Cytogenetic characterization of two laboratory colonies of Anopheles arabiensis Patton from Ethiopia.

Polytene chromosome analysis was carried out in samples of Anopheles arabiensis from two colonies originating from different sites in the Rift Valley, Ethiopia. Both colonies were found to maintain the main chromosomal inversion variations existing in wild populations. Changes in karyotype frequencies were documented as well as excesses of heterokaryotypes.     

Population genetic structure of Anopheles arabiensis mosquitoes in Ethiopia and Eritrea

This study examined the population genetic structure of the major malaria vector, Anopheles arabiensis mosquitoes, in Ethiopia and Eritrea. Ethiopia and Eritrea have great geographical diversity, with high mountains, rugged plateaus, deep gorges, and rolling plains. The plateau is bisected diagonally by the Great Rift Valley into the Northwestern Highlands and the Southeastern Highlands. Five A. arabiensis populations from the Northwestern Highlands region and two populations from high-altitude sites in the Great Rift Valley were genotyped using six microsatellite markers to estimate the genetic diversity and population genetic structure of A. arabiensis. We found that A. arabiensis populations from the Northwestern Highlands and the Great Rift Valley region showed a similar level of genetic diversity. The genetic differentiation (F(ST)) of the five mosquito populations within the Northwestern Highlands region was 0.038 (P <.001), while the two populations within the Great Rift Valley showed little genetic differentiation (F(ST) = 0.007, P <.01). The degree of genetic differentiation between the Northwestern Highlands region and the Great Rift Valley region was small but statistically significant (F(ST) = 0.017, P <.001). The population genetic structure of A. arabiensis in the study area did not follow the isolation-by-distance model (r(2) = 0.014, P >.05). The low F(ST) estimates for A. arabiensis populations in Ethiopia and Eritrea are consistent with the general population genetic structure of this species in East Africa based on other molecular markers.     

Insecticide resistance in the malarial mosquito Anopheles arabiensis and association with the kdr mutation

A colony of Anopheles arabiensis Patton (Diptera: Culicidae) from the Sennar region of Sudan was selected for resistance to dichlorodiphenyltrichloroethane (DDT). Adults from the F-16 generation of the resistant strain were exposed to all four classes of insecticides approved for use in malaria vector control and showed high levels of resistance to them all (24-h mortalities: malathion, 16.7%; bendiocarb, 33.3%; DDT, 12.1%; dieldrin, 0%; deltamethrin, 24.0%; permethrin, 0%). Comparisons between the unselected base colony and the DDT-resistant strain showed elevated glutathione-S-transferase (P<0.05) in both sexes and elevated esterases (P<0.05) in males only. The Leu-Phe mutation in the sodium channel gene was detected by polymerase chain reaction and sequencing, but showed no correlation with the resistant phenotype. These results do not provide any explanation as to why this colony exhibits such widespread resistance and further studies are needed to determine the precise mechanisms involved. The implications for malaria vector control in central Sudan are serious and resistance management (e.g. through the rotational use of different classes of insecticides) is recommended.     

Insecticide susceptibility of Anopheles coluzzii and Anopheles gambiae mosquitoes in Ibadan,Southwest Nigeria

The emergence of insecticide resistance in Anopheles (Diptera: Culicidae) mosquitoes has great implications for malaria control in Nigeria. This study aimed to determine the dynamics of insecticide susceptibility levels and the frequency of knock‐down resistance (kdr) mutations (L1014F) in wild Anopheles coluzzii Coetzee & Wilkerson sp. n. and Anopheles gambiae Giles from the Ojoo and Bodija areas of Ibadan, in southwest Nigeria. Insecticide susceptibility to pyrethroids, organophosphates, carbamates and organochlorines was assessed using World Health Organization (WHO) bioassays. A subset of the mosquitoes exposed to pyrethroids and DDT was used for species and molecular form identification; kdr genotyping was determined using the TaqMan real‐time polymerase chain reaction assay. The mosquitoes were resistant to pyrethroids and DDT but completely susceptible to organophosphates and carbamates. Bodija samples (n = 186) consisted of An. gambiae (91.4%) and An. coluzzii (8.1%) and included one An. coluzzii/An. gambiae hybrid specimen. All mosquitoes screened in Ojoo (n = 26) were An. gambiae. The 1014F kdr mutation was detected at frequencies of 24.5 and 5.8% in Bodija and Ojoo, respectively. No correlation was observed between kdr genotypes and resistance phenotypes. The results indicate that metabolic resistance probably plays an important role in the development of resistance and highlight the need to implement insecticide resistance management strategies.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Impacts of Agricultural Practices on Insecticide Resistance in the Malaria Vector Anopheles arabiensis in Khartoum State,Sudan

Background

Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan.

Methodology/Principal Findings

Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly.

Conclusions

Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.     

Cytogenetic studies on the malaria vector mosquito Anopheles arabiensis Patton in the Awash Valley, Ethiopia

Blood fed female mosquitoes were collected from human dwellings in the Awash Valley, Ethiopia. Those identified morphologically as A. gambiae s.l. were preserved in Carnoy's fixative for later ovarian polytene chromosomes examination. Only one member of the A. gambiae complex, A. arabiensis, was found by such examination. The polymorphic inversions identified were 2Rb and 3Ra. The frequencies of these inversions were variable in different localities and the former inversion was found to form an altitudinal cline.     

The distribution of two major malaria vectors, Anopheles gambiae and Anopheles arabiensis, in Nigeria.

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.     

Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence for larval selection

Abstract.  Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l . (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT₅₀ of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT₅₀ did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis . They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages.     

The relationship between female body size and survival rate of the malaria vector Anopheles arabiensis in Ethiopia

Abstract. The relationship between female mosquito body size and survival rate was studied in field populations of Anopheles arabiensis in the Awash valley, central Ethiopia. Body size was quantified by measuring the wing-length. Highly significant correlations were found between size, parousness and insemination. It was concluded that larger An.arabiensis females have a higher probability of survival, being inseminated and producing more egg batches than smaller adults. Implications for vectorial capacity and vector competence of mosquitoes are discussed.     

Islands and Stepping-Stones: Comparative Population Structure of Anopheles gambiae sensu stricto and Anopheles arabiensis in Tanzania and Implications for the Spread of Insecticide Resistance

Population genetic structures of the two major malaria vectors Anopheles gambiae s.s. and An. arabiensis, differ markedly across Sub-Saharan Africa, which could reflect differences in historical demographies or in contemporary gene flow. Elucidation of the degree and cause of population structure is important for predicting the spread of genetic traits such as insecticide resistance genes or artificially engineered genes. Here the population genetics of An. gambiae s.s. and An. arabiensis in the central, eastern and island regions of Tanzania were compared. Microsatellite markers were screened in 33 collections of female An. gambiae s.l., originating from 22 geographical locations, four of which were sampled in two or three years between 2008 and 2010. An. gambiae were sampled from six sites, An. arabiensis from 14 sites, and both species from two sites, with an additional colonised insectary sample of each species. Frequencies of the knock-down resistance (kdr) alleles 1014S and 1014F were also determined. An. gambiae exhibited relatively high genetic differentiation (average pairwise F_ST = 0.131), significant even between nearby samples, but without clear geographical patterning. In contrast, An. arabiensis exhibited limited differentiation (average F_ST = 0.015), but strong isolation-by-distance (Mantel test r = 0.46, p = 0.0008). Most time-series samples of An. arabiensis were homogeneous, suggesting general temporal stability of the genetic structure. An. gambiae populations from Dar es Salaam and Bagamoyo were found to have high frequencies of kdr 1014S (around 70%), with almost 50% homozygote but was at much lower frequency on Unguja Island, with no. An. gambiae population genetic differentiation was consistent with an island model of genetic structuring with highly restricted gene flow, contrary to An. arabiensis which was consistent with a stepping-stone model of extensive, but geographically-restricted gene flow.     

Early biting rhythm in the Afro-tropical vector of malaria, Anopheles arabiensis, and challenges for its control in Ethiopia

The biting cycle of the malaria vector Anopheles arabiensis Patton (Diptera: Culicidae) was assessed by hourly light trap collections in three villages in Tigray, northern Ethiopia. Hourly catches were conducted in two houses in each village, for four consecutive nights. Light traps were set from 18.00 hours to 07.00 hours in houses in which people slept under untreated bednets. Anopheles arabiensis showed early biting activities, which peaked between 19.00 hours and 20.00 hours in the three villages; over 70% of biting activity occurred before 22.00 hours, when people typically retire to bed. This early biting activity may have a negative impact on the efficiency of bednets to control malaria.     

Insecticide susceptibility status and major detoxifying enzymes activity in Anopheles subpictus from Kasur,Pakistan

Anopheles subpictus s.l. Grassi (Diptera: Culicidae) is a malaria vector in South Asia, where insecticides are the mainstay for vector control interventions. Information on any variation in metabolic enzyme levels in mosquitoes is helpful with respect to adapting alternative strategies for vector control. The scarce data on the biochemical basis of insecticide resistance in malaria vectors of Pakistan limit the available information for vector control interventions within the country. The insecticide susceptibility status and its biochemical basis against dichlorodiphenyltrichloroethane (DDT) (4%), deltamethrin (0.05%) and permethrin (0.75%) in An. subpictus s.l. collected from all Tehsils of district Kasur were evaluated. For this purpose, a World Health Organization susceptibility bioassay was performed followed by the detection of altered metabolic enzyme activity using biochemical assays. Similarly, a significant difference in knock‐down effect was observed among field collected and susceptible strain against all insecticides 24 h post exposure. The overall mean mortality rates of DDT, deltamethrin and permethrin were 27.86% [95% confidence interval (CI) = 29.65–26.06], 44.89% (95% CI = 46.23–43.54) and 78.82% (95% CI = 80.16–77.47), respectively. The biochemical assays revealed an elevated level of metabolic enzymes in the field population. The results provide evidence of resistance against organochlorine and pyrethroid groups in a field population of An. subpictus s.l. from district Kasur mediated by multiple metabolic mechanisms, including acetylcholinesterases, esterases, cytochrome P450 and glutathione S‐transferases.     

Genetic linkage between malathion and dieldrin resistance in Anopheles arabiensis

A strain of Anopheles arabiensis resistant to both malathion and dieldrin was crossed and backcrossed to a susceptible strain. The progeny were tested on each insecticide in turn. Less than 50% mortality in the second insecticide exposure among the backcross progeny indicated linkage between the resistance genes. In a backcross of A. gambiae X A. arabiensis hybrids a recombination rate of 7.5% was observed. A Y-translocation strain of A. arabiensis showed less than 2.8% recombination between the resistance genes. It is impossible to confirm the genotype of apparent recombinants using existing stocks, but the two resistance mechanisms are biochemically distinguishable. If the two genes are very closely linked, linkage disequilibrium could influence the consequences of switching to malathion spraying after dieldrin resistance has evolved.     

Characteristics of Anopheles arabiensis larval habitats in Tubu village,Botswana

Documented information on the ecology of larval habitats in Botswana is lacking but is critical for larval control programs. Therefore, this study determined the characteristics of these habitats and the influences of biotic and abiotic factors in Tubu village, Botswana. Eight water bodies were sampled between January and December, 2013. The aquatic vegetation and invertebrate species present were characterized. Water parameters measured were turbidity (NTU), conductivity (μS/cm), oxygen (mg/l), and pH. Larval densities of Anopheles arabiensis mosquitoes and their correlation with abiotic factors were determined. Larval breeding was associated with ‘short’ aquatic vegetation, a variety of habitats fed by both rainfall and flood waters and sites with predators and competitors. The monthly mean (± SE_mean) larval density was 8.16±1.33. The monthly mean (±SE_mean) pH, conductivity, oxygen, and turbidity were 7.65±0.13, 1152.834±69.171, 5.59±1.33, and 323.421±33.801, respectively. There was a significant negative correlation between larval density and conductivity (r = ‐0.839; p < 0.01), while a significant positive correlation occurred between turbidity and larval density (r = 0.685; p < 0.05). Oxygen (r = 0.140; p > 0.05) and pH (r = 0.252; p > 0.05) were not correlated with larval density. Floods and diversified breeding sites contributed to prolonged and prolific larval breeding. ‘Short’ aquatic vegetation and predator‐infested waters offered suitable environments for larval breeding. Turbidity and conductivity were good indicators for potential breeding places and can be used as early warning indices for predicting larval production levels.     

Identification and expression of odorant-binding proteins of the malaria-carrying mosquitoes Anopheles gambiae and Anopheles arabiensis

Host preference and blood feeding are restricted to female mosquitoes. Olfaction plays a major role in host-seeking behaviour, which is likely to be associated with a subset of mosquito olfactory genes. Proteins involved in olfaction include the odorant receptors (ORs) and the odorant-binding proteins (OBPs). OBPs are thought to function as a carrier within insect antennae for transporting odours to the olfactory receptors. Here we report the annotation of 32 genes encoding putative OBPs in the malaria mosquito Anopheles gambiae and their tissue-specific expression in two mosquito species of the Anopheles complex; a highly anthropophilic species An. gambiae sensu stricto and an opportunistic, but more zoophilic species, An. arabiensis. RT-PCR shows that some of the genes are expressed mainly in head tissue and a subset of these show highest expression in female heads. One of the genes (agCP1588) which has not been identified as an OBP, has a high similarity (40%) to the Drosophila pheromone-binding protein 4 (PBPRP4) and is only expressed in heads of both An. gambiae and An. arabiensis, and at higher levels in female heads. Two genes (agCP3071 and agCP15554) are expressed only in female heads and agC15554 also shows higher expression levels in An. gambiae. The expression profiles of the genes in the two members of the Anopheles complex provides the first step towards further molecular analysis of the mosquito olfactory apparatus.     

Insecticide susceptibility levels of Anopheles gambiae s.l. in the area of Ouagadougou, Burkina Faso

Tests to evaluate the susceptibility level in Anopheles gambiae s.1. from Ouagadougou and two nearby villages have been carried out. Anopheles gambiae s.1. larvae from Ouagadougou showed complete susceptibility to organophosphates and carbamates, and adults showed low-level resistance to DDT. Nine percent survival of adult An. gambiae s.1. to one-hour exposure of 4% DDT was observed in samples from Zagtouli village while in those from Koubri village, where dieldrin also was tested, resistance to both organochlorine insecticides was detected.     

Insecticide susceptibility of Ephemera orientalis (Ephemeroptera: Ephemeridae) and two mosquito species,Anopheles sinensis and Culex pipiens in the Republic of Korea

Field-collected populations of mayflies, Ephemera orientalis were tested for susceptibility to 10 different insecticides using a direct-contact mortality bioassay. Ephemera orientalis subimagoes were susceptible to the insecticides chlorpyrifos, fenitrothion and chlorfenapyr with LD₅₀ values of 69.7, 78.8 and 81.9 μg/♀, and adults had LD₅₀ values of 71.9, 78.8 and 85.4 μg/♀, respectively. Susceptibility ratios (SRs) of subimagoes and adults of E. orientalis to the 10 insecticides were 1.0 to1.2 folds. The mayflies showed higher susceptibility to organophosphates than to pyrethroids. The SRs of Anopheles sinensis to E. orientalis were 514 to 1438 folds higher for organophosphates (LD₅₀ values of 0.05 to 0.23 μg/♀) and 62 to 1155 folds higher for pyrethroids (LD₅₀ values of 0.13 to 2.41 μg/♀). The SRs of Culex pipiens to E. orientalis were 606 to 3595 folds higher for organophosphates with LD₅₀ values of 0.02–0.17 μg/♀ and 81 to 1365 folds higher for pyrethroids with LD₅₀ values of 0.11–1.83 μg/♀. These results indicate that the use of ineffective insecticides will result in unsatisfactory control against field populations of the subimagoes and adults of E. orientalis.     

Efficacy of three insect repellents against the malaria vector Anopheles arabiensis

Three commercial repellents marketed in South Africa: Bio-Skincare (BSC, oils of coconut, jojoba, rapeseed and vitamin E), Mosiguard towelletes with 0.574 g quwenling (p-menthane-3,8-diol, PMD) and the standard deet (15% diethyl-3-methylbenzamide, Tabard lotion), were compared against a laboratory colony of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae), the predominant malaria vector in South Africa. Human forearms were treated with 1.2 g BSC, 0.8 g PMD towelette or 0.5 g deet and exposed to 200 hungry An. arabiensis females for 1 min, at intervals of 1-6 h post-treatment. Tests were conducted by three adult male volunteers (aged 30-45 years, crossover controlled test design for 3 consecutive days), using their left arm for treatment and right arm for untreated control. Biting rates averaged 39-52 bites/min on untreated arms. All three repellents provided complete protection against An. arabiensis for up to 3-4 h post-application; deet and PMD gave 90-100% protection up to 5-6h, but BSC declined to only 52% protection 6h post-treatment. These results are interpreted to show that all three repellent products give satisfactory levels of personal protection against An. arabiensis for 4-5 h, justifying further evaluation in the field.