Female Anopheles Gambiae Mosquitoes Research Articles

Effects of Combretum micranthum, Xienmia americana and Aloysia citrodora Leaf Extracts against Oviposition and Egg Viability of Anopheles gambiae S.L. (Diptera: Culicidae)

The study investigated the oviposition and egg viability effects of leaf extracts from Combretum micranthum, Xienmia americana, and Aloysia citrodora on female Anopheles gambiae mosquitoes. Leaves were sourced from Goron-maje Town, Dambatta LGA, Kano State, and extracts were prepared using ethanol, methanol, and ethyl-acetate. Bioassays were conducted at ambient temperature and relative humidity, testing oviposition activity and egg viability. Ethyl-acetate extracts, particularly from A. citrodora, significantly reduced oviposition compared to controls, with a 42.85% decrease. Ethanol extract of C. micranthum also showed reduced oviposition (8.84%). Significant differences were observed in the effects of different concentrations (ppm/mL) of plant extracts compared to controls. X. americana ethanol leaf extract at 20.0 ml concentration showed a 10% decrease in egg viability, while Combretum micranthum extracts at 20.0 ml and 30.0 ml concentrations showed 40.0% and 26.0% reductions in viability respectively. Aloysia citrodora methanol extracts exhibited a lower effect (48.0%) on viability compared to controls. X. americana ethanol leaf extract at 10% concentration significantly reduced hatching ability (26±06.21). Similarly, ethanol and ethyl-acetate extracts of C. micranthum and A. citrodora at 20.0 ml and 30.0 ml concentrations showed decreased hatching abilities compared to controls. The study suggests that ethanol and ethyl-acetate extracts of X. americana, C. micranthum, and A. citrodora could be effective and safer methods for mosquito control.

Ovary Dissection Is a Sensitive Measure of Sterility in Anopheles gambiae Exposed to the Insect Growth Regulator Pyriproxyfen.

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

Behavioral responses of pyrethroid resistant and susceptible Anopheles gambiae mosquitoes to insecticide treated bed net.

Long-lasting insecticidal nets are an effective tool in reducing malaria transmission. However, with increasing insecticide resistance little is known about how physiologically resistant malaria vectors behave around a human-occupied bed net, despite their importance in malaria transmission. We used the Mbita bednet trap to assess the host-seeking behavior of insecticide-resistant Anopheles gambiae mosquitoes under semi-field conditions. The trap incorporates a mosquito netting panel which acts as a mechanical barrier that prevents host-seeking mosquitoes from reaching the human host baiting the trap. Susceptible and pyrethroid-resistant colonies of female Anopheles gambiae mosquitoes aged 3-5 days old were used in this study. The laboratory-bred mosquitoes were color-marked with fluorescent powders and released inside a semi-field environment where a human subject slept inside a bednet trap erected in a traditional African hut. The netting panel inside the trap was either untreated (control) or deltamethrin-impregnated. The mosquitoes were released outside the hut. Only female mosquitoes were used. A window exit trap was installed on the hut to catch mosquitoes exiting the hut. A prokopack aspirator was used to collect indoor and outdoor resting mosquitoes. In addition, clay pots were placed outside the hut to collect outdoor resting mosquitoes. The F1 progeny of wild-caught mosquitoes were also used in these experiments. The mean number of resistant mosquitoes trapped in the deltamethrin-impregnated bed net trap was higher (mean = 50.21± 3.7) compared to susceptible counterparts (mean + 22.4 ± 1.31) (OR = 1.445; P<0.001). More susceptible mosquitoes were trapped in an untreated (mean = 51.9 ± 3.6) compared to a deltamethrin-treated bed net trap (mean = 22.4 ± 1.3) (OR = 2.65; P<0.001). Resistant mosquitoes were less likely to exit the house when a treated bed net was present compared to the susceptible mosquitoes. The number of susceptible mosquitoes caught resting outdoors (mean + 28.6 ± 2.22) when a treated bed net was hanged was higher than when untreated bednet was present inside the hut (mean = 4.6 ± 0.74). The susceptible females were 2.3 times more likely to stay outdoors away from the treated bed net (OR = 2.25; 95% CI = [1.7-2.9]; P<0.001). The results show that deltamethrin-treatment of netting panels inside the bednet trap did not alter the host-seeking behavior of insecticide-resistant female An. gambiae mosquitoes. On the contrary, susceptible females exited the hut and remained outdoors when a treated net was used. However, further investigations of the behavior of resistant mosquitoes under natural conditions should be undertaken to confirm these observations and improve the current intervention which are threatened by insecticide resistance and altered vector behavior.

Molecular Detection of Insecticide Resistance Mutations in Anopheles gambiae from Sierra Leone Using Multiplex SNaPshot and Sequencing.

Vector control interventions including long-lasting insecticidal nets and indoor residual spraying are important for malaria control and elimination. And effectiveness of these interventions depends entirely on the high level of susceptibility of malaria vectors to insecticides. However, the insecticide resistance in majority of mosquito vector species across African countries is a serious threat to the success of vector control efforts with the extensive use of insecticides, while no data on insecticide resistance was reported from Sierra Leone in the past decade. In the present study, the polymerase chain reaction was applied for the identification of species of 757 dry adult female Anopheles gambiae mosquitoes reared from larvae collected from four districts in Sierra Leone during May and June 2018. And the mutations of kdr, rdl, ace-1 genes in An. gambiae were detected using SNaPshot and sequencing. As a result, one sample from Western Area Rural district belonged to Anopheles melas, and 748 An. gambiae were identified. Furthermore, the rdl mutations, kdr west mutations and ace-1 mutation were found. The overall frequency was 35.7%, 0.3%, 97.6% and 4.5% in A296G rdl, A296S rdl, kdrW and ace-1, respectively. The frequencies of A296G rdl mutation (P < 0.001), kdrW mutation (P = 0.001) and ace-1 mutation (P < 0.001) were unevenly distributed in four districts, respectively, while no statistical significance was found in A296S rdl mutation (P = 0.868). In addition, multiple resistance patterns were also found. In conclusion, multiple mutations involved in insecticide resistance in An. gambiae populations in Sierra Leone were detected in the kdrW, A296G rdl and ace-1 alleles in the present study. It is necessary to monitor vector susceptibility levels to insecticides used in this country, and update the insecticide resistance monitoring and management strategy.

Root exudate chemical cues of an invasive plant modulate oviposition behavior and survivorship of a malaria mosquito vector

Gravid female Anopheles gambiae mosquitoes identify suitable oviposition sites through a repertoire of cues, but the influence of allelochemicals, especially root phytochemicals in modulating this behavior and impacting subsequent progeny bionomics remains unexplored. We addressed these questions in the malaria vector Anopheles gambiae and its invasive host plant Parthenium hysterophorus. Using chemical analysis combined with laboratory behavioral assays, we demonstrate that a blend of terpenes, namely α-pinene, α-phellandrene, β-phellandrene, 3-carene and (E)-caryophyllene emitted from P. hysterophorus root exudate treated-water attracted gravid females. However, fewer eggs (55%) hatched in this treatment than in control water (66%). The sesquiterpene lactone parthenin, identified in both the natural aquatic habitat harboring P. hysterophorus and root exudate-treated water was found to be responsible for the ovicidal effect. Moreover, larvae exposed to parthenin developed 2 to 3 days earlier but survived 4 to 5 days longer as adults (median larval survival time = 9 days (all replicates);11 to 12 days as adults) than the non-exposed control (median larval survival time = 11 days (reps 1 & 2), 12 days (rep 3); 6 to 7 days as adults). These results improve our understanding of the risk and benefits of oviposition site selection by gravid An. gambiae females and the role root exudate allelochemicals could play on anopheline bionomics, with potential implications in malaria transmission.

Birds as a food base for mosquitoes – carriers of the causative agent of tropical malaria

The paper discusses the food supply of the vector of malaria mosquitoes of the genus Anopheles, which are birds of tropical regions of West Africa. Birds, as distant migrants, penetrate high latitudes and contribute to the spread of malaria in Europe and other countries of the northern hemisphere. The results of the studies show that the main role in the choice of prey objects by female Anopheles gambiae mosquitoes birds is played by the nesting and forage biotopes of birds, which are comfortable for mosquito breeding. Probably, female mosquitoes use non-feathered parts of the body of adult birds for feeding unfeathered or weakly feathered chicks in nests. The circulation of Plasmodium falciparum includes populations of birds, primarily water, near-water and marsh complexes, as well as birds, the development of which takes place in specific conditions of a closed space in holes, hollows and closed nests. The Anopheles gambiae mosquito in this system plays the role of a carrier of Plasmodium falciparum not only among populations of birds and mammals, but also among humans, which determines the range of tropical malaria, which is a natural focal vector-borne disease. The authors have identified 37 species of birds carriers of malaria in natural and anthropogenic biocoenoses of Mali (West Africa). The most numerous during the migration and nesting period are birds of the aquatic, near-water and meadow-bog complexes (herons, herons, waders) distant migrants on the territory of Russia and neighboring countries. The risk areas include, first of all, the southern regions Astrakhan Region, Rostov Region and Krasnodar Region.

Dynamic miRNA-mRNA interactions coordinate gene expression in adult Anopheles gambiae.

microRNAs (miRNAs) are increasingly recognized as important regulators of many biological processes in mosquitoes, vectors of numerous devastating infectious diseases. Identification of bona fide targets remains the bottleneck for functional studies of miRNAs. In this study, we used CLEAR-CLIP assays to systematically analyze miRNA-mRNA interactions in adult female Anopheles gambiae mosquitoes. Thousands of miRNA-target pairs were captured after direct ligation of the miRNA and its cognate target transcript in endogenous Argonaute–miRNA–mRNA complexes. Using two interactions detected in this manner, miR-309-SIX4 and let-7-kr-h1, we demonstrated the reliability of this experimental approach in identifying in vivo gene regulation by miRNAs. The miRNA-mRNA interaction dataset provided an invaluable opportunity to decipher targeting rules of mosquito miRNAs. Enriched motifs in the diverse targets of each miRNA indicated that the majority of mosquito miRNAs rely on seed-based canonical target recognition, while noncanonical miRNA binding sites are widespread and often contain motifs complementary to the central or 3’ ends of miRNAs. The time-lapse study of miRNA-target interactomes in adult female mosquitoes revealed dynamic miRNA regulation of gene expression in response to varying nutritional sources and physiological demands. Interestingly, some miRNAs exhibited flexibility to use distinct sequences at different stages for target recognition. Furthermore, many miRNA-mRNA interactions displayed stage-specific patterns, especially for those genes involved in metabolism, suggesting that miRNAs play critical roles in precise control of gene expression to cope with enormous physiological demands associated with egg production. The global mapping of miRNA-target interactions contributes to our understanding of miRNA targeting specificity in non-model organisms. It also provides a roadmap for additional studies focused on regulatory functions of miRNAs in Anopheles gambiae.

Behavioural and Electrophysiological Responses of Female Anopheles gambiae Mosquitoes to Volatiles from a Mango Bait

Attractive Toxic Sugar Baits (ATSB) are used in a “lure-and-kill” approach for management of the malaria vector Anopheles gambiae, but the active chemicals were previously unknown. Here we collected volatiles from a mango, Mangifera indica, juice bait which is used in ATSBs in Tanzania and tested mosquito responses. In a Y-tube olfactometer, female mosquitoes were attracted to the mango volatiles collected 24–48 h, 48–72 h and 72–96 h after preparing the bait but volatiles collected at 96–120 h were no longer attractive. Volatile analysis revealed emission of 23 compounds in different chemical classes including alcohols, aldehydes, alkanes, benzenoids, monoterpenes, sesquiterpenes and oxygenated terpenes. Coupled GC-electroantennogram (GC-EAG) recordings from the antennae of An. gambiae showed robust responses to 4 compounds: humulene, (E)-caryophyllene, terpinolene and myrcene. In olfactometer bioassays, mosquitoes were attracted to humulene and terpinolene. (E)-caryophyllene was marginally attractive while myrcene elicited an avoidance response with female mosquitoes. A blend of humulene, (E)-caryophyllene and terpinolene was highly attractive to females (P < 0.001) when tested against a solvent blank. Furthermore, there was no preference when this synthetic blend was offered as a choice against the natural sample. Our study has identified the key compounds from mango juice baits that attract An. gambiae and this information may help to improve the ATSBs currently used against malaria vectors.

&lt;i&gt;Anopheles gambiae s.l.&lt;/i&gt; insecticide susceptibility status in Umudike, Ikwuano LGA, Abia State, Nigeria

This study was conducted in order to determine the insecticide susceptibility status of Anopheles gambiae in Umudike, Abia State, Nigeria. Larval and pupal stages of An. gambiae mosquitoes were collected from different points within Umudike, and reared to adult stage in the laboratory. The adults that emerged were tested on four types of WHO bioassay test papers impregnated with recommended diagnostic concentrations of 4% DDT (Organochlorines); 0.1% bendiocarb (Carbamates); 0.25% Primiphos-methyl (Organophosphates); and 0.05% Deltamethrin (Pyrethroids) procured from National Abovirus and Vector Research Institute Enugu. Twenty 10% sugar solution fed female Anopheles gambiae mosquitoes aged 3-5days were used for the bioassay which was replicated four times with two control of same population without insecticide treatment. Knockdown was recorded at five minutes post-exposure, and then at 10 minutes interval for 1 hour and then maintained for 24 hours post-exposure on 10% sugar solution, after which a final mortality was recorded. The Knockdown Times (KDT50 and KDT90) were determined by Probit analysis. An. gambiae was resistant to DDT, primiphos-methyl and Deltamethrin with 24 hours post exposure percentage mortalities of 4.65, 11.85, and 24.83 respectively.In contrary the mosquitoes were susceptible to bendiocarb which killed 98.88% of the mosquitoes 24 hours after exposure. Bendiocarb is hence recommended as the most effective insecticide for the control of Anopheles gambiae in Umudike, while there were need for the investigation of the mechanism behind the resistance displayed by this mosquito. Furthermore, routine surveillance of insecticide susceptibility/resistance in mosquito population is advocated in line with integrated vector control strategy.&#x0D; Keywords: Insecticide; susceptibility; Anopheles gambiae; Umudike; Abia State; Nigeria

A testing cascade to identify repurposed insecticides for next-generation vector control tools: screening a panel of chemistries with novel modes of action against a malaria vector.

Background: With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved to date by long-lasting insecticidal nets and indoor residual sprays, used as part of an insecticide resistance management strategy. Screening existing registered pesticides, predominantly those developed for use in agriculture, may provide a more rapid and less logistically challenging route to identifying active ingredients of value to public health than screening and chemical synthesis programmes for novel compounds. Methods: Insecticides and acaricides from all IRAC classes, including those with unclassified modes of action, were assessed for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae mosquitoes. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and likely hurdles to registration for public health use. An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal contact assay identified those with activity at an appropriate concentration. Compounds of interest were ranked by relative potency using dose response assays and discriminating dose calculations. Results: Inclusion of an adjuvant enhanced the tarsal efficacy of several compounds, facilitating the promotion of chemistries with great potential, given suitable formulation, which would not progress based on activity of compound alone. Comparison of data between stages in the testing cascade suggest that a more streamlined approach, topical application to test for intrinsic activity and determining the discriminating dose to compare relative potency of compounds, may be sufficient to identify compounds with potential value for use in long lasting insecticidal nets and indoor residual spray products. Conclusions: Identified were 11 compounds of interest as vector control agents (in descending order of potency): clothianidin, spinetoram, metaflumizone, dinotefuran, indoxacarb, abamectin, sulfoxaflor, oxazosulfyl, triflumezopyrim, fenpyroximate, and tolfenpyrad.

A testing cascade to identify repurposed insecticides for next-generation vector control tools: screening a panel of chemistries with novel modes of action against a malaria vector

Background: With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved to date by long-lasting insecticidal nets and indoor residual sprays, used as part of an insecticide resistance management strategy. Screening existing registered pesticides, predominantly those developed for use in agriculture, may provide a more rapid and less logistically challenging route to identifying active ingredients of value to public health than screening and chemical synthesis programmes for novel compounds. Methods: Insecticides and acaricides from all IRAC classes, including those with unclassified modes of action, were assessed for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae mosquitoes. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and likely hurdles to registration for public health use. An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal contact assay identified those with activity at an appropriate concentration. Compounds of interest were ranked by relative potency using dose response assays and discriminating dose calculations. Results: Inclusion of an adjuvant enhanced the tarsal efficacy of several compounds, facilitating the promotion of chemistries with great potential, given suitable formulation, which would not progress based on activity of compound alone. Comparison of data between stages in the testing cascade suggest that a more streamlined approach, topical application to test for intrinsic activity and determining the discriminating dose to compare relative potency of compounds, may be sufficient to identify compounds with potential value for use in long lasting insecticidal nets and indoor residual spray products. Conclusions: Identified were 11 compounds of interest as vector control agents (in descending order of potency): clothianidin, spinetoram, metaflumizone, dinotefuran, indoxacarb, abamectin, sulfoxaflor, oxazosulfyl, triflumezopyrim, fenpyroximate, and tolfenpyrad.

A comprehensive testing cascade to identify resistance breaking repurposed insecticides for next-generation vector control tools: screening a panel of chemistries against a malaria vector.

Background: With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved to date by long-lasting insecticidal nets and indoor residual sprays, used as part of an insecticide resistance management strategy. Screening existing registered pesticides, predominantly those developed for use in agriculture, may provide a more rapid and less logistically challenging route to identifying active ingredients of value to public health than screening and chemical synthesis programmes for novel compounds. Methods: Insecticides and acaricides from all IRAC classes, including those with unclassified modes of action, were assessed for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae mosquitoes. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and likely hurdles to registration for public health use. An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal contact assay identified those with activity at an appropriate concentration. Compounds of interest were ranked by relative potency using dose response assays and discriminating dose calculations. Results: Inclusion of an adjuvant enhanced the tarsal efficacy of several compounds, facilitating the promotion of chemistries with great potential, given suitable formulation, which would not progress based on activity of compound alone. Comparison of data between stages in the testing cascade suggest that a more streamlined approach, topical application to test for intrinsic activity and determining the discriminating dose to compare relative potency of compounds, may be sufficient to identify compounds with potential value for use in long lasting insecticidal nets and indoor residual spray products. Conclusions: Identified were 11 compounds of interest as vector control agents (in descending order of potency): clothianidin, spinetoram, metaflumizone, dinotefuran, indoxacarb, abamectin, sulfoxaflor, oxazosulfyl, triflumezopyrim, fenpyroximate, and tolfenpyrad.

Exposing Anopheles mosquitoes to antimalarials blocks Plasmodium parasite transmission.

Every year the bites of Anopheles mosquitoes kill hundreds of thousands of people, mostly young African children, by transmitting deadly Plasmodium falciparum malaria parasites. Since the turn of the century, efforts to prevent transmission of these parasites via the mass distribution of insecticide-treated bed nets have been extremely successful, causing an unprecedented reduction in malaria deaths1. However, resistance to insecticides has become widespread in Anopheles populations2–4, threatening a global resurgence of the disease and making the generation of effective new malaria control tools an urgent public health priority. Here, we show that development of P. falciparum can be rapidly and completely blocked when Anopheles gambiae females uptake low concentrations of specific antimalarials from treated surfaces, simulating contact with a bed net. Mosquito exposure to atovaquone prior to or shortly after P. falciparum infection causes full parasite arrest in the female midgut, preventing transmission of infection. Similar transmission-blocking effects are achieved with other cytochrome B inhibitors, demonstrating that parasite mitochondrial function is a good target for parasite killing. Incorporating these effects into a model of malaria transmission dynamics predicts that the inclusion of Plasmodium inhibitors on mosquito nets would significantly mitigate the global health impact of insecticide resistance. This study identifies a powerful new strategy for blocking Plasmodium transmission by Anopheles females, with promising implications for malaria eradication efforts.

CRISPR hack wipes out caged mosquitoes

Scientists are recruiting CRISPR gene editing to fight malaria, and at least in the lab, one group at Imperial College London has just devised a strategy that wipes out the disease’s carriers—mosquitoes—for good. Researchers led by biologist Andrea Crisanti used CRISPR/Cas9 to generate a mutant gene that leaves female Anopheles gambiae mosquitoes unable to reproduce but allows males to continue spreading the female infertility mutation to offspring. Crisanti’s team also made use of a genetic trick called a gene drive, which propagates the infertility mutation at unnaturally high rates. In two experiments, each with thousands of mosquitoes, the team showed that the gene drive spreads through the entire population, leaving all the insects infertile after a dozen generations or less (Nat. Biotechnol. 2018, DOI: 10.1038/nbt.4245). “The power of the drive was unexpected and beyond our most optimistic expectations,” Crisanti says. “Overall this is a remarkable study,” says Gaeten Burgio, a

State-dependent domicile leaving rates in Anopheles gambiae

BackgroundTransmission of Plasmodium greatly depends on the foraging behaviour of its mosquito vector (Anopheles spp.). The accessibility of blood hosts and availability of plant sugar (i.e., nectar) sources, together with mosquito energy state, have been shown to modulate blood feeding (and thus biting rates) of anopheline mosquitoes. In this study, the influence of mosquito starvation status and availability of nectar on the decision of female Anopheles gambiae mosquitoes to leave a bed net-protected blood host was examined.MethodsTwo small-scale mesocosm experiments were conducted using female mosquitoes starved for 0, 24 or 48 h, that were released inside a specially constructed hut with mesh-sealed exits and containing a bed net-protected human volunteer. Floral cues were positioned on one side of the hut or the other. Several biologically plausible exponential decay models were developed that characterized the emigration rates of mosquitoes from the huts. These varied from simple random loss to leaving rates dependent upon energy state and time. These model fits were evaluated by examining their fitted parameter estimates and comparing Akaike information criterion.ResultsStarved mosquitoes left domiciles at a higher rate than recently fed individuals however, there was no difference between 1- and 2-day-starved mosquitoes. There was also no effect of floral cue placement. The best fitting emigration model was one based on both mosquito energy state and time whereas the worst fitting model was one based on the assumption of constant leaving rates, independent of time and energy state.ConclusionsThe results confirm that mosquito-leaving behaviour is energy-state dependent, and provide some of the first evidence of state-dependent domicile emigration in An. gambiae, which may play a role in malarial transmission dynamics. Employment of simple, first-principle, mechanistic models can be very useful to our understanding of why and how mosquitoes leave domiciles.

English

Midgut symbiotic bacteria are known to play fundamental roles in the biology of mosquitoes, however knowledge of midgut bacterial communities associated with mosquitoes is scanty due to limitation of the isolation techniques based on culturing. In this study, the composition and diversity of midgut bacteria in field collected and lab reared adult female Anopheles gambiae and Culex quinquefasciatus mosquitoes was explored using the Illumina sequencing. Deoxyribonucleic acid was isolated from the pooled midgut extracts and their 16S rRNA gene sequenced using Illumina sequencing platform. Operational taxonomic units (OTUs) were analyzed using QIIME 1.8.0; taxonomy was assigned using BLASTn against SILVA 119 and hierarchical clustering was done using R program software. Out of the total number of sequence reads obtained, 145 OTUs were realized at 3% genetic distance. The 145 OTUs spanned 12 phyla; Proteobacteria, Firmicutes, Cyanobacteria, Euryarchaeota, Gemmatimonadetes, Spirochaetae, Archeabacteria Verrucomicrobia, Chloroflexi, Bacteriodetes, Acidobacteria and Actinobacteria. Microbial community composition based on OTUs showed significant difference between field collected and lab reared mosquitoes (aµa2 = 45.0799, p = 3.2 × 10-5). Similarly, there was a significant difference in community composition at OTU level between Anopheles gambiae and Culex quinquefasciatus (aµa2 = 31.2257, p = 7.7 × 10-4). The bacterial composition and diversity appeared to be influenced by the environment and the species of the mosquitoes. Key words: Anopheles gambiae, Culex quinquefasciatus, midgut, DNA, diversity.

How do mosquitoes smell us? The answers could help eradicate disease

In the coastal Rabai region of southeast Kenya, two populations of mosquitoes live within a half mile of each other but lead separate and curiously different lifestyles. Entomologists in the mid-20th century found that color distinguished the two subspecies of Aedes aegypti ( Aedes aegypti aegypti are brown and Aedes aegypti formosa are black) as well as the hosts they chose for blood meals. The “domestic” brown form frequented people’s homes, preferentially feeding on people and laying their eggs in indoor water receptacles. Meanwhile, the black-colored “forest” form roamed the nearby woods preferring to bite nonhuman animals and lay its eggs in tree trunk holes. This light sheet microscope image of an adult female Anopheles gambiae mosquito includes genetic labeling to show olfactory neurons (green). The antennae, maxillary palp, and labella on the proboscis contain many such neurons. Image courtesy of Olena Riabinina, Courtney Akitake, and Chris Potter (Johns Hopkins University School of Medicine, Baltimore). Following up on these reports some 50 years later as a postdoctoral fellow at The Rockefeller University in New York, Lindy McBride saw an opportunity to examine this natural case study with modern genetic tools. The feeding habits of Aedes aegypti aegypti make it a dangerously efficient vector for spreading yellow fever, dengue, Zika, and other diseases between humans. The other subspecies poses much less of a threat. To understand why, McBride traveled to Rabai to collect mosquito larvae and pupae from people’s homes and the nearby woods. She wanted to compare the two mosquitoes genetically and discover what changes had led the more recently evolved domestic form to hunt humans. Back at Rockefeller in the laboratory of her then-postdoctorate advisor Leslie Vosshall, McBride raised 30 strains of mosquitoes from the samples and used RNA sequencing to compare the genes expressed in their antennae (a major …

A Comparative Study of Anopheles Gambiae Male Mosquito’s Response to Frequency Modulated [FM] and Pulse Modulated [PM] Waves at Different Acoustic Frequencies and Distances

The efficiency of an electronic device meant to disrupt acoustic communication between male and female Anopheles gambiae mosquitoes was tested by recording approaches of the males towards the device as a function of distance and frequency. Different bioassays were conducted with broadcasts from the device and taped mimics of the female species wing beats at different distances and frequencies. A large number of the males, 85% remained near the broadcast speaker for a significantly longer period when the broadcast sounds of the device were at 380 Hz than at any other frequency range. The taped mimic sounds of the female mosquitoes in flight elicited a maximum response rate of 75 %. A test involving a choice between the device and taped sound mimics by male mosquitoes revealed that a high number (75 %) of them flew towards the device while 10% were attracted by the taped sounds whereas 15 % were undecided and made oscillatory motion between the two sources. Additional bioassays conducted to examine the effect of the device on non-flying males showed that almost 75 % were disturbed and approached the speaker within a distance of 0.5 m, which confirmed a radiative near-field communication for these insects.

Age and prior blood feeding of Anopheles gambiae influences their susceptibility and gene expression patterns to ivermectin-containing blood meals.

BackgroundIvermectin has been proposed as a novel malaria transmission control tool based on its insecticidal properties and unique route of acquisition through human blood. To maximize ivermectin’s effect and identify potential resistance/tolerance mechanisms, it is important to understand its effect on mosquito physiology and potential to shift mosquito population age-structure. We therefore investigated ivermectin susceptibility and gene expression changes in several age groups of female Anopheles gambiae mosquitoes.MethodsThe effect of aging on ivermectin susceptibility was analyzed in three age groups (2, 6, and 14-days) of colonized female Anopheles gambiae mosquitoes using standard survivorship assays. Gene expression patterns were then analyzed by transcriptome sequencing on an Illumina HiSeq 2500 platform. RT-qPCR was used to validate transcriptional changes and also to examine expression in a different, colonized strain and in wild mosquitoes, both of which blood fed naturally on an ivermectin-treated person.ResultsMosquitoes of different ages and blood meal history died at different frequencies after ingesting ivermectin. Mortality was lowest in 2-day old mosquitoes exposed on their first blood meal and highest in 6-day old mosquitoes exposed on their second blood meal. Twenty-four hours following ivermectin ingestion, 101 and 187 genes were differentially-expressed relative to control blood-fed, in 2 and 6-day groups, respectively. Transcription patterns of select genes were similar in membrane-fed, colonized, and naturally-fed wild vectors. Transcripts from several unexpected functional classes were highly up-regulated, including Niemann-Pick Type C (NPC) genes, peritrophic matrix-associated genes, and immune-response genes, and these exhibited different transcription patterns between age groups, which may explain the observed susceptibility differences. Niemann-Pick Type 2 genes were the most highly up-regulated transcripts after ivermectin ingestion (up to 160 fold) and comparing phylogeny to transcriptional patterns revealed that NPCs have rapidly evolved and separate members respond to either blood meals or to ivermectin.ConclusionWe present evidence of increased ivermectin susceptibility in older An. gambiae mosquitoes that had previously bloodfed. Differential expression analysis suggests complex midgut interactions resulting from ivermectin ingestion that likely involve blood meal digestion physiological responses, midgut microflora, and innate immune responses. Thus, the transcription of certain gene families is consistently affected by ivermectin ingestion, and may provide important clues to ivermectin’s broad effects on malaria vectors. These findings contribute to the growing understanding of ivermectin’s potential as a transmission control tool.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2029-8) contains supplementary material, which is available to authorized users.

Identification of morphological and chemical markers of dry- and wet-season conditions in female Anopheles gambiae mosquitoes.

BackgroundIncreased understanding of the dry-season survival mechanisms of Anopheles gambiae in semi-arid regions could benefit vector control efforts by identifying weak links in the transmission cycle of malaria. In this study, we examined the effect of photoperiod and relative humidity on morphologic and chemical traits known to control water loss in mosquitoes.MethodsAnopheles gambiae body size (indexed by wing length), mesothoracic spiracle size, and cuticular hydrocarbon composition (both standardized by body size) were examined in mosquitoes raised from eggs exposed to short photoperiod and low relative humidity, simulating the dry season, or long photoperiod and high relative humidity, simulating the wet-season.ResultsMosquitoes exposed to short photoperiod exhibited larger body size and larger mesothoracic spiracle length than mosquitoes exposed to long photoperiod. Mosquitoes exposed to short photoperiod and low relative humidity exhibited greater total cuticular hydrocarbon amount than mosquitoes exposed to long photoperiod and high relative humidity. In addition, total cuticular hydrocarbon amount increased with age and was higher in mated females. Mean n-alkane retention time (a measure of cuticular hydrocarbon chain length) was lower in mosquitoes exposed to short photoperiod and low relative humidity, and increased with age. Individual cuticular hydrocarbon peaks were examined, and several cuticular hydrocarbons were identified as potential biomarkers of dry- and wet-season conditions, age, and insemination status.ConclusionsResults from this study indicate that morphological and chemical changes underlie aestivation of Anopheles gambiae and may serve as biomarkers of aestivation.