Entomological survey in two communes with residual malaria transmission in Gia Lai Province in the central highlands of Vietnam

Background In 2018, the National Malaria Control Programme in Vietnam switched from prioritizing malaria control to elimination. However, with the ongoing elimination programme, there are still areas where residual malaria transmission persists, including the central highlands. This entomological survey was conducted to evaluate Anopheles diversity and host-seeking activity of Anopheles vectors in two communes with very low malaria transmission in Gia Lai Province. Methods Anopheles species were collected in Ia DReh commune and Ia KDam commune, Gia Lai Province in the central highlands of Vietnam. Collections were conducted using human-baited double net trap, light trap and manual aspiration collections around cattle sheds, in the dry and rainy season. Mosquito specimens were identified morphologically, and members of species complexes were distinguished molecularly. Mosquito night-feeding patterns were investigated during the dry and rainy seasons. Results Overall, 18,835 specimens including 19 taxa were collected in Ia KDam and Ia DReh communes. These included the primary malaria vectors, Anopheles dirus and Anopheles minimus, and other secondary vector species. Anopheles dirus was observed to be an anthropophilic species, whereas An. minimus and a number of secondary vectors were observed to be zoophilic. Anopheles vagus was the dominant species, followed by Anopheles sinensis and Anopheles peditaeniatus. The majority of specimens were collected in the rainy season due to the relatively large number of An. vagus, while An. peditaeniatus, An. dirus, Anopheles kochi, Anopheles monstrosus and Anopheles tessellatus were collected in greater numbers during the dry season. The peak of host-seeking activity for An. dirus, An. sinensis, and An. vagus was between 18.00 and 19.00 h. Conclusion This study provided information on the diversity, seasonal prevalence and behaviour of Anopheles at the study sites. Identifying the diverse mosquito fauna in the central highlands of Vietnam allows species-specific control measures to be implemented by the National Programme to reduce malaria in areas of very low malaria transmission. The peak Anopheles host-seeking activity observed in this study was between 18.00 and 23.00 h, which highlights the need to better characterize Anopheles behaviour in this region of Vietnam to inform on vector control strategies.

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.

Cananga odorata (Magnoliales: Annonaceae) Essential Oil Produces Significant Avoidance Behavior in Mosquitoes

Essential oil of Cananga odorata Hook. F. & Tomson is a source of insect repellent, but contact irritancy and noncontact repellency actions that stimulate insect’s avoidance behavior (escape away from chemical source after direct physical contact or without making physical contact, respectively) have not been investigated. Therefore, an excito-repellency test chamber was used for measuring avoidance behavior of four insectary-reared mosquito species (Diptera: Culicidae) that escape from esposure to four concentrations (0.5, 1.0, 2.5, and 5.0% v/v) of C. odorata oil. The oil strongly repelled both Culex quinquefasciatus Say (85–97% escape) and Anopheles minimus Theobald (97–99%) at high concentrations (2.5–5.0%). For Anopheles dirus Peyton & Harrison and Aedes aegypti (L.), highest repellency (64 and 39% escape, respectively) was demonstrated at 2.5% concentration. For contact irritancy, the oil produced relatively high percent escape found in Cx. quinquefasciatus (90–100% escape) and An. minimus (83–100%). Whereas moderate contact irritancy was observed against An. dirus (40–50% escape) and Ae. aegypti (51–59%). The percent escape was then adjusted with repellency to estimate the effect of contact irritancy alone. We found that highest contact irritancy was presented at 0.5% concentration against An. minimus (67% escape). Knockdown and toxic actions were only found in Anopheles mosquitoes at 5.0% concentration. The results revealed that An. minimus and Cx. quinquefasciatus were more prone to be repelled by C. odorata oil. Detailed analysis of oil identified primary compounds as methyl benzoate (14.6%), α-gurjunene (12.8%), p-methyl-anisole (11.3%), and benzyl acetate (9.9%). Further investigations are needed to assess excito-repellency actions of these compounds alone or in combination.

Evaluation of two Plasmodium vivax sexual stage antigens as transmission-blocking vaccine candidates

Background Plasmodium vivax transmission-blocking vaccines (TBVs) are receiving increasing attention. Based on excellent transmission-blocking activities of the PbPH (PBANKA_0417200) and PbSOP26 (PBANKA_1457700) antigens in Plasmodium berghei, their orthologs in P. vivax, PVX_098655 (PvPH) and PVX_101120 (PvSOP26), were selected for the evaluation of their potential as TBVs. Methods Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain antisera. The transmission-reducing activities of these antisera were evaluated using the direct membrane feeding assay (DMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. The DMFA showed that the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0 and 84.1% in two parasite isolates, respectively, whereas the anti-PvPH sera did not show evident transmission-reducing activity. The variation in the DMFA results was unlikely due to the genetic polymorphisms of the two genes since their respective sequences were identical in the clinical P. vivax isolates. Conclusion PvSOP26 could be a promising TBV candidate for P. vivax, which warrants further evaluation. Graphical Abstract

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle

Background Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. Methods In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5′ UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. Results Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. Conclusion All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Ivermectin Treatment for Cattle Reduced the Survival of Two Malaria Vectors, Anopheles dirus and Anopheles epiroticus, Under Laboratory Conditions in Central Vietnam

Ivermectin is a low-cost and nontoxic mosquitocide that may have a role in malaria elimination. However, the extent to which this drug impacts the mortality of Anopheles dirus and Anopheles epiroticus, two important malaria vectors in Southeast Asia, is unknown. This study compared quantified anopheline mortality after feeding on ivermectin-treated cattle and control cattle in Vietnam. Local anopheles colonies fed on cattle 1 to 3, 6 to 8, 13 to 15, 20 to 22, and 28 to 30 days after injection (DAI) with ivermectin (intervention) or saline (control). An. dirus that fed on ivermectin-treated cattle had higher mortality rates than controls for up to 20 DAI (P < 0.05); An. epiroticus that fed on ivermectin-treated cattle had consistently higher mortality rates than controls for up to 8 DAI (P < 0.05). Feeding on ivermectin-treated cattle increased the mortality rate of these vector species for biologically relevant time periods. Therefore, ivermectin has the potential to become an important tool for integrated vector management.

Anopheles Bionomics in a Malaria Endemic Area of Southern Thailand

Background: Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles in Surat Thani province, Thailand.Methods: Mosquitoes were collected via human landing collections, February - October of 2019. Anopheles were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status and a subset evaluated for molecular identification and Plasmodium detection.Results: A total of 17,348 mosquitoes were collected. Of 5,777 Anopheles mosquitoes, 15 species were identified morphologically. The most abundant Anopheles were Anopheles minimus s.l. (87.87%, n = 5,035), Anopheles dirus s.l. (7.05%, n = 407), and Anopheles barbirostris s.l. (2.86%, n = 165). Molecular identification confirmed that of An. minimus s.l., 99.80% were An. minimus s.s. (n=484) and 0.2% An. aconitus (n = 1), of An. dirus s.l., 100% were An. baimaii (n = 348), and of An. maculatus s.l., 93.62% were An. maculatus s.s. (n = 44) and 6.38% An. sawadwongporni (n = 3). No Anopheles were Plasmodium positive (n = 879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection (Kruskal-Wallis = 80.89, P < 0.0001, n = 5,666), with more An. barbirostris s.l. and An. maculatus s.l. caught earlier compared to An. minimus s.l. (P = 0.0001, P < 0.0001, respectively) and An. dirus s.l. (P = 0.0082, P < 0.001, respectively). The proportion of parous An. minimus s.l. captured by hour increased throughout the night (Wald Chi-square = 17.31, P=0.000, odds ratio = 1.0535 [1.0279 – 1.0796] 95% CI (n = 3,400). Overall, An. minimus s.l. parity was 67.68% (2,375/3,509) with an intra cluster correlation of 0.0378. A power calculation determined that an An. minimus s.l. parity reduction treatment effect size = 34%, with four clusters per treatment arm, a minimum of 300 mosquitoes dissected per cluster at an α= 0.05 will provide 82% power to detect a significant difference following ivermectin MDA. Conclusions: The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on An. minimus parity.

The Microbiota of Three Anopheles Species in China

ABSTRACT Microbiota are vital for the development, physiology, and vectorial capacity of mosquitoes. The composition and role of microbiota in Anopheles species, especially Anopheles gambiae and Anopheles stephensi, have been extensively studied, but little is known about the microbiota of Anopheles species in China. We characterized the microbial communities of Anopheles dirus, Anopheles sinensis, and Anopheles lesteri by 16S rRNA sequencing. There were distinct differences in the composition of microbiota in An. lesteri and the other 2 species. The discriminatory genera in the 3 species were analyzed by the linear discriminant analysis effect size method. Our results provide an overview of the population structure of microbiota in 3 native Anopheles species and will pave the way for further understanding of their role in mosquito physiology and vector competence.