Nobuko Tuno

Survivorship of Anopheles gambiae sensu stricto (Diptera: Culicidae) Larvae in Western Kenya Highland Forest

Abstract The western Kenya highland has been experiencing dramatic landuse changes in the past three decades. Landuse change has been hypothesized to be one of the mechanisms for malaria epidemics in African highlands because it can alter the physical and chemical characteristics of mosquito breeding habitats. One important landuse change in western Kenya highland is deforestation. The current study examined the effects of forestation or deforestation on the survivorship of Anopheles gambiae larvae and colonization of other aquatic insects in larval habitats in Kakamega forest (elevation 1,500–1,700 m above sea level), western Kenya. We found that the survivorship of An. gambiae larvae was reduced from 55 to 57% in habitats fully exposed to sunlight (open habitats) to 1–2% in habitats with full forest canopy coverage (forest habitats) and partial canopy coverage (forest edge habitats) in two out of three trials. The average daily water temperature of the open habitats was ≈3–3.4°C higher than the forest habitats. Insect species in the orders of Diptera, Coleoptera, and Odonata colonized the larval habitats, but the three habitat types differed greatly in the animal assemblage. Canonical correspondence analysis found that water temperature and amount of leaf litter were the significant variables associated with animal assemblages. Redundancy analysis revealed that openness and the presence of predatory animals were significantly related to An. gambiae survivorship. This result suggests that deforestation facilitates the survival of the immature stage of An. gambiae in the highland.

Heterogeneity in the host preference of Japanese encephalitis vectors in Chiang Mai, northern Thailand.

Experiments, using the capture-mark-release-recapture technique inside large nets, were carried out in Chiang Mai, northern Thailand, to examine heterogeneity in the host preference of Japanese encephalitis (JE) vectors. A significantly higher proportion of the vector species that were initially attracted to a cow fed when released into a net with a cow than when released into a net containing a pig. However, Culex vishnui individuals that had been attracted to a pig had a higher feeding rate in a net containing a pig rather than a cow. When mosquitoes were given a choice by being released into a net containing both animals, they exhibited a tendency to feed on the host to which they had originally been attracted. This feeding preference was, however, not shown by the offspring of pig-fed individuals. We have therefore shown evidence of physiological/behavioural conditioning in the host preference of JE vectors rather than genetic variability. Our results suggest that effective control of JE might be achieved by increasing the availability of cows (the dead-end hosts of JE virus) to deflect the vectors from pigs (the amplifying host). The behavioural imprinting which we have found would tend to re-inforce the initial tendency of the vectors to bite cows.

Effects of temperature and diet on development and interspecies competition in Aedes aegypti and Aedes albopictus

We asked whether climate change might affect the geographic distributions of Aedes aegypti (L.) and Aedes albopictus (Skuse) (Diptera: Culicidae). We tested the effects of temperature, diet and the presence of congeneric species on the performance of immature stages of these two aedine species in the laboratory. Mosquitoes in three different species‐density combinations were reared at four constant temperatures (20 °C, 25 °C, 30 °C, 35 °C) on low‐ or high‐level diets. Of the four temperatures tested, mortality increased only at 35 °C in both species. Mortality was higher on the high‐level diet than on the low‐level diet at 35 °C, but not at other temperatures. The presence of congeneric species had a significant positive effect on mortality in Ae. albopictus, but not in Ae. aegypti. Both species developed more quickly at higher temperatures within the range of 20–30 °C; development was not enhanced at 35 °C. Population growth of Ae. albopictus was more stable, regardless of diet and temperature; that of Ae. aegypti varied more according to these two factors. These species‐specific attributes may help to explain the latitudinal distribution of the mosquitoes and degree of species dominance where they are sympatric.

Multiple blood feeding and host-seeking behavior in aedes aegypti and aedes albopictus (diptera: Culicidae)

ABSTRACT The body size of mosquitoes can influence a number of bionomic factors, such as their blood-feeding ability, host attack rate, and fecundity. All of these traits are important determinants of their potential to transmit diseases. Among abiotic and biotic factors, high temperature and low nutrition in the developing stages of mosquitoes generally result in small adults. We studied the relationship between body size and multiple feeding in a gonotrophic cycle and some fecundity attributes by using three strains of two competent vector species, Aedes aegypti (L.) and Aedes albopictus (Skuse). We raised small and large mosquitoes under low and high food conditions in the laboratory to measure parameters of fecundity and blood-feeding behavior. Fecundity was positively correlated with body size in both species, whereas the number of bloodmeals, the frequency of host-seeking behavior, and egg retention were negatively correlated with body size in the Ae. albopictus Nagasaki strain. We found that multiple feeding and host-seeking behavior were negatively correlated with body size, i.e., small mosquitoes tended to have more contact with hosts. We found that two mechanisms that inhibit engorged mosquitoes from seeking out hosts, distension-induced and oocyte-induced inhibition, were not strong enough to limit host-seeking behavior, and multiple feeding increased fecundity. Size-dependent multiple feeding and host-seeking behavior affect contact frequency with hosts and should be considered when predicting how changes in mosquito body size affect disease transmission.

Diving Ability of Anopheles gambiae (Diptera: Culicidae) Larvae

Abstract Anopheles gambiae Giles larvae usually live near the surface of shallow and temporary aquatic habitats. How deep the larvae can dive and how long they can submerge may be related to feeding efficiency and predator avoidance. This study examined diving behavior of An. gambiae larvae in the laboratory. We recorded diving depths and larval mortality of second and fourth instars in clean water and muddy water by using deep water (32-cm) and shallow water (20-cm) columns. In deep water columns with clean water, we found that 2% of second instars and 6% of fourth instars died from diving, whereas 3% of second instars and 11% of fourth instars died in muddy water. The fourth instars dived deeper in muddy water than in clean water. The mortality rates of the fourth instars subjected to diving stimulations were significantly higher than those in the shallow water columns. Therefore, larval diving behavior may offer the benefits of predator avoidance and food acquisition but also incur energetic costs and increased mortality.

Gut content identification of larvae of the Anopheles gambiae complex in western Kenya using a barcoding approach

Although larvae feeding and food source are vital to the development, survival and population regulation of African malaria vectors, the prey organisms of Anopheles gambiae larvae in the natural environment have not been well studied. This study used a molecular barcoding approach to investigate the natural diets of Anopheles gambiae s.l. larvae in western Kenya. Gut contents from third‐ and fourth‐instar larvae from natural habitats were dissected and DNA was extracted. The 18S ribosomal DNA gene was amplified, the resulting clones were screened using a restriction fragment length polymorphism method and nonmosquito clones were sequenced. Homology search and phylogenetic analyses were then conducted using the sequences of non‐mosquito clones to identify the putative microorganisms ingested. The phylogenetic analyses clustered ingested microorganisms in four clades, including two clades of green algae (Chlorophyta, Chlorophyceae Class, Chlamydomonadales and Chlorococcales families), one fungal clade, and one unknown eukaryote clade. In parallel, using the same approach, an analysis of the biodiversity present in the larval habitats was carried out. This present study demonstrated the feasibility of the barcoding approach to infer the natural diets of Anopheles gambiae larvae. Our analysis suggests that despite the wide range of microorganisms available in natural habitats, mosquito larvae fed on specific groups of algae. The novel tools developed from this study can be used to improve our understanding of the larval ecology of African malaria vectors and to facilitate the development of new mosquito control tools.

A laboratory and field evaluation of Macrocyclops distinctus, Megacyclops viridis and Mesocyclops pehpeiensis as control agents of the dengue vector Aedes albopictus in a peridomestic area in Nagasaki, Japan

Abstract The use of the cyclopoid copepods Macrocyclops distinctus (Richard) Megacyclops viridis (Jurine) and Mesocyclops pehpeiensis Hu (Cyclopoida: Cyclopidae) as biological control agents against the dengue vector Aedes albopictus (Skuse) (Diptera: Culicidae) was evaluated. In the laboratory their predatory ability was highest against the younger instars of Ae. albopictus and none of the three copepods killed the fourth instar. Except for M. viridis, predatory ability was affected by the size of the container: the smaller the container, the higher the predation. A 4‐month field test was conducted to examine the impact of these predators on wild Ae. albopictus. Thirty artificial containers were placed in a peridomestic area to allow Ae. albopictus colonization. We showed continuous and similar oviposition responses in treated and control containers. The densities of Ae. albopictus showed considerable short‐term changes and were much reduced by the copepod species. Macrocyclops and the mixture of all three provided better Ae. albopictus control than either Megacyclops or Mesocyclops alone. When larval densities peaked in the control containers in August and September, the overall reduction due to the copepods was nearly complete. Mesocyclops inoculated alone had the highest population survival. However, the growth and survival of all the copepod species was poor when the three genera were mixed. Based on their performance and survival in the trial, Macrocyclops and Mesocyclops merit consideration as bio‐control agents of Ae. albopictus.

The association between the phytoplankton, Rhopalosolen species (Chlorophyta; Chlorophyceae), and Anopheles gambiae sensu lato (Diptera: Culicidae) larval abundance in western Kenya

Algae are important food resources of the larvae of the African malaria vectors, Anopheles gambiae Giles and Anopheles arabiensis Patton (Anopheles gambiae sensu lato), and other zooplankton, but empirical evidence remains meager about the agal flora in ephemeral water bodies. The animals present in natural aquatic habitats in western Kenya were sampled from July to November 2002 to study abiotic and biotic environmental factors determining A. gambiae sl larval abundance. The five highest concentrations of third and fourth instars and pupae (hereafter referred to as old-stage larvae) were sampled in conjunction with the unicellular epizoic green algae, Rhopalosolen species (Chlorophyta; Chlorophyceae). Canonical correspondence analysis revealed that the presence of Rhopalosolen species was the most important determinant of the animal assemblage. The density of old-stage A. gambiae sl larvae was positively correlated with the presence of Rhopalosolen species, but the density of first and second instars of A. gambiae sl was not. The water bodies with Rhopalosolen sp. yielded larger mosquitoes in spite of the higher density of larvae. We demonstrated that the productivity of water bodies in terms of the larvae of malaria vectors can differ in magnitude depending on the agal flora. We discuss phytoplankton as a regulator of mosquito larval populations.

Seasonal abundance of Anopheles mosquitoes and their association with meteorological factors and malaria incidence in Bangladesh

BackgroundThe relationship between climatic factors and mosquito abundance is very important to determine parasite activity levels and, therefore, disease risk. Therefore, this study was conducted to investigate the seasonal abundance of anophelines and their association with meteorological variables and disease transmission in two malaria endemic areas of Bangladesh.MethodsMonthly sampling was done from both indoors and outdoors in 12 selected houses using light traps (LTs) and pyrethrum spray (PS) during January, 2011 to January, 2012 in two malaria endemic areas of Bangladesh. Outdoor rainfall, temperature, and relative humidity data of the study areas were collected from the meteorological department of Bangladesh. Mosquitoes were killed with chloroform and identified morphologically under stereoscopic microscopes using taxonomic keys. Samples were tested for CSP of P. falciparum, P. vivax 210 and P. vivax 247 using ELISA. Pearson correlation and canonical correspondence analyses (CCA) were computed to investigate the associations with species abundance and rainfall, temperature, humidity and malaria cases.ResultsA total of 2,443 female anophelines, representing 22 species were captured. Every female Anopheles were tested for P. falciparum, P. vivax 210 and P. vivax 247 CSP, of which 10 species were found positive. The CSP positive species were An. annularis, An. baimaii, An. barbirostris, An. jeyporiensis, An. karwari, An. minimus s.l., An. philippinensis, An. umbrosus, An. vagus and An. wilmori. Anopheles vagus and An. philippinensis were the dominant species present almost throughout the year with highest peaks in March and smallest peaks in September but An. baimaii and An. willmori were found during monsoon (July -September) only. Lag rainfall and relative humidity were the most significant variables influencing An. baimaii, An. willmori, An. vagus, and An. subpictus density in Kumari area. Abundance of these four species positively related to malaria cases. The effects of temperature were not found as a significant variable on the abundance of anophelines mosquitoes in Bangladesh.ConclusionsOur study demonstrates that the nature of relationship between malaria vector and climatic variables were multifaceted. Detailed studies of vector bionomics, continuous monitoring and malaria transmission dynamics is essential for predicting disease outbreaks and vector control in the region.

False positivity of circumsporozoite protein (CSP)–ELISA in zoophilic anophelines in Bangladesh

Circumsporozoite protein enzyme-linked immunosorbent assays (CSP-ELISAs) are widely used for malaria vector identification throughout the world. However, several studies have reported false-positive results when using this method. The present study was conducted to estimate the frequency of false positives among anopheline species in malaria endemic areas of Bangladesh. In total, 4724 Anopheles females belonging to 25 species were collected and tested for Plasmodium falciparum, Plasmodium vivax-210, and P. vivax-247 CSP. Initially, 144 samples tested positive using routine CSP-ELISA, but the number of positive results declined to 85 (59%) when the samples were tested after heating at 100°C for 10min to remove false-positive specimens. Ten species, Anopheles annularis, Anopheles baimaii, Anopheles barbirostris, Anopheles jeyporiensis, Anopheles karwari, Anopheles kochi, Anopheles minimus s.l., Anopheles peditaeniatus, Anopheles philippinensis, and Anopheles vagus were CSP-positive. The highest and lowest infection rates were found in An. baimaii (4/25, 16.0%) and An. jeyporiensis (1/139, 0.67%), respectively. A significant correlation was found (regression analysis, R(2)=0.49, F=8.25, P<0.05) between human blood index results and the true CSP-positive ratios in 15 Anopheles species. We confirmed that false-positive reactions occurred more frequently in zoophilic species. The relatively high proportion of false positives (40%) that was found in this study should warn malaria epidemiologists working in the field to be cautious when interpreting ELISA results.