Adult Anopheline Mosquitoes Research Articles

Molecular and morphological identification of suspected Plasmodium vivax vectors in Central and Eastern Sudan

BackgroundIn spite of the global effort to eliminate malaria, it remains the most significant vector-borne disease of humans. Plasmodium falciparum is the dominant malaria parasite in sub-Saharan Africa. However, Plasmodium vivax is becoming widely spread throughout Africa. The overuse of vector control methods has resulted in a remarkable change in the behaviour of mosquito that feeds on human as well as on vector composition. The aim of this study was to identify Anopheles mosquito species in vivax malaria endemic regions and to investigate their role in P. vivax circumsporozoite protein (Pvcsp) allele diversity.MethodsMosquito samples were collected from Central Sudan (Rural Khartoum and Sennar) and Eastern Sudan (New Halfa, Kassala state) using pyrethrum spray catch (PSC) and CDC light traps. Mosquitoes were identified using appropriate morphological identification keys and Anopheles gambiae complex were confirmed to species level using molecular analysis. A subset of blood-fed anopheline mosquitoes were dissected to determine the presence of natural infection of malaria parasites. In addition, the rest of the samples were investigated for the presence of Pvcsp gene using nested-PCR.ResultsA total of 1037 adult anopheline mosquitoes were collected from New Halfa (N = 467), Rural Khartoum (N = 132), and Sennar (N = 438). Morphological and molecular identification of the collected mosquitoes revealed the presence of Anopheles arabiensis (94.2%), Anopheles funestus (0.5%), and Anopheles pharoensis (5.4%). None of the dissected mosquitoes (N = 108) showed to be infected with malaria parasite. Overall P. vivax infectivity rate was 6.1% (63/1037) by Pvcsp nested PCR. Co-dominance of An. arabiensis and An. pharoensis is reported in Sennar state both being infected with P. vivax.ConclusionThis study reported P. vivax infection among wild-caught anopheline mosquitoes in Central and Eastern Sudan. While An. arabiensis is the most abundant vector observed in all study areas, An. funestus was recorded for the first time in New Halfa, Eastern Sudan. The documented Anopheles species are implicated in Pvcsp allele diversity. Large-scale surveys are needed to identify the incriminated vectors of P. vivax malaria and determine their contribution in disease transmission dynamics.

Pyrethroid resistance in the major malaria vector Anopheles arabiensis in Nouakchott, Mauritania

BackgroundMauritania is one of the African countries with ongoing malaria transmission where data on insecticide resistance of local malaria vectors are limited despite an increasing use of long-lasting insecticide-treated nets (LLINs) as the main intervention for vector control. This study presents an evaluation of the level of insecticide resistance of Anopheles arabiensis in Nouakchott.MethodsAnopheles gambiae (s.l.) larvae were collected in breeding sites during the rainy season (August-September) in 2015 and 2016 from two selected sites in Nouakchott and reared until emergence. Adult anopheline mosquitoes were tested against malathion (5%), bendiocarb (0.1%), permethrin (0.75%) and deltamethrin (0.05%) using standard World Health Organization (WHO) insecticide-impregnated papers. PCR assays were used for the identification of An. gambiae (s.l.) sibling species as well as knockdown resistance (kdr).ResultsThe mean knockdown times 50% (KDT50) and 95% (KDT95) were 66 ± 17 and 244 ± 13 min, respectively, for permethrin in 2015. The KDT50 and the KDT95 were 39 ± 13 and 119 ± 13 min, respectively, for deltamethrin. The KDT50 and the KDT95 doubled for both molecules in 2016. The mortality rates 24 h post-exposure revealed that An. arabiensis populations in Nouakchott were fully susceptible to bendiocarb and malathion in 2015 as well as in 2016, while they were resistant to permethrin (51.9% mortality in 2015 and 24.1% mortality in 2016) and to deltamethrin (83.7% mortality in 2015 and 39.1% mortality in 2016). The molecular identification showed that Anopheles arabiensis was the only malaria vector species collected in Nouakchott in 2015 and 2016. Both the West and East African kdr mutant alleles were found in An. arabiensis mosquitoes surviving exposure to pyrethroid insecticide, with a high rate of homozygous resistant genotypes (54.3% for the West African kdr mutation and 21.4% for the East African kdr mutation) and a significant departure from Hardy-Weinberg proportions (χ2 = 134, df = 3, P < 0.001).ConclusionsThe study showed high levels of pyrethroid resistance in An. arabiensis populations in Nouakchott and presence of both West and East African kdr alleles in the resistant phenotype. These results highlight a need for routine monitoring of susceptibility of malaria vector populations to insecticides used in public health programs.

Larvivorous fish for preventing malaria transmission.

Larvivorous fish for preventing malaria transmission.

Benchmarking insecticide resistance intensity bioassays for Anopheles malaria vector species against resistance phenotypes of known epidemiological significance

BackgroundInsecticide use via indoor residual spraying (IRS) or treated nets is the primary method for controlling malaria vector populations. The incidence of insecticide resistance in vector populations is burgeoning globally making resistance management key to the design of effective malaria control and elimination strategies. Vector populations can be assessed for insecticide resistance using a binary (susceptible or resistant) classification based on the use of the standard WHO insecticide susceptibility assay for adult anopheline mosquitoes. However, the recent scaling up of vector control activities has necessitated a revision of the WHO bioassay protocol to include the production of information that not only diagnoses resistance but also gives information on the intensity of expression of resistance phenotypes detected. This revised protocol is expected to inform on the range of resistance phenotypes in a target vector population using discriminating/diagnostic insecticide concentrations (DC) as well as their potential operational significance using 5× DC and 10× DC assays. The aim of this project was to use the revised protocol to assess the intensity of pyrethroid resistance in a range of insecticide resistant Anopheles strains with known resistance mechanisms and for which there is evidence of operational significance in the field setting from which these colonies were derived.MethodsDiagnostic concentration (DC) bioassays followed by 5× DC and 10× DC assays using the pyrethroid insecticides permethrin and deltamethrin were conducted according to the standard WHO bioassay method against pyrethroid resistant laboratory strains of Anopheles funestus, An. arabiensis and An. gambiae.ResultsLow to moderate resistance intensities were recorded for the An. arabiensis and An. gambiae strains while moderate to high intensities were recorded for the An. funestus strains.ConclusionsIt is evident that resistance intensity assays can add predictive value to the decision making process in vector control settings, although more so in an IRS setting and especially when bench-marked against resistance phenotypes of known operational significance.

Mosquito genomics. Extensive introgression in a malaria vector species complex revealed by phylogenomics.

Introgressive hybridization is now recognized as a widespread phenomenon, but its role in evolution remains contested. Here, we use newly available reference genome assemblies to investigate phylogenetic relationships and introgression in a medically important group of Afrotropical mosquito sibling species. We have identified the correct species branching order to resolve a contentious phylogeny and show that lineages leading to the principal vectors of human malaria were among the first to split. Pervasive autosomal introgression between these malaria vectors means that only a small fraction of the genome, mainly on the X chromosome, has not crossed species boundaries. Our results suggest that traits enhancing vectorial capacity may be gained through interspecific gene flow, including between nonsister species.

Comparative entomological study on ecology and behaviour of Anopheles mosquitoes in highland and lowland localities of Derashe District, southern Ethiopia.

BackgroundChange in climatic and socio-economic situations is paving the way for the spread of malaria in highland areas which were generally known to be malaria free. Despite this, information regarding highland malaria transmission is scarce. Thus, the present study investigated entomological parameters linked to malaria transmission in the highlands of Southern Ethiopia.MethodsA longitudinal entomological study was conducted in three localities situated at different altitudes ranging between 1300 and 2650m above sea level in Derashe district, Southern Ethiopia. Larval and adult anopheline mosquitoes were collected between October 2011 and February 2012.ResultsAn. arabiensis and An. funestus s.l existed at significantly higher densities in the lowland (Wozeka) in contrast to An. christyi and An. Demeilloni, which were more abundant in the highland localities (P < 0.01). Conversely, An. pharoensis and An. cinereus were scarce and only found in the lowland and highlands, respectively. Habitats of larvae of An. arabiensis were characterized as clear, sun-lit, permanent, still water (streams) without vegetation and situated close to human habitations. On the other hand, habitats of An. christyi are shaded, still, turbid and contain natural water (rain pools) with vegetation and mats of algae. The relative abundance of An. Arabiensis, which is the primary malaria vector in Ethiopia is significantly and positively correlated with water temperature, pH and average depth (P < 0.05). An. arabiensis, An. funestus s.l and An. demeilloni showed zoophilic and exophilic tendencies. None of the anophelines tested for P. falciparum and P. vivax sporozoite infections were positive.ConclusionIn conclusion, malaria parasites and vectors existed in the highlands of Derashe District. Therefore, appropriate disease and vector control strategies must be designed and implemented to prevent potential outbreaks.

Comparative entomological study on ecology and behaviour of Anopheles mosquitoes in highland and lowland localities of Derashe District, southern Ethiopia

Change in climatic and socio-economic situations is paving the way for the spread of malaria in highland areas which were generally known to be malaria free. Despite this, information regarding highland malaria transmission is scarce. Thus, the present study investigated entomological parameters linked to malaria transmission in the highlands of Southern Ethiopia. A longitudinal entomological study was conducted in three localities situated at different altitudes ranging between 1300 and 2650m above sea level in Derashe district, Southern Ethiopia. Larval and adult anopheline mosquitoes were collected between October 2011 and February 2012. An. arabiensis and An. funestus s.l existed at significantly higher densities in the lowland (Wozeka) in contrast to An. christyi and An. Demeilloni, which were more abundant in the highland localities (P < 0.01). Conversely, An. pharoensis and An. cinereus were scarce and only found in the lowland and highlands, respectively. Habitats of larvae of An. arabiensis were characterized as clear, sun-lit, permanent, still water (streams) without vegetation and situated close to human habitations. On the other hand, habitats of An. christyi are shaded, still, turbid and contain natural water (rain pools) with vegetation and mats of algae. The relative abundance of An. Arabiensis, which is the primary malaria vector in Ethiopia is significantly and positively correlated with water temperature, pH and average depth (P < 0.05). An. arabiensis, An. funestus s.l and An. demeilloni showed zoophilic and exophilic tendencies. None of the anophelines tested for P. falciparum and P. vivax sporozoite infections were positive. In conclusion, malaria parasites and vectors existed in the highlands of Derashe District. Therefore, appropriate disease and vector control strategies must be designed and implemented to prevent potential outbreaks.

Larvivorous fish for preventing malaria transmission.

Larvivorous fish for preventing malaria transmission.

Vector ecology and susceptibility in a malariaendemic focus in southern Islamic Republic of Iran

This study aimed to carry out a malaria situation analysis, species composition and susceptibility levels of the main malaria vector, Anopheles stephensi, to different insecticides in Bashagard. A longitudinal survey was conducted in 2 randomly selected villages in Bashagard. Malaria vectors were sampled by dipping method for the larvae and hand catch, night-biting catch, total catch, and shelter pit collection for the adults. Standard WHO susceptibility tests were used for a variety of insecticides on F1 progeny of An. stephensi reared from wild-caught females. In total, 693 adult anopheline mosquitoes and 839 third and fourth-instar larvae were collected and identified. They comprised 7 species; the most abundant adult and larvae anopheline mosquito was An. dthali (40.7% and 30.5% respectively). An. culicifacies (24.2%) and An. stephensi (16.7%) were the next most common species for adult mosquitoes. An. stephensi was fully susceptible to malathion and pyrethroid insecticides but resistant to DDT and tolerant to dieldrin.

Species diversity and biting activity of Anopheles dirus and Anopheles baimaii (Diptera: Culicidae) in a malaria prone area of western Thailand

BackgroundA survey of adult anopheline mosquito diversities, collected from September 2009 to August 2010, was conducted in a malaria endemic area of western Thailand. Two anopheline species complexes, Dirus and Minimus, along with the Maculatus group were observed. Of several species documented from within each complex and group, four important malaria vectors were identified, including An. dirus, An. baimaii, An. minimus, and An. sawadwongporni. Information on biting activity and host preference for any single species within the Dirus complex has never been assessed. Using specific molecular identification assays, the trophic behavior and biting activity of each sibling species within the Dirus complex were observed and analyzed for the Kanchanaburi Province, Thailand.MethodsAdult female mosquitoes were collected for two consecutive nights each month during a one year period. Three collection methods, human landing indoor (HLI), human landing outdoor (HLO), and cattle baited collections (CBC) were applied. Each team of collectors captured mosquitoes between 1800 and 0600 h.ResultsFrom a total of 9,824 specimens, 656 belong to the Dirus complex (An. dirus 6.09% and An. baimaii 0.59%), 8,802 to the Minimus complex (An. minimus 4.95% and An. harrisoni 84.65%) and 366 to the Maculatus group (An. maculatus 2.43% and An. sawadwongporni 1.29%). Both An. dirus and An. baimaii demonstrated exophagic and zoophilic behaviors. Significantly greater numbers of An. dirus and An. baimaii were collected from cattle as compared to humans (P = 0.003 for An. dirus and P = 0.048 for An. baimaii).ConclusionsSignificantly greater numbers of An. dirus and An. baimaii were collected from cattle baited traps as compared to human landing collections (P < 0.05), demonstrating that both species show a strong zoophilic behavior. Knowledge of host-seeking behavior helps to define a species' capacity to acquire and transmit malaria and its contribution to the overall risk for disease transmission in the human population, as well as, assisting in the design and implementation of appropriate vector prevention and control strategies.

Field evaluation of permethrin long-lasting insecticide treated nets (Olyset®) for malaria control in an endemic area, southeast of Iran

Long lasting insecticide treated nets (LLINs) have been advocated as an effective tool for prevention and control of malaria. Olyset net was the first LLINs which became commercially available and obtained WHO approval. According to the national strategic plan on evaluation of Olyset net, a field trial was conducted to determine the efficacy of these nets against malaria vectors in an endemic area in the southeast of Iran.Fourteen villages with similar topographical and epidemiological situations were selected and randomly assigned to two clusters of the study: Olyset net and untreated net. Distribution of nets was carried out to cover 100% of the population in Olyset net and untreated net cluster.Anopheline mosquitoes were collected monthly using different WHO standard methods in both areas to determine their abundance, feeding pattern and resting behaviour. Human blood index was determined using ELISA test. Additionally, Olyset nets were evaluated for their biological activity using WHO cone bioassay test by susceptible colony of Anopheles stephensi (Beech strain) and then for insecticide residues by employing high performance thin layer chromatography. Malaria incidence was measured by passive and active case detection from all study population.In total 2115 adult anopheline mosquitoes were collected and identified using morphological characters. They comprised of seven species: Anopheles dthali (Liston), A. culicifacies (Giles), A. stephensi (Liston), A. superpictus (Grassi), A.fluviatilis (James), A. moghulensis (Christophers) and A. turkhudi (Liston). A. dthali, A. culicifacies and A. stephensi were most prevalent species in both areas.In the Olyset net study area, there was a significant reduction of 41.1%, 54.4%, 59.39% and 64.1% in the indoor-resting density of A. culicifacies, A. stephensi, A. dthali and A. superpictus, respectively, with an overall reduction of 39.3% in total mosquitoes in comparison with untreated net area. A significant reduction was also observed in human blood index of vector species in the Olyset net villages. Bioefficacy test results of Olyset nets showed that the median knockdown time was 1.48 and 3.25min, while the average mortality rate was 100% and 72.3%±7.07 in baseline and after 1 year of intervention, respectively. The average permethrin content reached to 68.31% (683.1mg/m2) of the initial insecticide dose of 937±21.69mg/m2 (nearly 1000mg/m2) at the end of intervention.Malaria incidence was reduced by 96.6% and 64.8% in the village with Olyset nets and in the villages with untreated nets, respectively. During intervention period, there was a reduction of 93.2% in malaria incidence in Olyset net area as compared to the untreated area.This study indicated that Olyset nets have a major impact on malaria vectors and disease burden; therefore it could be recommended as an effective personal protection tool for malaria control in malarious areas.

Seasonal Trends in Epidemiological and Entomological Profiles of Malaria Transmission in North Central Nigeria

The influence of seasonal changes on epidemiological and entomological indices of malaria transmission in North Central Nigeria was elucidated in a series of studies carried out between January 2004 and December 2009. The climate in the study area was divided into three seasonal periods namely, rainy (May-October), dry (December-March) and transitional (April and November), during which larval and adult anopheline mosquito collections were carried out and assessed for densities, sporozoite infection and parity rates and potentials for malaria transmission. The results indicated that the climate in the study area was clearly seasonal, with close similarities in the patterns of distribution of the climatic factors in the study sites. Mosquito densities, both at the adult and larval stages (i.e., 29.35 +/- 5.10 adult mosquitoes/man/night and 10.36 +/- 3.34 larvae/dip, respectively), were significantly (p<0.05) highest during the rainy season. However, while the former varied significantly in the three seasonal periods, the latter was not significantly different during the dry and transitional seasons. Malaria transmission risks, in terms of sporozoite rates and entomological inoculation rates, was significantly (p<0.05) least in the dry season (i.e., 2.89 +/- 1.25% and 0.37 +/- 0.21 infective bite/man/night, respectively) but the two variables were not significantly (p>0.05) different during the transitional and rainy seasons. Adult mosquito daily survival rate and adult longevity were least in the dry season (26.52 +/- 11.80% and 6.80 days, respectively) and significantly (p<0.05) highest during the rainy season (72.28 +/- 4.00% and 16.95 +/- 4.20 days, respectively). Parous rates of the mosquitoes and duration of sporogony had distinct distribution pattern from the other variables investigated. While, significantly highest parous rates were recorded in the transitional season (86.00 +/- 4.30%), duration of sporogony was not significantly (p>0.05) different during the three seasons. The epidemiology of urban malaria in North Central Nigeria was discussed from the view points of the these results and concluded that the findings should promote the development of informed temporally-targeted vector control programs for the area.

Pyrethroid resistance in the major malaria vector Anopheles arabiensis from Gwave, a malaria-endemic area in Zimbabwe

BackgroundInsecticide resistance can present a major obstacle to malaria control programmes. Following the recent detection of DDT resistance in Anopheles arabiensis in Gokwe, Zimbabwe, the underlying resistance mechanisms in this population were studied.MethodsStandard WHO bioassays, using 0.75% permethrin, 4% DDT, 5% malathion, 0.1% bendiocarb and 4% dieldrin were performed on wild-collected adult anopheline mosquitoes and F1 progeny of An. arabiensis reared from wild-caught females. Molecular techniques were used for species identification as well as to identify knockdown resistance (kdr) and ace-1 mutations in individual mosquitoes. Biochemical assays were used to determine the relative levels of detoxifying enzyme systems including non-specific esterases, monooxygenases and glutathione-S-transferases as well as to detect the presence of an altered acetylcholine esterase (AChE).ResultsAnopheles arabiensis was the predominant member of the Anopheles gambiae complex. Of the 436 An. arabiensis females, 0.5% were positive for Plasmodium falciparum infection. WHO diagnostic tests on wild populations showed resistance to the pyrethroid insecticide permethrin at a mean mortality of 47% during February 2006 and a mean mortality of 68.2% in January 2008. DDT resistance (68.4% mean mortality) was present in February 2006; however, two years later the mean mortality was 96%. Insecticide susceptibility tests on F1 An. arabiensis families reared from material from two separate collections showed an average mean mortality of 87% (n = 758) after exposure to 4% DDT and 65% (n = 587) after exposure to 0.75% permethrin. Eight families were resistant to both DDT and permethrin. Biochemical analysis of F1 families reared from collections done in 2006 revealed high activity levels of monooxygenase (48.5% of families tested, n = 33, p < 0.05), glutathione S-transferase (25.8% of families tested, n = 31, p < 0.05) and general esterase activity compared to a reference susceptible An. arabiensis colony. Knockdown resistance (kdr) and ace-IR mutations were not detected.ConclusionThis study confirmed the presence of permethrin resistance in An. arabiensis populations from Gwave and emphasizes the importance of periodic and ongoing insecticide susceptibility testing of malaria vector populations whose responses to insecticide exposure may undergo rapid change over time.

Malaria transmission and rice cultivation in Lagdo, northern Cameroon

Cross-sectional entomological surveys were carried out during the 2006 dry and rainy seasons in Lagdo, Cameroon to measure the impact of rice cultivation on malaria transmission and to monitor vector susceptibility to insecticides. Adult anopheline mosquitoes were captured on human volunteers and by pyrethrum spray collections. A total of 4740 mosquitoes was collected during the study. Anopheles arabiensis was the major species and the main malaria vector in all study sites, followed by A. funestus. Malaria transmission was high in the non-irrigated zone of Mayo Mbocki, whereas in the irrigated area of Gounougou it was below detection level during the dry season and high during the rainy season. Insecticide susceptibility tests performed on A. gambiae s.l. populations detected resistance to lambdacyhalothrin and to a lower extent to deltamethrin. All survivors were A. arabiensis. None of the surviving mosquitoes carried the kdr mutation, suggesting an alternative resistance mechanism.

Entomopathogenic fungi as biological insecticides to control malaria

Malaria is arguably the most serious vector-borne disease worldwide. The already-alarming number of deaths caused by malaria is increasing, caused in part by the increase in mosquito resistance to chemical insecticides. In two recent articles, the use of an approach was reported that could open a new front in the fight against malaria. Laboratory and field studies demonstrate that entomopathogenic fungi can efficiently kill adult anopheline mosquitoes, the females of which are the obligatory vectors for malaria parasites.

Identification and characterization of larval and adult anopheline mosquito habitats in the Republic of Korea: potential use of remotely sensed data to estimate mosquito distributions

BackgroundPlasmodium vivax malaria reemerged in the Republic of Korea in 1993, with more than 2,000 cases reported in the northwestern part of the country over the last 10 years. To better assess the risk of malaria transmission we conducted a surveillance study to identify and characterize the habitats that produce potential Anopheles vector mosquitoes. Immature and adult mosquito collection data were incorporated into a Geographic Information System (GIS) along with remotely sensed satellite imagery, and imagery classified to land use to determine if remote sensing data could be used to estimate mosquito habitats.ResultsMore than 2,100 anopheline larvae were collected and mapped from 186 immature habitats, which were categorized into 9 types. Anopheles sinensis was the most commonly collected species, representing more than 97% of the specimens, followed by Anopheles pullus 1.0%, Anopheles lesteri 1.0%, and Anopheles sineroides 0.8%. Anopheles sinensis, pullus, and lesteri were found most frequently in rice paddies followed by: ditches, flooded areas, ground pools, wheel tracks, swamps, irrigation canals, and stream margins. Anopheles sineroides was found most commonly in flooded areas. Supervised classification was conducted on a LANDSAT 7 ETM+ image to identify 5–6 land-use classes that were considered to be of interest. The spatial distribution of the larval mosquito collections was overlaid on the land-use image derived from the LANDSAT image, and the number of mosquitoes habitats in each class calculated. On Ganghwa Island 67% of the immature habitats containing Anopheles sinensis were in the Paddy land-use class, although the class only represented 17% of the land area. In Paju District 53% of the immature habitats containing Anopheles sinensis were in the Paddy land-use class which represented only 9 % of the area of the district. There was significant (p < 0.05) correlation between habitats containing all four species and land use on Ganghwa Island; however, only Anopheles sinensis was significantly correlated with land-use in Paju District. In Ganghwa Island almost 50% of the Anopheles sinensis adults were collected in the paddy land-use class, and there was a positive correlation between larval and adult distributions. However, in Paju District adult Anopheles sinensis adults were most commonly collected in the Bare land-use class and only 17% collected in the Paddy class. There was a negative correlation between larval and adult distributions.ConclusionImmature habitats most commonly associated with Anopheles sinensis, Anopheles pullus, Anopheles sineroides and Anopheles lesteri were flooded rice paddies. Adult Anopheles sinensis, the most commonly collected anopheline, were found closely associated with rice habitats on Ganghwa Island but not in Paju District. Both QuickBird and LANDSAT satellite data were used to display spatial data in the form of geographic coverage and descriptive information in the form of relational databases associated with the mapped features. Supervised classification of LANDSAT imagery permitted good separation between Paddy, Forest, and Water land-use classes. The immature collections of Anopheles sinensis were significantly correlated with land-use as determined in the land-use classification in both Ganghwa Island and Paju District. These data suggest that classified remotely sensed data could potentially be used to estimate the distribution of immature and adult mosquito populations in the Republic of Korea.

Adult anopheline ecology and malaria transmission in irrigated areas of South Punjab, Pakistan.

Surface irrigation in the Punjab province of Pakistan has been carried out on a large scale since the development of the Indus Basin Irrigation System in the late 19th century. The objective of our study was to understand how the population dynamics of adult anopheline mosquitoes (Diptera: Culicidae) could be related to malaria transmission in rural areas with intensive irrigation and a history of malaria epidemics. In this paper we present our observations from three villages located along an irrigation canal in South Punjab. The study was carried out from 1 April 1999 to 31 March 2000. Mosquitoes were collected from bedrooms using the pyrethroid spraycatch method and from vegetation and animal sheds using backpack aspirators. Overall, Anopheles subpictus Grassi sensu lato predominated (55.6%), followed by An. stephensi Liston s.l. (41.4%), An. culicifacies Giles s.l. (2.0%), An. pulcherrimus Theobald (1.0%) and An. peditaeniatus Leicester (0.1%). Most mosquitoes (98.8%) were collected from indoor resting-sites whereas collections from potential resting-sites outdoors accounted for only 1.2% of total anopheline densities, confirming the endophilic behaviour of anophelines in Pakistan. Anopheles stephensi, An. culicifacies and An. subpictus populations peaked in August, September and October, respectively. High temperatures and low rainfall negatively affected seasonal abundance in our area. There were interesting differences in anopheline fauna between villages, with An. culicifacies occurring almost exclusively in the village at the head of the irrigation canal, where waterlogged and irrigated fields prevailed. Monthly house-to-house fever surveys showed that malaria transmission remained low with an overall slide positivity rate of 2.4% and all cases were due to infection with Plasmodium vivax. The most plausible explanation for low transmission in our study area seems to be the low density of Pakistan's primary malaria vector, An. culicifacies. The role of other species such as An. stephensi is not clear. Our observations indicate that, in South Punjab, irrigation-related sites support the breeding of anopheline mosquitoes, including the vectors of malaria. As our study was carried out during a year with exceptionally hot and dry climatic conditions, densities and longevity of mosquitoes would probably be higher in other years and could result in more significant malaria transmission than we observed. To assess the overall importance of irrigation-related sites in the epidemiology of malaria in the Punjab, more studies are needed to compare irrigated and non-irrigated areas.

Water management for controlling the breeding of Anopheles mosquitoes in rice irrigation schemes in Kenya

An experiment to assess the impact of intermittent irrigation on Anopheles larval populations, rice yields and water use was conducted in the Mwea rice irrigation scheme in Kenya. Four water regimes including intermittent irrigation were tested in a complete randomized block experimental design. Intermittent irrigation was carried out on a weekly schedule, with flooded conditions from Saturday through Tuesday morning. Larval sampling at each plot was conducted every Monday and prior to draining of intermittently irrigated subplots on Tuesday. All the adult anopheline mosquitoes emerging from larvae collected in the experimental plots were identified as being An. arabiensis. By far the highest numbers of An. arabiensis 1st instar larvae were found in the intermittently irrigated subplots, indicating that the water regime provided the most attractive environment for egg laying. However, the ratio between the 4th and 1st instar larvae in the subplots was only 0.08, indicating very low survival rates. In contrast, the 4th/1st instar ratio for subplots with other water management regimes ranged between 0.27 and 0.68, suggesting a correspondingly higher survival than observed with intermittent irrigation. The total number of 4th instars was almost the same in the intermittently irrigated subplots and the irrigation system normally practised by the farmers. The failure to eliminate larval development up to the 4th instar in the former method was attributed to residual pools of water. Larval abundance fluctuated throughout the 12-week sampling period. The highest larval densities were recorded in the 3 weeks after transplanting the rice seedlings. Afterwards, larval numbers dropped dramatically as the height of rice plants increased. Rice yields at harvest did not show statistically significant differences among subplots with different water regimes. The average yield per hectare ranged from 4.8–5.3 metric tonnes. The average daily water percolation/seepage rate was 3.6 mm and did not significantly differ among different water management regimes. Further research is necessary to, among other things, determine whether rice yields could be increased by having flooded and drained intervals that were different from those used in this study. It would likewise be important to assess on a wider scale the feasibility of implementing intermittent irrigation with respect to farmer acceptance and required changes in irrigation system design and management.

Isolation and characterization of chromosomal proteins from the mosquito Anopheles albimanus Weidemann.

1. Nuclei were isolated from adult anopheline mosquitoes and fractionated into nucleolar chromatin, nucleoplasmic chromatin and ribonucleoprotein particles by sucrose density gradients. 2. Histones and nonhistone proteins were selectively dissociated from chromatin by treatment with sodium chloride, urea and guanidine HC1. 3. A special class of nonhistone proteins (tight proteins) were extracted from chromatin with Na4P2O7. 4. The electrophoretic properties of the histones, nonhistone proteins and ribonucleoprotein particles were examined by isoelectric focusing and SDS multiphase polyacrylamide gel electrophoresis. 5. By contrast to the histones, the nonhistone proteins displayed considerable heterogeneity. 6. Possible functional implications of the chromosomal proteins are discussed.

Seasonal trends in population size and survivorship of Anopheles culicifacies, An. stephensi and An. subpictus (Diptera: Culicidae) in rural Punjab province, Pakistan.

Adult anopheline mosquitoes were collected resting inside houses and cattle sheds at the rural village of Khano-Harni, Punjab Province, Pakistan, during 1978. Anopheles stephensi was collected throughout the year, and showed bimodal population increases during the spring and postmonsoon seasons. Anopheles culicifacies and An. subpictus were first collected at Khano-Harni at the onset of the monsoon season when populations of both species exhibited sharp initial increases, followed by decreases related to flooding and then increases during the postmonsoon season. An. subpictus populations subsequently declined with the onset of cold weather. Absolute population size estimated by Bailey's modification of the Lincoln Index did not differ significantly from concurrent estimates calculated by lto's modification of Jackson's positive and negative methods. Temporal patterns in population size agreed with changes in the relative abundance (number collected per man-hour) of An. culicifacies and An. stephensi , but not An. subpictus . Collection of relative-abundance data was hampered, in part, by temperature-related changes in mosquito resting behavior, which affected sampling efficiency. The regression of the number of females in each gonotrophic cycle on female age provided the most appropriate estimate of survivorship for epidemiological purposes. Davidson's method provided slightly higher estimates, since survivorship during the nulliparous period was measured. Horizontal survivorship based on the recapture rate of marked specimens provided lower estimates, since losses due to emigration and sampling were included.