Evaluation of Mosquito Attractant Candidates Using a High-Throughput Screening System for Aedes aegypti (L.), Culex quinquefasciatus Say. and Anopheles minimus Theobald (Diptera: Culicidae)

Several types of olfactometers have been used to evaluate mosquito responses to agents that mimic natural volatiles that repel or attract. The Y-tube olfactometer has been widely used to study repellents and attractants, while the high-throughput screening system assay has only been used to study repellents. Whether the high-throughput screening system assay is suitable for evaluating attractants is unknown. We evaluated the responses to four lactic-acid-based mixtures and two non-lactic-acid-based chemical lure candidates using the high-throughput screening system (HITSS) for three mosquito species (laboratory strains and field populations of both Aedes aegypti (L.) and Culex quinquefasciatus Say.; laboratory strain of Anopheles minimus Theobald) under laboratory-controlled conditions. HITSS assay results showed that KU-lure #1 elicited the greatest percent attraction for pyrethroid-resistant and -susceptible Ae. aegypti. KU-lure #6 elicited the strongest attractive response for pyrethroid-susceptible and -resistant Cx. quinquefasciatus and pyrethroid-susceptible An. minimus. The response to the lures from each species was independent of the pyrethroid susceptibility status (Ae. aegypti, p = 0.825; Cx. quinquefasciatus, p = 0.056). However, a significant difference in attraction to KU-lure #6 was observed between diurnal and nocturnal mosquitoes (Cx. quinquefasciatus vs. Ae. aegypti, p = 0.014; An. minimus vs. Ae. aegypti, p = 0.001). The laboratory-level HITSS assay effectively selects potential lure candidates. Because the host-seeking behavior differs between mosquito species, further studies are needed to develop species-specific attractants. Additional studies in semi-field screen houses using commercial traps are necessary to evaluate the accuracy of these laboratory assay results.

Download Full-text

Dose–Response Assay for Synthetic Mosquito (Diptera: Culicidae) Attractant Using a High-Throughput Screening System

Insects ◽

10.3390/insects12040355 ◽

2021 ◽

Vol 12 (4) ◽

pp. 355

Author(s):

Dae-Yun Kim ◽

Theerachart Leepasert ◽

Michael J. Bangs ◽

Theeraphap Chareonviriyaphap

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Egg Production ◽

Critical Role ◽

Laboratory Strain ◽

Screening System ◽

Significant Difference ◽

Blood Source ◽

Parallel Control ◽

Spatial Repellency

Natural volatile host cues play a critical role for mosquito orientation and locating a blood source for egg production. Similar olfactory activation responses have allowed the use and development of artificial chemical attractants to lure mosquitoes to trapping devices. Using a pre-formulated commercial product mixture of different attractant chemicals, a high-throughput screening system (HITSS) is used to screen varying doses of chemical required to activate behavioral responses. Two strains of Aedes aegypti (L.): permethrin-susceptible (USDA) and -resistant (Pu Teuy) phenotypes and one Culex quinquefasciatus Say. (NIH) laboratory strain were tested. Overall, mosquitoes showed repellency between 1.0 g and to 10.0 g dose of each compound. However, by progressively reducing the dose, Cx. quinquefasciatus showed a greater positive percent attraction (88.9%) at 0.025 g, whereas the USDA and Pu Teuy Ae. aegypti produced optimum attractant activation at 0.005 g (72.6% and 58.9%, respectively) without significant difference within species (p > 0.05). In parallel control assays, Cx. quinquefasciatus was significantly attracted to 1 g of dry ice (carbon dioxide) (76%) more than Ae. aegypti (USDA) (12.2%). The HITSS was originally designed to measure three chemical actions to sublethal concentrations of chemicals by mosquitoes: toxicity and the two primary behavior avoidance responses (contact excitation and spatial repellency). These findings demonstrate that the HITSS assay, with only minor modifications, allows comparison screening of candidate compounds as potential attractants for anemotactic responses under laboratory-controlled conditions. Further investigations will be required to equate measurements obtained from controlled laboratory assays to more varied field conditions for attracting natural mosquito populations.

Download Full-text

Laboratory evaluation of novel long-lasting insecticidal nets on Aedes aegypti L., using a high-throughput screening system

Agriculture and Natural Resources ◽

10.34044/j.anres.2021.55.2.08 ◽

2021 ◽

Vol Volume 55 issue 2 ◽

Keyword(s):

Aedes Aegypti ◽

High Throughput ◽

High Throughput Screening ◽

Laboratory Evaluation ◽

Screening System ◽

Long Lasting Insecticidal Nets

Download Full-text

High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting

RSC Advances ◽

10.1039/c8ra09684h ◽

2019 ◽

Vol 9 (8) ◽

pp. 4507-4513 ◽

Cited By ~ 12

Author(s):

Xu-Dong Zhu ◽

Xiang Shi ◽

Shu-Wen Wang ◽

Ju Chu ◽

Wei-Hong Zhu ◽

...

Keyword(s):

Flow Cytometry ◽

Lactic Acid ◽

High Throughput ◽

Cell Sorting ◽

High Throughput Screening ◽

Bacillus Coagulans ◽

Screening System ◽

Fluorescence Activated Cell Sorting ◽

Microfluidic Sorting

A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans.

Download Full-text

A High Throughput Screening System for Determining the Three Actions of Insecticides Against Aedes aegypti (Diptera: Culicidae) Populations in Thailand

Journal of Medical Entomology ◽

10.1603/me09246 ◽

2010 ◽

Vol 47 (5) ◽

pp. 833-841 ◽

Cited By ~ 8

Author(s):

Kanutcharee Thanispong ◽

Nicole L. Achee ◽

John P. Grieco ◽

Michael J. Bangs ◽

Wannapa Suwonkerd ◽

...

Keyword(s):

Aedes Aegypti ◽

High Throughput ◽

High Throughput Screening ◽

Screening System

Download Full-text

Evaluation of Perma Net® 2.0 in the Control of Culex quinquefasciatus and Aedes aegypti from Awka, Anambra State, Nigeria

NIGERIAN ANNALS OF PURE AND APPLIED SCIENCES ◽

10.46912/napas.66 ◽

2019 ◽

Vol 1 ◽

pp. 68-75

Author(s):

C M Egbuche ◽

P U Samuel ◽

C K Ezihe ◽

C B Ukonze ◽

O A Chukwuzoba ◽

...

Keyword(s):

Aedes Aegypti ◽

Culex Quinquefasciatus ◽

Mosquito Species ◽

Family Health ◽

Residual Effect ◽

Physical Barrier ◽

Physical Integrity ◽

Mortality Effect ◽

Significant Difference ◽

Who Cone Bioassay

Long Lasting Insecticide Nets (LLINs) reduce human-mosquito contact through the provision of physical barrier, repellent effect or mortality effect. This study evaluated the physical integrity and residual effect of insecticides in Perma Net® 2.0 of 6 months, 1 year, 2 years, 3 years and 4 years duration of use. Perma Net®2.0 used were obtained from systematically picked households within Ifite Awka and new Perma Net® 2.0 serving as control were collected from Society for Family Health. Adult mosquitoes were collected from picked households using Pyrethrum knockdown Collection method before the Perma Net® 2.0 were removed. The top and four sides of the nets were examined for presence of holes. Also, net pieces measuring 25cm × 25cm were obtained from Perma Net® 2.0 of different duration and subjected to WHO cone bioassay using Randomized Block Experimental Design. The Culex quinquefasciatus and Aedes aegypti used for the bioassay were collected from breeding sites within Ifite Awka, reared and maintained in an insectary. There was at least one hole in 10%, 30% and 40% of LLINs with 2 years, 3years and 4 years duration of use respectively. Significant differences were found in the physical integrity of the nets (P < 0.05). Culexquinquefasciatus and Aedes aegypti constituted 40.9% and 27.0% of all mosquitoes collected indoors respectively. There was no significant difference in the indoor abundance of the two mosquito species (P >0.05). However, their abundance depended on the duration of LLINs usage (P < 0.05). The mortality effects of16%, 8%, 7%, 5%, 5% and 2% on Culex quinquefasciatus were recorded for Perma Net® 2.0 of 0 month, 6 months, 1 year, 2 years, 3 years and 4 years respectively. The mortality effects of 23%, 12%, 9%, 6%, 5% and 3% on Aedes aegypti were recorded for Perma Net® 2.0 of 0 month, 6 months, 1 year, 2 years, 3 years and 4years respectively. The new LLINs produced significantly highest mortality effect (P < 0.05) on both mosquito species than nets of other duration of usage. The mortality effect of Perma Net® 2.0 on Culex quinquefasciatus, and that on Aedes aegypti were similar (P > 0.05). It shows that Perma Net® 2.0 offer physical barrier more than repellency and mortality effect against Culex quinquefasciatus, and Aedes aegypti.

Download Full-text

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

International Journal of Molecular Sciences ◽

10.3390/ijms22063041 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3041

Author(s):

Gheorghita Menghiu ◽

Vasile Ostafe ◽

Radivoje Prodanović ◽

Rainer Fischer ◽

Raluca Ostafe

Keyword(s):

High Throughput ◽

Cell Sorting ◽

High Throughput Screening ◽

Screening Method ◽

Enzymatic Reaction ◽

Hydrolytic Enzymes ◽

Screening System ◽

Fluorescence Activated Cell Sorting ◽

Fungal Cell ◽

Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Download Full-text

Ligusticum sinense Nanoemulsion Gel as Potential Repellent against Aedes aegypti, Anopheles minimus, and Culex quinquefasciatus (Diptera: Culicidae)

Insects ◽

10.3390/insects12070596 ◽

2021 ◽

Vol 12 (7) ◽

pp. 596

Author(s):

Anuluck Junkum ◽

Wanchai Maleewong ◽

Atiporn Saeung ◽

Danita Champakaew ◽

Arpaporn Chansang ◽

...

Keyword(s):

Aedes Aegypti ◽

Culex Quinquefasciatus ◽

Disease Transmission ◽

Skin Irritation ◽

Mosquito Vectors ◽

Plant Family ◽

Anopheles Minimus ◽

Human Volunteers ◽

Nanoemulsion Gel ◽

Fresh Preparation

Ligusticum sinense Oliv. cv. is a species of Umbelliferae (Apiaceae), a large plant family in the order Apiales. In this study, L. sinense hexane extract nanoemulsion gel (LHE-NEG) was investigated for mosquito repellency and compared to the standard chemical, N,N-diethyl-3-methylbenzamide (DEET), with the goal of developing a natural alternative to synthetic repellents in protecting against mosquito vectors. The results demonstrated that LHE-NEG afforded remarkable repellency against Aedes aegypti, Anopheles minimus, and Culex quinquefasciatus, with median protection times (MPTs) of 5.5 (4.5–6.0), 11.5 (8.5–12.5), and 11.25 (8.5–12.5) h, respectively, which was comparable to those of DEET-nanoemulsion gel (DEET-NEG: 8.5 (7.0–9.0), 12.0 (10.0–12.5), and 12.5 (10.0–13.5) h, respectively). Evaluation of skin irritation in 30 human volunteers revealed no potential irritant from LHE-NEG. The physical and biological stability of LHE-NEG were determined after being kept under heating/cooling cycle conditions. The stored samples of LHE-NEG exhibited some changes in appearance and differing degrees of repellency between those kept for 3 and 6 heating/cooling cycles, thus providing slightly shorter MPTs of 4.25 (4.0–4.5) and 3.25 (2.5–3.5) h, respectively, when compared to those of 5.0 (4.5–6.0) h in fresh preparation. These findings encourage commercially developed LHE-based products as an alternative to conventional synthetic repellents in preventing mosquito bites and helping to interrupt mosquito-borne disease transmission.

Download Full-text

Behavioural response of mosquito vectors Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus to synthetic pyrethroid and organophosphorus-based slow-release insecticidal paint

Parasites & Vectors ◽

10.1186/s13071-021-04746-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Sunil Dhiman ◽

Kavita Yadav ◽

B. N. Acharya ◽

Raj Kumar Ahirwar ◽

D. Sukumaran

Keyword(s):

Aedes Aegypti ◽

Culex Quinquefasciatus ◽

Anopheles Stephensi ◽

Slow Release ◽

Disease Risk ◽

Mosquito Species ◽

Escape Time ◽

Behavioural Avoidance ◽

Vector Mosquito ◽

Contact Exposure

Abstract Background The direct toxicological impact of insecticides on vector mosquitoes has been well emphasized; however, behavioural responses such as excito-repellency and physical avoidance as a result of insecticide exposure have not been much studied. We have demonstrated the excito-repellency and behavioural avoidance in certain vector mosquito species on exposure to a slow-release insecticidal paint (SRIP) formulation in addition to direct toxicity. Methods A SRIP formulation developed by the Defence Research and Development Establishment, Gwalior, contains chlorpyriphos, deltamethrin and pyriproxyfen as active insecticides. Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti mosquitoes were used to study the excito-repellency response of the formulation. The experiments were performed in a specially designed dual-choice exposure and escape chamber made of transparent polymethyl methacrylate. For the experiments, the SRIP formulation was applied undiluted at a rate of 8 m2 per kg on 15 cm2 metallic surfaces. Mosquitoes were introduced into the exposure chamber, and observations of the movement of mosquitoes into the escape chamber through the exit portal were taken at 1-min intervals for up to 30 min. Results The evaluated formulation displayed strong excito-repellency against all three tested vector mosquito species. Results showed that the ET50 (escape time 50%) for Ae. aegypti, An. stephensi and Cx. quinquefasciatus was 20.9 min, 14.5 min and 17.9 min for contact exposure (CE) respectively. Altogether in CE, the escape rates were stronger in An. stephensi mosquitoes at different time intervals compared to Ae. aegypti and Cx. quinquefasciatus mosquitoes. The probit analysis revealed that the determined ET did not deviate from linearity for both non-contact exposure (NCE) and placebo exposure (PE) (χ2 ≤ 7.9; p = 1.0) for Ae. aegypti mosquitoes and for NCE (χ2 = 8.3; p = 1.0) and PE (χ2 = 1.7; p = 1.0) treatments in Cx. quinquefasciatus. Mortality (24 h) was found to be statistically higher (F = 6.4; p = 0.02) in An. stephensi for CE but did not vary for NCE (p ≥ 0.3) and PE (p = 0.6) treatments among the tested mosquito species. Survival probability response suggested that all the three tested species displayed similar survival responses for similar exposures (χ2 ≤ 2.3; p ≥ 0.1). Conclusion The study demonstrates the toxicity and strong behavioural avoidance in known vector mosquito species on exposure to an insecticide-based paint formulation. The combination of insecticides in the present formulation will broaden the overall impact spectrum for protecting users from mosquito bites. The efficacy data generated in the study provide crucial information on the effectiveness of the tested formulation and could be useful in reducing the transmission intensity and disease risk in endemic countries.

Download Full-text