TY - JOUR
AU - Yan, G
AB - Abstract The population sizes of Anopheles gambiae Giles (Diptera: Culicidae) and Anopheles arabiensis Patton (Diptera: Culicidae) increase dramatically with the onset of the rainy season in sub-Saharan Africa, but the ecological mechanisms underlying the increases are not well understood. As a first step toward to understand, we investigated the proliferation of algae, the major food of mosquito larvae, in artificial fresh water bodies exposed to sunlight for a short period, and old water bodies exposed to sunlight for a long period, and the effects thereof on the development of these anopheline larvae. We found that an epizoic green algal species of the genus Rhopalosolen (Chlorophyta: Chlorophyceae) proliferated immediately after water freshly taken from a spring was placed in sunlight. This alga proliferated only briefly (for ~10 d) even if the water was repeatedly exposed to sunlight. However, various algal species were observed in water that remained under sunlight for 40 d or longer (i.e., in old water bodies). The growth performance of larvae was higher in sunlight-exposed (alga-rich) water than in shade-stored (alga-poor) water. Stable isotope analysis suggested that these two anopheline species fed on Rhopalosolen algae in fresh water bodies but hardly at all on other algae occurring in the old water bodies. We concluded that freshly formed ground water pools facilitate high production of anopheline species because of the proliferation of Rhopalosolen algae therein, and the increase in the number of such pools in the rainy season, followed by rapid increases in A. gambiae and A. arabiensis numbers. malaria vector, larval diet, epizoic algae, stable isotope analysis The most prevalent malarial vectors in sub-Saharan Africa are Anopheles funestus Giles (Diptera: Culicidae) of the A. funestus group, and Anopheles gambiae Giles (Diptera: Culicidae) and Anopheles arabiensis Patton (Diptera: Culicidae) of the A. gambiae group. Larvae of A. funestus are found mainly in permanent water bodies, such as ponds and lakes, and their densities remain relatively constant throughout the year. In contrast, A. gambiae, Anopheles coluzzii (formerly A. gambiae M form), and A. arabiensis larvae mainly inhabit temporal shallow-water bodies and exhibit much larger seasonal fluctuations; thus, their densities are very low in the dry season but dramatically increase shortly after the onset of the rainy season (Smith et al. 1995; Mbogo et al. 1995, Fontenille et al. 1997, Lindblade et al. 2000). It is assumed that the increase in the number of temporary pools in the rainy season is a major cause of the increase in the numbers of these two anopheline species (Muirhead-Thomson 1948, Gillies and De Meillon 1968, Service 1977). However, it is not well-understood why such temporal pools allow rapid proliferation of these anopheline species. In this respect, we earlier reported that a green algal species, Rhopalosolen sp. (Chlorophyta; Chlorophyceae), occurring in temporal pools was an important food resource for A. gambiae and A. arabiensis larvae (Tuno et al. 2006). Members of the genus Rhopalosolen, which was previously included in the genus Characium, are epizoic; they grow on the surface of animals such as crustaceans or insects. Iyengar and Iyengar (1932) first reported the occurrence of such epizoic algae on the body surface of anopheline larvae, and described the algal species as Characium anophelesii (later, this species was transferred to the genus Characiellopsis by Komárek and Fott (1983)). It has not been determined whether Characium (or Characiellopsis) anophelesii and Rhopalosolen sp. are conspecific or congeneric, but they have similar life cycles; the mother cells grow on the body surfaces of anopheline larvae and release a large number of zoospores containing single chloroplasts after maturation (Iyengar and Iyengar 1932). We earlier showed (Tuno et al. 2006) that A. gambiae and A. arabiensis larvae completed development without any reduction in body size, even when they attain high densities in temporary pools where the algal species was present, and we found a positive association between the abundance of A. gambiae and A. arabiensis larvae and the presence of Rhopalosolen algae. These observations suggested that A. gambiae and A. arabiensis larvae fed on mother cells and zoospores of the algal species, and that proliferation of the alga explained the increase in the numbers of these two anopheline species. To further understand the importance of Rhopalosolen algae, we explored proliferation of this algal species in newly formed and old water bodies, and the effect thereof on the development and survival of A. arabiensis and A. gambiae larvae. It is not known whether A. arabiensis and A. gambiae larvae differ in their feeding habits. However, A. gambiae larvae dive into water and crawl on the bottom or vegetation more frequently than do A. arabiensis larvae (Tuno et al. 2007), suggesting that the larvae differ in the use of food resources such as microbial layers occurring on the bottom or vegetation. To explore this possibility, we compared the growth of A. gambiae and A. arabiensis larvae in the presence or absence of microbial layers on the surface of internal walls, and the bottom of rearing cups. In addition, we explored whether A. gambiae and A. arabiensis larvae differed in terms of nutritional sources by measuring the stable isotope ratios of artificially raised mosquitoes. Over the past few decades, the methods used to trace organic matter flow through food webs have improved; the levels of naturally occurring stable isotopes, such as C (13C/12C) and N (15N/14N), have been measured (Michener and Schell 1994). This approach is based on the characteristic behaviors of stable isotopes in food webs (DeNiro and Epstein 1978, 1981). There is little (~1‰) or no change in the relative abundance of 13C when the trophic levels change (Hobson and Welch 1992); this isotope is thus useful to identify primary food sources in systems where only a few isotopically distinct food sources are present. However, the concentration of 15N increases in a stepwise manner as the trophic level rises, at a relatively constant rate of 3‰–4‰ (Michener and Schell 1994). The combined use of stable C and N isotope analyses enables investigation of trophic relationships and the feeding ecology of mosquitoes, as well as other organisms. Mosquito larvae engage in filter-feeding, thus consuming water-borne food materials indiscriminately. Stable isotope analysis allows us to understand what nutrients mosquito larvae actually assimilate from indiscriminately collected materials. Materials and Methods Mosquito Rearing Blood-fed females of A. arabiensis resting indoors were collected using aspirators from several houses at Ahero (0°09′S, 34°55′E, 1,190 m altitude), and blood-fed A. gambiae females were collected from several houses at Iguhu (0°09′N, 34°44′E, 1,420 m altitude), Western Kenya. Blood-fed females were placed into vials with wet cotton wool to allow us to collect eggs, which were soaked in water to the time of hatching. Two larvae of every female were used for species confirmation via polymerase chain reaction (PCR) (Scott et al. 1993). We thus confirmed that all A. gambiae sensu lato collected at Ahero were A. arabiensis and all A. gambiae sensu lato collected at Iguhu were A. gambiae. We did not conduct further experiments to distinguish between A. coluzzii and A. gambiae because the former distributes in West Africa but not in East Africa (Wiebe et al. 2017). The populations were maintained at the Vector Biology Control and Research Center, Kenya Medical Research Institute (Kisumu, Kenya). Mosquito larvae were reared in basins (40 cm in diameter, 10 cm in height) and fed ground yeast tablets (Pharmadass Ltd., Harrow, UK). The basins were exposed to sunlight from 9:00 a.m. to 5:00 p.m. Second-generation offspring of field-collected mosquitoes were used in experiments. Experimental Design We conducted rearing experiments in March (experiment 1) and August (experiment 2) 2005. In experiment 1, the water used to rear mosquito larvae mimicked a freshly formed water body, whereas the water used in experiment 2 mimicked an old water body, i.e., a natural ground pool exposed to sunlight for 40 d (Fig. 1). We chose 40 d by reference to our preliminary observations of algal succession in natural ground pools. The algal species composition of freshly formed water bodies was poor (or simple), with only a few algal species blooming, but that of older water was rich, without any predominating species evident when the water bodies were exposed to sunlight for longer periods (Tuno et al. 2006). Fig. 1. View largeDownload slide Experimental design. Experiment 1: We mimicked a freshly formed ground pool in which to breed larvae. Spring water was exposed to sunlight for 1–12 d or was kept in light-shielded tanks in a room used to raise the larvae of two mosquito species to pupation. Half of the rearing cups were wiped inside daily to remove microbial layers. Thus, we established four experimental conditions: alga-rich water with microbial layers, alga-rich water without microbial layer, alga-poor water with microbial layers, and alga-poor water without microbial layers. For each condition, six replicates (cups) were prepared. A total of 960 larvae (20 larvae per cup × 6 cups × 2 types of water × 2 treatments for microbial layers × 2 species) were reared simultaneously. Experiment 2: We mimicked an old ground pool exposed to sunlight for longer than 40 d to breed larvae. Rain water was exposed to sunlight for 41–58 d or kept in light-shielded tanks in the room used to raise larvae of two mosquito species to pupation. The other steps were the same as those of experiment 1. Fig. 1. View largeDownload slide Experimental design. Experiment 1: We mimicked a freshly formed ground pool in which to breed larvae. Spring water was exposed to sunlight for 1–12 d or was kept in light-shielded tanks in a room used to raise the larvae of two mosquito species to pupation. Half of the rearing cups were wiped inside daily to remove microbial layers. Thus, we established four experimental conditions: alga-rich water with microbial layers, alga-rich water without microbial layer, alga-poor water with microbial layers, and alga-poor water without microbial layers. For each condition, six replicates (cups) were prepared. A total of 960 larvae (20 larvae per cup × 6 cups × 2 types of water × 2 treatments for microbial layers × 2 species) were reared simultaneously. Experiment 2: We mimicked an old ground pool exposed to sunlight for longer than 40 d to breed larvae. Rain water was exposed to sunlight for 41–58 d or kept in light-shielded tanks in the room used to raise larvae of two mosquito species to pupation. The other steps were the same as those of experiment 1. In experiment 1, rearing water was obtained from a spring located 10 km south-east of Kisumu City. The water was stored in light-shielded plastic tanks in a dark room, and was therefore alga-poor. To prepare alga-rich water, ~60 liter of water was transferred from tanks to a basin (40 cm in diameter and 10 cm in depth) and exposed to sunlight for 10 d (from the start until the end of the experiment) from 8:00 a.m. to 5:00 p.m.; the basin was covered with a lid from 5:00 p.m. to 8:00 a.m. Thus, the water conditions in the basin changed during the experiment (Fig. 2, Table 1). Fig. 2. View largeDownload slide Changes in chlorophyll a levels throughout the experiments. Fig. 2. View largeDownload slide Changes in chlorophyll a levels throughout the experiments. Table 1. Physicochemical characteristics of water used for larval rearing (see text and Figure 1 for explanation) Water sample Chlorophyll a (µg/ml) Turbidity (NTU) NH4+ (µg/ml) COD (µg/ml) NO2− (µg/ml) PO4− (µg/ml) pH Experiment 1 (with Rhopalosolen algae)  Original 0.0 4.9 <0.2 <8 <0.02 <0.2 7.2  Shade stored 0.0 2.7 ± 0.7 <0.2 <8 <0.02 <0.2 7.2  Sunlight exposed 20.3 ± 0.4 15.9 ± 0.3 <0.2 26 ± 0.8 <0.02 <0.2 7.2 Experiment 2 (with other algal species)  Original 0.0 3.7 <0.2 <8 <0.02 <0.2 6.6  Shade stored 0.0 3.0 ± 0.9 <0.2 <8 <0.02 <0.2 6.7 ± 0.2  Sunlight exposed 29.9 ± 0.4 10.6 ± 0.4 0.3 ± 0.6 40.1 ± 0.4 <0.02 <0.2 6.8 ± 0.0 Water sample Chlorophyll a (µg/ml) Turbidity (NTU) NH4+ (µg/ml) COD (µg/ml) NO2− (µg/ml) PO4− (µg/ml) pH Experiment 1 (with Rhopalosolen algae)  Original 0.0 4.9 <0.2 <8 <0.02 <0.2 7.2  Shade stored 0.0 2.7 ± 0.7 <0.2 <8 <0.02 <0.2 7.2  Sunlight exposed 20.3 ± 0.4 15.9 ± 0.3 <0.2 26 ± 0.8 <0.02 <0.2 7.2 Experiment 2 (with other algal species)  Original 0.0 3.7 <0.2 <8 <0.02 <0.2 6.6  Shade stored 0.0 3.0 ± 0.9 <0.2 <8 <0.02 <0.2 6.7 ± 0.2  Sunlight exposed 29.9 ± 0.4 10.6 ± 0.4 0.3 ± 0.6 40.1 ± 0.4 <0.02 <0.2 6.8 ± 0.0 Mean ± SD is shown when more than one samples are present. View Large Table 1. Physicochemical characteristics of water used for larval rearing (see text and Figure 1 for explanation) Water sample Chlorophyll a (µg/ml) Turbidity (NTU) NH4+ (µg/ml) COD (µg/ml) NO2− (µg/ml) PO4− (µg/ml) pH Experiment 1 (with Rhopalosolen algae)  Original 0.0 4.9 <0.2 <8 <0.02 <0.2 7.2  Shade stored 0.0 2.7 ± 0.7 <0.2 <8 <0.02 <0.2 7.2  Sunlight exposed 20.3 ± 0.4 15.9 ± 0.3 <0.2 26 ± 0.8 <0.02 <0.2 7.2 Experiment 2 (with other algal species)  Original 0.0 3.7 <0.2 <8 <0.02 <0.2 6.6  Shade stored 0.0 3.0 ± 0.9 <0.2 <8 <0.02 <0.2 6.7 ± 0.2  Sunlight exposed 29.9 ± 0.4 10.6 ± 0.4 0.3 ± 0.6 40.1 ± 0.4 <0.02 <0.2 6.8 ± 0.0 Water sample Chlorophyll a (µg/ml) Turbidity (NTU) NH4+ (µg/ml) COD (µg/ml) NO2− (µg/ml) PO4− (µg/ml) pH Experiment 1 (with Rhopalosolen algae)  Original 0.0 4.9 <0.2 <8 <0.02 <0.2 7.2  Shade stored 0.0 2.7 ± 0.7 <0.2 <8 <0.02 <0.2 7.2  Sunlight exposed 20.3 ± 0.4 15.9 ± 0.3 <0.2 26 ± 0.8 <0.02 <0.2 7.2 Experiment 2 (with other algal species)  Original 0.0 3.7 <0.2 <8 <0.02 <0.2 6.6  Shade stored 0.0 3.0 ± 0.9 <0.2 <8 <0.02 <0.2 6.7 ± 0.2  Sunlight exposed 29.9 ± 0.4 10.6 ± 0.4 0.3 ± 0.6 40.1 ± 0.4 <0.02 <0.2 6.8 ± 0.0 Mean ± SD is shown when more than one samples are present. View Large Experimental rearing was performed using cups (7 cm in diameter and 100 ml in volume) filled with 50 ml of alga-rich or alga-poor water (2 cm in depth). Twenty second-instar larvae (obtained 1 d after hatching) were placed in each rearing cup. Water in the cups was refreshed every day at 8:00 a.m. with water from the basin. In this experiment, 1.2 liter of water was used every day (see below). To keep the water level in the basin constant, 1.2 liter of water was transferred from a storage tank to the basin every day. As a control, mosquitoes were reared in alga-poor water that had been stored in light-shielded tanks. To render the temperature of rearing water uniform, water drawn from the basin or the tanks was kept in the same room for ~30 min before transferring to the cups. Powdered yeast tablets were served as food; 25 mg of yeast was added to each cup with second-instar larvae, and 37.5 mg to each cup with older larvae. The experimental cups were kept in a box in a dark room to prevent algal proliferation. During rearing, algae and other microbes grew to form layers on the internal walls and bottoms of the rearing cups. To explore the effect of these layers on the development and survival of mosquito larvae, mosquitoes were reared in cups from which the microbial layers were removed. To remove the layers, the insides of rearing cups were wiped with clean cotton every day when the rearing water was refreshed. Control cups were untreated. Thus, we used four experimental conditions: alga-rich water with microbial layers, alga-rich water without microbial layers, alga-poor water with microbial layers, and alga-poor water without microbial layers. For each condition, six replicates (cups) were prepared. Thus, a total of 960 larvae (20 larvae per cup × 6 cups × 2 types of water × 2 treatments for microbial layers × 2 species) were reared simultaneously. In experiment 2, artificially prepared old water was used. In this experiment, it was difficult to avoid the inflow of rainwater into the basin entirely because the basin was kept outdoors for a long period. If spring water had been used, the quality of the water may have changed when it rained. In this experiment, therefore, rainwater was used instead of spring water. Rainwater was collected from the rooves of buildings and kept in light-shielded tanks. This was alga-poor water. To prepare an alga-rich old water-like water body, ~60 liter of rainwater was transferred from the tank to a basin and exposed to sunlight for 40 d. Using these alga-rich and alga-poor waters, experimental rearing was performed as described for experiment 1. As the alga-rich water in the basin decreased in level during rearing, rainwater stored in the tanks was used to top up the basin to a constant water level. Stable Isotope Analysis Adult mosquitoes obtained in experiments 1 and 2 were dried and stored on filter paper with silica gel in 1.5-ml centrifuge tubes at room temperature until the analyses were performed. The individual that pupated earliest in each rearing cup was selected (9–10 March 2005) and inserted into tin capsules. Yeast tablets, used as mosquito feed, were also inserted into the tin capsules. Nitrogen and carbon isotope ratios were measured using an isotope ratio mass spectrometer (Delta S; Finnigan MAT, Bremen, Germany) connected to an elemental analyzer (EA1108; Fisons Instruments, Milan, Italy) via an interface (Conflo II; Finnigan MAT) in the Center for Ecological Research of Kyoto University, Japan in December 2005. The precision of the online procedure was better than ±0.1‰ for the 15N and 13C ratios. The natural abundances of 15N and 13C were expressed per mil (‰) based on international standards; δ15N or δ13C = Rsample/Rstandard − 1, where R in δ15N and δ13C is 15N/14N and 13C/12C, respectively. Atmospheric N and Vienna Pee Dee Belemnite were used as international standards for N and C, respectively; the internal standards were CERKU-08 and CERKU-09 (Tayasu et al. 2011). N and C concentrations were measured using a thermal conductivity detector attached to an elemental analyzer. Measurements Mortality and duration (in days) of development from second instar to adult emergence were recorded. Adult mosquitoes were killed in a freezer within 12 h of emergence, and their wing lengths were measured with a micrometer under a stereomicroscope. One of the two wings was soaked in 20 µl of PCR reaction cocktail to distinguish species of the A. gambiae complex (Scott et al. 1993). After wing removal, the insect bodies were used for stable isotope analysis. The water temperatures in the experimental cups (two cups with water 2 cm in depth) and the basin were recorded hourly using a HOBO Tidbit data logger (Onset Computer Corp., Pocasset, MA, USA). The physicochemical characteristics of the rearing water were measured every morning; chlorophyll a levels and turbidity were measured using an Aquafluor instrument (Turner Designs, Sunnyvale, CA, USA), and NH4+, NO2−, and PO43− concentrations and the chemical oxygen demand (COD) were assessed using test kits (Kyoritsu Chemical Co., Osaka, Japan). Data Analysis We evaluated larval growth in each rearing cup by modifying the population performance parameter I of Livdahl and Sugihara (1984): I=ln(∑wx3)/(∑xwx3/∑wx3) where wx is the wing length of females that emerged on day x. The cubic value of the female wing length, wx3, a dimensionless expression of body volume, was used as a surrogate of fecundity in the original equation of Livdahl and Sugihara (1984). We calculated I for each rearing cup (n = 6 per treatment). In this study, the I values were compared among the four experimental conditions for each species in each experiment. However, I was not compared between species or experiments because the number of explanatory variables becomes too large in relation to the number of replications (6) to allow robust results to be obtained. Fit model analysis (least squares) was applied to evaluate the effects of multiple factors on the parameter I. The effect of treatment on survival from the second instar to the adult stage was analyzed using a nominal logistic fit model. The effect of treatment on developmental time was analyzed using Cox’s regression. The effect of treatment on adult size was determined using analysis of variance (ANOVA) followed by the Tukey–Kramer honestly significant difference (HSD) test. The sequential Bonferroni correction was applied to the results of these multiple comparisons, except those of the Tukey–Kramer HSD test. Multivariate analysis of variance (MANOVA) was used to evaluate the effect of various parameters (species, sex, and water type) on the N and C stable isotope ratios. The mean values of stable isotope ratios were compared between the groups by Kruskal–Wallis test. Statistical analyses were performed using JMP software (ver. 11.2.1; SAS Institute, Cary, NC, USA). Results Experiment 1 Effects of Water Type and Microbial Treatment on Larval Growth The temperature of rearing water in the experimental cups placed in the shade ranged from 19.9 to 30.3°C (average 24.9°C). However, the temperature of water in the basin (i.e., water exposed to sunlight) ranged from 19.1 to 43.8°C (average 27.8°C). The temperature of water in tanks placed in the shade was not measured, but would not differ greatly from that of experimental cups placed in the shade. The physicochemical characteristics of water changed little when the water was kept in light-shielded tanks for 10 d; chlorophyll a was not detectable either in the original or shade-stored water (Table 1). The chlorophyll a level, turbidity, and the COD increased greatly when water in the basin was exposed to sunlight (Table 1), probably reflecting increased algal levels. In sunlight-exposed water, the chlorophyll a level peaked (58.45 mg/liter) on the first day after exposure to sunlight, declined to the minimum value (3.0 mg/liter) on day 6, and then increased again to day 10 (Fig. 2). In this water, algal cells and zoospores were evident, and were identified as Rhopalosolen species based on morphological characteristics reported by Iyengar and Iyengar (1932) as well as Komárek and Fott (1983). All mosquito specimens were subjected to PCR and the absence of species contamination was confirmed. In both anopheline species, mortality was lowest when they were reared in unwiped cups containing sunlight-exposed water from the basin (i.e., water containing Rhopalosolen sp.) and highest when reared in wiped cups with shade-stored water (Table 2). The whole nominal logistic fit model was significant for both species (χ2 = 13.09, P < 0.01 for A. arabiensis; χ2 = 50.42, P < 0.001 for A. gambiae; Table 3). In both species, the effect of water type (i.e., sunlight-exposed or shade-stored) was significant (χ2 = 7.58, P < 0.01 for A. arabiensis; χ2 = 15.7, P < 0.001 for A. gambiae), and the nested effect of microbial treatment (i.e., wiped or unwiped) within water type was significant (χ2 = 4.96, P < 0.05 for A. arabiensis; χ2 = 23.3, P < 0.001 for A. gambiae). Table 2. Mortality of A. arabiensis and A. gambiae under four rearing conditions in experiments 1 and 2 Species Water Treatmenta No. of larvae Mortality rate Experiment 1 (with Rhopalosolen algae)  A. arabiensis Sunlight exposedb Unwiped 120 0.08 Wiped 120 0.18 Shade stored Unwiped 120 0.20 Wiped 120 0.25  A. gambiae Sunlight exposedb Unwiped 120 0.10 Wiped 120 0.18 Shade stored Unwiped 120 0.17 Wiped 120 0.47 Experiment 2 (with other algal species)  A. arabiensis Sunlight exposedb Unwiped 120 0.40 Wiped 120 0.18 Shade stored Unwiped 120 0.38 Wiped 120 0.30  A. gambiae Sunlight exposedb Unwiped 120 0.30 Wiped 120 0.52 Shade stored Unwiped 120 0.48 Wiped 120 0.42 Species Water Treatmenta No. of larvae Mortality rate Experiment 1 (with Rhopalosolen algae)  A. arabiensis Sunlight exposedb Unwiped 120 0.08 Wiped 120 0.18 Shade stored Unwiped 120 0.20 Wiped 120 0.25  A. gambiae Sunlight exposedb Unwiped 120 0.10 Wiped 120 0.18 Shade stored Unwiped 120 0.17 Wiped 120 0.47 Experiment 2 (with other algal species)  A. arabiensis Sunlight exposedb Unwiped 120 0.40 Wiped 120 0.18 Shade stored Unwiped 120 0.38 Wiped 120 0.30  A. gambiae Sunlight exposedb Unwiped 120 0.30 Wiped 120 0.52 Shade stored Unwiped 120 0.48 Wiped 120 0.42 aWall and bottom of cups were wiped with cotton to remove microbial layer or unwiped. bWater was exposed to sunlight for 2–9 d (experiment 1) or 40–52 d (experiment 2) before use. View Large Table 2. Mortality of A. arabiensis and A. gambiae under four rearing conditions in experiments 1 and 2 Species Water Treatmenta No. of larvae Mortality rate Experiment 1 (with Rhopalosolen algae)  A. arabiensis Sunlight exposedb Unwiped 120 0.08 Wiped 120 0.18 Shade stored Unwiped 120 0.20 Wiped 120 0.25  A. gambiae Sunlight exposedb Unwiped 120 0.10 Wiped 120 0.18 Shade stored Unwiped 120 0.17 Wiped 120 0.47 Experiment 2 (with other algal species)  A. arabiensis Sunlight exposedb Unwiped 120 0.40 Wiped 120 0.18 Shade stored Unwiped 120 0.38 Wiped 120 0.30  A. gambiae Sunlight exposedb Unwiped 120 0.30 Wiped 120 0.52 Shade stored Unwiped 120 0.48 Wiped 120 0.42 Species Water Treatmenta No. of larvae Mortality rate Experiment 1 (with Rhopalosolen algae)  A. arabiensis Sunlight exposedb Unwiped 120 0.08 Wiped 120 0.18 Shade stored Unwiped 120 0.20 Wiped 120 0.25  A. gambiae Sunlight exposedb Unwiped 120 0.10 Wiped 120 0.18 Shade stored Unwiped 120 0.17 Wiped 120 0.47 Experiment 2 (with other algal species)  A. arabiensis Sunlight exposedb Unwiped 120 0.40 Wiped 120 0.18 Shade stored Unwiped 120 0.38 Wiped 120 0.30  A. gambiae Sunlight exposedb Unwiped 120 0.30 Wiped 120 0.52 Shade stored Unwiped 120 0.48 Wiped 120 0.42 aWall and bottom of cups were wiped with cotton to remove microbial layer or unwiped. bWater was exposed to sunlight for 2–9 d (experiment 1) or 40–52 d (experiment 2) before use. View Large Table 3. Summary of nominal nested logistic fit for mortality in A. arabiensis and A. gambiae A. arabiensis A. gambiae df χ2 P-value df χ2 P-value Experiment 1 (with Rhopalosolen algae)  Whole model 3, 476 13.09 0.0045 3, 476 50.42 <0.0001  Parameter estimates   Water type 1 7.58 0.006 1 15.70 <0.0001   Treatment (sunlight-exposed)a 1 4.96 0.026 1 23.30 <0.0001   Treatment (shade-stored)a 1 0.86 >0.1 1 3.34 0.068 Experiment 2 (with other algal species)  Whole model 3, 476 6.54 0.088 3, 476 25.21 <0.0001  Parameter estimates   Water type 1 3.79 0.052 1 7.85 0.005   Treatment (sunlight-exposed)a 1 0.73 >0.1 1 9.82 0.002   Treatment (shade-stored)a 1 2.72 0.099 1 1.23 >0.1 A. arabiensis A. gambiae df χ2 P-value df χ2 P-value Experiment 1 (with Rhopalosolen algae)  Whole model 3, 476 13.09 0.0045 3, 476 50.42 <0.0001  Parameter estimates   Water type 1 7.58 0.006 1 15.70 <0.0001   Treatment (sunlight-exposed)a 1 4.96 0.026 1 23.30 <0.0001   Treatment (shade-stored)a 1 0.86 >0.1 1 3.34 0.068 Experiment 2 (with other algal species)  Whole model 3, 476 6.54 0.088 3, 476 25.21 <0.0001  Parameter estimates   Water type 1 3.79 0.052 1 7.85 0.005   Treatment (sunlight-exposed)a 1 0.73 >0.1 1 9.82 0.002   Treatment (shade-stored)a 1 2.72 0.099 1 1.23 >0.1 aNested effect of treatment (wiped or unwiped) with water type (sunlight-exposed or shade-stored). View Large Table 3. Summary of nominal nested logistic fit for mortality in A. arabiensis and A. gambiae A. arabiensis A. gambiae df χ2 P-value df χ2 P-value Experiment 1 (with Rhopalosolen algae)  Whole model 3, 476 13.09 0.0045 3, 476 50.42 <0.0001  Parameter estimates   Water type 1 7.58 0.006 1 15.70 <0.0001   Treatment (sunlight-exposed)a 1 4.96 0.026 1 23.30 <0.0001   Treatment (shade-stored)a 1 0.86 >0.1 1 3.34 0.068 Experiment 2 (with other algal species)  Whole model 3, 476 6.54 0.088 3, 476 25.21 <0.0001  Parameter estimates   Water type 1 3.79 0.052 1 7.85 0.005   Treatment (sunlight-exposed)a 1 0.73 >0.1 1 9.82 0.002   Treatment (shade-stored)a 1 2.72 0.099 1 1.23 >0.1 A. arabiensis A. gambiae df χ2 P-value df χ2 P-value Experiment 1 (with Rhopalosolen algae)  Whole model 3, 476 13.09 0.0045 3, 476 50.42 <0.0001  Parameter estimates   Water type 1 7.58 0.006 1 15.70 <0.0001   Treatment (sunlight-exposed)a 1 4.96 0.026 1 23.30 <0.0001   Treatment (shade-stored)a 1 0.86 >0.1 1 3.34 0.068 Experiment 2 (with other algal species)  Whole model 3, 476 6.54 0.088 3, 476 25.21 <0.0001  Parameter estimates   Water type 1 3.79 0.052 1 7.85 0.005   Treatment (sunlight-exposed)a 1 0.73 >0.1 1 9.82 0.002   Treatment (shade-stored)a 1 2.72 0.099 1 1.23 >0.1 aNested effect of treatment (wiped or unwiped) with water type (sunlight-exposed or shade-stored). View Large Developmental duration (number of days from second instar to adult emergence) and adult wing lengths are shown in Supp Appendix 1 (online only). No significant effect of rearing conditions on developmental duration was observed in A. arabiensis (ANOVA with Tukey’s HSD test, P > 0.05). In contrast, the developmental duration was significantly shorter when A. gambiae was reared in sunlight-exposed versus shade-stored water (ANOVA with Tukey’s HSD test, P < 0.05). Wing length was largest when both sexes of both species were reared in unwiped cups containing sunlight-exposed water (Supp Appendix 1 [online only]). The values of I were calculated for mosquito populations reared in experimental cups (Fig. 3) and were highest for A. arabiensis reared in wiped cups with sunlight-exposed water than for A. gambiae reared in unwiped cups with sunlight-exposed water. For both species, the I values were lowest upon rearing in unwiped cups with shade-stored water. A summary of the least squares fit model data for I is provided in Table 4. The whole model was significant in both species (P < 0.001). The effect of water type was significant in both species (P < 0.001, Table 4). The microbial treatment by water type interaction effect was not significant for A. arabiensis (P > 0.05), but was significant for A. gambiae (P < 0.001). Thus, the exposure of water to sunlight (which triggered proliferation of Rhopalosolen) contributed positively to larval growth in both species, while the presence of a microbial layer on the internal walls and bottoms of cups contributed positively to the growth only of A. gambiae. Fig. 3. View largeDownload slide Growth index (I) values Mosquito larvae were reared under four conditions: unwiped cups (i.e., with microbial layers on the internal walls and bottoms) with sunlight-exposed water; wiped cups (i.e., without microbial layers) with sunlight-exposed water; unwiped cups with shade-stored water; and wiped cups with shade-stored water. Columns represent the means, and error bars are the 95% CIs. Letters on columns indicate the results of statistical testing (Steel–Dwass method); different letters indicate significant differences (α < 0.05). Fig. 3. View largeDownload slide Growth index (I) values Mosquito larvae were reared under four conditions: unwiped cups (i.e., with microbial layers on the internal walls and bottoms) with sunlight-exposed water; wiped cups (i.e., without microbial layers) with sunlight-exposed water; unwiped cups with shade-stored water; and wiped cups with shade-stored water. Columns represent the means, and error bars are the 95% CIs. Letters on columns indicate the results of statistical testing (Steel–Dwass method); different letters indicate significant differences (α < 0.05). Table 4. Summary of fit model analysis (least squares) on growth index I of A. arabiensis and A. gambiae A. arabiensis A. gambiae Experiment 1 Experiment 2 Experiment 1 Experiment 2 df F ratio P-value df F ratio P-value df F ratio P-value df F ratio P-value Whole model 3, 20 14.8 <0.0001 3, 20 60.0 <0.0001 3, 20 13.2 <0.0001 3, 20 38.1 <0.0001 Effect test  Water type 1 38.5 <0.0001 1 41.3 <0.0001 1 22.7 0.0001 1 59.3 <0.0001  Treatment (Water type)a 2 2.9 0.076 2 69.4 <0.0001 2 8.4 0.002 2 27.5 <0.0001 A. arabiensis A. gambiae Experiment 1 Experiment 2 Experiment 1 Experiment 2 df F ratio P-value df F ratio P-value df F ratio P-value df F ratio P-value Whole model 3, 20 14.8 <0.0001 3, 20 60.0 <0.0001 3, 20 13.2 <0.0001 3, 20 38.1 <0.0001 Effect test  Water type 1 38.5 <0.0001 1 41.3 <0.0001 1 22.7 0.0001 1 59.3 <0.0001  Treatment (Water type)a 2 2.9 0.076 2 69.4 <0.0001 2 8.4 0.002 2 27.5 <0.0001 aNested effect of treatment (wiped or unwiped) with water type (sunlight-exposed or shade-stored). View Large Table 4. Summary of fit model analysis (least squares) on growth index I of A. arabiensis and A. gambiae A. arabiensis A. gambiae Experiment 1 Experiment 2 Experiment 1 Experiment 2 df F ratio P-value df F ratio P-value df F ratio P-value df F ratio P-value Whole model 3, 20 14.8 <0.0001 3, 20 60.0 <0.0001 3, 20 13.2 <0.0001 3, 20 38.1 <0.0001 Effect test  Water type 1 38.5 <0.0001 1 41.3 <0.0001 1 22.7 0.0001 1 59.3 <0.0001  Treatment (Water type)a 2 2.9 0.076 2 69.4 <0.0001 2 8.4 0.002 2 27.5 <0.0001 A. arabiensis A. gambiae Experiment 1 Experiment 2 Experiment 1 Experiment 2 df F ratio P-value df F ratio P-value df F ratio P-value df F ratio P-value Whole model 3, 20 14.8 <0.0001 3, 20 60.0 <0.0001 3, 20 13.2 <0.0001 3, 20 38.1 <0.0001 Effect test  Water type 1 38.5 <0.0001 1 41.3 <0.0001 1 22.7 0.0001 1 59.3 <0.0001  Treatment (Water type)a 2 2.9 0.076 2 69.4 <0.0001 2 8.4 0.002 2 27.5 <0.0001 aNested effect of treatment (wiped or unwiped) with water type (sunlight-exposed or shade-stored). View Large Effects of Water Type and Microbial Treatment on Stable Isotope Values The N and C isotope ratios of A. arabiensis and A. gambiae adults are shown in Table 5 along with the results of Tukey HSD pairwise comparisons. We applied MANOVA to test the influence of species, sex, water type, and microbial treatment on the isotope ratios in adults. The whole model was significant (F2.45, P < 0.001), but the only factor exhibiting significance was water type (F2.29, P < 0.001). Figure 4 shows δ15N versus δ13C plots for individuals of A. gambiae and A. arabiensis and for the yeast tablets used as larval food. In both species, δ15N was higher in individuals reared in shade-stored water than in the yeast tablets, probably because of the step-up in trophic level (i.e., mosquitoes feeding on yeasts). Also in both species, δ13C was lower in individuals reared in sunlight-exposed versus shade-stored water, suggesting that the food differed between these conditions. Thus, the individuals reared in sunlight-exposed water fed on Rhopalosolen species in addition to the yeast tablets. Table 5. Nitrogen and carbon isotope ratios of A. arabiensis and A. gambiae Experiment 1 (Rhopalosolen sp. blooms) Experiment 2 (Other algae grow) Water Material N δ15N (‰) δ13C (‰) N δ15N (‰) δ13C (‰) Yeast Tablet 2.59 −19.28 2.59 −19.28 Sunlight-exposed A. arabiensis 15 5.34 a −20.12 a 5 5.03 a −18.23 a A. gambiae 13 5.10 a −20.22 a 6 4.95 a −18.42 a Shade-stored A. arabiensis 22 4.23 b −19.12 b 6 5.11 a −18.64 b A. gambiae 16 4.53 b −19.12 b 6 4.87 a −18.66 b Experiment 1 (Rhopalosolen sp. blooms) Experiment 2 (Other algae grow) Water Material N δ15N (‰) δ13C (‰) N δ15N (‰) δ13C (‰) Yeast Tablet 2.59 −19.28 2.59 −19.28 Sunlight-exposed A. arabiensis 15 5.34 a −20.12 a 5 5.03 a −18.23 a A. gambiae 13 5.10 a −20.22 a 6 4.95 a −18.42 a Shade-stored A. arabiensis 22 4.23 b −19.12 b 6 5.11 a −18.64 b A. gambiae 16 4.53 b −19.12 b 6 4.87 a −18.66 b Different letters indicate significant difference, P < 0.05 (Tukey’s HSD test). View Large Table 5. Nitrogen and carbon isotope ratios of A. arabiensis and A. gambiae Experiment 1 (Rhopalosolen sp. blooms) Experiment 2 (Other algae grow) Water Material N δ15N (‰) δ13C (‰) N δ15N (‰) δ13C (‰) Yeast Tablet 2.59 −19.28 2.59 −19.28 Sunlight-exposed A. arabiensis 15 5.34 a −20.12 a 5 5.03 a −18.23 a A. gambiae 13 5.10 a −20.22 a 6 4.95 a −18.42 a Shade-stored A. arabiensis 22 4.23 b −19.12 b 6 5.11 a −18.64 b A. gambiae 16 4.53 b −19.12 b 6 4.87 a −18.66 b Experiment 1 (Rhopalosolen sp. blooms) Experiment 2 (Other algae grow) Water Material N δ15N (‰) δ13C (‰) N δ15N (‰) δ13C (‰) Yeast Tablet 2.59 −19.28 2.59 −19.28 Sunlight-exposed A. arabiensis 15 5.34 a −20.12 a 5 5.03 a −18.23 a A. gambiae 13 5.10 a −20.22 a 6 4.95 a −18.42 a Shade-stored A. arabiensis 22 4.23 b −19.12 b 6 5.11 a −18.64 b A. gambiae 16 4.53 b −19.12 b 6 4.87 a −18.66 b Different letters indicate significant difference, P < 0.05 (Tukey’s HSD test). View Large Fig. 4. View largeDownload slide δ15N versus δ13C plots for A. arabiensis and A. gambiae reared in sunlight-exposed water (sun) or shade-stored water (shade), and the yeast tablets provided as food (crosses). Fig. 4. View largeDownload slide δ15N versus δ13C plots for A. arabiensis and A. gambiae reared in sunlight-exposed water (sun) or shade-stored water (shade), and the yeast tablets provided as food (crosses). Experiment 2 Effects of Water Type and Microbial Treatment on Larval Growth The temperature of rearing water in experimental cups placed in the shade ranged from 18.2 to 26.3°C (average 22.0°C). The temperature of water in the basin (i.e., exposed to sunlight) ranged from 17.1 to 37.8°C, (average 25.1°C). Physicochemical data are shown in Table 1. The chlorophyll a level of water in the basin exposed to sunlight was 29.9 ± 0.4 mg/liter and remained near-constant throughout the experimental period (Fig. 2). The algae in the water were identified as Volvox spp., Chlamydomonas spp., Chlorella spp., Oedogonium spp., Oscillatoria spp., and Gomphonema spp. In water stored in shade, chlorophyll a was not detectable (Table 1), suggesting the absence of algal growth. Mortality of A. arabiensis was lowest in wiped cups with sunlight-exposed water and highest in unwiped cups with sunlight-exposed water; in contrast, the mortality of A. gambiae was lowest in unwiped cups with sunlight-exposed water and highest in wiped cups with sunlight-exposed water (Table 2). The whole nominal logistic fit model for mortality was not significant for A. arabiensis (χ2 = 6.54, P > 0.05) but was significant for A. gambiae (χ2 = 25.21, P < 0.001) (Table 3). In A. gambiae, the effect of water type was significant (χ2 = 7.85, P < 0.01); the nested effect of microbial treatment within sunlight-exposed water was also significant (χ2 = 9.82, P < 0.01) (Table 3). Developmental durations and adult wing lengths are listed in Supp Appendix 1 (online only). No significant effect of rearing conditions on developmental duration was observed in A. arabiensis (ANOVA with Tukey’s HSD test, P > 0.05), whereas developmental duration was significantly longer when both sexes of A. gambiae were reared in wiped cups with shade-stored water than under other conditions (ANOVA with Tukey’s HSD test, P < 0.05). There was no significant effect of rearing conditions on wing length in A. arabiensis females (ANOVA with Tukey’s HSD test, P > 0.05), whereas wing length was significantly larger when A. arabiensis males and both sexes of A. gambiae were reared in unwiped cups with sunlight-exposed water versus in wiped cups with shade-stored water (ANOVA with Tukey’s HSD test, P > 0.05). The value of I for A. arabiensis was highest on rearing in wiped cups with sunlight-exposed water, but for A. gambiae the value was highest on rearing in unwiped cups with sunlight-exposed water (Fig. 3). By fitting standard least squares models, we found that the effect of water type and the nested effect of microbial treatment within water type were significant for both species (P < 0.01, Table 4). Effects of Water Type and Microbial Treatment on Stable Isotope Values MANOVA revealed that the whole model was nonsignificant (F2.49, P > 0.05). Figure 4 shows δ15N versus δ13C plots for A. gambiae and A. arabiensis adults, and the yeast tablets supplied as larval food. All individual mosquito values were closely clustered irrespective of rearing conditions. As in experiment 1, δ15N was higher in mosquitoes reared on yeast tablets, suggesting that they fed on yeasts. However, δ13C was slightly higher in mosquitoes than yeast, suggesting that they also fed on algae or microorganisms in the rearing water. There was a small but significant difference in δ13C values between water types in both species (Table 5). This suggests that larvae differed slightly in food acquisition when they were reared in sunlight-exposed or shade-stored water. However, this difference was much smaller than the difference observed in experiment 1. The effect of microbial treatment on the stable isotope values was significant for A. gambiae but not for A. arabiensis (Table 5), suggesting that A. gambiae fed on microbes of the internal walls and bottoms of cups whereas A. arabiensis did not (or at least seldom did so). No such effect of microbial treatment was observed in experiment 1. Discussion A. gambiae and A. arabiensis larvae mainly inhabit freshly formed, small, sunlit water pools, and the increase in such pools in the rainy season is assumed to be a major cause of the rapid increases in the numbers of these two anopheline species (Gillies and De Meillon 1968, Service 1977). The productivity of such pools can be extremely high for anopheline species; high concentrations of late-stage larvae and pupae have occasionally been observed (Muirhead-Thomson 1948, Gillies and De Meillon 1968, Service 1977). We have also encountered highly productive pools in Kisumu, Western Kenya (Tuno et al. 2006), Kumasi, Ghana, and Lombok Island, Indonesia (N. T. Tuno, unpublished data). It is not well understood why such pools are so productive, but algal proliferation has been suggested to be an important factor (Tuno et al. 2005, Low et al. 2016). In productive pools in Kisumu, we found epizoic small green algae (Rhopalosolen sp.) on the body surfaces of anopheline larvae (Tuno et al. 2006). According to Iyengar and Iyengar (1932), mother cells of epizoic algal species of the genus Characium, in which the genus Rhopalosolen was previously included, grow on the body surfaces of anopheline larvae and release large numbers of zoospores containing single chloroplasts after maturation. These zoospores would be expected to be a major food resource for anopheline larvae. Using chlorophyll a assays, we proposed in this study that blooming of Rhopalosolen zoospores commences on the first day after exposure of water to sunlight, in agreement with our field observations (Tuno et al. 2006). In addition, we showed in rearing experiments, and using stable isotope assays, that A. arabiensis and (particularly) A. gambiae exhibited enhanced growth when reared in Rhopalosolen-rich water (i.e., the sunlight-exposed water of experiment 1) and both species fed on Rhopalosolen algae. After peaking on the first day after exposure to sunlight, the chlorophyll a level declined to day 6 and then increased again to day 10. This fluctuation pattern coincided well with the pattern observed in a culture of Characiellopsis anophelesii, a species previously termed Characium anophelesii (Komárek and Fott 1983) and possibly identical with Rhopalosolen sp. in this study. It is assumed that the decline after the first peak is attributable to the fact that the chloroplasts of most zoospores degenerated as the zoospores entered dormancy, while the second peak is attributable to proliferation of the offspring of nondormant individuals of the first generation (Komárek and Fott 1983). The relationship between anopheline species and the epizoic algae Rhopalosolen and Characiellopsis may be mutualistic, as has been suggested for a zooplankton, Daphnia pulicaria Forbus (Diplostraca: Daphniidae), and its algal epibiont (Barea-Arco et al. 2001, Bertolo et al. 2015). Anopheline larvae are usually found in surface water, and epizoic algae occurring on their body surfaces may thus receive adequate sunlight even in muddy water. However, these anopheline larvae may feed on zoospores released by epizoic stem cells. Such mutualistic associations may allow mosquitoes to attain large body sizes despite high larval densities in freshly formed pools (Tuno et al. 2006). In water exposed to sunlight for a long period (experiment 2), no Rhopalosolen sp. proliferated; rather, other algal species such as Volvox spp., Chlamydomonas spp., Chlorella spp., Oedogonium spp., Oscillatoria spp., and Gomphonema spp. showed proliferation. These algal species seem to be a less suitable food for the anopheline species studied because larval developmental performance of both A. gambiae and A. arabiensis was minimally enhanced in the presence of these algal species (experiment 2). These results agree with our field observations to the effect that the density of anopheline larvae is usually low in older ground pools (Tuno et al. 2006). In general, phytoplankton blooming is caused by high water temperature, nutrient-rich conditions, and exposure to intense light (Jimenez et al. 1995, Asaeda and Van 1997, Gonulol and Obali 1998, Kishimoto et al. 2001, Lee and Choi 2001, Briand et al. 2002). In addition, the germination of some resting cysts is triggered by exposure to intense light or high temperature (Kishimoto et al. 2001, Briand et al. 2002). Therefore, newly formed ground pools lacking emergent vegetation would favor algal proliferation caused by intense light and high temperature, thus becoming favorable for the growth of mosquito larvae that feed on algae. In this study, we also aimed to clarify differences in the nutritional requirements of A. arabiensis and A. gambiae. Stable isotope analysis revealed no clear difference in the nutritional sources of the two mosquitoe species. However, A. gambiae performed better in unwiped cups (i.e., with microbial layers) than wiped cups, at least in experiment 1, but A. arabiensis did not. In the present experiments, Rhopalosolen mother cells or other algal or bacterial species formed layers on the walls and bottoms of unwiped experimental cups. A. gambiae may feed on these microbial layers but A. arabiensis may not (or seldom). In this respect, we observed that A. gambiae larvae voluntary dive into water and crawl on the bottom or vegetation more frequently than do A. arabiensis (Tuno et al. 2007). Such diving does not cause immediate mortality (Tuno et al. 2004, 2007), but may retard larval development by consuming energy. In compensation, diving may allow A. gambiae to feed on underwater microbial layers. The difference in this behavior between the two mosquito species may be related to a difference in habitat preference. A. gambiae prefers temporary shallow water bodies (Gimnig et al. 2001, Low et al. 2016), and acquires resources from the bottom of such bodies by diving. However, A. arabiensis prefers comparatively deeper water bodies, such as paddy fields (Service 1977), and dives less frequently. In this study, we showed that the larval growth of A. gambiae and A. arabiensis was accelerated by Rhopalosolen species, but not (or less so) by Volvox spp., Chlamydomonas spp., Chlorella spp., Oedogonium spp., Oscillatoria spp., or Gomphonema spp. Thus, the algal species composition of pools is a major factor determining pool productivity, in addition to environmental conditions such as duration of sunshine, water temperature, and water depth. Supplementary Data Supplementary data are available at Journal of Medical Entomology online. Acknowledgments We thank Prof. M. T. Kimura for the help with manuscript preparation. Isotope analysis was conducted using Joint Usage/Research Grant of Center for Ecological Research, Kyoto University. References Cited Asaeda , T. , and B. T. Van . 1997 . Modelling the effects of macrophytes on algal blooming in eutrophic shallow lakes . Ecol. Model . 104 : 261 – 287 . Google Scholar CrossRef Search ADS Barea-Arco , J. , C. Perez-Martinez , and R. 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TI - An Algal Diet Accelerates Larval Growth of Anopheles gambiae (Diptera: Culicidae) and Anopheles arabiensis (Diptera: Culicidae)
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjx244
DA - 2018-01-21
UR - https://www.deepdyve.com/lp/oxford-university-press/an-algal-diet-accelerates-larval-growth-of-anopheles-gambiae-diptera-bCp5YEMeVS
SP - 1
EP - 608
VL - Advance Article
IS - 3
DP - DeepDyve
ER -