Population genetic structure of the malaria vector Anopheles minimus in Thailand based on mitochondrial DNA markers

Background The malaria vector Anopheles minimus has been influenced by external stresses affecting the survival rate and vectorial capacity of the population. Since An. minimus habitats have continuously undergone ecological changes, this study aimed to determine the population genetic structure and the potential gene flow among the An. minimus populations in Thailand. Methods Anopheles minimus was collected from five malaria transmission areas in Thailand using Centers for Disease Control and Prevention (CDC) light traps. Seventy-nine females from those populations were used as representative samples. The partial mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and cytochrome b (Cytb) gene sequences were amplified and analyzed to identify species and determine the current population genetic structure. For the past population, we determined the population genetic structure from the 60 deposited COII sequences in GenBank of An. minimus collected from Thailand 20 years ago. Results The current populations of An. minimus were genetically divided into two lineages, A and B. Lineage A has high haplotype diversity under gene flow similar to the population in the past. Neutrality tests suggested population expansion of An. minimus, with the detection of abundant rare mutations in all populations, which tend to arise from negative selection. Conclusions This study revealed that the population genetic structure of An. minimus lineage A was similar between the past and present populations, indicating high adaptability of the species. There was substantial gene flow between the eastern and western An. minimus populations without detection of significant gene flow barriers. Graphical abstract

Time of Test Periods Influence the Behavioral Responses of Anopheles minimus and Anopheles dirus (Diptera: Culicidae) to DEET

Information on factors influencing the behavioral responses of mosquitoes to repellents is lacking and poorly understood, especially in the Anopheles species, night-biting mosquitoes. Our goal was to investigate the impact of different time periods on circadian activity and behavioral responses of two malaria vectors, Anopheles minimus and An. dirus, to 5% DEET using an excito-repellency test system. Each mosquito species was exposed to the repellent during the daytime (06.00–18.00) and nighttime (18.00–06.00), and time of observation was further divided into four 3-h intervals. Significant escape responses were observed between daytime and nighttime for An. minimus in both noncontact and contact tests. An. dirus showed statistical differences in contact irritancy escape response, whereas no significant difference was found in noncontact repellency tests. Both mosquito species showed more significantly higher escape responses when exposed to DEET during the afternoon and late in the night. This finding indicates that the time of testing may affect the behavioral responses of mosquitoes to repellents, especially in An. minimus and An. dirus. A better understanding of nocturnally active mosquito behavioral responses spanning from dusk to dawn would assist in optimizing product development, screening, and effective evaluation.

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

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.

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.

Potential malaria vector Anopheles minimus (species A) still persisting in North East India

ABSTRACTBackgroundVector borne infectious diseases affect two third of the world’s human population and cause mortality in millions each year. Malaria remains one of the major killers in the Indian sub-continent and transmitted uninterruptedly by many efficient vectors and their sibling species. In North East India (NE), Anopheles minimus has been recognized as an important vector which shares majority of malaria cases. This study primarily focuses on to recognize the presence and distribution of sibling species of An. minimus in certain endemic area of NE India.MethodsAnopheles species were collected and identified using available morphological keys. The genomic DNA was extracted from the mosquito specimen and used to perform species specific PCR (ss PCR) for molecular identification of major malaria vector An. minimus sibling speciesResultMorphological identification suggested the presence of An. minimus sl in low density in the study area. The specimen of An. minimus subjected to ss PCR confirmed the prevalence of only one sibling species namely, An. minimus A in Sialmari and Chandubi.ConclusionThough in low density, but malaria vector An. minimus is still present in certain endemic areas of NE India. The ss PCR assay employed presently suggested that An. minimus sibling species A is prevailing in the region. Presently used ss PCR assay was simpler, faster, cheaper and more readily interpreted than earlier assays. This information could be useful in understanding of current prevalence and distribution of An. minimus sibling species complex in NE region of India.

The impact of transfluthrin on the spatial repellency of the primary malaria mosquito vectors in Vietnam: Anopheles dirus and Anopheles minimus

Background The complexity of mosquito-borne diseases poses a major challenge to global health efforts to mitigate their impact on people residing in sub-tropical and tropical regions, to travellers and deployed military personnel. To supplement drug- and vaccine-based disease control programmes, other strategies are urgently needed, including the direct control of disease vectors. Modern vector control research generally focuses on identifying novel active ingredients and/or innovative methods to reduce human-mosquito interactions. These efforts include the evaluation of spatial repellents, which are compounds capable of altering mosquito feeding behaviour without direct contact with the chemical source. Methods This project examined the impact of airborne transfluthrin from impregnated textile materials on two important malaria vectors, Anopheles dirus and Anopheles minimus. Repellency was measured by movement within taxis cages within a semi-field environment at the National Institute of Hygiene and Epidemiology in Hanoi, Vietnam. Knockdown and mortality were measured in adult mosquito bioassay cages. Metered-volume air samples were collected at a sub-set of points in the mosquito exposure trial. Results Significant differences in knockdown/mortality were observed along a gradient from the exposure source with higher rates of knockdown/mortality at 2 m and 4 m when compared with the furthest distance (16 m). Knockdown/mortality was also greater at floor level and 1.5 m when compared to 3 m above the floor. Repellency was not significantly different except when comparing 2 m and 16 m taxis cages. Importantly, the two species reacted differently to transfluthrin, with An. minimus being more susceptible to knockdown and mortality. The measured concentrations of airborne transfluthrin ranged from below the limit of detection to 1.32 ng/L, however there were a limited number of evaluable samples complicating interpretation of these results. Conclusions This study, measuring repellency, knockdown and mortality in two malaria vectors in Vietnam demonstrates that both species are sensitive to airborne transfluthrin. The differences in magnitude of response between the two species requires further study before use in large-scale vector control programmes to delineate how spatial repellency would impact the development of insecticide resistance and the disruption of biting behaviour.