, metabolic detoxification enzyme activity assays showed upregulated glutathione S-transferases, α-esterases, and β-esterases. Continued monitoring and assessment of the Cx. quinquefasciatus population is necessary to determine levels of resistance.     

Changes in malathion and pyrethroid resistance after cypermethrin selection of Culex quinquefasciatus field populations of Cuba

Abstract. Use. of the organophosphorus insecticide malathion for mosquito control in Cuba, for 7 years up to 1986, selected elevated non-specific esterase and altered acetylcholinesterase (AChE) resistance mechanisms in Culex quinquefasciatus. In central Havana space-spraying of malathion was replaced by the pyrethroid cypermethrin in 1987: alternate cycles of malathion and cypermethrin were applied in some of the more rural areas of Havana district during 1987-91. Consequently, populations of Cx quinquefasciatus in the central area of Havana developed resistance to cypermethrin, but there is no evidence of pyrethroid resistance in the outlying areas. Malathion resistance levels declined significantly after 1986, measured both by bioassay and the frequency of the elevated esterase resistance mechanism, and then stabilized with no measurable decline during 1990 in any of the populations tested. These populations had less than 10% frequency of susceptible homozygotes for both the esterase and AChE resistance mechanisms, indicating that organophosphate resistance is still prevalent in Cuban Cx quinquefasciatus. These two mechanisms appear to be in linkage equilibrium, suggesting that current selection for double resistance is not strong. In the central Havana region, pirimiphos-methyl, an organophosphorus insecticide unaffected by the two common malathion resistance mechanisms, is now being used in a resistance management strategy designed to avoid pyrethroid resistance spreading.     

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 .     

Monitoring temporal fluctuations of Culex quinquefasciatus using oviposition traps containing attractant and larvicide in an urban environment in Recife, Brazil

The use of attractants and larvicides in oviposition traps is of practical interest for the surveillance and control of urban mosquitoes. In addition to increasing the safety of the traps, this combination is essential for an attract-and-kill control strategy based on trapping mosquito eggs. The combination of Bacillus thuringiensis var. israelensis (Bti) and grass infusion (GI) vs. GI alone were tested for their ability to attract in paired BR-OVT traps in the backyards of 10 houses in Recife, Brazil, for a period of 45 days. Results show that females prefer to oviposit in traps containing Bti (363 compared with 251 egg rafts over 45 days). Results from a one-year trial on the efficacy of BR-OVT traps loaded with GI and Bti as a sampling tool to monitor temporal fluctuations in the population densities of Culex quinquefasciatus in an urban environment are also reported. From December 2006-January 2007, one trap per home was installed and maintained for 348 consecutive days in 134-151 houses located in three urban blocks. Throughout the one-year field trial a total of 43,151 Culex egg rafts were collected in the traps. The data show that BR-OVT loaded with GI and Bti is sensitive enough to demonstrate continuous reproductive activity of Cux. quinquefasciatus in the study area throughout the year and to monitor temporal fluctuations in population density.     

致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用

为探讨致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用,通过病毒分离、逆转录聚合酶链反应、透射电镜等技术进行了相关研究。结果表明：吸食感染性血液后,登革Ⅱ型病毒能侵染白纹伊蚊中肠上皮细胞并大量复制,但不能侵染致倦库蚊中肠上皮细胞。以上研究证明致倦库蚊对登革Ⅱ型病毒存在中肠感染屏障。     

Changes in the haemocyte population of the mosquito, Culex quinquefasciatus, following infection with the filarial parasite, Wuchereria bancrofti

The mosquito Culex quinquefasciatus Say (Diptera: Culicidae) is the vector of the filarial parasite Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae), which causes human bancroftian filariasis. Information on the mosquito humoral response against the filarial parasite during the process of its infection and development is important, as it decides the vector competence of the mosquito. Visible changes in the haemocyte population of mosquito, if any, will be an indicator of the possible humoral factors. The present study was aimed at investigating changes in the populations of various types of haemocytes of Cx. quinquefasciatus following infection with W. bancrofti. On day 2 post-feeding on microfilaraemic blood, the haemolymph perfusate of infected mosquitoes with L1 stage of the parasite showed 44.1% granulocytes, 42% prohaemocytes and 13.9% plasmatocytes, whereas that of the control mosquitoes fed on amicrofilaraemic blood showed 63.4% plasmatocytes, 22.2% prohaemocytes and 14.4% granulocytes. Differences in the population numbers of haemocyte types between the infected and control were significant (P > 0.05). However, the mosquitoes examined on day 6 post-feeding, when the parasite was in L2 stage, did not show any such changes. But, similar changes reappeared on day 12 in mosquitoes with L3 stage of the parasite. The observed haemocyte population changes indicate the possibility of some amount of humoral immune response, through the production of certain immune molecules, in Cx. quinquefasciatus infected with W. bancrofti. The nature and exact role of such a response on the filarial parasite development need further investigation.     

The arbovirus vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe

Two species of arbovirus vector, Culex torrentium and Culex pipiens (Diptera: Culicidae), occur in several European countries, but difficulties in their accurate identification and discrimination have hampered both detailed and large‐scale distribution and abundance studies. Using a molecular identification method, we identified to species 2559 larvae of Cx. pipiens/torrentium collected from 138 sites in 13 European countries ranging from Scandinavia to the Mediterranean coast. In addition, samples of 1712 males of Cx. pipiens/torrentium collected at several sites in the Czech Republic were identified to species based on the morphology of their hypopygia. We found that the two species occur together in large areas of Europe, and that Cx. torrentium dominates in northern Europe and Cx. pipiens dominates south of the Alps. The transition in dominance occurs in central Europe, where both species are roughly equally common. There was a strong correlation between the length of the growing season at different sites and occurrences of the two species. As the growing season increases, the proportion and detection of Cx. torrentium decrease, whereas those of Cx. pipiens increase. The present findings have important consequences for the interpretation of the results of studies on major enzootic and link‐vectors of mosquito‐borne bird‐associated viruses (i.e. Sindbis, West Nile and Usutu viruses), especially in central Europe and Scandinavia.     

Effect of fenthion treatment on larval densities of insecticide-resistant Culex quinquefasciatus in an urban area of Sri Lanka

Abstract. In Sri Lanka the national Anti-Filaria Campaign (AFC) has routinely employed fenthion since 1974 for larvicidal control of Culex quinquefasciatus , the vector of Bancroftian filariasis in urban areas, where this mosquito breeds prolifically in polluted waters. During 1994 the efficacy of AFC fenthion treatment against organophosphate-resistant Cx quinquefasciatus was investigated at Dehiwela, near Colombo.
The AFC target rate of fenthion application was 1 mg a.i./l, but the actual concentrations of fenthion in freshly treated pits ranged from 0.64 to 63.2 mg a.i./l. There was significant suppression of larval densities in treated soakage pits, the predominant breeding site of Cx quinquefasciatus , although the mosquito population was 6-fold resistant to fenthion at the LC_W level. Production of pupae was almost completely prevented in soakage pits which were sprayed weekly with fenthion, indicating that adult mosquito emergence from this source was minimal. The rapid decline in concentration of fenthion detected in the water of soakage pits indicated that a weekly treatment schedule is essential for effective control. With the rapid recolonization of treated sites, the weekly schedule must be strictly implemented in order to achieve control of resistant larvae. Fenthion activity levels detected in treated pits suggest that a 7–10 day schedule of retreatment would completely suppress susceptible Cx quinquefasciatus.     

Differential protein expression in the midgut of Culex quinquefasciatus mosquitoes induced by the insecticide temephos

Mosquitoes are vectors for pathogens of malaria, lymphatic filariasis, dengue, chikungunya, yellow fever and Japanese encephalitis. Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) is a known vector of lymphatic filariasis. Its control in Brazil has been managed using the organophosphate temephos. Studies examining the proteins of Cx. quinquefasciatus that are differentially expressed in response to temephos further understanding of the modes of action of the insecticide and may potentially identify resistance factors in the mosquito. In the present study, a comparative proteomic analysis, using 2‐dimensional electrophoresis coupled with matrix‐assisted laser desorption/ionization (MALDI) time of flight (TOF)/TOF mass spectrometry, and bioinformatics analyses were performed to identify midgut proteins in Cx. quinquefasciatus larvae that were differentially expressed in response to exposure to temephos relative to those in untreated controls. A total of 91 protein spots were differentially expressed; 40 were upregulated and 51 were downregulated by temephos. A total of 22 proteins, predominantly upregulated, were identified as known to play a role in the immune response, whereas the downregulated proteins were involved in energy and protein catabolism. This is the first proteome study of the midgut of Cx. quinquefasciatus and it provides insights into the molecular mechanisms of insecticide‐induced responses in the mosquito.     

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.     

Reverse and conventional chemical ecology approaches for the development of oviposition attractants for Culex mosquitoes

Synthetic mosquito oviposition attractants are sorely needed for surveillance and control programs for Culex species, which are major vectors of pathogens causing various human diseases, including filariasis, encephalitis, and West Nile encephalomyelitis. We employed novel and conventional chemical ecology approaches to identify potential attractants, which were demonstrated in field tests to be effective for monitoring populations of Cx. p. quinquefasciatus in human dwellings. Immunohistochemistry studies showed that an odorant-binding protein from this species, CquiOBP1, is expressed in trichoid sensilla on the antennae, including short, sharp-tipped trichoid sensilla type, which house an olfactory receptor neuron sensitive to a previously identified mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide. CquiOBP1 exists in monomeric and dimeric forms. Monomeric CquiOBP1 bound MOP in a pH-dependent manner, with a change in secondary structure apparently related to the loss of binding at low pH. The pheromone antipode showed higher affinity than the natural stereoisomer. By using both CquiOBP1 as a molecular target in binding assays and gas chromatography-electroantennographic detection (GC-EAD), we identified nonanal, trimethylamine (TMA), and skatole as test compounds. Extensive field evaluations in Recife, Brazil, a region with high populations of Cx. p. quinquefasciatus, showed that a combination of TMA (0.9 microg/l) and nonanal (0.15 ng/microl) is equivalent in attraction to the currently used infusion-based lure, and superior in that the offensive smell of infusions was eliminated in the newly developed synthetic mixture.