Molecular diversity of genes related to biological rhythms (period and timeless) and insecticide resistance (Na V and ace-1) in Anopheles darlingi

BACKGROUND Malaria is a public health concern in the Amazonian Region, where Anopheles darlingi is the main vector of Plasmodium spp. Several studies hypothesised the existence of cryptic species in An. darlingi, considering variations in behaviour, morphological and genetic aspects. Determining their overall genetic background for vector competence, insecticide resistance, and other elements is essential to better guide strategies for malaria control. OBJECTIVES This study aimed to evaluate the molecular diversity in genes related to behaviour and insecticide resistance, estimating genetic differentiation in An. darlingi populations from Amazonian localities in Brazil and Pacific Colombian region. METHODS We amplified, cloned and sequenced fragments of genes related to behaviour: timeless (tim) and period (per), and to insecticide resistance: voltage-gated sodium channel (Na V ) and acetylcholinesterase (ace-1) from 516 An. darlingi DNA samples from Manaus, Unini River, Jaú River and Porto Velho - Brazil, and Chocó - Colombia. We discriminated single nucleotide polymorphisms (SNPs), determined haplotypes and evaluate the phylogenetic relationship among the populations. FINDINGS The genes per, tim and ace-1 were more polymorphic than Na V . The classical kdr and ace-1 R mutations were not observed. Phylogenetic analyses suggested a significant differentiation between An. darlingi populations from Brazil and Colombia, except for the Na V gene. There was a geographic differentiation within Brazilian populations considering per and ace-1. CONCLUSIONS Our results add genetic data to the discussion about polymorphisms at population levels in An. darlingi. The search for insecticide resistance-related mechanisms should be extended to more populations, especially from localities with a vector control failure scenario.

Studies on Malaria Vector Population Density in Three Selected Areas in Sokoto Metropolitan, Sokoto State - Nigeria

This study is conducted to determine the population density of mosquitoes in three selected area (Kofar Taramniya, Mabera and Bello Way) in Sokoto metropolis. To identified and count the population of mosquito, different breeding sites were investigated for the preferences of mosquitoes. Mosquito larvae were collected using dipper, ladle spoon from drainages, stagnant water, gutters, open wells, local pots, water tanks and cattle sheds. A total population of 4,764 mosquito larvae were collected and identified From the three (3) selected areas larvae and pupae were encounters in abundance. In Mabera area, a total of 1799 (37.7%) larvae are collected in which 1199 (45.8%) belonging to Anophelesgenera and 600 (27.9%) belong to culexgenera. In kofar Taramniya a total of 1649 (34.6%) larvae are collected in which 1000 (38.2%) belong to Anopheles genera and 649 (30.2%) belong to culexgenera, in Bello Way, A total of 1317 (27.5%) larvae were collected,417 (15.9%) belong to Anophelesgenera and 900 (41.9%)belongto culexgenera. Statistical analysis conducted indicated a significant difference (p<0.05) between the three collections. Mosquitoes encountered belong to two genera of Anophelesand Culex. The predominant genus was Anopheleswith a total of 2,615 larvaewhile Culexhas a total of 2149 larvae in all the three study areas. Considering the medical importance of mosquitoes, the information provided from this study will serve as a basis for understanding the implication of mosquitoes nuisance and provide effective control strategies against the vector and to reduce mosquitoes born disease

Description of an automatic copulation induction system used to establish a free-mating laboratory colony of Nyssorhynchus deaneorum from Brazil

BACKGROUND Nyssorhynchus deaneorum is a potential malaria vector because it has been shown to be competent to transmit Plasmodium vivax and Plasmodium falciparum, and because it exhibits antropophilic and endophilic behaviors in some regions of the Amazon. This profile makes Ny. deaneorum a useful mosquito for experiments that model Plasmodium-vector interactions in the Amazon. OBJECTIVE Herein we describe how a free-mating colony of Ny. deaneorum has been established using an automated light stimulation system. METHODS Mosquitoes were captured in São Francisco do Guaporé, Rondônia. The F1 generation was reared until adult emergence at which point copulation was induced using an automatic copulation induction system (ACIS). FINDINGS After four generations, natural mating and oviposition began to occur without light stimulation. The number of pupae and adult mosquitoes increased from the F5 to F10 generations. The new Ny. deaneorum colony exhibited susceptibility to P. vivax. MAIN CONCLUSIONS Automated light stimulation is an effective method for establishing an Ny. deaneorum colony under laboratory conditions as it produces enough adults to create a stenogamic colony. The establishment of a stable, P. vivax-susceptible colony of Ny. deaneorum makes it possible to model parasite-vector interactions and to test novel drug therapies that target parasite development in mosquitoes.

Efficacy of Tithonia diversifolia and Momordica charantia Leaves Extracts against Malaria Vector, Anopheles gambiae Gile (Diptera: Culicidae)

The gastrointestinal tracts were cut open and the contents of the various sections were put into separate Petri dishes containing normal saline. The skin and the bladder were observed directly under a dissecting microscope for the presence of cysts and monogeneans. The parasites were fixed and preserved in 70% alcohol following standard procedure. Parasites recovered from the gastrointestinal tracts of the anurans include Cosmocerca ornata, Deising, 1861,Cosmocerca cummutata, Diesing, 1851 Paracosmocerca mucronata, Kung and Wu, 1945, Ampliceacum africanum, Taylor, 1924, Gendria liberrei Bain and Philipon, 1969 and Chenospirura asturiHsu, 1957Others were Procamallus brevis Kung, 1948 and Camallanus dimitrovi Durette-Desset and Batcharov, 1974. Some of the parasites are zoonotic while a few others are established parasites of African fishes and water Birds raising probable public health concerns from the findings. Further works aimed at unravelling the biodiversity of hosts and parasites in thelush ecosystem of Ondo state, as well as identification of organisms involved in the life cycle are noted. Methodology: Larvae and pupae of Anopheles gambiaewere reared inthe laboratory at ambient temperature of 28±2°Cand relative humidity of 75±5%. The leave extracts of T. diversifolia and M. charantia were extracted with methanol and were prepared at concentrations, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%. The larvae and pupae of A. gambiae were exposed to these concentrations of the plant extract for 24 hours. Mortality of the larvae and pupae was monitored and recorded. Probit analysis was used to determine the LC50. Results: Date of this research revealed that at all levels of concentrations, mortality of both the larvae and pupae of this insect increased with increase in the concentrations regardless the type of plant extract used. The leave extract of T. diversifoliahavinga lower value of LC50(larvae: 0.20%; Pupae: 0.27%) was more potent than extract from M. charantiahaving a higher value of LC50(larvae: 0.31%; Pupae: 0.44%) after 24 hours Post Treatment of larvae and pupae of A. gambiae. T. diversifoliahad significanteffect on the larvae of A. gambiaewith percentage mortality ranges from 23.33-100% within 24hrs of exposure when compared with M. charantiathat had 16.67-100% of mortality larvae of A. gambiae.Conclusion: The obtained results from this research revealed that extracts from the two plants exhibited great insecticidal properties against larvae and pupae of A. gambiae. Therefore, more exploration on the use of these plants for the development of insecticides at commercial level should be done

Brazil's first free-mating laboratory colony of Nyssorhynchus darlingi

Abstract INTRODUCTION: The lack of highly-productive Nyssorhynchus darlingi laboratory colonies limits some studies. We report the first well-established laboratory colony of Ny. darlingi in Brazil. METHODS: Mosquitoes were collected from Porto Velho and were reared at the Laboratory of Fiocruz/RO. After induced mating by light stimulation in the F1 to F6, the subsequent generations were free mating. Larvae were reared in distilled water and fed daily until pupation. RESULTS: In 11 generations, the colony produced a high number of pupae after the F5 generation. CONCLUSIONS: These results demonstrate the potential for permanently establishing Ny. darlingi colonies for research purposes in Brazil.

Characterization of artificial larval habitats of Anopheles darlingi (Diptera: Culicidae) in the Brazilian Central Amazon

ABSTRACT Mosquitoes belonging to the Anopheles genus are of great relevance in the epidemiology and transmission of malaria, with their larval phase developing in clean waters in the presence of organic matter. However, the human presence in the Amazon has increasingly influenced the emergence of new breeding sites and larval habitats, such as clay pits, fish ponds and dams, among others. The objective of the study was to characterize mosquito larval habitats using the biotic and abiotic parameters in the metropolitan area of Manaus. We collected in 23 artificial larval habitats in Manaus, classified in dams, fish ponds and clay pits. Water samples, Anopheles larvae, aquatic macrophytes and limnological parameters were collected from each artificial larval habitat. The Larvae Index per Man/Hour and canonical correspondence analysis were used for data analysis. Results indicate that artificial larval habitats with characteristics similar to natural sites present higher larval density, displaying a high abundance of An. triannulatus and An. darlingi. More than 90% of the determined limnological parameters were in agreement with the environmental resolution stipulated by the Brazilian environmental resolution, while pH, dissolved oxygen and phosphorus levels were below the established limits at some of the larval habitats. Conductivity, total suspended solids and phosphorus were positively correlated to the presence of An. albitarsis, An. peryassui and An. nuneztovari in fish ponds, and An. trianulatus and An. braziliensis in dams. Thus, the evaluated limnological variables and habitat structure explain Anopheles species distribution in artificial larval habitats in the metropolitan Manaus region.

Fitness cost in field Anopheles labranchiae populations associated with resistance to the insecticide deltamethrin

Abstract We evaluated in the present study the effect of deltamethrin resistance on the fitness cost of the filed populations of Anopheles labranchiae. A susceptible population was used as reference to do different comparisons. We selected the most resistant larvae population collected from northern Tunisia. Eggs were used for study of life history traits including developmental time, larvae mortality, fertility, hatchability and adult sex-ratio. Our results showed that deltamethrin resistance affected negatively (p < 0.05) the developmental time with the median range of 70 h, mortality with the rate of 7 folds in resistant population and hatchability which are lower than in susceptible population. Whereas, no significant differences were detected in adult sex-ratio and fertility of the two studied populations. Our results could help to determine the evolution of population dynamics of the resistant studied population in the areas where insecticide resistance is reported and resistance management is needed.

Astrodaucus persicus as a new source of bioinsectisides against malaria vector, Anopheles stephensi

OBJECTIVE@#To determine the larvicidal activities of petroleum ether, chloroform, ethyl acetate and methanol fractions of roots and fruits extracts of Astrodaucus persicus from Apiaceae family against malaria vector, Anopheles stephensi (An. stephensi).@*METHODS@#Twenty five third instar larvae of An. stephensi were exposed to various concentrations (10-160 g/L) of fractions and were assayed according to World Health Organization protocol. The larval mortality was calculated after 24 h treatment.@*RESULTS@#Among tested fractions, the highest larvicidal efficacy was observed from ethyl acetate fraction of fruits extract with 50% and 90% mortality values (LC and LC) of 34.49 g/L and 108.61 g/L, respectively. Chloroform fraction of fruits extract was the second larvicidal sample with LC of 45.11 g/L and LC of 139.36 g/L. Petroleum ether fractions of fruits and roots and methanol fraction of fruits showed moderate toxicity against An. stephensi.@*CONCLUSIONS@#Astrodaucus persicus is a potential source of valuable and natural larvicidal compounds against malaria vector, An. stephensi and can be used in mosquitoes control programs as an alternative to synthetic insecticides.

Astrodaucus persicus as a new source of bioinsectisides against malaria vector, Anopheles stephensi

Objective To determine the larvicidal activities of petroleum ether, chloroform, ethyl acetate and methanol fractions of roots and fruits extracts of Astrodaucus persicus from Apiaceae family against malaria vector, Anopheles stephensi (An. stephensi). Methods Twenty five third instar larvae of An. stephensi were exposed to various concentrations (10–160 g/L) of fractions and were assayed according to World Health Organization protocol. The larval mortality was calculated after 24 h treatment. Results Among tested fractions, the highest larvicidal efficacy was observed from ethyl acetate fraction of fruits extract with 50% and 90% mortality values (LC

Composition, abundance and aspects of temporal variation in the distribution of Anopheles species in an area of Eastern Amazonia

Introduction The diverse and complex environmental conditions of the Amazon Basin favor the breeding and development of Anopheles species. This study aimed to describe the composition, abundance and temporal frequency of Anopheles species and to correlate these factors with precipitation, temperature and relative humidity. Methods The study was conducted in the District of Coração, State of Amapá, Brazil. Samples were collected monthly during three consecutive nights, from 6:00 PM to 10:00 PM, from December 2010 to November 2011. In addition, four 12-hour collections (i.e., 6:00 PM to 6:00 AM) were performed during this period. Results A total of 1,230 Anopheles specimens were collected. In the monthly collections, Anopheles darlingi was the predominant species, followed by An. braziliensis and An. albitarsis s.l., whereas An. darlingi, An. peryassui and An. braziliensis were the most frequent species collected in the 12-hour collections. The greatest number of anophelines was collected in September (the dry season). The highest frequency of anophelines was observed for An. darlingi during September, when there were the least rainfalls of the year, along with lower relative humidity and higher temperatures. There was little variation in the abundance of this species in other months, with the exception of slight increases in February, July and August. Conclusions The major malaria vectors, An. darlingi and An. albitarsis s.l. (likely An. marajoara), were the most abundant species collected in the study area. Consequently, prevention and control measures should be taken to prevent malaria outbreaks in the District of Coração. .

Effect of swamp cultivation on distribution of anopheline larval habitats in Western Kenya.

Background & objectives: Malaria resurgence in highland regions of East Africa has been on increase. The spatio-temporal distribution of larval habitats of malaria vectors determines the distribution of adult vectors, hence, disease transmission. Vector’s ecology is necessary for strategic vector control through effective plan for source reduction. Mapping of the larval habitats is necessary for targeted control measures. The purpose of this study is to assess and compare the spatial and seasonal variations in anopheline larval habitats in Western Kenya. Methods: A comparative study was conducted on spatial distribution of GPS geo-located anopheline larval habitats in relation to highland and lowland environments. Land use types were categorized and all potential aquatic habitats of malaria vectors were examined in February, May, August and November 2004. Data analyses were performed using SAS JMP software. Results & discussion: Results showed a higher percentage of Anopheles gambiae s.s. (70.9%) than An. funestus (29.1%) in highland. In the lowland, An. gambiae s.l. comprised 60.1% while An. funestus represented 39.9%. The distribution of larval breeding is confined to the valley bottom in the highland while it was dispersed in the lowland. Land use type influenced the occurrence of positive breeding habitats in the highland. In the lowland, distribution was due to seasonality. We found high proportion of potential and positive breeding sites in cultivated swamps and farmlands at the highland site. These results suggest that swamp cultivation increases the availability and suitability of larval breeding habitats of malaria vectors, thus malaria transmission in the Western Kenya highlands environment.

Incrimination of Anopheles (Anopheles) intermedius Peryassú, An. (Nyssorhynchus) nuneztovari Gabaldón, An. (Nys.) oswaldoi Peryassú as natural vectors of Plasmodium falciparum in French Guiana

Anopheles darlingi Root is the major vector of human malaria in the Neotropics and has been considered to be the sole malaria vector in French Guiana. The presence of other potential vectors suggests that malaria may be transmitted by other species under certain conditions. From 2006-2011, all anopheline specimens collected from 11 localities were assayed to determine if the Plasmodium circumsporozoite protein was present. In addition to An. darlingi, we found Anopheles oswaldoi, Anopheles intermedius and Anopheles nuneztovari specimens that were infected with Plasmodium sp. Further investigations on the behaviour and ecology of An. oswaldoi, An. intermedius and An. nuneztovari are necessary to determine their role in malaria transmission in French Guiana.

Persistence and residue activity of deltamethrin on indoor residual spraying surfaces against malaria vectors in southeastern Iran

Objective:To evaluate the efficacy of deltamethrin and find a relation between persistence and residue of this insecticide on the prevalent surfaces against malaria vectors in southeastern Iran. Methods:After indoor residual spraying on prevalent surfaces in studied areas (plaster and mud as absorbent surfaces, wood as non absorbent surface and filter paper as control) for malaria control, conical tests as a bioassay method and chromatographic method as an analytical method were used for evolution of persistence and residue of deltamethrin insecticide. Results were investigated statistically by ANOVA and Tukey-HSD tests for determining relations or differences between residue and persistence of deltamethrin. Results:According to the results, there was no significant difference between mortality rates from bioassay tests on different surfaces, and deltamethrin kept its utility to malaria vector control until 120 days after indoor residual spraying on these surfaces. In the case of residue, there was no significant relation between residue amounts and mortality rates on different surfaces, whereas this relation existed between residual amounts on filter papers and mortality rates from bioassay tests. Conclusions: This study shows that measurement of residue in filter papers is a suitable tool for evolution and dictum of efficiency of deltamethrin insecticide in indoor residual spraying for malaria control.

Evaluation of Tribulus terrestris Linn (Zygophyllaceae) Acetone extract for Larvicidal and Repellence Activity against Mosquito Vectors.

Acetone extracts of leaves and seeds from the Tribulus terrestris (Zygophyllaceae) were tested against mature and immature different mosquito vectors under laboratory condition. The extract showed strong larvicidal, properties 100 per cent mortality in the 3rd-instar larvae was observed in the bioassays with An. culicifacies Giles species A, An. stephensi Liston , Culex quinquefasciatus Say and Aedes aegypti Linn ,against 200ppm of the leaf acetone extract and 100 ppm seed acetone extract. The LC values 50 of leaf acetone extract estimated for 3rd-instars An. culicifacies species A, An. stephensi, Cx. quinquefasciatus and Ae. aegypti after 24 hour of exposure were 117, 124, 168 and 185 ppm respectively. The LC values of seed 50 acetone extract estimated for 3rd-instars An. culicifacies species A, An. stephensi, Cx. quinquefasciatus and Ae. aegypti after 24 hour of exposure were 100, 72, 91 and 91 ppm respectively. It is confirmed from the LC values 50 that the seed acetone extract of T. terrestris is more effective compared to leaf extracts. A significant (P<0.004) higher concentration of acetone extract leaf was required to kill equal number of larvae i.e. against acetone extract of seed. The seed acetone extract showed strong repellent activity against adults mosquitoes. Per cent protection obtained against Anopheles culicifacies species A 100% repellency in 1h, 6h; Anopheles stephensi 100% repellency in 0h, 4h, 6h; and Culex quinquefasciatus 100% repellency in 0h, 2h, 4h, at 10% concentration respectively. Against Deet- 2.5% An. culicifacies Giles species A has shown 100% repellency in 1h,2h,6h , An. stephensi Liston 99% repellency in 4h , and Culex quinquefasciatus Say has shown 100% repellency in 1h,2h.

Structure of Anopheles (Diptera: Culicidae) population in areas with different degrees of human settlement: Cantá - Roraima - Brazil / Estrutura da população de Anopheles (Diptera: Culicidae) em áreas com diferentes graus de colonização humana: Cantá - Roraima - Brasil

Malaria has still been one of the most important endemic diseases in the Amazonian region. This study presents the impact of human settlements on the structure of Anopheles population. Diversity, abundance, richness and distribution of the genus Anopheles were observed in two areas with different levels of human settlement in the Cantá city, Roraima State, Northern Brazil. The influence of the dry and rainy seasons on mosquito populations was also observed. Mosquito captures were performed between 6:00 and 10:00 pm during the dry (February and November) and rainy (May and August) seasons at four different sites of each area. Among the 11 species of Anopheles identified through the adults' characteristics, An. albitarsis s.l. (45.5 percent) and An. darlingi (19.2 percent) were the most abundant in the more intensively anthropized area while An. triannulatus (19.2 percent) was more common in the less modified area. Other species found were An. nuneztovari (10.9 percent), An. oswaldoi (2.0 percent), An. evansae (1.7 percent), An. brasiliensis (0.6 percent), An. intermedius (0.3 percent), An. mediopunctatus (0.5 percent), An. periassui (0.08 percent) and An. argyritarsis (0.04 percent). The highest mosquitoes' population density was observed in May and the lowest one was observed in February and November. These results demonstrate the existence of a high diversity of anophelines in the study areas, showing that anthropic changes in the environment and climate variability affect both the population density and relative abundance of these vectors.

Malária continua sendo uma das mais importantes doenças endêmicas da região Amazônica. Esse estudo mostra o impacto da colonização humana sobre a estrutura populacional de Anopheles. Diversidade, abundância, riqueza e distribuição do gênero Anopheles foi observado em duas áreas com diferentes graus de antropização no município do Cantá, Estado de Roraima, Norte do Brasil. A influência das estações seca e chuvosa sobre a população dos mosquitos também foram investigadas. A captura dos mosquitos foi realizada entre 6:00 e 10:00 pm na estação seca (Fevereiro e Novembro) e estação chuvosa (Maio e Agosto) em quatro locais diferentes. Entre as 11 espécies de Anopheles identificadas através das características dos adultos, An. albitarsis s.l. (45.5 por cento) e An. darlingi (19.2 por cento) foram as mais abundantes nas áreas mais intensamente antropizadas, enquanto An. triannulatus (19.2 por cento) foi a espécie mais comum na área menos modificada. As outras espécies encontradas foram An. nuneztovari (10.9 por cento), An. oswaldoi (2.0 por cento), An. evansae (1.7 por cento), An. brasiliensis (0.6 por cento), An. intermedius (0.3 por cento), An. mediopunctatus (0.5 por cento), An. periassui (0.08 por cento) e An. argyritarsis (0.04 por cento). A mais alta densidade populacional de mosquitos foi observada em Maio e as mais baixas em Fevereiro e Novembro. Esses resultados demonstram a existência de uma alta diversidade de anofelinos na área estudada e que mudanças antrópicas no ambiente e variabilidade climática afetam a densidade populacional desses vetores.

Population genetic structure of the major malaria vector Anopheles darlingi (Diptera: Culicidae) from the Brazilian Amazon, using microsatellite markers

The population genetic structure of Anopheles darlingi, the major human malaria vector in the Neotropics, was examined using seven microsatellite loci from nine localities in central and western Amazonian Brazil. High levels of genetic variability were detected (5-25 alleles per locus; H E = 0.519-0.949). There was deviation from Hardy-Weinberg Equilibrium for 59.79 percent of the tests due to heterozygote deficits, while the analysis of linkage disequilibrium was significant for only two of 189 (1.05 percent) tests, most likely caused by null alleles. Genetic differentiation (F ST = 0.001-0.095; Nm = 4.7-363.8) indicates that gene flow is extensive among locations < 152 km apart (with two exceptions) and reduced, but not absent, at a larger geographic scale. Genetic and geographic distances were significantly correlated (R² = 0.893, P < 0.0002), supporting the isolation by distance (IBD) model. The overall estimate of Ne was 202.4 individuals under the linkage disequilibrium model, and 8 under the heterozygote excess model. Analysis of molecular variance showed that nearly all variation (~ 94 percent) was within sample locations. The UPGMA phenogram clustered the samples geographically, with one branch including 5/6 of the state of Amazonas localities and the other branch the Acre, Rondônia, and remaining Amazonas localities. Taken together, these data suggest little genetic structure for An. darlingi from central and western Amazonian Brazil. These findings also imply that the IBD model explains nearly all of the differentiation detected. In practical terms, populations of An. darlingi at distances < 152 km should respond similarly to vector control measures, because of high gene flow.