To hatch or not to hatch? Egg hatch response to larval density and to larval contact in a treehole mosquito

Abstract.

• 1 We investigated the effect on egg hatch of exposure to: (1) varying larval density, and (2) larval contact in Aedes triseriatus Say (Diptera: Culicidae). For 2 days in the laboratory we submerged eggs into a treehole water medium containing 0 (control), 4, 12 or 24 larvae that could either contact the eggs directly or were separated from them by a screen. Following treatment, abundance of microorganisms on the egg surfaces, a food source for newly hatched larvae and a proposed hatching stimulus, was assessed by counts made from serial dilutions of samples.
• 2 We discovered a complex hatching response to larval contact and to larval density, and an interaction between these two factors in their effect on microbial growth. Hatching was inhibited in the 0-larva control, even though microorganisms grew abundantly on the eggs. Hatch rate, as well as microbial counts, were high for eggs in direct contact with 4 larvae. As density increased in the larval contact treatment, microorganisms disappeared from the egg surfaces and hatch rate declined.
• 3 When protected from larval grazing, eggs supported numerous microbial colonies irrespective of larval density. In contrast to the contact treatment group, egg hatch increased with increasing larval density. These observations suggest that the combination of microbial growth and a larval factor stimulates hatch. This hatching response may have evolved because both abundant microorganisms and numerous larvae reflect a habitat of good quality.

     

Influence of soil quality in the larval habitat on development of Anopheles gambiae Giles.

Larval ecology is an important aspect of the population dynamics of anopheline mosquitoes (Diptera: Culicidae), the vectors of malaria. Anopheles larvae live in pools of stagnant water and adult fitness may be correlated with the nutritional conditions under which larvae develop. A study was conducted in Mbita, Western Kenya, to investigate how properties of the soil substrate of Anopheles gambiae breeding pools can influence development of this mosquito species. An. gambiae eggs from an established colony were dispensed into experimental plastic troughs containing soil samples from a range of natural Anopheles larval habitats and filtered Lake Victoria water. The duration of larval development (8-15 days), pupation rate (0-79%), and adult body size (20.28-26.91 mm3) varied among different soil types. The total organic matter (3.61-21.25%), organic carbon (0.63-7.18%), and total nitrogen (0.06-0.58%) levels of the soils were positively correlated with pupation rate and negatively correlated with development time and adult body size.     

The effects of blood feeding and exogenous supply of tryptophan on the quantities of xanthurenic acid in the salivary glands of Anopheles stephensi (Diptera: Culicidae)

Xanthurenic acid (XA), produced as a byproduct during the biosynthesis of insect eye pigment (ommochromes), is a strong inducer of Plasmodium gametogenesis at very low concentrations. In previous studies, it was shown that XA is present in Anopheles stephensi (Diptera: Culicidae) mosquito salivary glands and that during blood feeding the mosquitoes ingested their own saliva into the midgut. Considering these two facts together, it is therefore likely that XA is discharged with saliva during blood feeding and is swallowed into the midgut where it exerts its effect on Plasmodium gametocytes. However, the quantities of XA in the salivary glands and midgut are unknown. In this study, we used high performance liquid chromatography with electrochemical detection to detect and quantify XA in the salivary glands and midgut. Based on the results of this study, we found 0.28+/-0.05 ng of XA in the salivary glands of the mosquitoes, accounting for 10% of the total XA content in the mosquito whole body. The amounts of XA in the salivary glands reduced to 0.13+/-0.06 ng after mosquitoes ingested a blood meal. Approximately 0.05+/-0.01 ng of XA was detected in the midgut of nonblood fed An. stephensi mosquitoes. By adding synthetic tryptophan as a source of XA into larval rearing water (2 mM) or in sugar meals (10 mM), we evaluated whether XA levels in the mosquito (salivary glands, midgut, and whole body) were boosted and the subsequent effect on infectivity of Plasmodium berghei in the treated mosquito groups. A female specific increase in XA content was observed in the whole body and in the midgut of mosquito groups where tryptophan was added either in the larval water or sugar meals. However, XA in the salivary glands was not affected by tryptophan addition to larval water, and surprisingly it reduced when tryptophan was added to sugar meals. The P. berghei oocyst loads in the mosquito midguts were lower in mosquitoes fed tryptophan treated sugar meals than in mosquitoes reared on tryptophan treated larval water. Our results suggest that mosquito nutrition may have a significant impact on whole body and midgut XA levels in mosquitoes. We discuss the observed parasite infectivity results in relation to XA's relationship with malaria parasite development in mosquitoes.     

Reduced susceptibility to permethrin and its relationship to DDT resistance in larvae of Anopheles stephensi

Permethrin selection of DDT resistant Anopheles stephensi Liston mosquito larvae produced a reduction in susceptibility to knockdown (2 h exposure) of 17-fold, but only 1.6-fold to kill (24 h exposure). Genetic analysis, incorporating visible mutant markers, was interpreted as indicating that, through multigenic inheritance, several interacting genetic factors were collectively responsible for reduced larval susceptibility to knockdown. These were maintained together only under selection pressure, as the effect was lost quickly in the absence of selection or with outcrossing. The 30-40-fold DDT-resistance found in the parental strain was barely altered by permethrin selection, suggesting no relationship with the major source of DDT resistance. This was confirmed in single family studies. Some evidence for an additional tolerance to DDT was found to be associated with reduced larval susceptibility to permethrin.     

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 field study of potential ecological costs of resistance by ‘stem ducking’ in tall goldenrod,Solidago altissima

Culex quinquefasciatus Say (Diptera: Culicidae) is an abundant urban mosquito that is the vector of filariasis. Breeding in septic tanks, where there are very high levels of bacterial food, it is likely to have a different reaction to crowding compared with other mosquitoes. To test for the presence and type of crowding effects, four larval densities of C. quinquefasciatus varying from 0.4 to 3.2 larvae ml^?1 of water were reared in tubes. Mortality was found to greatly increase at densities above 0.8 larvae, whereas larval duration increased even above 0.4 larvae ml^?1. Changing the water in the tubes daily gave a small (but significant) response in reducing mortality and larval duration. However, when larvae kept at a low density shared the same water with larvae at high density, there was no chemical influence on their growth rate and mortality. The effect of crowding was primarily due to physical disturbances between larvae. When larvae were kept at a high density in the same volume of water, but in shallow trays with a large surface area and therefore much less contact between them, mortality was the same as for the lowest density. There was still, however, a significant increase in larval duration from 8.6 days at 0.4 larvae ml^?1 to 12.1 days at 3.2 larvae ml^?1. It is therefore concluded that the larvae respond to physical rather than chemical factors by prolonging larval development and having some increase in mortality.     

Utilization of larval and pupal detritus by Aedes aegypti and Aedes albopictus

The utilization of detritus sources by mosquito larvae during development may significantly affect adult life history traits and mosquito population growth. Many studies have shown invertebrate carcasses to be an important detritus source in larval habitats, but little is known regarding how invertebrate carcasses are utilized by mosquito larvae. We conducted two studies to investigate the rate of detritus consumption and its effect on larval development and life history traits. Overall, we found that Aedes aegypti and Aedes albopictus larvae rapidly consumed larval detritus, while pupal detritus was consumed at a significantly slower rate. We also found that the consumption of larval detritus significantly increased larval survivorship and decreased male development time but did not significantly influence female development time or pupal cephalothorax length for either sex. Our results suggest that the direct consumption of larval detritus can support the production of adults in larval habitats that lack allochthonous detritus inputs or where such organic inputs are insufficient. These studies indicate that different forms of invertebrate detritus are utilized in distinct ways by mosquito larvae, and therefore different forms of invertebrate detritus may have distinct effects on larval development and adult life history traits.     

Mosquitoes and other aquatic insects in fallow field biotopes and rice paddy fields

Fallow field biotopes that develop from abandoned rice fields are man‐made wetlands that provide new habitats for various aquatic animals. Although consideration of such biotopes generally focuses on their positive aspects, this study evaluated the negative aspects of establishing fallow field biotopes with regard to mosquito breeding sites. To determine whether fallow field biotopes become breeding habitats for vector mosquitoes, we evaluated mosquito fauna in fallow field biotopes and adjacent rice fields. We found larvae of Anopheles lesteri, Anopheles sinensis and Culex tritaeniorhynchus (all: Diptera: Culicidae) in the biotopes. Although abundances of mosquito larvae in the biotopes and rice fields were statistically similar, mosquito abundances in rice fields increased dramatically in August when the water level reduced after the rainy season. The abundance and variety of the mosquitoes' natural predators were greater in biotopes than in rice fields because the former are a permanent and stable aquatic environment. A generalized linear mixed model showed a negative effect of predator diversity on mosquito larvae abundance in both habitats. Although fallow field biotopes become breeding habitats for vector mosquitoes, establishing biotopes from fallow fields in order to protect various aquatic animals, including mosquito insect predators, may help to control mosquito breeding.     

Effects of interspecific competition, predation, and their interaction on survival and development time of immature Anopheles quadrimaculatus.

We examined the effect of predation by the backswimmer (Notonecta undulata; Hemiptera: Notonectidae), competition by zooplankton and snails, and both predation and competition on the survival and development time of larval Anopheles quadrimaculatus mosquitoes in experimental mesocosms. We found that both predation and interspecific competition greatly reduced the survivorship of larvae and the number of larvae emerging into adulthood. Treatments with both predators and competitors had fewer larvae transitioning among instars and into adulthood but not in an additive way. In addition, mosquito larvae in the presence of predators and competitors took two days longer to emerge than where predators and competitions were absent. Our work provides evidence that biotic interactions, such as predation and competition, can strongly regulate the number of mosquito larvae by reducing the number of larvae that survive through instars and to emergence and by increasing the generation time.     

Delayed larval development in <Emphasis Type="Italic">Anopheles</Emphasis> mosquitoes deprived of <Emphasis Type="Italic">Asaia</Emphasis>bacterial symbionts

BACKGROUND: In recent years, acetic acid bacteria have been shown to be frequently associated with insects, but knowledge on their biological role in the arthropod host is limited. The discovery that acetic acid bacteria of the genus Asaia are a main component of the microbiota of Anopheles stephensi makes this mosquito a useful model for studies on this novel group of symbionts. Here we present experimental results that provide a first evidence for a beneficial role of Asaia in An. stephensi. RESULTS: Larvae of An. stephensi at different stages were treated with rifampicin, an antibiotic effective on wild-type Asaia spp., and the effects on the larval development were evaluated. Larvae treated with the antibiotic showed a delay in the development and an asynchrony in the appearance of later instars. In larvae treated with rifampicin, but supplemented with a rifampicin-resistant mutant strain of Asaia, larval development was comparable to that of control larvae not exposed to the antibiotic. Analysis of the bacterial diversity of the three mosquito populations confirmed that the level of Asaia was strongly decreased in the antibiotic-treated larvae, since the symbiont was not detectable by PCR-DGGE (denaturing gradient gel electrophoresis), while Asaia was consistently found in insects supplemented with rifampicin plus the antibiotic-resistant mutant in the diet, and in those not exposed to the antibiotic. CONCLUSIONS: The results here reported indicate that Asaia symbionts play a beneficial role in the normal development of An. stephensi larvae.     

Aquatic microfauna alter larval food resources and affect development and biomass of West Nile and Saint Louis encephalitis vector Culex nigripalpus (Diptera: Culicidae)

Ciliate protists and rotifers are ubiquitous in aquatic habitats and can comprise a significant portion of the microbial food resources available to larval mosquitoes, often showing substantial declines in abundance in the presence of mosquito larvae. This top‐down regulation of protists is reported to be strong for mosquitoes inhabiting small aquatic containers such as pitcher plants or tree holes, but the nature of these interactions with larval mosquitoes developing in other aquatic habitats is poorly understood. We examined the effects of these two microbial groups on lower trophic level microbial food resources, such as bacteria, small flagellates, and organic particles, in the water column, and on Culex larval development and adult production. In three independent laboratory experiments using two microeukaryote species (one ciliate protist and one rotifer) acquired from field larval mosquito habitats and cultured in the laboratory, we determined the effects of Culex nigripalpus larval grazing on water column microbial dynamics, while simultaneously monitoring larval growth and development. The results revealed previously unknown interactions that were different from the top‐down regulation of microbial groups by mosquito larvae in other systems. Both ciliates and rotifers, singly or in combination, altered other microbial populations and inhibited mosquito growth. It is likely that these microeukaryotes, instead of serving as food resources, competed with early instar mosquito larvae for microbes such as small flagellates and bacteria in a density‐dependent manner. These findings help our understanding of the basic larval biology of Culex mosquitoes, variation in mosquito production among various larval habitats, and may have implications for existing vector control strategies and for developing novel microbial‐based control methods.     

Effects of a larval mosquito biopesticide and Culex larvae on a freshwater nanophytoplankton (Selenastrum capricornatum) under axenic conditions

The effects of microbial biopesticides used for mosquito control on autotrophic microorganisms such as nanophytoplankton are equivocal. We examined impacts of mosquito biopesticides and mosquito larvae on primary producers in two independent experiments. In the first experiment, we examined the effects of a commonly used microbial biopesticide formulation (VectoMax^® CG) on a unicellular microalga, Selenastrum capricornatum Printz, under axenic laboratory conditions. The biopesticide treatments included two concentrations (0.008 and 0.016 g liter^?1) of VectoMax^® CG and two controls (one untreated and another with autoclaved 0.016 g VectoMax^® CG liter^?1) in replicated axenic experimental microcosms. Spectrophotometric analysis of chlorophyll a (proxy for algal biomass) and direct enumeration of algal cells following the treatments revealed no significant effects of the microbial biopesticide on algal population growth during the four‐week study. In the second experiment, we tested the effects of different densities of Culex larvae on the population of S. capricornatum. Effects of mosquito larvae feeding on S. capricornatum were significant with a curvilinear relationship between larval density and algal abundance in the water column. Together, these studies demonstrated a lack of direct cytological/toxicological effects of Bacillus‐based microbial pesticides on freshwater primary production and support the hypothesis that the reduction in algal primary production previously reported when Bti products were applied to aquatic environments was likely independent of the Bacillus‐based larvicidal toxins. Instead, it was likely mediated by microbial interactions in the water column and the trophic cascade effects that resulted from the removal of larval mosquitoes. These studies suggest that mosquito larvae independent of pesticide application can influence primary production. Our method of evaluating biopesticides against small photoautotrophs can be very useful for studying the unintended effects on autotrophic microorganisms of other pesticides, including herbicides and pesticides applied to aquatic environments.     

The effect of water turbidity on the near-surface water temperature of larval habitats of the malaria mosquito <Emphasis Type="Italic">Anopheles gambiae</Emphasis>

Water temperature is an important determinant in many aquatic biological processes, including the growth and development of malaria mosquito (Anopheles arabiensis and A. gambiae) immatures. Water turbidity affects water temperature, as suspended particles in a water column absorb and scatter sunlight and hence determine the extinction of solar radiation. To get a better understanding of the relationship between water turbidity and water temperature, a series of semi-natural larval habitats (diameter 0.32 m, water depth 0.16 m) with increasing water turbidity was created. Here we show that at midday (1300 hours) the upper water layer (thickness of 10 mm) of the water pool with the highest turbidity was on average 2.8°C warmer than the same layer of the clearest water pool. Suspended soil particles increase the water temperature and furthermore change the temperature dynamics of small water collections during daytime, exposing malaria mosquito larvae, which live in the top water layer, longer to higher temperatures.     

Pathogenicity of Spiroplasma taiwanense for larval Aedes aegypti mosquitoes

Helical replicative forms, but not the persistent non-replicative forms, of Spiroplasma taiwanense Abalain-Colloc et al. (isolated from the mosquito Anopheles sinensis Wiedemann in Taiwan) were shown to reduce significantly the survival of Aedes aegypti (L.) mosquito larvae reared in 10 ml of water with 0.3 ml of S.taiwanense suspensions added on days 0 and 3. The suspensions contained, respectively, helical forms at a concentration of 10(9) Colour Change Units (CCU)/ml and persistent forms at 10(6) CCU/ml. It is suggested that S.taiwanense, or toxins produced from it, are potentially useful for use in integrated mosquito control programmes.     

Breeding of Anopheles mosquitoes in irrigated areas of South Punjab, Pakistan

As part of investigations on potential linkages between irrigation and malaria transmission, all surface water bodies in and around three villages along an irrigation distributary in South Punjab, Pakistan, were surveyed for anopheline mosquito larvae (Diptera: Culicidae) from April 1999 to March 2000. Samples were characterized according to exposure to sunlight, substratum, presence of vegetation, fauna, inorganic matter and physical water condition (clear/turbid/foul). Also water temperature, dissolved oxygen (DO), electroconductivity (EC) and pH of sites were recorded. A total of 37982 Anopheles larvae of six morphological types were collected from 2992 samples taken from irrigation/agricultural and village/domestic aquatic habitats. Anopheles subpictus Grassi sensu lato was by far the most abundant (74.3%), followed by An. culicifacies Giles s.l. (4.1%), An. stephensi Liston s.l. (2.6%), An. pulcherrimus Theobald (1.8%), An. peditaeniatus Leicester (0.3%) and An. nigerrimus Giles (0.1%). The four most abundant species were significantly associated with waterlogged fields and communal village drinking-water tanks. Habitat characteristics most correlated with occurrence of anophelines were the physical water condition and the absence/presence of fauna, particularly predators. Occurrence and abundance of Anopheles immatures were not significantly correlated with water temperature, DO, EC or pH. Malaria vectors of the Anopheles culicifacies complex occurred at relatively low densities, mainly in irrigated and waterlogged fields. In South Punjab, where rainfall is very low, it should be possible to reduce anopheline breeding through water management, as larvae develop mainly in water bodies that are directly or indirectly related to the extensive canal-irrigation system.     

Larval habitat for the avian malaria vector Culex quinquefasciatus (Diptera: Culicidae) in altered mid‐elevation mesic‐dry forests in Hawai'i

Effective management of avian malaria (Plasmodium relictum) in Hawai'i's endemic honeycreepers (Drepanidinae) requires the identification and subsequent reduction or treatment of larval habitat for the mosquito vector, Culex quinquefasciatus (Diptera: Culicidae). We conducted ground surveys, treehole surveys, and helicopter aerial surveys from 2001–2003 to identify all potential larval mosquito habitat within two 100+ ha mesic‐dry forest study sites in Hawai'i Volcanoes National Park, Hawai'i; ‘Ainahou Ranch and Mauna Loa Strip Road. At ‘Ainahou Ranch, anthropogenic sites (43%) were more likely to contain mosquitoes than naturally occurring (8%) sites. Larvae of Cx. quinquefasciatus were predominately found in anthropogenic sites while Aedes albopictus larvae occurred less frequently in both anthropogenic sites and naturally‐occurring sites. Additionally, moderate‐size (≈20–22,000 liters) anthropogenic potential larval habitat had >50% probability of mosquito presence compared to larger‐ and smaller‐volume habitat (<50%). Less than 20% of trees surveyed at ‘Ainahou Ranch had treeholes and few mosquito larvae were detected. Aerial surveys at ‘Ainahou Ranch detected 56% (95% CI: 42–68%) of the potential larval habitat identified in ground surveys. At Mauna Loa Strip Road, Cx. quinquefasciatus larvae were only found in the rock holes of small intermittent stream drainages that made up 20% (5 of 25) of the total potential larval habitat. The volume of the potential larval habitat did not influence the probability of mosquito occurrence at Mauna Loa Strip Road. Our results suggest that Cx. quinquefasciatus abundance, and subsequently avian malaria, may be controlled by larval habitat reduction in the mesic‐dry landscapes of Hawai'i where anthropogenic sources predominate.     

Leaf-Associated Bacterial and Fungal Taxa Shifts in Response to Larvae of the Tree Hole Mosquito, <Emphasis Type="Italic">Ochlerotatus triseriatus</Emphasis>

Larvae of the eastern tree hole mosquito, Ochlerotatus triseriatus (Say), and related container-breeding species are known to feed upon substrate-associated microorganisms. Although the importance of these microbial resources to larval growth has been established, almost nothing is known about the taxonomic composition and dynamics of these critical microbial food sources. We examined bacterial and fungal community compositional changes on oak leaves tethered in natural tree hole habitats of O. triseriatus. We eliminated larvae experimentally in a subset of the tree holes and examined 16S rDNA gene sequences for bacteria and ergosterol concentrations and 18S rRNA gene sequences for fungi collected from leaf material subsamples. Leaf ergosterol content varied significantly with time, but not treatment. Principal component analysis (PCA) was used to compare microbial taxonomic patterns found in leaves incubated with or without larvae present, and we found that larval presence affected both bacterial and fungal groups, either from loosely attached or strongly adherent categories. Bacterial communities generally grouped more tightly when larvae were present, and class level taxa proportions changed when larvae were present, suggesting selection by larval feeding or activities for particular taxa such as members of the Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria classes. Fungal taxa composite scores also separated along PC axes related to the presence of larvae and indicated larval feeding effects on several higher taxonomic groups, including Saccharomycetes, Dothideomycetes, and Chytridiomycota. These results support the hypothesis that larval mosquito feeding and activities altered microbial communities associated with substrate surfaces, potentially leading to decreased food value of the resource and affecting decomposition of particulate matter in the system.     

Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mosquitoes in the laboratory

The insect growth regulator (IGR) pyriproxyfen is highly active against mosquitoes (Diptera: Culicidae). Through continuous emersion of large larvae (instars 3-4) the concentration causing 50% inhibition of adult emergence (EI50) was determined as 0.200 p.p.b. for Aedes albopictus (Skuse) and 3.5 to 7 times less for Ochlerotatus triseriatus (Say): IE50 0.0288 p.p.b. As a possible method of application to larval microcosms of these species that oviposit in water containers and phytotelmata, the horizontal transfer of pyriproxyfen to larval microcosms by adult mosquitoes was evaluated under laboratory conditions. Gravid females were forced to walk on surfaces treated with pyriproxyfen (tarsal contact exposure) and then allowed to oviposit in larval microcosms. Using replicate bioassay cages, each with an oviposition container, and a factorial experimental design, we assessed Ae. albopictus for the effects of (i) pyriproxyfen concentration (0.2, 0.3 and 0.4 mg/cm2) contacted by gravid females, and (ii) the number of treated gravid females added to bioassay cages (one, three or five females/cage), on the mortality of larvae in oviposition containers. Only 0.2 mg/cm2 treatment rate was tested on Oc. triseriatus. A significant (P < 0.05) curvilinear response in inhibition of emergence (IE) was achieved on both species. Densities of one or three treated Oc. triseriatus females/cage yielded IE rates of only 21-27%, whereas five treated females/cage resulted in 70% inhibition. With Ae. albopictus, densities of three or five treated females/cage yielded 48-67% and 59-73% IE, respectively, whereas one treated female/cage gave only 4-30% inhibition. Use of IGR-treated oviposition containers to achieve horizontal transfer of pyriproxyfen to mosquito oviposition sites could be a field management technique based on mosquito biology and behaviour. In binary choice tests with Ae. albopictus, horizontal transfer of pyriproxyfen from a container with a treated ovistrip (0.3 or 0.4 mg/cm2) to an untreated microcosm resulted in 14-38% inhibition. In larval bioassays, pyriproxyfen activity declined markedly within 10 days. Forcibly exposing gravid female mosquitoes to pyriproxyfen-treated paper surface did not affect their fecundity. However, from the 1st to 2nd gonotrophic cycles the egg hatch rate declined by 30% (P < 0.05). Some variation of results could be due to interactions between females at the oviposition site, possibly causing disproportionate transfer of pyriproxyfen to larval microcosms. Comparative studies of the oviposition behaviour of each mosquito are warranted and would potentially provide information needed to improve the technique.     

Spatial clustering and longitudinal variation of Anopheles darlingi (Diptera: Culicidae) larvae in a river of the Amazon: the importance of the forest fringe and of obstructions to flow in frontier malaria

Deforestation has been linked to a rise in malaria prevalence. In this paper, we studied longitudinally 20 spots, including forested and deforested portions of a temporary river in a malarigenous frontier zone. Larval habitat parameters influencing distribution of Anopheles darlingi (Diptera: Culicidae) larvae were studied. We observed that larvae were clustered in forested-deforested transitions. For the first time in the literature, it was verified that parameters determining larval distribution varied from deforested to forested areas. The proximity to human dwellings was also a significant factor determining distribution, but larvae was most importantly associated with a previously undescribed parameter, the presence of small obstructions to river flow, such as tree trunks within the river channel, which caused pooling of water during the dry season ('microdams'). In deforested areas, the most important factor determining distribution of larvae was shade (reduced luminance). Larvae were absent in the entire studied area during the wet season and present in most sites during the dry season. During the wet-dry transition, larvae were found sooner in areas with microdams, than in other areas, suggesting that flow obstruction prolongs the breeding season of An. darlingi. Adult mosquito densities and malaria incidence were higher during the dry season. Our data correlate well with the published literature, including the distribution of malaria cases near the forest fringes, and has permitted the creation of a model of An. darlingi breeding, where preference for sites with reduced luminance, human presence and microdams would interact to determine larval distribution.     

The mosquitoes of Sardinia: species records 35 years after the malaria eradication campaign

1. Thirty-five years ago malaria was eradicated from Sardinia by massive application of DDT directed against the mosquito vector species, Anopheles labranchiae, but the long-term effects on the mosquito fauna have not been investigated previously. 2. A 5-year mosquito survey (1980-85) involved in the inspection of 348 sites (plus 173 reinspections) and the processing of nearly 60,000 specimens. 3. Twenty-four species in six genera were identified from larval and adult collections. Species of the genera Culex (41%) and Aedes (22%) were more commonly encountered than Culiseta (18%) and Anopheles (17%) species. Orthopodomyia (1.6%) and Coquillettidia (0.6%) were far less frequent. 4. The species composition was similar to that described for the years of the eradication campaign, except for the genus Anopheles. Of the eight Anopheles species previously recorded, only four species were identified in collections of 1159 larvae from eighty-five positive sites. 5. Anopheles labranchiae was the most frequently collected species of its genus (49%), but was represented in only 12% of the total collections. Comparison with previous records indicated a progressive increase in sites positive for An.labranchiae over the past 35 years. 6. Anopheles hispaniola was not found, although it had apparently replaced An.labranchiae soon after the eradication campaign. An. labranchiae was common in the sites where the replacement phenomenon has been observed.