Important Malaria Vector Research Articles

Oviposition and Development of Anopheles coluzzii coetzee and Wilkerson in Salt Water.

Anopheles coluzzii is an important vector of malaria in sub-Saharan Africa particularly of the most dangerous malaria parasite. It completes its life cycle in water and a change in physicochemical properties particularly that of salinity of water may affect egg laying and perhaps the development of eggs to maturity. Studies have shown that climate change may alter the transmission of many vector-borne diseases in different parts of the world and global warming will also raise sea levels which will lead to an increase in saline and brackish water body in coastal areas. This study investigated the salinity tolerance level of An. coluzzii. It involved creation of artificial environments of different salinity gradients using rainwater and sea water and the subsequent exposure of the media to An. coluzzii for laying of eggs and development of larvae to adult. Anopheles coluzzii showed ovipositional preference for less saline media as there was significant negative correlation between number of eggs laid and salinity of oviposition media. Effect of salinity was evident in egg development and larval survival, as no egg hatched in >30% sea water, all L3 larvae died in >40% seawater, and the maximum seawater concentration for L4 survival was 30%. An LC50 of 17.51% (95% CI: 9.31–24.56)% and 23.4% (95% CI: 16.76–22.30)% were calculated for L3 and L4 larvae respectively. Adults emerging from fresh and low saline water of 10% seawater had greater energy reserve than those emerging from 20% and 30% seawater. Increasing salinity did not affect wing length of the emerging adult. Despite the increased stress on larval development, some individuals survived and went on to emerge as adults in conditions that seem to be representative of brackish water. This may imply that an increase in brackish water sites caused by rising sea levels might create more suitable breeding sites for this species.

Mitochondrial genes associated with pyrethroid resistance revealed by mitochondrial genome and transcriptome analyses in the malaria vector Anopheles sinensis (Diptera: Culicidae)

Insecticides are still the main method of mosquito control, but mosquito resistance presents a large obstacle. The function of mitochondrial genes in the evolution of insecticide resistance is still poorly understood. Pyrethroid is the most commonly used insecticide, and Anopheles sinensis is an important malaria vector in China and Southeast Asia. In this study, we investigated the mitochondrial genes associated with pyrethroid resistance through their genetic and expression variation based on analyses of transcriptomes and 36 individuals with resequencing in three geographical populations in China. The nucleotide diversity (Pi) in 18 resistant individuals was much lower than that in 18 susceptible individuals, which suggests that some sites experienced purifying selection subject to pyrethroid stress. Ka/Ks and amino acid analyses showed that ND4 experienced positive selection and had 23 amino acid mutations due to pyrethroid stress. These mutations might change the ND4 structure and function and thus alter the efficiency of the respiratory chain. ND5 was significantly upregulated, and ATP8 was significantly downregulated in these three pyrethroid resistant populations, which suggests that these two genes function in the production and maintenance of pyrethroid resistance. There are differences in mitochondrial genes involved in pyrethroid resistance among these three populations. This is the first study to reveal the association of mitochondrial genes in the evolution of insecticide resistance through amino acid mutation and expression patterns and can help us further understand insecticide resistance mechanisms. © 2019 Society of Chemical Industry.

Identification of novel Glutathione S-Transferases epsilon 2 mutation in Anopheles maculipennis s.s. (Diptera: Culicidae)

Anopheles maculipennis complex comprises some important malaria vectors in Iran, Middle East, and Europ. The principal way to control of malaria remains on the use of chemical insecticides against its vectors because there is no vaccine for malaria prevention. Extensive use of organophosphate compounds has caused to emergence and distribution of insecticide resistance in Anopheles species in Asia. The current study aimed to the detection of three well-known amino acid substitutions (I114T, L119F, and F120L) in the Glutathione S-Transferases epsilon 2 (GSTe2) gene are associated with DDT and organophosphate insecticides resistance in an Anopheles maculipennis population collected from Iran. Adult samples of An. maculipennis were collected by hand and Total catch in Animal and Human Shelters from Azerbaijan-Gharbi and Zanjan provinces. Following morphological identification, DNA was extracted by YTA Genomic DNA Extraction Mini Kit for amplification of rDNA-ITS2 and GSTe2 fragments. ∼500 bp fragment was amplified using F rDNA-ITS2 and GSTe2 primers. rDNA-ITS2 sequence analysis showed 100% similarity with An. maculipennis. GSTe2 nucleotide sequence similarity within species was 99–100%, while, it was 95–96 % when compared with Anopheles sacharovi GSTe2 sequences available in GenBank. Amino acid sequence comparisons showed a novel amino acid substitution in N148D position with 15.79% frequency. The current study reports new GSTe2 amino acid substitution in An. maculipennis s.s., for the first time. The function of the mutation N148D and its association with resistance phenotype need to validate. However, the integration of these data into the malaria control program still remains a challenge.

Genetic homogeneity of Anopheles maculatus in Indonesia and origin of a novel species present in Central Java

BackgroundAnopheles maculatus (s.s.) is an important vector of malaria in Indonesia. Previously it was considered the only member of the Maculatus Group present in Indonesia. A novel species was recently identified in the Kulon Progo District in Central Java. Until recently, few investigations have been conducted looking at An. maculatus genetic diversity in Indonesia, including allopatric island populations.MethodsIndonesian An. maculatus (s.l.) samples were collected in several locations in Java, Lesser Sunda Island group, Sumatra and in Kulon Progo (Yogyakarta, central Java) where a novel species has been identified. Samples from a 30-year-old colony of the Kulon Progo population were also included in the analysis. Maximum-likelihood analysis established the phylogenies of the ITS2 (nuclear) and cox1 (mitochondrial) markers. Putative times of separation were based on cox1 genetic distances.ResultsTwo species of the Maculatus Group are present in Indonesia. The novel sibling species is more closely related to Anopheles dispar than to An. maculatus (s.s.). Anopheles maculatus (s.s.) samples are homogeneous based on the ITS2 sequences. Indonesian samples and An. dispar belong to the same cox1 maternal lineage and differ from all other known members of the Maculatus Group. Divergence time between the different populations found in Java was estimated using an established cox1 mutation rate.ConclusionsA novel species within the Maculatus Group, most closely related to An. dispar, is confirmed present in the Kulon Progo area of Central Java. The divergence of this species from An. maculatus (s.s.) is explained by the stable refugia in the Kulon Progo area during the quaternary period of intense volcanic activity throughout most of Java. This novel species awaits detailed morphological description before applying a formal species name. For the interim, it is proposed that the Kulon Progo population be designated An. maculatus var. menoreh to distinguish it from An. maculatus (s.s.).

Effect of acute and chronic exposure to ammonia on different larval instars of Anopheles darlingi (Diptera: Culicidae)

Anopheles darlingi (Diptera: Culicidae) is the most important vector of malaria in South America and has already been found in peri-urban areas that commonly contain toxic nitrogenous compounds, such as ammonia. The adaptation of mosquitoes to polluted breeding sites can increase their distribution and affect the dynamics of vector-borne diseases such as malaria. Therefore, the present study investigated the tolerance of larval instars of An. darlingi to ammonia under acute and chronic exposure conditions. Anopheles darlingi larval mortality, development time, and pupal and adult production using larvae of the 1st (L1) and 3rd (L3) instar were assessed as both acute and chronic effects of exposure to different concentrations of ammonia. Lethal concentrations (LCs) for L1 larvae were lower than LCs for L3 larvae. In general, higher ammonia concentrations caused an increase in larval mortality, especially in chronically exposed L1 larvae. The larval development time in L1 and L3 was longer with chronic treatment and decreased with increasing concentrations of ammonia. The number of pupae was very low for acutely exposed L1 and L3 larvae. Likewise, the probability of adult production decreased with increasing ammonia concentrations. This is the first report on the tolerance of An. darlingi to pollutants.

Is Genetic Continuity Between Anopheles sinensis (Diptera: Culicidae) and its Sibling Species Due to Gene Introgression or Incomplete Speciation?

The Anopheles mosquito Hyrcanus Group is widely distributed geographically across both Palearctic and Oriental regions and comprises 26 valid species. Although the species Anopheles sinensis Wiedemann (1828) is the most common in China and has a low potential vector rank, it has nevertheless long been thought to be an important natural malaria vector within the middle and lower reaches of the Yangtze River. A number of previous research studies have found evidence to support the occurrence of natural hybridization between An. sinensis and Anopheles kleini Rueda, 2005 (a competent malaria vector). We, therefore, collected a sample series of An. sinensis and morphologically similar species across China and undertook ribosomal and mitochondrial DNA analyses in order to assess genetic differentiation (Fst) and gene flow (Nm) amongst different groups. This enabled us to evaluate divergence times between morphologically similar species using the cytochrome oxidase I (COI) gene. The results of this study reveal significant genetic similarities between An. sinensis, An. kleini, and Anopheles belenrae Rueda, 2005 and therefore imply that correct molecular identifications will require additional molecular markers. As results also reveal the presence of gene flow between these three species, their taxonomic status will require further work. Data suggest that An. kleini is the most basal of the three species, while An. sinensis and An. belenrae share the closest genetic relationship.

Characterization of Bacterial Communities in Breeding Waters of Anopheles darlingi in Manaus in the Amazon Basin Malaria-Endemic Area

The microbiota in mosquito breeding waters can affect ovipositing mosquitoes, have effects on larval development, and can modify adult mosquito-gut bacterial composition. This, in turn, can affect transmission of human pathogens such as malaria parasites. Here, we explore the microbiota of four breeding sites for Anopheles darlingi, the most important malaria vector in Latin America. The sites are located in Manaus in the Amazon basin in Brazil, an area of active malaria transmission. Using 16S rRNA gene sequencing by MiSeq, we found that all sites were dominated by Proteobacteria and Firmicutes and that 94% of the total number of reads belonged to 36 operational taxonomic units (OTUs) identified in all sites. Of these, the most common OTUs belonged to Escherichia/Shigella, Staphylococcus, and Pseudomonas. Of the remaining 6% of the reads, the OTUs found to differentiate between the four sites belonged to the orders Burkholderiales, Actinomycetales, and Clostridiales. We conclude that An. darlingi can develop in breeding waters with different surface-water bacteria, but that the common microbiota found in all breeding sites might indicate or contribute to a suitable habitat for this important malaria vector.

Dry Season Composition and Abundance of the Anopheles, Culex and Aedes Genera in Makrudi Benue State

Mosquitoes are very important vectors of malaria, yellow fever, dengue and other diseases. Their unmitigated availability is key to the spread of parasites spread by their bites. This research was designed to find out the abundance of mosquitoes of the Anopheles, Culex and Aedes Genera in Makurdi Local Government of Benue state. Larva was collected from different water bodies in three (3) different locations that cover Makurdi representatively with the aid of a scoop spoon. Collected larva was stored in transparent plastic plates and transported to the insectarium, filtered into cleaner water by decantation, fed with baker’s yeast and grown into adult Mosquitoes in premade 16x16x16 inches cages. Adults were fed with sugar solution in cotton balls. Dead mosquitoes were stored in vials containing 70% ethanol. Using the Photographic Guide to common mosquitoes of Florida, Mosquitoes were identified by microscopy. 537 Mosquitoes were identified of different Genera. Aedesgenera recorded the highest abundance of 34.64%, while Anophelesgenera has the least abundance with 31.10%. Culexgenera recorded an abundance of 34.26% in makurdi. NorthBank with 40.78%, recorded the highest number of mosquitoes of the three Genera under consideration. Logo II/Kanshio follows with 32.22% and Karmen, 27.00%, showed the least abundance. Based on this research, Mosquitoes showed relatively high abundance in Makurdi in the dry and windy harmattan season. This could be one reason malaria persists even in the dry seasons in makurdi.

Geometric morphometrics approach towards discrimination of three member species of Maculatus group in Thailand

Members of the Maculatus group are important malaria vectors in the border regions of Thailand. However, the role of each species in malaria transmission remains unclear because of their highly similar morphologies, making them difficult to be differentiated. Whereas An. pseudowillmori may be identified by the color pattern of some scales on abdomen and wings, the distinction between An. maculatus and An. sawadwongporni relies on the wings only. Scales are labile structures, as they may be accidentally removed during capture and transportation to the laboratory. To discriminate among the species of this group, we tested the suitability of geometric techniques. Shape variables were used as input for discriminant analyses and validated reclassification. Both landmark- and outline-based geometric techniques disclosed significant differences between the three species. For the delicate An. maculatus - An. sawadwongporni distinction, the outline-based approach appeared as the most promising, with validated reclassification scores reaching 93%, as compared to 77% obtained by landmark data. For An. pseudowillmori, reclassification scores were 100% and 94%, respectively. Geometric morphometrics may provide an alternative and useful complement for discriminating members of the Maculatus group.

Susceptibility to common insecticides and detoxifying enzyme activities in Anopheles sundaicus (sensu lato) after cessation of indoor residual spraying of insecticides in the Jaffna Peninsula and its surroundings in northern Sri Lanka

BackgroundSri Lanka has been malaria-free since 2013 but re-introduction of malaria transmission by infected overseas travelers is possible due to a prevalence of potent malaria vectors. Knowledge of the insecticide resistance status among Anopheles vectors is important if vector control has to be reintroduced in the island. The present study investigated the insecticide susceptibility levels and resistance mechanisms of Anopheles sundaicus (sensu lato) (previously classified as Anopheles subpictus species B) an important malaria vector in the Jaffna Peninsula and it surroundings in northern Sri Lanka after indoor residual spraying of insecticides was terminated in 2013.ResultsSpecies-specific PCR assays identified An. sundaicus (s.l.) in four locations in the Jaffna and adjacent Kilinochchi districts. Bioassays confirmed that An. sundaicus (s.l.) collected in Kilinochchi were completely susceptible to 0.05% deltamethrin and 5% malathion and resistant to 4% dichlorodiphenyltrichloroethane (DDT), whereas those from Jaffna were relatively susceptible to all three insecticides. Kilinochchi populations of An. sundaicus (s.l.) showed significantly higher glutathione S-transferase activity than population from Jaffna. However, Jaffna An. sundaicus (s.l.) had significantly higher Propoxur-resistant acetylcholinesterase activity. Activities of non-specific esterases and monooxygenases were not significantly elevated in An. sundaicus (s.l.) collected in both districts.ConclusionsThe susceptibility to malathion and deltamethrin in An. sundaicus (s.l.) suggests that they can be still used for controlling this potential malaria vector in the Jaffna Peninsula and adjacent areas. Continuing country-wide studies on other malaria vectors and their insecticide susceptibilities are important in this regard.

Mosquitoes at the University of Port Harcourt Teaching Hospital Nigeria: A Threat to Caregivers and Patients

Anopheles gambiae sensu lato is the most important malaria vector in Nigeria. Referral hospitals have a mandate to provide patients with the best health care. The persistent complaints of the nuisance caused to patients, professionals, and visitors by mosquitoes at the University of Port Harcourt Teaching Hospital (UPTH), a referral Hospital, prompted the design of this study to assess the species composition, density, and man-biting rates of endophilic mosquitoes at the UPTH. A longitudinal entomological survey covered dry and wet seasons, February–July 2017, with sampling done thrice weekly, using the aspiration method. Caught mosquitoes were identified by standard keys using a dissecting microscope and classified according to their gonotrophic status. A total of 1,307 mosquitoes in two genera (Culex, Anopheles) and three species (Culex quinguefasciatus, Anopheles gambiae s.l. and Anopheles mouchetti) were caught, comprising 150 (11.5%) males and 1,157 females. The most abundant species was Culex quinguefasciatus (98.01%), An. gambiae s.l. (1.64%), and the least abundant, An. mouchetti (0.35%). Gonotrophic examinations of caught females revealed 52.8% blood-fed and 40.1% unfed. Culex quinquefasciatus had higher man-biting rates (1.26 bites/patient/night) than Anopheles (0.2 bites/patient/night). The Accident and Emergency ward, 595 (45.5%), and the Obstetrics and Gynecology ward, 328 (25.1%), had the highest records of caught mosquitoes among the four wards visited for collections. No significant difference (Fcal = 1.0722, Ftab = 13, p > 0.05, df = 2) existed between wet and dry seasons' collections. Because of the high numbers of blood-fed Anopheles, an urgent need for intervention is required to reduce the case of vector/human contacts; notably, larval source management will be a principal approach toward control.

Entomological status of Anopheles sergentii and the first molecular investigation of its insecticide-resistant genes, kdr and ace-1 in Morocco.

Prior to their elimination in 1974 and 2004, respectively, Plasmodium falciparum and P. vivax were the main native malaria parasites involved in disease transmission in Morocco. Imported cases of human malaria are still reported from the country. Anopheles labranchiae in northern Morocco and An. sergentii in the southern regions are the main malaria vectors. The bionomics and insecticide susceptibility of An. sergentii are poorly understood and need to be further studied to enhance the epidemiological surveillance of this important malaria vector. The adults and larvae of Anopheles sergentii were collected during the mosquito breeding season in 2015 and 2016 and environmental characteristics of their breeding sites were recorded. Blood meals were analyzed using PCR-RFLP. Alongside, the WHO routine susceptibility tests with DDT (4%) and malathion (5%) were conducted and An. sergentii specimens were screened for knockdown resistance (kdr) and acetyl cholinesterase encoding (ace-1) gene mutations. Anopheles sergentii was observed during the summer and autumn seasons, feeding mainly on sheep, cows and also on humans. The WHO bioassays revealed complete susceptibility to DDT and malathion. Analysis of the sequences of the voltage-gated sodium channel gene revealed the absence of the kdr "Leu-Phe" mutation and PCR-RFLP revealed the absence of the G119S mutation in the ace-1. With the increasing number of imported cases of human malaria in Morocco, the indiscriminate feeding behavior of this species may pose an infectious medical threat. Fortunately, the absence of insecticide resistance can ensure, for now, the efficiency of insecticides, as a part of the vector control program in controlling An. sergentii in Morocco.

A transgenic tool to assess Anopheles mating competitiveness in the field

BackgroundMalaria parasites, transmitted by the bite of an anopheline mosquito, pose an immense public health burden on many tropical and subtropical regions. The most important malaria vectors in sub-Saharan Africa are mosquitoes of the Anopheles gambiae complex including An. gambiae (sensu stricto). Given the increasing rates of insecticide resistance in these mosquitoes, alternative control strategies based on the release of genetically modified males are being evaluated to stop transmission by these disease vectors. These strategies rely on the mating competitiveness of release males, however currently there is no method to determine male mating success without sacrificing the female. Interestingly, unlike other insects, during mating An. gambiae males transfer their male accessory glands (MAGs) seminal secretions as a coagulated mating plug which is deposited in the female atrium.ResultsHere we exploit this male reproductive feature and validate the use of a MAG-specific promoter to fluorescently label the mating plug and visualize the occurrence of insemination in vivo. We used the promoter region of the major mating plug protein, Plugin, to control the expression of a Plugin-tdTomato (PluTo) fusion protein, hypothesizing that this fusion protein could be incorporated into the plug for sexual transfer to the female. Anopheles gambiae PluTo transgenic males showed strong red fluorescence specifically in the MAGs and with a pattern closely matching endogenous Plugin expression. Moreover, the fusion protein was integrated into the mating plug and transferred to the female atrium during mating where it could be visualized microscopically in vivo without sacrificing the female. PluTo males were equally as competitive at mating as wild type males, and females mated to these males did not show any reduction in reproductive fitness.ConclusionThe validation of the first MAG-specific promoter in transgenic An. gambiae facilitates the live detection of successful insemination hours after copulation has occurred. This provides a valuable tool for the assessment of male mating competitiveness not only in laboratory experiments but also in semi-field and field studies aimed at testing the feasibility of releasing genetically modified mosquitoes for disease control.

Vertical transmission of naturally occurring Bunyamwera and insect-specific flavivirus infections in mosquitoes from islands and mainland shores of Lakes Victoria and Baringo in Kenya.

BackgroundMany arboviruses transmitted by mosquitoes have been implicated as causative agents of both human and animal illnesses in East Africa. Although epidemics of arboviral emerging infectious diseases have risen in frequency in recent years, the extent to which mosquitoes maintain pathogens in circulation during inter-epidemic periods is still poorly understood. This study aimed to investigate whether arboviruses may be maintained by vertical transmission via immature life stages of different mosquito vector species.MethodologyWe collected immature mosquitoes (egg, larva, pupa) on the shores and islands of Lake Baringo and Lake Victoria in western Kenya and reared them to adults. Mosquito pools (≤25 specimens/pool) of each species were screened for mosquito-borne viruses by high-resolution melting analysis and sequencing of multiplex PCR products of genus-specific primers (alphaviruses, flaviviruses, phleboviruses and Bunyamwera-group orthobunyaviruses). We further confirmed positive samples by culturing in baby hamster kidney and Aedes mosquito cell lines and re-sequencing.Principal findingsCulex univittatus (2/31pools) and Anopheles gambiae (1/77 pools) from the Lake Victoria region were positive for Bunyamwera virus, a pathogenic virus that is of public health concern. In addition, Aedes aegypti (3/50), Aedes luteocephalus (3/13), Aedes spp. (2/15), and Culex pipiens (1/140) pools were positive for Aedes flaviviruses at Lake Victoria, whereas at Lake Baringo, three pools of An. gambiae mosquitoes were positive for Anopheles flavivirus. These insect-specific flaviviruses (ISFVs), which are presumably non-pathogenic to vertebrates, were found in known medically important arbovirus and malaria vectors.ConclusionsOur results suggest that not only ISFVs, but also a pathogenic arbovirus, are naturally maintained within mosquito populations by vertical transmission, even in the absence of vertebrate hosts. Therefore, virus and vector surveillance, even during inter-epidemics, and the study of vector-arbovirus-ISFV interactions, may aid in identifying arbovirus transmission risks, with the potential to inform control strategies that lead to disease prevention.

Speculation on the possibility for introducing Anopheles stephensi as a species complex: preliminary evidence based on odorant binding protein 1 intron I sequence

BackgroundAnopheles stephensi is considered an important malaria vector in Iran, Asia, and recently in the Horn of Africa. Recently, Ansteobp1 intron I sequence has been introduced a new molecular marker for identification of its biological forms including, mysorensis, intermediate and type, using insectary colony specimens.MethodsIn the current study, new marker ability in molecular identification of biological forms has been evaluated with An. stephensi specimens collected from Iran and Afghanistan malarious provinces. Following DNA extraction and PCR amplification, sequence analysis and constructed phylogenetic tree revealed that type and intermediate forms are distributed in Iran.ResultsThe specimens collected from Afghanistan identified as intermediate and mysorensis forms. Therefore, intermediate form is sympatric species in both countries. Based on the results of Ansteobp1 intron I sequences, An. stephensi could be suggested as new Anopheles complex species including An. stephensi sibling A (type form), An. stephensi sibling B (intermediate form) and An. stephensi sibling C (mysorensis form). This is the first report on the presence of An. stephensi biological forms in Afghanistan.ConclusionsIran is going to eliminate malaria transmission from the country, precise species identification, especially in complex species will be helpful in the prevention of malaria resurgence in the country, mainly because of common fauna of Anopheles species and through border malaria and population movement within Afghanistan, Pakistan, and Iran.

Exploring malaria vector diversity on the Amazon Frontier

BackgroundDeforestation in the Amazon and the social vulnerability of its settler communities has been associated with increased malaria incidence. The feeding biology of the most important malaria vectors in the region, notably Nyssorhynchus darlingi, compounds efforts to control vectors and reduce transmission of what has become known as “Frontier Malaria”. Exploring Anophelinae mosquito diversity is fundamental to understanding the species responsible for transmission and developing appropriate management and intervention strategies for malaria control in the Amazon River basin.MethodsThis study describes Anophelinae mosquito diversity from settler communities affected by Frontier Malaria in the states of Acre, Amazonas and Rondônia by analysing COI gene data using cluster and tree-based species delimitation approaches.ResultsIn total, 270 specimens from collection sites were sequenced and these were combined with 151 reference (GenBank) sequences in the analysis to assist in species identification. Conservative estimates found that the number of species collected at these sites was between 23 (mPTP partition) and 27 (strict ABGD partition) species, up to 13 of which appeared to be new. Nyssorhynchus triannulatus and Nyssorhynchus braziliensis displayed exceptional levels of intraspecific genetic diversity but there was little to no support for putative species complex status.ConclusionsThis study demonstrates that Anophelinae mosquito diversity continues to be underestimated in poorly sampled areas where frontier malaria is a major public health concern. The findings will help shape future studies of vector incrimination and transmission dynamics in these areas and support efforts to develop more effective vector control and transmission reduction strategies in settler communities in the Amazon River basin.

The Development of Cytogenetic Maps for Malaria Mosquitoes.

Anopheline mosquitoes are important vectors of human malaria. Next-generation sequencing opens new opportunities for studies of mosquito genomes to uncover the genetic basis of a Plasmodium transmission. Physical mapping of genome sequences to polytene chromosomes significantly improves reference assemblies. High-resolution cytogenetic maps are essential for anchoring genome sequences to chromosomes as well as for studying breakpoints of chromosome rearrangements and chromatin protein localization. Here we describe a detailed pipeline for the development of high-resolution cytogenetic maps using polytene chromosomes of malaria mosquitoes. We apply this workflow to the refinement of the cytogenetic map developed for Anopheles beklemishevi.

Repellence Activity of Cymbopogon Citratus (DC) Extracts on Anopheles Mosquitoes using Swiss Albino Rat and Human Volunteer

Introduction:Insect-transmitted disease remains a major source of illness and death worldwide.Anopheles stephensiandAnopheles culicifaciesare the important vectors of malaria, malaria continues to be a major public health problem in the tropical world. This study is aimed at carrying out repellence activity ofCymbopogon citratus(Lemon grass) extracts onAnophelesmosquitoes using swiss albino rat and human volunteers.Methods:Methanol, chloroform and water were used to extract the bioactive compounds of plant leaves, qualitative and quantitative phytochemical components of plant extracts were determined, twenty-five laboratory-reared 3 day oldAnophelesmosquitoes which had been starved for 5 hours were used, extracts and the commercial insecticide N, N-diethyl-3-methylbenzamide (DEET) were applied topically on the skin of uncovered portions of the hand of the human volunteers and abdomen of swiss albino rat after the abdominal hairs has been shaved and the landing rate of mosquitoes were observed to calculate percentage repellency.Results:The result revealed that methanol extract had the highest percentage yield of 10.3%, tannin and alkaloid were present in all extracts. DEET had 100% repellency toAnophelesmosquitoes on both human volunteers and rats for 300 minutes post application, in human volunteers, water, chloroform and methanol extracts, has percentage repellency reduced from 100% to 94% after 60 minutes, 100% to 94% after 120 minutes and 100% to 83% after 150 minutes post application respectively while in swiss albino rat, water, chloroform and methanol extracts, percentage repellency reduce from 100% to 87% after 90 minutes, 100% to 87% 150 minutes and 100% to 90% after 180 minutes post application respectively.Conclusion:Methanol extracts ofC. citratusleaves could be used for the development of topical cream that repels mosquitoes for effective control of malaria.

Blood feeding behaviour comparison and contribution of Anopheles coluzzii and Anopheles gambiae, two sibling species living in sympatry, to malaria transmission in Alibori and Donga region, northern Benin, West Africa

BackgroundThe main goal of this study was to assess the blood feeding behaviour and the contribution Anopheles coluzzii and Anopheles gambiae, 2 sibling species of An. gambiae sensu stricto. present and living in sympatry in 2 regions of northern Benin targeted for indoor residual spraying (IRS).MethodsThe study was carried out in 6 districts of 2 regions of Benin (Alibori and Donga). Human landing catches (HLC) performed inside and outside of the households and pyrethrum spray captures (PSC) carried out in bedrooms were used to sample vector populations (An. gambiae and An. coluzzii). Collected mosquitoes were analysed to estimate the human biting rate indoors and outdoors, the circumsporozoite antigen positivity, and the anthropophagic index using ELISA methodology. Polymerase chain reaction was used to estimate the frequency of the knockdown resistance (kdr) L1014F and the ace-1 mutations, 2 markers associated respectively with pyrethroids and carbamate/organophosphate insecticide resistance.ResultsA higher blood feeding rate was observed in An. gambiae compared to An. coluzzii as well as, a non-pronounced outdoor biting behavior in both species. The latter showed similar anthropophagic and sporozoite rates. However the analysis indicates a seasonal difference in the contribution of each species to malaria transmission associated with shifts in resting behaviour. Anopheles coluzzii females accounted for most of the detected infections: 86% in Alibori and 79% in Donga, during the dry season versus 14.4% and 21.2%, respectively for An. gambiae during the same period. This relationship was reversed in Donga during the rainy season (66% for An. gambiae against 34% for An. coluzzii). Results also indicated lower frequencies of kdr L1014F and ace-1 in An. coluzzii versus An. gambiae.ConclusionDespite similarity in some parameters related to malaria transmission in both surveyed species, An. coluzzii is potentially a more important malaria vector because of high density in the region. It is also characterized by lower frequencies of the ace-1 mutation than is An. gambiae. The ongoing use of pirimiphos methyl (organophosphate) for IRS should continue to show a good impact in Alibori and Donga because of the very low level of the ace-1 mutation in both species.

Optimal Discriminating Concentrations of Six Synthetic Pyrethroids for Monitoring Insecticide Susceptibility in Anopheles minimus (Diptera: Culicidae), a Primary Malaria Vector in Thailand.

Malaria is a serious mosquito-borne disease transmitted to humans by Anopheles mosquitoes. Seven species of Anopheles are important malaria vectors in Thailand and all remain susceptible to pyrethroid insecticides, despite decades of use in public health mosquito control programs. However, for a more refined and accurate approach to temporal monitoring of susceptibility patterns to commonly used pyrethroids to control adult Anopheles mosquitoes, it is preferred to have specific concentrations for more discriminating testing. A laboratory strain of Anopheles minimus Theobald was used to establish baseline concentrations for susceptibility to six different synthetic pyrethroids-deltamethrin, permethrin, bifenthrin, cypermethrin, α-cypermethrin, and λ-cyhalothrin using the World Health Organization (WHO) adult bioassay system. Final discriminating concentrations of each active ingredient were determined based on doubling LC99 for each chemical to arrive at the following percent concentrations: deltamethrin (0.009%), permethrin (0.551%), bifenthrin (0.12%), cypermethrin (0.104%), α-cypermethrin (0.034%), and λ-cyhalothrin (0.019%). For permethrin, deltamethrin, α-cypermethrin, and λ-cyhalothrin, all revised percentages were lower than currently recommended discriminating concentrations by WHO. Using the newly derived concentrations, insecticide susceptibility tests were followed in a field population of An. minimus s.l. from Kanchanaburi Province. Deltamethrin, bifenthrin, and λ-cyhalothrin produced 100% kill, whereas permethrin, cypermethrin, and α-cypermethrin gave between 92.9 and 97.7% mortality, indicating lower sensitivity (tolerance). Routine monitoring of insecticide susceptibility in An. minimus wild populations throughout Thailand should continue to ensure that insecticides in use are effective to control this vector species.