Oviposition habitat selection for a predator refuge and food source in a mosquito

Abstract.  1. The influence of filamentous algae on oviposition habitat selection by the mosquito Anopheles pseudopunctipennis and the consequences of oviposition decisions on the diet, development, body size, and survival of offspring were examined.
2. A natural population of An. pseudopunctipennis in Chiapas, Mexico, oviposited almost exclusively in containers with filamentous algae. Algae represented 47% of the gut contents of mosquito larvae sampled from the natural population. Mosquito larvae fed on an exclusive diet of algae developed as quickly and achieved the same adult body size (wing length) as their conspecifics fed on a standard laboratory diet.
3. Multiple regression of survival of mosquito larvae on percentage surface area cover of algae (0–99%) and the density of predatory fish (zero to four fish per container) was best described by a second-order polynomial model. Increasing fish densities resulted in a reduction in mosquito survival in all algal treatments. The highest incidence of survival was observed at intermediate (66%) algal cover in all treatments.
4. The presence of fish significantly extended larval development times whereas algal cover had no significant effect. The presence of fish resulted in emergence of smaller adults due to reduced feeding opportunities and predator avoidance behaviour. Algal cover also affected mosquito wing length but differently at each fish density.
5. Oviposition habitat selection improves survival in the presence of predators and feeding opportunities for An. pseudopunctipennis larvae.     

Impact of environmental manipulation for Anopheles pseudopunctipennis Theobald control on aquatic insect communities in southern Mexico.

Extraction of filamentous algae from river pools is highly effective for the control of Anophelespseudopunctipennis in southern Mexico. We determined the magnitude of changes to the aquatic insect community following single annual perturbations performed over two years. In 2001, algae were manually removed from all the pools in a 3 km long section of the River Coatán, Mexico, while an adjacent section was left as an untreated control. In 2002, the treatments of both zones were switched and algal extraction was repeated. The abundance of An. pseudopunctipennis larvae + pupae was dramatically reduced by this treatment and remained depressed for two to three months. A total of 11,922 aquatic insects from ten orders, 40 families, and 95 genera were collected in monthly samples taken over five months of each year. Algal extraction did not reduce the overall abundance of aquatic insects in river pools, but a greater abundance and a greater richness of taxa were observed in 2002 compared to the previous year. This was associated with reduced precipitation and river discharge in 2002 compared to 2001. Shannon diversity index values were significantly depressed following algal extraction for a period of three months, in both years, before returning to values similar to those of the control zone. However, differences between years were greater than differences between treatments within a particular year. When insects were classified by functional feeding group (FFG), no significant differences were detected in FFG densities between extraction and control zones over time in either year of the study. Similarly, percent model affinity index values were classified as "not impacted" by the extraction process. Discriminant function analysis identified two orders of insects (Diptera and Odonata), water temperature, dissolved oxygen and conductivity, and river volume (depth, width, and discharge) as being of significant value in defining control and treatment groups in both years. We conclude that habitat manipulation represents an effective and environmentally benign strategy for control of An. pseduopunctipennis. Variation in precipitation and river discharge between years was much more important in determining aquatic insect community composition than variation generated by the filamentous algal extraction treatment.     

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.     

Comparative use of bendiocarb and DDT to control Anopheles pseudopuncitpennis in a malarious area of Mexico

The state of Sinaloa has one of the highest and most persistent malaria transmission levels in Mexico. Due to this situation, with resistance of the vector Anopheles pseudopunctipennis Theobald to DDT, the carbamate insecticide bendiocarb was evaluated as an alternative to DDT for residual house-spraying in village-scale trials during 1985-87. Application rates of the active ingredient per square metre of sprayable surface (ai/m2) were 0.4 g bendiocarb 80% wettable powder (80WP) and 2 g DDT 75% WP. Both insecticides failed to control mosquito populations. Human-bait mosquito densities were not altered as a result of insecticide spraying and human-bait collected mosquito mortality rates were low, suggesting little pre-biting insecticide contact due to avoidance or insufficient resting time indoors. Lower densities of indoor-resting mosquitoes were observed with DDT as opposed to bendiocarb treated houses. Anopheline mortality was higher (98-100%) when exposed for 1 h to 1% bendiocarb in standard WHO susceptibility tests and wall bioassays. Mortality-rates of 15-48% due to 1 h exposure to 4% DDT indicated that this insecticide may continue to be partially effective. House curtain and mark-recapture mosquito studies indicated that DDT produced higher excito-repellency than bendiocarb, as reflected by more mosquito landings but lower feeding rates, shorter resting period and earlier exit time from DDT sprayed houses. In the absence of insecticide, more than 50% of blood-fed An.pseudopunctipennis females exited from houses within 2-4 h of release, showing exophilic behaviour. The outdoor/indoor density ratio indicated that the majority were exophagic. These behavioural characteristics limit the usefulness of any residual insecticide against An.pseudopunctipennis.     

Local adaptation and vector-mediated population structure in Plasmodium vivax malaria

Plasmodium vivax in southern Mexico exhibits different infectivities to 2 local mosquito vectors, Anopheles pseudopunctipennis and Anopheles albimanus. Previous work has tied these differences in mosquito infectivity to variation in the central repeat motif of the malaria parasite's circumsporozoite (csp) gene, but subsequent studies have questioned this view. Here we present evidence that P. vivax in southern Mexico comprised 3 genetic populations whose distributions largely mirror those of the 2 mosquito vectors. Additionally, laboratory colony feeding experiments indicate that parasite populations are most compatible with sympatric mosquito species. Our results suggest that reciprocal selection between malaria parasites and mosquito vectors has led to local adaptation of the parasite. Adaptation to local vectors may play an important role in generating population structure in Plasmodium. A better understanding of coevolutionary dynamics between sympatric mosquitoes and parasites will facilitate the identification of molecular mechanisms relevant to disease transmission in nature and provide crucial information for malaria control.     

Development of Exon-Primed Intron-Crossing (EPIC) PCR primers for the malaria vector Anopheles pseudopunctipennis (Diptera: Culicidae)

Using the Anopheles gambiae Giles genome as a template, we designed, screened and identified 14 novel Exon-Primed Intron-Crossing (EPIC) PCR primer pairs for Anopheles pseudopunctipennis Theobald 1901, a major vector of human Plasmodium sp. in South America. These primers were designed to target the conserved regions flanking consecutive exons of different genes and enabled the amplification of 17 loci of which nine were polymorphic. Polymorphisms at these loci ranged from two to four alleles. Intron length polymorphism analysis is a useful tool, which will allow the study of the population structure of this mosquito species, which remains poorly understood.     

Plasmodium vivax: ookinete destruction and oocyst development arrest are responsible for Anopheles albimanus resistance to circumsporozoite phenotype VK247 parasites

Anopheles albimanus and An. pseudopunctipennis differ in their susceptibilities to Plasmodium vivax circumsporozoite phenotypes. An. pseudopunctipennis is susceptible to phenotype VK247 but almost refractory to VK210. In contrast, An. albimanus is almost refractory to VK247 but susceptible to VK210. To investigate the site in the mosquito and the parasite stage at which resistance mechanisms affect VK247 development in An. albimanus, parasite development was followed in a series of experiments in which both mosquitoes species were simultaneously infected with blood from patients. Parasite phenotype was determined in mature oocysts and salivary gland sporozoites by use of immunofluorescence and Western blot assays and/or gene identification. Ookinete maturation and their densities within the bloodmeal bolus were similar in both mosquito species. Ookinete densities on the internal midgut surface of An. albimanus were 4.7 times higher than those in An. pseudopunctipennis; however, the densities of developing oocysts on the external midgut surface were 6.12 times higher in the latter species. Electron microscopy observation of ookinetes in An. albimanus midgut epithelium indicated severe parasite damage. These results indicate that P. vivax VK247 parasites are destroyed at different parasite stages during migration in An. albimanus midguts. A portion, accumulated on the internal midgut surface, is probably destroyed by the mosquito's digestive enzymes and another portion is most likely destroyed by mosquito defense molecules within the midgut epithelium. A third group, reaching the external midgut surface, initiates oocyst development, but over 90% of them interrupt their development and die. The identification of mechanisms that participate in parasite destruction could provide new elements to construct transgenic mosquitoes resistant to malaria parasites.     

Population interactions between Culex vishnui mosquitoes and their natural enemies in Pondicherry, India.

Population interactions among mosquitoes in the Culex vishnui subgroup, which are vectors of Japanese Encephalitis, and their natural enemies were studied in Pondicherry, India. We tested the hypothesis that the breakdown of interactions between the larvae and their natural enemies due to drought followed by rain was responsible for the sudden increase in the vector population above the threshold for disease transmission during the heavy rainy period. We randomly sampled mosquito larvae and their predators in different breeding habitats and subjected the mean densities of prey, predator, and mosquito larvae infected with parasites/pathogens to covariate analysis to understand the interaction between prey and their natural enemies in relation to environmental factors. In rice fields, neither prey nor predator showed any positive correlation with temperature, RH, or the number of rainy days. However, the pathogen/parasite of mosquito immatures showed a positive correlation with RH. Among the mosquito predators, notonectids exhibited a significant positive correlation with Cx. vishnui larvae. The parasitic Romanomermis iyengari and pathogenic Coelomomyces anopheliscus also showed positive correlations with immatures. No parasites and pathogens of mosquito larvae were recorded in shallow water pools (SWP) or cement tanks (CT) during the study period. Important predators recorded in SWP were notonectids, damselfly nymphs, Diplonychus indicus, and hydrophilids. Dragonfly nymphs, gerrids, and tadpole shrimps were recorded in CT. In CT, prey and their predators were positively correlated with RH and rainy days. In SWP, there was a highly significant correlation between prey, predators and environmental factors. We conclude that rice fields are a stable ecosystem where regular interaction occurs between larvae and their natural enemies and a sudden increase in mosquito populations is uncommon. In transient habitats, no such stability is present and they become more important as breeding habitats in terms of seasonality and number. Shallow water pools should be seriously considered for the control of these vectors.     

Mosquito immunity against Plasmodium

Understanding the molecular mechanisms of the innate immune responses of Anopheles gambiae against Plasmodium parasites is of great importance for current efforts to develop novel strategies for malaria disease control. The parasite undergoes substantial stage-specific losses during its development in the mosquito, which in some cases lead to complete refractoriness of the mosquito against the parasite. The underlying genetics of refractoriness are complex and multifactorial. Completion of the genome sequence of An. gambiae 2 years ago, together with the development of DNA microarrays in this species and the extension of the RNAi technique to adult mosquitoes, has allowed comparative and functional genomic approaches of the mosquito innate immune system. A variety of factors were shown to negatively affect the development of Plasmodium parasites in the mosquito, in some cases leading to complete transmission blockage. In addition, mosquito factors have been identified that play positive roles and are required for successful transmission of the parasite. These findings indicate a highly complex interplay between parasite and vector. Research is continuing to identify new factors involved in this interaction and to decipher the interplay of these molecules and their regulation.     

The multifaceted mosquito anti-Plasmodium response

Plasmodium development within its mosquito vector is an essential step in malaria transmission, as illustrated in world regions where malaria was successfully eradicated via vector control. The innate immune system of most mosquitoes is able to completely clear a Plasmodium infection, preventing parasite transmission to humans. Understanding the biological basis of this phenomenon is expected to inspire new strategies to curb malaria incidence in countries where vector control via insecticides is unpractical, or inefficient because insecticide resistance genes have spread across mosquito populations. Several aspects of mosquito biology that condition the success of the parasite in colonizing its vector begin to be understood at the molecular level, and a wealth of recently published data highlights the multifaceted nature of the mosquito response against parasite invasion. In this brief review, we attempt to provide an integrated view of the challenges faced by the parasite to successfully invade its mosquito host, and discuss the possible intervention strategies that could exploit this knowledge for the fight against human malaria.     

PbCap380, a novel oocyst capsule protein, is essential for malaria parasite survival in the mosquito

An essential requisite for transmission of Plasmodium, the causative agent of malaria, is the successful completion of a complex developmental cycle in its mosquito vector. Of hundreds of ookinetes that form in the mosquito midgut, only few transform into oocysts, a loss attributed to the action of the mosquito immune system. However, once oocysts form, they appear to be resistant to mosquito defences. During oocyst development, a thick capsule forms around the parasite and appears to function as a protective cover. Little information is available about the composition of this capsule. Here we report on the identification and partial characterization of the first Plasmodium oocyst capsule protein (PbCap380). Genetic analysis indicates that the gene is essential and that PbCap380(-) mutant parasites form oocysts in normal numbers but are gradually eliminated. As a result, mosquitoes infected with PbCap380(-) parasites do not transmit malaria. Targeting of the oocyst capsule may provide a new strategy for malaria control.     

A comparison of mosquito densities,weather and infection rates of Aedes aegypti during the first epidemics of Chikungunya (2014) and Zika (2016) in areas with and without vector control in Puerto Rico

In Puerto Rico, the first records of the transmission of Chikungunya (CHIKV) and Zika (ZIKV) viruses were confirmed in May 2014 and December 2015, respectively. Transmission of CHIKV peaked in September 2014, whereas that of ZIKV peaked in August 2016. The emergence of these mosquito‐transmitted arboviruses in the context of a lack of human population immunity allowed observations of whether the outbreaks were associated with Aedes aegypti (Diptera: Culicidae) densities and weather. Mosquito density was monitored weekly in four communities using sentinel autocidal gravid ovitraps (AGO traps) during 2016 in order to provide data to be compared with the findings of a previous study carried out during the 2014 CHIKV epidemic. Findings in two communities protected against Ae. aegypti using mass AGO trapping (three traps per house in most houses) were compared with those in two nearby communities without vector control. Mosquito pools were collected to detect viral RNA of ZIKV, CHIKV and dengue virus. In areas without vector control, mosquito densities and rates of ZIKV detection in 2016 were significantly higher, similarly to those observed for CHIKV in 2014. The density of Ae. aegypti in treated sites was less than two females/trap/week, which is similar to the putative adult female threshold for CHIKV transmission. No significant differences in mosquito density or infection rates with ZIKV and CHIKV at the same sites between years were observed. Although 2016 was significantly wetter, mosquito densities were similar.     

Can Wolbachia be used to control malaria?

Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites transmitted by the infectious bite of Anopheles mosquitoes. Vector control of malaria has predominantly focused on targeting the adult mosquito through insecticides and bed nets. However, current vector control methods are often not sustainable for long periods so alternative methods are needed. A novel biocontrol approach for mosquito-borne diseases has recently been proposed, it uses maternally inherited endosymbiotic Wolbachia bacteria transinfected into mosquitoes in order to interfere with pathogen transmission. Transinfected Wolbachia strains in Aedes aegypti mosquitoes, the primary vector of dengue fever, directly inhibit pathogen replication, including Plasmodium gallinaceum, and also affect mosquito reproduction to allow Wolbachia to spread through mosquito populations. In addition, transient Wolbachia infections in Anopheles gambiae significantly reduce Plasmodium levels. Here we review the prospects of using a Wolbachia-based approach to reduce human malaria transmission through transinfection of Anopheles mosquitoes.     

Exceptional diversity, maintenance of polymorphism, and recent directional selection on the APL1 malaria resistance genes of Anopheles gambiae

The three-gene APL1 locus encodes essential components of the mosquito immune defense against malaria parasites. APL1 was originally identified because it lies within a mapped QTL conferring the vector mosquito Anopheles gambiae natural resistance to the human malaria parasite, Plasmodium falciparum, and APL1 genes have subsequently been shown to be involved in defense against several species of Plasmodium. Here, we examine molecular population genetic variation at the APL1 gene cluster in spatially and temporally diverse West African collections of A. gambiae. The locus is extremely polymorphic, showing evidence of adaptive evolutionary maintenance of genetic variation. We hypothesize that this variability aids in defense against genetically diverse pathogens, including Plasmodium. Variation at APL1 is highly structured across geographic and temporal subpopulations. In particular, diversity is exceptionally high during the rainy season, when malaria transmission rates are at their peak. Much less allelic diversity is observed during the dry season when mosquito population sizes and malaria transmission rates are low. APL1 diversity is weakly stratified by the polymorphic 2La chromosomal inversion but is very strongly subdivided between the M and S "molecular forms." We find evidence that a recent selective sweep has occurred at the APL1 locus in M form mosquitoes only. The independently reported observation of a similar M-form restricted sweep at the Tep1 locus, whose product physically interacts with APL1C, suggests that epistatic selection may act on these two loci causing them to sweep coordinately.     

Fitness of anopheline mosquitoes expressing transgenes that inhibit Plasmodium development

One potential strategy for the control of malaria and other vector-borne diseases is the introduction into wild vector populations of genetic constructs that reduce vectorial capacity. An important caveat of this approach is that the genetic construct should have minimal fitness cost to the transformed vector. Previously, we produced transgenic Anopheles stephensi expressing either of two effector genes, a tetramer of the SM1 dodecapeptide or the phospholipase A2 gene (PLA2) from honeybee venom. Mosquitoes carrying either of these transgenes were impaired for Plasmodium berghei transmission. We have investigated the role of two effector genes for malaria parasite blockage in terms of the fitness imposed to the mosquito vector that expresses either molecule. By measuring mosquito survival, fecundity, fertility, and by running population cage experiments, we found that mosquitoes transformed with the SM1 construct showed no significant reduction in these fitness parameters relative to nontransgenic controls. The PLA2 transgenics, however, had reduced fitness that seemed to be independent of the insertion site of the transgene. We conclude that the fitness load imposed by refractory gene(s)-expressing mosquitoes depends on the effect of the transgenic protein produced in that mosquito. These results have important implications for implementation of malaria control via genetic modification of mosquitoes.     

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell

Plasmodium parasites are fertilized in the mosquito midgut and develop into motile zygotes, called ookinetes, which invade the midgut epithelium. Here we show that a calcium-dependent protein kinase, CDPK3, of the rodent malarial parasite (Plasmodium berghei) is produced in the ookinete stage and has a critical role in parasite transmission to the mosquito vector. Targeted disruption of the CDPK3 gene decreased ookinete ability to infect the mosquito midgut by nearly two orders of magnitude. Electron microscopic analyses demonstrated that the disruptant ookinetes could not access midgut epithelial cells by traversing the layer covering the cell surface. An in vitro migration assay showed that these ookinetes lack the ability to migrate through an artificial gel, suggesting that this defect caused their failure to access the epithelium. In vitro migration assays also suggested that this motility is induced in the wild type by mobilization of intracellular stored calcium. These results indicate that a signalling pathway involving calcium and CDPK3 regulates ookinete penetration of the layer covering the midgut epithelium. Because humans do not possess CDPK family proteins, CDPK3 is a good target for blocking malarial transmission to the mosquito vector.     

Performance of Five Food Regimes on Anopheles gambiae Senso Stricto Larval Rearing to Adult Emergence in Insectary

Background

Rearing of Anopheles gambiae s.s mosquitoes in insectary with quality cheap food sources is of paramount importance for better and healthy colony. This study evaluated larval survival and the development rate of aquatic stages of An.gambiae s.s under five food regimes; tetramin fish food (a standard insectary larval food), maize pollen, Cerelac, green filamentous algae and dry powdered filamentous algae.

Methods

Food materials were obtained from different sources, cerelac was made locally, fresh filamentous algae was taken from water bodies, dry filamentous algae was ground to powder after it was dried under shade, and maize pollen was collected from the flowering maize. Each food source type was used to feed three densities of mosquito larvae 20, 60, and 100 in six replicates each. Larval age structure was monitored daily until pupation and subsequently adult emergence. Tetramin was used and taken as a standard food source for An. gambiae s.s. larvae feeding in Insectary.

Results

Larval survivorship using maize pollen and Tetramin fish food was statistically insignificant (P = 0.564). However when compared to other food regime survivorship was significantly different with Tetramin fish food performing better than cerelac (P<0.001), dry algae (P<0.001) and fresh algae (P<0.001). The pupation rates and sex ratio of emerging adults had significant differences among the food regimes.

Conclusion

The findings of this study have shown that maize pollen had closely similar nutritional value for larval survivorship to tetramin fish food, a standard larvae food in insectary. Further studies are required to assess the effect of food sources on various life traits of the emerged adults.     

The developmental migration of Plasmodium in mosquitoes

Migration of the protozoan parasite Plasmodium through the mosquito is a complex and delicate process, the outcome of which determines the success of malaria transmission. The mosquito is not simply the vector of Plasmodium but, in terms of the life cycle, its definitive host: there, the parasite undergoes its sexual development, which results in colonization of the mosquito salivary glands. Two of the parasite's developmental stages in the mosquito, the ookinete and the sporozoite, are invasive and depend on gliding motility to access, penetrate and traverse their host cells. Recent advances in the field have included the identification of numerous Plasmodium molecules that are essential for parasite migration in the mosquito vector.     

Colonization of abandoned swimming pools by larval mosquitoes and their predators following Hurricane Katrina

Thousands of flooded swimming pools were abandoned in New Orleans following Hurricane Katrina and provided a natural experiment to examine colonization of a novel aquatic habitat by mosquito larvae and their aquatic predators. We conducted a randomized survey of flooded swimming pools in two neighborhoods in January 2006 and found that 64% contained mosquito larvae, 92% contained predatory invertebrates, and 47% contained fishes. We collected 12,379 immature mosquitoes representing five species, primarily Culiseta inornata, and secondarily, the arboviral vector Culex quinquefasciatus. Dragonfly nymphs in the families Aeshnidae and Libellulidae were the most common predatory invertebrates collected among a total of 32 non-mosquito invertebrate species. Eleven species of fishes were collected, with Gambusia affinis accounting for 76% of the catch. Diversity of fishes in swimming pools was positively correlated with proximity to a levee breach and the fish assemblage found in swimming pools was similar to that found along shorelines of Lake Pontchartrain and drainage canals that flooded the study area. Mosquito larvae were rare or absent from pools containing fishes; however, path analysis indicated that the presence of top predators or abundant competitors may somewhat mitigate the effect of Gambusia affinis on mosquito presence.