Surveillance of mosquitoes harnessing their buzzing sound.

Mosquito surveillance for vector-borne disease management relies on traditional morphological and molecular techniques, which are tedious, time-consuming, and costly. The present study describes a simple and efficient recording device that analyzes mosquito sound to estimate species composition, male-female ratio, fed-unfed status, and harmonic convergence interaction using fundamental frequency (F0) bandwidth, harmonics, amplitude, and combinations of these parameters. The study examined a total of 19 mosquito species, including 3 species of Aedes, 7 species of Anopheles, 1 species of Armigeres, 5 species of Culex, 1 species of Hulecoetomyia, and 2 species of Mansonia. Among them, the F0 ranges between 269.09 ± 2.96 Hz (Anopheles culiciformis) to 567.51 ± 3.82 Hz (Aedes vittatus) and the harmonic band (hb) number ranges from 5 (An. culiciformis) to 12 (Ae. albopictus). In terms of species identification, the success rate was 95.32 % with F0, 84.79 % with F0-bandwidth, 84.79 % with harmonic band (hb) diversity, and 49.7 % with amplitude (dB). The species identification rate has gone up to 96.50 % and 97.66 % with the ratio and multiplication of F0 and hb, respectively. This is because of the matrices that combine multiple sound attributes. Comparatively, combinations of the amplitude of the F0 and the higher harmonic frequency band were non-significant for species identification (60.82 %). The fed females have shown a considerable increase in F0 in comparison to the unfed. The males of all the species possessed significantly higher frequencies with respect to the females. Interestingly, the presence of male-female of Ae. vittatus together showed harmonic convergence between the 2nd and 3rd harmonic bands. In conclusion, the sound-based technology is simple, precise, and cost-effective and provides better resolution for species, sex, and fed-unfed status detection in comparison to conventional methods. Real-time surveillance of mosquitoes could potentially utilize this technology.

Genetic differentiation among Aedes aegypti populations from different eco-geographical zones of India.

The present study explicitly evaluated the genetic structure of Aedes aegypti Linn, the vector of dengue, chikungunya, and Zika viruses, across different geo-climatic zones of India and also elucidated the impact of ecological and topographic factors. After data quality checks and removal of samples with excess null alleles, the final analysis was performed on 589 individual samples using 10 microsatellite markers. Overall findings of this study suggested that, Ae. aegypti populations are highly diverse with moderate genetic differentiation between them. Around half of the populations (13 out of 22) formed two genetic clusters roughly associated with geographical regions. The remaining nine populations shared genetic ancestries with either one or both of the clusters. A significant relationship between genetic and geographic distance was observed, indicating isolation by distance. However, spatial autocorrelation analysis predicted the signs of long-distance admixture. Post-hoc environmental association analysis showed that 52.7% of genetic variations were explained by a combination of climatic and topographic factors, with latitude and temperature being the best predictors. This study indicated that though overall genetic differentiation among Ae. aegypti populations across India is moderate (Fst = 0.099), the differences between the populations are developing due to the factors associated with geographic locations. This study improves the understanding of the Ae. aegypti population structure in India that may assist in predicting mosquito movements across the geo-climatic zones, enabling effective control strategies and assessing the risk of disease transmission.

Ecological variations in adult life table attributes of Aedes aegypti (L.) from the desert and coastal regions of India.

The ecological variation in biological and adult life-table attributes of two populations of Aedes aegypti (Diptera: Culicidae) from the desert (Jodhpur) and coastal (Kolkata) regions of India are assessed to understand the reproductive and survival strategies. The results showed that females lived longer than males in both strains. The desert strain was more r-strategist because of its higher intrinsic rate of increase (rm  = 0.23), finite rate of increase (λ = 1.25), lower life expectancy of males (7.9 days) and females (14.4 days), mean generation time (T = 19.2 days) and doubling time (DT = 3.0 days). However, there was no difference in net reproductive rate (R0 ) between the desert and coastal strains. The coastal strain showed a longer female life expectancy (22.0 days) than the desert strain (14.4 days). However, the fecundity (eggs/female/day) was lower in the coastal strain (11.4) than in the desert strain (15.1). Conclusively, the desert (Jodhpur) strain is adapted to a better r-strategy than the coastal (Kolkata) strain of Ae. aegypti, which might be helpful to flourish in harsh environmental conditions. This study may provide accurate predictions of Ae. aegypti population dynamics for vector management.

Life table, survival and fecundity parameters of Aedes albopictus (Diptera: Culicidae) strains from desert and coastal regions of India.

The Asian tiger mosquito, Aedes albopictus (Skuse, 1895) is the anthropophilic and day-biter mosquito, residing in peridomestic habitats and is responsible for vector-borne diseases, i.e., chikungunya and dengue. The knowledge of mosquito life demographics is very important to provide a foundation for a successful vector control program. In the present study, a comparative adult life table, survival, and fecundity parameters were evaluated on desert (Jodhpur, Rajasthan) and coastal (Kolkata, West Bengal) strains of Aedes albopictus (Diptera: Culicidae) from India under the standard laboratory conditions. The results showed significant differences between these strains for the life expectancy of males, fecundity (total eggs/cohort and eggs/female life span), oviposition days, intrinsic rate of increase (rm), finite rate of increase (λ), and doubling time (DT). Female mosquitoes lived longer than the males in both strains. Significant differences were observed in the net reproductive rate (R0) between Kolkata and Jodhpur strains. The coastal (Kolkata) strain showed a longer male life expectancy (5.6 days) than the desert strain (4.0 days). However, the fecundity (eggs/female lifespan) was higher in Kolkata (360.2) than the Jodhpur strain (229.7). The Kolkata strain showed higher R0, rm, λ, and the lower T, DT than the Jodhpur strain. Conclusively, the results indicate that the coastal strain of Ae. albopictus is more r-strategist than the desert strain. This study may help in the precise prediction of Ae. albopictus population dynamics surviving in these geographical areas for vector management.

Prevalence of mouthpart sensilla and protease producing symbiotic gut bacteria in the forensic fly Chrysomya megacephala (Fabricius, 1794): Insight from foraging to digestion.

The blow fly, Chrysomya megacephala (Fabricius, 1794) is a globally prevalent forensically important species that helps to estimate accurate postmortem interval since the death. This fly occasionally causes cutaneous myiasis and transmits several pathogenic bacteria. To understand their ability of corpse detection and digestion of protein-rich meal, the present study describes the mouthpart sensilla and assessment of protease producing symbiotic gut bacteria. Scanning electron microscopy (SEM) showed the prevalence of trichoid sensilla (Tr), basiconic sensilla (Ba) and microtrichia (Mr) on labellar lobes, haustellum and maxillary palps of mouthparts. Bacterial particles of both rod (small and large) and spherical shaped were detected in the gut of C. megacephala using SEM. The bacterial density was higher on the foregut and midgut in comparison to the hindgut. From 72 bacterial isolates, 10 isolates from the foregut region showed considerable protease-producing efficacy ranging between 3.98 - 6.83 GHR and 9.73 - 34.68 U/ml protease. Among these, the most promising protease-producing bacterial isolate showed 16S rDNA sequence similarity (99.85%) with Chryseobacterium artocarpi DNA. This bacterium was the first report from flies. The findings of the study might help in better understanding of the role of sensilla in host perception and foregut symbiotic bacterial association in protein digestion in C. megacephala.

A membrane and blood-free approach to rear adult Aedes albopictus.

Most artificial dietary systems for feeding mosquitoes require a membrane feeder, host cues, phosphate saline buffers and a phagostimulant. These multicomponent feeders are complex, expansive and cumbersome that requires fully trained personnel. The objective of the present is to develop a simple sugar assisted protein (SAP) diet for the egg production of Aedes albopictus. The present study assessed the potential use of SAP dietary system on the engorgement, fecundity, preference of diet components and production of multiple generations of Ae. albopictus. Our data show that the female mosquitoes have strongly preferred a diet with: (i) a combination of sugar and protein over the individual component, and (ii) water over PBS (phosphate buffered saline) buffer as a carrier, whereas adenosine triphosphate (ATP) was not required as a phagostimulant. Based on our optimization data, the SAP diets (10-20% bovine serum albumin in 5% sucrose aqueous solution) do not require chemo-attractive lure, phagostimulant ATP, temperature and membrane feeding components. Female mosquitoes readily engorge on SAP diets and show similar rates of survival and fecundity compared to those when blood-fed on live animals. In addition, the number of eggs produced by female mosquitoes fed on SAP diets kept consistent for 10 consecutive generations. Our results indicate that SAP diet is a potential alternative against blood feeding that is simple and cost-effective diets for Ae. albopictus colony maintenance and to support large scale mass- production for experimental and other purposes.

Evaluation of enhanced oviposition attractant formulations against Aedes and Culex vector mosquitoes in urban and semi-urban areas.

Surveillance is not only an important tool to assess the population dynamics of vector mosquitoes, but it can also be used to control vector-borne diseases. Mosquito vectors that belong to several genera such as Anopheles, Aedes, and Culex play a crucial role in the transmission of malaria, dengue, chikungunya, Zika, and elephantiasis diseases worldwide. We tested the efficacy of two commercial-grade oviposition attractant formulations that were developed for the container-inhabiting Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus mosquitoes present in urban or semi-urban environments. These attractants can lure gravid females. Field trials were conducted in residential yards during a post-rainy season in September and October. Our data showed considerable efficacy for both attractants. Aedes-attractant collected 1.6-fold more larvae (101.2 ± 10.5 larvae/trap) than the control, and Culex-attractant collected 1.27-fold more larvae (151.2 ± 12.5 larvae/trap) than the control, resulting in 0.8 and 0.7 oviposition attraction indices (OAIs), respectively. Regression analysis indicated that the Aedes-attractant was more stable than the Culex-attractant. Location and time did not alter the efficacy of these attractants. Our experiment suggests that these attractants can be used for the development of species-specific gravid traps to detect, estimate, and control the mosquito population in urban and semi-urban areas.

Seasonal field efficacy of pyriproxyfen autodissemination stations against container-inhabiting mosquito Aedes albopictus under different habitat conditions.

BACKGROUND: Control of the container-inhabiting mosquito, Aedes albopictus, is difficult using conventional methods due to its selection of cryptic peri-domestic habitats. We evaluated whether autodissemination stations can deliver sufficient pyriproxyfen to sentinel containers to produce significant pupal mortality in different habitats such as competing oviposition sites, peri-domestic habitats, junkyards and tire piles. We also tested how far the pesticide could be transferred over a 200-m range. RESULTS: Autodissemination stations performed effectively for 8-12 weeks under field conditions. Pupal mortality was reduced in sentinel cups with high-competing oviposition habitats (5 versus 20) in isolated plots; however, similar results were not seen in residential areas. Increasing the number of stations per plot (from 1 to 4) enhanced the efficacy. Peri-domestic habitat trials showed the highest pupal mortality (50.4%) and site contamination with pyriproxyfen (82.2%) among the trials. Autodissemination stations were able to contaminate habitats in a junkyard (50.0%) and tire piles (40.2%). Pyriproxyfen was detected in sentinel cups up to 200 m from stations. Detection of pyriproxyfen by residue analysis (0.005-0.741 µg L-1 ) in field samples confirmed the transfer of the insect growth regulator. CONCLUSION: Autodissemination stations have shown promising potential as a novel pest management tool against container mosquitoes in field trials in different habitats confronted by mosquito control personnel. © 2017 Society of Chemical Industry.

Targeting a Hidden Enemy: Pyriproxyfen Autodissemination Strategy for the Control of the Container Mosquito Aedes albopictus in Cryptic Habitats.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a vector of dengue, Chikungunya, and Zika viruses. This mosquito inhabits a wide range of artificial water-holding containers in urban and suburban areas making it difficult to control. We tested the hypothesis that female-driven autodissemination of an insect growth regulator could penetrate cryptic oviposition habitats difficult to treat with conventional insecticidal sprays. METHODOLOGY: Oviposition preferences of Ae. albopictus females for open and cryptic cups were tested in semi-field experiments. Two conventional larvicidal sprayers were tested to determine droplet penetration and larvicidal efficacy in open and cryptic habitats using Bacillus thuringiensis var. israelensis (Bti) in the field. Finally, the efficacy of pyriproxyfen autodissemination stations was assessed in cryptic and open cups in residential areas during 2013 and 2014. PRINCIPAL FINDINGS: Gravid females strongly preferred cryptic (53.1±12.9 eggs/cup) over open (10.3±4.3 eggs/cup) cups for oviposition. Cryptic cups showed limited droplet penetration and produced 0.1-0.3% larval mortality with a conventional backpack and low-volume sprays of Bti. The autodissemination stations effectively contaminated these cryptic cups (59.3-84.6%) and produced 29.7-40.8% pupal mortality during 2013-2014. Significant pupal mortality was also observed in open cups. CONCLUSIONS: The autodissemination station effectively exploits the oviposition behavior of wild gravid females to deliver pyriproxyfen to targeted oviposition habitats. Although the pupal mortality in cryptic cups was relatively lower than expected for the effective vector control. Autodissemination approach may be a suitable supporting tool to manage Ae. albopictus immatures in the cryptic habitats those are less accessible to conventional larvicidal sprays.

Effects of larval habitat substrate on pyriproxyfen efficacy against Aedes albopictus (Diptera: Culicidae).

Pyriproxyfen, a juvenile hormone analog insect growth regulator (IGR), is a recommended insecticide for the control of container-inhabiting mosquitoes. The effects of eight container substrates (three plastics, wood, tire rubber, clay pot, concrete, and glass) were studied on reduction of bio-efficacy of pyriproxyfen for Aedes albopictus (Skuse) in the laboratory. Insect growth regulator-bioassay of third instars showed significant increase of LC50 with some substrates compared with control probably because of pyriproxyfen adsorption to the substrate. The LC50 for the control substrate (glass mug) was 0.029 microg/liter. In comparison to the control, LC50 increased 150.1 times with tire rubber (4.354 microg/liter) and 8-10 times with wood and high-density polyethylene plastic containers. Slight increases of LC50 were detected for clay pots, polypropylene, and polystyrene plastics (1.9-2.7 times). Pyriproxyfen efficacy with concrete substrate was higher than with glass containers, which might be caused by high pH. The pH of water with wood (5.6) and concrete (9.8) substrates differed from the other substrates tested (6.3-7.1). There was no pupal mortality at pH 5-11 without pyriproxyfen; however, interaction of pH (5-11) with pyriproxyfen (0.05 microg/liter) showed additive effects at both low and high pH values. Different substrates have variable impacts on pyriproxyfen efficacy, which might be associated with the failure of larval control strategies for the container mosquitoes under certain field conditions.

Effects of insect growth regulators on the mosquito-parasitic nematode Romanomermis iyengari.

Pyriproxyfen, a juvenile hormone analogue, diflubenzuron, a chitin synthesis inhibitor, and azadirachtin, an ecdysone agonist, are three insect growth regulators (IGRs) considered as selective and effective insecticides for mosquitoes. Romanomermis iyengari (Welch) is a mosquito-parasitic mermithid that can provide biological control against many medically important mosquito species. The compatibility of these two control tactics was tested by evaluating the sublethal effects of exposure to IGR on nematode developmental stages (preparasitic, parasitic, and preparasitic + parasitic) using Culex pipiens larvae as the host. Sublethal concentrations of IGRs were 90 % emergence inhibition of host mosquito. Preparasitic exposure to pyriproxyfen, azadirachtin, and diflurbenzuron had no effect on infectivity, parasite load, sex ratio, or male size but reduced nematode female length and increased male sex ratio at one parasite/larva. When IGRs treatments were made against the parasitic and preparasitic + parasitic stages, pyriproxyfen and azadirachtin reduced R. iyengari infectivity, parasite load, and male nematode length, whereas pyriproxyfen exposure increased male sex ratio and reduced the female R. iyengari length. Thus, IGRs have significant negative impacts on different stages of mosquito mermithid that can destabilize the balance of host-parasite population interaction. Therefore, IGRs should be used with caution in mosquito habitats where these parasites have established.

Differentiation of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) with egg surface morphology and morphometrics using scanning electron microscopy.

Aedes aegypti and Aedes albopictus are potential arboviral vectors leading to high human fatality worldwide. Efforts in the present study were made to differentiate the eggs of A. aegypti and A. albopictus morphologically and morphometrically using scanning electron microscopy (SEM). Morphometrically, these species' eggs were 48.48% significantly different of the 33 attributes including egg dimensions, micropylar apparatus, dimensions and density of outer chorionic cells (OCCs), tubercles and width of exochorionic network. In comparison to A. aegypti eggs, A. albopictus eggs were significantly smaller and more tapered at the posterior end; however, the micropylar disc of A. aegypti was wider and had incomplete circular sectors whereas it was a narrower polygon without sectors in A. albopictus. These species were also significantly different with regards to OCC which enclose both large central and small peripheral tubercles. Specifically, the exochorionic networks in A. aegypti were interwoven, reticulated and extensively wide whereas they were narrow, prominent and solid-wall-like in A. albopictus. This feature may strengthen A. albopictus eggs against desiccation, when they are laid in containers. The morphometrical and morphological analysis of the egg's attributes of A. aegypti and A. albopictus may be helpful in understanding egg biology as well as in species confirmation.