Natural Mosquito Populations Research Articles

Efficacy of Three Attractant Blends Tested in Combination with Carbon Dioxide Against Natural Populations of Mosquitoes and Biting Flies at the Lower Suwannee Wildlife Refuge

Synthetic blends of chemicals identified previously from human skin emanations were evaluated against mosquito and biting fly populations at the Lower Suwannee Wildlife Refuge near Cedar Key, FL. Mosquito Magnet-Experimental traps were baited with the Red (400 ml acetone: 10 ml 1-hexen-3-ol:10 ml 1-octen-3-ol), Blue (400 ml acetone: 1 g/liter lactic acid:20 ml glycolic acid), or Green blend (400 ml acetone:1.5 g/liter lactic acid:20 ml dimethyl disulfide) plus CO2 or with CO2 alone (control). A relative index of efficacy was determined by dividing each mean blend trap catch by the mean control trap catch. Five mosquitoes (Aedes infirmatus, Ae. taeniorhynchus, Ae. triseriatus, Anopheles crucians, and Culex nigripalpus), 2 ceratopogonid (Culicoides floridensis and C. furens), and 1 tabanid (Diachlorus ferrugatus) and phlebotomine (Lutzomyia shannoni) species were trapped. The Red blend + CO2 treatment significantly increased collections of Ae. taeniorhynchus (3.4x), An. crucians (2.8X), total mosquitoes (2.7x), C. furens (17.6x), and L. shannoni (10.8x) compared with control traps. Traps baited with either the Blue or Green blends generally captured fewer insects than traps baited with the other 2 treatments. However, traps baited with the Green blend caught 7 x as many C. furens as the control traps. Responses clearly varied according to species; therefore, "one size does not fit all" when it comes to attractant blends.

Temperature-Mediated Differential Expression of Immune and Stress-Related Genes in Aedes aegypti Larvae

The mechanisms by which natural populations of vector mosquitoes cope with daily and seasonal fluctuations in temperature are poorly understood. We examined the effect of water temperature on expression of stress and immune-related genes in Aedes aegypti larvae. Aedes aegypti 3rd instars were exposed for 24 h to one of 7 constant temperatures (10 degrees C, 15 degrees C, 20 degrees C, 25 degrees C [control], 32 degrees C, 36 degrees C, or 40 degrees C) and expression of antimicrobial peptides (cecropin, defensin), transferrin, and heat shock proteins (HSP70 and HSP83) quantified by real-time reverse-transcriptase polymerase chain reaction. Cecropin, defensin, and transferrin were overexpressed at 36 degrees C and underexpressed at 15 degrees C and 32 degrees C. HSP83 was overexpressed at 10 degrees C and 40 degrees C and underexpressed at 20 degrees C, while HSP70 was underexpressed at 15 degrees C, 32 degrees C, and 36 degrees C. These findings suggest that antimicrobial peptides can serve as biomarkers of thermal stress and that HSP83 may buffer mosquito larvae against extreme temperatures.

A portable approach for the surveillance of dengue virus-infected mosquitoes

Dengue virus is the most significant human viral pathogen spread by the bite of an infected mosquito. With no vaccine or antiviral therapy currently available, disease prevention relies largely on surveillance and mosquito control. Preventing the onset of dengue outbreaks and effective vector management would be considerably enhanced through surveillance of dengue virus prevalence in natural mosquito populations. However, current approaches to the identification of virus in field-caught mosquitoes require relatively slow and labor intensive techniques such as virus isolation or RT-PCR involving specialized facilities and personnel. A rapid and portable method for detecting dengue virus-infected mosquitoes is described. Using a hand held battery operated homogenizer and a dengue diagnostic rapid strip the viral protein NS1 was detected as a marker of dengue virus infection. This method could be performed in less than 30 min in the field, requiring no downstream processing, and is able to detect a single infected mosquito in a pool of at least 50 uninfected mosquitoes. The method described in this study allows rapid, real-time monitoring of dengue virus presence in mosquito populations and could be a useful addition to effective monitoring and vector control responses.

Molecular evolution of the insect-specific flaviviruses.

There has been an explosion in the discovery of ‘insect-specific’ flaviviruses and/or their related sequences in natural mosquito populations. Herein we review all ‘insect-specific’ flavivirus sequences currently available and conduct phylogenetic analyses of both the ‘insect-specific’ flaviviruses and available sequences of the entire genus Flavivirus. We show that there is no statistical support for virus–mosquito co-divergence, suggesting that the ‘insect-specific’ flaviviruses may have undergone multiple introductions with frequent host switching. We discuss potential implications for the evolution of vectoring within the family Flaviviridae. We also provide preliminary evidence for potential recombination events in the history of cell fusing agent virus. Finally, we consider priorities and guidelines for future research on ‘insect-specific’ flaviviruses, including the vast potential that exists for the study of biodiversity within a range of potential hosts and vectors, and its effect on the emergence and maintenance of the flaviviruses.

Mosquitoes attacked from within

Infection with a harmless bacterium makes the mosquitoes that transmit dengue virus resistant to viral infection. The resistant population can rapidly replace the natural, susceptible population. See Letters p.450 & p.454 The mosquito-borne viral disease dengue fever is an increasing problem in tropical and subtropical regions. Traditional control measures aimed at reducing populations of the main transmission vector, Aedes aegypti, have had little success. Two papers in this issue report an alternative approach to mosquito population control using the bacterium Wolbachia pipientis, natural insect symbionts that facilitate their own transmission through a process called cytoplasmic incompatibility. In the first paper, Scott O'Neill and colleagues describe a Wolbachia strain derived from fruitflies that significantly reduces dengue virus carriage in mosquitoes without imposing a fitness cost. In the second paper, they demonstrate in a controlled field trial that the release of colonized mosquitoes leads to successful invasion of natural mosquito populations. These results suggest a viable strategy to control dengue fever.

Impact of West Nile Virus Dose and Incubation Period on Vector Competence ofCulex nigripalpus(Diptera: Culicidae)

Female Culex nigripalpus were fed blood containing a low dose (6.3±0.01 logs plaque-forming units (PFU)/mL) or high dose (7.3±0.1 logs PFU/mL) of West Nile virus (WNV) and maintained at 28°C for incubation periods (IPs) of 6 or 12 days. Vector competence was measured using rates of infection (% with WNV-positive bodies), dissemination (% infected with WNV-positive legs), and transmission (% infected with WNV-positive saliva). Infection rates were not influenced by dose or IP. Dissemination rates were significantly higher at the high dose, and this was dependent on IP. Despite 100% infection and 90% dissemination in the most permissive treatment of high dose and 12 days, only 11% transmission was observed. Virus titers in body and leg tissues were significantly lower at the low dose and the titers were not influenced by IP. We show that not all mosquitoes with infections and/or disseminated infections transmit WNV under the conditions of this test. Therefore, characterizing the transmission ability of a vector population using infection or dissemination as indicators of transmission may provide inaccurate information. The complex relationships between infection, dissemination, and transmission must be evaluated under a variety of biological and environmental conditions to begin to assess the epidemiological risk of natural mosquito populations.

Evidence of co-infection of chikungunya and densonucleosis viruses in C6/36 cell lines and laboratory infected Aedes aegypti (L.) mosquitoes.

BackgroundDensonucleosis viruses are the etiological agents of insect's disease. We have reported the isolation of densovirus from India and its distribution among the natural populations of Aedes aegypti mosquitoes across the country. Since densonucleosis virus persistently infects mosquito populations, and is demonstrated to negatively affect multiplication of dengue virus in Aedes albopictus, it would be interesting to study if this virus has a role in determining the susceptibility of the vector mosquito Ae. aegypti to chikugunya virus.MethodsMosquito cell lines and adult Ae. aegypti mosquitoes infected with densovirus were superinfected with Chikungunya virus and both the viruses were quantitated by determining their genomic copy number by real time amplification. Comparison was made between the log of genomic copy numbers of the viruses in the presence and absence of each other.ResultsThe log of copy number of the viruses did not vary due to co-infection. Even though the RNA copy number of chikungunya virus increased over the period of time, no change was observed in the RNA copy number between the control and the co-infected group on any given day. Similarly, DNA copy number of densovirus also remained unchanged between the control and the co-infected groups.ConclusionChikungunya virus neither stimulates the replication of densovirus nor is its own replication suppressed due to co-infection. Ae. aegypti mosquitoes with densovirus infection were as susceptible to infection by chikungunya virus as the uninfected mosquitoes.

Hydric stress-dependent effects of Plasmodium falciparum infection on the survival of wild-caught Anopheles gambiae female mosquitoes

BackgroundWhether Plasmodium falciparum, the agent of human malaria responsible for over a million deaths per year, causes fitness costs in its mosquito vectors is a burning question that has not yet been adequately resolved. Understanding the evolutionary forces responsible for the maintenance of susceptibility and refractory alleles in natural mosquito populations is critical for understanding malaria transmission dynamics.MethodsIn natural mosquito populations, Plasmodium fitness costs may only be expressed in combination with other environmental stress factors hence this hypothesis was tested experimentally. Wild-caught blood-fed Anopheles gambiae s.s. females of the M and S molecular form from an area endemic for malaria in Mali, West Africa, were brought to the laboratory and submitted to a 7-day period of mild hydric stress or kept with water ad-libitum. At the end of this experiment all females were submitted to intense desiccation until death. The survival of all females throughout both stress episodes, as well as their body size and infection status was recorded. The importance of stress, body size and molecular form on infection prevalence and female survival was investigated using Logistic Regression and Proportional-Hazard analysis.ResultsFemales subjected to mild stress exhibited patterns of survival and prevalence of infection compatible with increased parasite-induced mortality compared to non-stressed females. Fitness costs seemed to be linked to ookinetes and early oocyst development but not the presence of sporozoites. In addition, when females were subjected to intense desiccation stress, those carrying oocysts exhibited drastically reduced survival but those carrying sporozoites were unaffected. No significant differences in prevalence of infection and infection-induced mortality were found between the M and S molecular forms of Anopheles gambiae.ConclusionsBecause these results suggest that infected mosquitoes may incur fitness costs under natural-like conditions, they are particularly relevant to vector control strategies aiming at boosting naturally occurring refractoriness or spreading natural or foreign genes for refractoriness using genetic drive systems in vector populations.

Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: Culicidae)

Chemical composition and larvicidal activity of edible plant-derived essential oils against the pyrethroid-susceptible and -resistant strains of Aedes aegypti (Diptera: Culicidae)

Genome scan in the mosquito Aedes rusticus: population structure and detection of positive selection after insecticide treatment

Identification of genes involved in local adaptation is particularly challenging for species functioning as a network of interconnected populations undergoing frequent extinctions-recolonizations, because populations are submitted to contrasted evolutionary pressures. Using amplified fragment length polymorphism markers, population genetic structure of the mosquito Aedes rusticus was analysed in five geographical areas of the French Rhône-Alpes region. We included a number of sites that were treated with the bio-insecticide Bacillus thuringiensis israelensis (Bti) for more than 15 years. Analysis of molecular variance revealed that most of the genetic variability was found within populations (96%), with no significant variation among geographical areas, although variation among populations within areas (4%) was significant. The global genetic differentiation index F(ST) was low (0.0366 +/- 0.167). However, pairwise F(ST) values were significant and no isolation-by-distance at the regional level was observed, suggesting a metapopulation structure in this species. Bti-treatment had no effect on genetic structure and on within-population genetic diversity. Potential signatures of positive selection associated with Bti-treatment were detected for five loci, even though toxicological bioassays performed on field-collected larvae showed no significant difference in mortality between Bti-treated and nontreated sites. The difficulty of detecting moderate resistance in field-collected larvae together with possible differential persistence of toxins in the environment may explain our inability to detect a toxicological response to Bti in treated sites. The evidence for positive selection occurring at several genomic regions suggests a first step towards Bti resistance in natural mosquito populations treated with this bio-insecticide. Furthermore, this signal was detectable using genomic tools before any toxicological evidence for resistance could be identified.

Laboratory and Field Evaluation of Spinosad, a Biorational Natural Product, Against Larvae of Culex Mosquitoes

Spinosad, a fermentation product from the naturally occurring soil actinomycete bacterium Saccharopolyspora spinosa, has been reported to have a high level of activity against phytophagous insects and insects impacting human and animal health. It has low mammalian toxicity and a favorable environmental profile, including low persistence and no toxicity to fish and wildlife at mosquito larvicidal rates. In order to determine the activity and efficacy of spinosad against larvae of Culex mosquitoes, technical powder and liquid formulations of spinosad were tested against mosquito larvae under laboratory and field conditions. In the laboratory, spinosad powder was highly active against 2nd and 4th instars of Culex quinquefasciatus after 24 h of exposure. The extent of mortality increased slightly after 48 h of exposure. Second instars were slightly more susceptible than 4th instars. The liquid formulation showed somewhat higher activity (about 2x) than the technical powder material at both the LCs50 and LC90 levels. In field microcosm tests against natural populations of mosquitoes, the liquid formulation yielded excellent control of immature Culex spp. for 21 days at concentrations of 0.05 mg (AI)/liter and 35 days at 0.1 to 0.5 mg (AI)/liter in outdoor tubs. This formulation also yielded excellent control of natural Culex mosquitoes for 14 days or longer at 0.025 to 0.1 mg (AI)/liter in outdoor ponds. From our data, it appears that spinosad as a new mode of action has a good potential for controlling mosquito larvae.

Isolation of a novel species of flavivirus and a new strain of Culex flavivirus (Flaviviridae) from a natural mosquito population in Uganda.

The genus Flavivirus, which contains approximately 70 single-stranded, positive-sense RNA viruses, represents a unique model for studying the evolution of vector-borne disease, as it includes viruses that are mosquito-borne, tick-borne or have no known vector. Both theoretical work and field studies suggest the existence of a large number of undiscovered flaviviruses. Recently, the first isolation of cell fusing agent virus (CFAV) was reported from a natural mosquito population in Puerto Rico, and sequences related to CFAV have been discovered in mosquitoes from Thailand. CFAV had previously been isolated from a mosquito cell line in 1975 and represented the only known ‘insect-only’ flavivirus, appearing to replicate in insect cells alone. A second member of the ‘insect-only’ group, Kamiti River virus (KRV), was isolated from Kenyan mosquitoes in 2003. A third tentative member of the ‘insect-only’ group, Culex flavivirus (CxFV), was first isolated in 2007 from Japan and further strains have subsequently been reported from the Americas. We report the discovery, isolation and characterization of two novel ‘insect-only’ flaviviruses from Entebbe, Uganda: a novel lineage tentatively designated Nakiwogo virus (NAKV) and a new strain of CxFV. The individual mosquitoes from which these strains were isolated, identified retrospectively by using a reference molecular phylogeny generated using voucher specimens from the region, were Mansonia africana nigerrima and Culex quinquefasciatus, respectively. This represents the first isolation, to our knowledge, of a novel insect-only flavivirus from a Mansonia species and the first isolation of a strain of CxFV from Africa.

Field trials on the efficacy of DEET-impregnated anklets, wristbands, shoulder, and pocket strips against mosquito vectors of disease

A field trial was undertaken in order to determine the efficacy of DEET (N,N-diethyl-m-toluamide) impregnated anklets, wristbands, shoulder and pocket fabric strips against mosquito vectors of disease. The present study was conducted in the urban locality of Pondicherry, India. Human test subjects were exposed to natural populations of mosquitoes for a 12 h (18.00-06.00) night time period. The fabric strips (anklets, wristbands, shoulder, and pocket strips) were impregnated with DEET at two different concentrations of 1.5 mg/cm(2) and 2.0 mg/cm(2). The results clearly revealed that DEET-impregnated anklets, wristbands, shoulder and pocket fabric strips were found more effective against mosquitoes remarkably. The DEET-impregnated anklets, wristbands, shoulder and pocket fabric strips at a concentration of 2 mg/cm(2) provided 5 h complete protection against mosquitoes bites and the reduction of man-landing rate varied between 65.85 and 100%. However, DEET-impregnated fabric strips at a concentration of 1.5 mg/cm(2) provided 4 h complete protection against mosquito bites and the reduction of man-landing rate varied between 51.21 and 100%. The final results clearly demonstrate that repellent activity of DEET-impregnated anklets, wristbands, shoulder, and pocket strips were dose-dependent. Certainly, the DEET-impregnated fabric strips can be used as an effective potential personal protection measure in order to avoid those insects/mosquitoes that prefer to feed outdoors or those that feed in the early evening.

Field evaluation of G10, a celery (Apium graveolens)-based topical repellent, against mosquitoes (Diptera: Culicidae) in Chiang Mai province, northern Thailand

The potential of G10, a celery (Apium graveolens)-based topical product, as a repellent against natural mosquito populations was evaluated in comparison to commercial (Insect Block 28) and standard (25% DEET) repellents in Chiang Mai province, northern Thailand. These repellent products afforded encouragingly excellent personal protection against a broad range of mosquito species belonging to various genera, including Aedes, Anopheles, Armigeres, Culex, and Mansonia. No mosquito bite was observed on the volunteers treated with G10 and Insect Block 28 throughout the field study, whereas two species, i.e., six A. barbirostris and two A. subalbatus, came to bite or land on 25% DEET-treated volunteers. Thus, it can be concluded that while G10 and Insect Block 28 exhibited similarly powerful repellent activities with complete (100%) protection, 25% DEET was effective in minimizing bites with 99.68% protection. G10 formula was also studied for physical properties and biological stability after being kept under two conditions; a heating and cooling cycle, and varying temperature and time storage. Most samples of stored G10 not only demonstrated a similarity in appearance and physical properties, but also provided comparable repellency to that of the fresh preparation. These findings encourage commercial development of G10 formula as an alternative to conventional synthetic repellents.

Field Evaluation of Boric Acid- and Fipronil-Based Bait Stations Against Adult Mosquitoes

The effectiveness of boric acid (1%) and fipronil (0.1%) bait stations in reducing the number of laboratory-reared female Aedes aegypti and Ochlerotatus taeniorhynchus mosquitoes released in outdoor screened cages was evaluated. Both toxicants significantly reduced landing rates of the 2 mosquito species on a human subject. However, no significant reduction in the number of adult mosquitoes caught by counterflow geometry (CFG) traps baited with CO2 was observed in the boric acid- and fipronil-treated cages compared with control cage. The 2 bait stations were further evaluated against natural populations of adult mosquitoes in 3 residential yards (each ca. 0.8 ha) in St. Johns County, FL. Both bait stations failed to significantly reduce counts of female mosquitoes landing on a human subject; and the capture rates of adult mosquitoes by CFG traps baited with dry ice in bait-treated and control experimental yards were not significantly different. Further field research is needed on the density and placement of bait stations in order to determine their utility in adult mosquito control programs.

Amino-acid substitutions in acetylcholinesterase 1 involved in insecticide resistance in mosquitoes

In natural populations of mosquitoes, high level of resistance to carbamates (CX) and organophosphates (OP) is provided by insensitive acetylcholinesterase (AChE1). Different alleles conferring resistance have been identified at the ace1 locus. They differ from the wild-type by only one amino-acid substitution. The comparison of the biochemical characteristics of mutated recombinant proteins and AChE1 in resistant mosquito extracts confirmed the role of each substitution in insensitivity. Selection of these different resistant alleles in field populations depends likely on the insecticides used locally. Theoretical modelling studies are initiated to develop novel strategies of mosquito control.

Evaluation of the enantiomers of 1‐octen‐3‐ol and 1‐octyn‐3‐ol as attractants for mosquitoes associated with a freshwater swamp in Florida, U.S.A.

Field studies were conducted at wooded wetlands in Gainesville, FL, U.S.A., to assess responses of natural populations of adult mosquitoes (Diptera: Culicidae) to American Biophysics MM-X and Coleman MD-2500 traps baited with enantiomers of 1-octen-3-ol, a naturally occurring compound, and 1-octyn-3-ol, a closely related synthetic compound. Overall, the same species of mosquitoes were attracted by all enantiomers, although the (R)-(+) isomer of octenol generally attracted more species, and it is the isomer produced in greatest proportion in nature. Traps baited with the R-enantiomer caught greater numbers of mosquitoes than those baited with the S-enantiomer of each compound, whereas traps baited with S-enantiomers were equally or slightly less attractive than those baited with carbon dioxide only.

Do mosquitoes acquire organophosphate resistance by functional changes in carboxylesterases?

Carboxylesterase-based metabolic resistance to organophosphates (OPs) in insects has been shown to originate either from mutations in esterase-encoding sequences or from amplification of esterase genes. This study aimed to test the hypothesis that mosquitoes can acquire OP resistance by functional changes in carboxylesterases. Mutations were introduced into the esterase B1 of mosquito Culex pipiens by site-directed mutagenesis at positions 110 and 224. Three single mutants (G110D, W224L, and W224S) and two double mutants (G110D/W224L and G110D/W224S) were expressed and purified. All five mutants lost native carboxylesterase activity. Mutation W224L converted esterase B1 to an OP hydrolase and increased its malathion carboxylesterase activity. No obvious OP hydrolysis was observed by G110D or W224S. Our data strongly support our hypothesis and suggest that mutation W224L might occur in natural populations of mosquitoes. Sequence comparison shows that the site 224 is especially highly conserved among various insect carboxylesterases. This leads to another hypothesis: that the position 224 plays a key role in insect carboxylesterases' switching from their native physiological functions to other functional niches under selection pressure exerted by insecticides.

Effectiveness of Zanthoxylum piperitum-derived essential oil as an alternative repellent under laboratory and field applications

Recently, there were considerable efforts made to promote the use of environmentally friendly and biodegradable natural insecticides and repellents, particularly from botanical sources. In this study, Zanthoxylum piperitum-derived essential oil isolated by steam distillation was investigated and compared to the standard synthetic repellent, N,N-diethyl-3-methylbenzamide (DEET), for repellency against mosquitoes under laboratory and field conditions. The oil of Z. piperitum alone and also with 5% vanillin added repelled laboratory-reared female Aedes aegypti with the median protection times of 1.5 and 2.5 h, respectively. These repellency times were proven to be significantly lower than those of DEET-based products: 25% DEET and 25% DEET + 5% vanillin demonstrating 3.5 and 5.5 h, respectively. However, when applied under field conditions, Z. piperitum oil + 5% vanillin was found to provide better protection against a wide range of natural mosquito populations than 25% DEET + 5% vanillin. While the product of DEET was effective in reducing bites with 99.7% protection, the protective effect of Z. piperitum oil product appeared complete (100%). It had a protective effect against Aedes gardnerii, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelidus, Culex vishnui group, and Mansonia uniformis. The better repellency against a wide range of field mosquitoes derived from Z. piperitum oil products suggested an advantage of efficacy by providing a broad spectrum of activity. Therefore, Z. piperitum could have potential for use in the development of combined repellents as a natural active ingredient, synergist, or additive to conventional synthetic chemicals, particularly in situations when DEET is ineffective and impractical.

Isolation of a new strain of the flavivirus cell fusing agent virus in a natural mosquito population from Puerto Rico

The genus Flavivirus contains approximately 70 single-stranded, positive-sense RNA viruses that are mosquito-borne, tick-borne or have no known vector. Two discoveries support previous suggestions of the existence of a large number of unsampled flaviviruses: (i) a new flavivirus, Kamiti River virus, was recently isolated from Kenyan mosquitoes, and (ii) sequences with high similarity to those of flaviviruses have been found integrated into the genome of Aedes mosquitoes, suggesting a past infection with a virus (or viruses) that has yet to be discovered. These sequences were related most closely to a flavivirus that infects insects alone, cell fusing agent virus (CFAV). CFAV was originally isolated in the laboratory from an Aedes aegypti cell line. To date, this virus had not been found in the wild. In the present study, over 40 isolates of a novel strain of CFAV were discovered from mature mosquitoes sampled from the wild in Puerto Rico. The viral strain was present in a range of mosquito species, including Aedes aegypti, Aedes albopictus and Culex sp., from numerous locations across the island and, importantly, in mosquitoes of both sexes, suggesting vertical transmission. Here, results from viral screening, and cell culture and molecular identification of the infected mosquitoes are presented. Experimental-infection tests were also conducted by using the original CFAV strain and a highly efficient reverse-transcription mechanism has been documented, in which initiation of copying occurs at the 3' terminus of either the genomic RNA or the intermediate of replication, potentially elucidating the mechanism by which flaviviral sequences may have integrated into mosquito genomes.