Influence of temperature on microsporidia infections in a natural population of Simulium pertinax Kollar, 1832 (Diptera; Simuliidae).

During the studies involving the correlation between the water temperature of the breeding site of Simulium pertinax larvae and the infection prevalence by microsporidia, developed in the Andorinhas river, Magé, RJ, weekly samples of blackfly larvae were taken within a two-year period (2001-2002 and 2003-2004), and it was noticed that the infections by Amblyospora sp. were more prevalent when compared to infections by Polydispyrenia sp. in larvae. It was also observed that the infections do not follow the same pattern, since the genus Amblyospora was recorded almost every month during the study with the exception of December, 2001. In the results of correlation between the environmental water temperature and the microsporidia infection rates, it was observed that for the first period studied, there was a high negative correlation, while during the second period there was absence correlation. On the other hand, the Amblyospora sp. infection rates prove that the correlation was high and significant in the first period, but was not significant in the second sampling period and Polydispyrenia sp. showed absence correlation in both periods.

DsRNA-mediated targeting of ribosomal transcripts RPS6 and RPL26 induces long-lasting and significant reductions in fecundity of the vector Aedes aegypti.

Ribosomal transcripts produce critical proteins that are involved in most cellular production processes. Targeting ribosomal transcripts has produced mortality in mites and ticks but the effect of ribosomal transcript knockdown has not been thoroughly examined in mosquitoes. We examine the effects of triggers targeting four ribosomal proteins (RP) transcripts. Although no significant mortality was observed after dsRNA microinjection and subsequent blood feeding, significant contrasts were observed on fecundity. Triggers targeting RPS6 and RPL26 effectively reduced gene expression but more importantly, reduced reproductive output by more than 96% and 91% at the first oviposition while triggers targeting RPL1 and RPS2 did not cause a reduction although gene expression was reduced. Significantly reduced fecundity continued through a second oviposition cycle in dsRPS6 and dsRPL26 cohorts, although the effect was not as strong. Relative gene expression levels confirmed specific transcript knockdown up to 20days post-injection in mosquitoes that did not oviposit or produced reduced clutch sizes. Dissections at 36h post-blood meal indicated defects in oocyte provisioning. The strong phenotype produced by dsRPS6 allowed us to examine the effects in various tissues as well as the dose response, trigger format, delivery method and trigger specificity in Aedes aegypti. Strong knockdown was observed in the abdomen and the ovaries. Greater than 50ng of dsRPS6 significantly reduced fecundity but not when delivered in a sugar meal or as an siRNA. Similar bioassays with mutated dsRPS6 triggers indicates that up to three mismatches per possible siRNA are still effective in reducing fecundity. These studies indicate that while active and effective triggers can be developed for vector species, the lack of an efficient delivery method is the biggest barrier to use as a potential control method.

Microsporidia - Emergent Pathogens in the Global Food Chain.

Intensification of food production has the potential to drive increased disease prevalence in food plants and animals. Microsporidia are diversely distributed, opportunistic, and density-dependent parasites infecting hosts from almost all known animal taxa. They are frequent in highly managed aquatic and terrestrial hosts, many of which are vulnerable to epizootics, and all of which are crucial for the stability of the animal-human food chain. Mass rearing and changes in global climate may exacerbate disease and more efficient transmission of parasites in stressed or immune-deficient hosts. Further, human microsporidiosis appears to be adventitious and primarily associated with an increasing community of immune-deficient individuals. Taken together, strong evidence exists for an increasing prevalence of microsporidiosis in animals and humans, and for sharing of pathogens across hosts and biomes.

Edhazardia aedis (Microspora: Culicosporidae) effects on the reproductive capacity of Aedes aegypti (Diptera: Culicidae).

The influence of the microsporidium Edhazardia aedis (Kudo) on the survival and reproduction of its mosquito host, Aedes aegypti (L.), was studied in the laboratory. Survival, fecundity, egg hatch, and percentage of emergence for 4 gonotrophic cycles were compared for control and infected mosquitoes. Control females oviposited an average of 123.1 eggs over 4 gonotrophic cycles, 86.1% of which hatched, whereas infected females laid an average of 38.0 eggs with a 69.3% hatch. Emergence in progeny of infected female Ae. aegypti was significantly less than for control mosquitoes in all gonotrophic cycles. The reproductive capacity (Ro) for control and infected adults was 168.4 and 4.1, respectively, representing a decrease of 98.2%. Overall infection levels in progeny of infected females was 95.7%, of which 46.6% were lethal, larval infections. Infected female survivors were capable of initiating infections in F2 progeny. Wing length, often used as an indicator of fitness, was not significantly different between infected and control adults.

Ultrastructure of Culicosporella lunata (Microsporida: Culicosporellidae fam. n.) in the mosquito Culex pilosus (Diptera: Culicidae) with new information on the developmental cycle.

The ultrastructural cytology of Culicosporella lunata is described from larvae of its mosquito host Culex pilosus. Diplokaryotic sporonts entered 1 of 12 fundamentally different sporulation sequences that occurred concurrently, each producing morphologically distinctive spores. One involved abortive meiosis and rarely ended with meiospores; the other, the predominant one, involved diplokaryotic sporogonial plasmodia and produced lanceolate, binucleate spores. A previously unknown sporulation sequence in the adult mosquito was found to result in a second type of binucleate spore. The taxonomic affinities of Culicosporella lunata are discussed and because of its distinctive characters, Culicosporellidae fam. n. is created.

Safety of Edhazardia aedis (Microspora: Amblyosporidae) for nontarget aquatic organisms.

The susceptibility of common nontarget aquatic organisms to the microsporidium Edhazardia aedis was investigated in the laboratory. Eight predacious species along with 9 scavengers and filter feeders were tested. The nontarget organisms were not susceptible to infection by E. aedis and there was no appreciable mortality. To measure the relative safety of E. aedis to nontarget organisms, a simple mathematical expression was employed where risk is defined as the product of the probability of exposure and the result of exposure (infection) expressed as P(e)P(i). In these laboratory tests, the probability of exposure was fixed at 1 (maximum challenge) and the probability of infection was determined to be 0. Therefore, the risk associated with release of E. aedis into the environment is considered to be negligible under these conditions. The true risk for nontarget organisms to E. aedis can only be determined by careful evaluation of controlled field studies in the natural habitat of the target host.

A device for monitoring populations of larval mosquitoes in container habitats.

A device was developed for repetitive sampling of mosquito larvae without undue disruption of the larval habitat. The sampler is a 3-oz. (ca. 100-ml capacity) transparent plastic cup with a hole in the center of its convex bottom. The device is buoyed by corks so that the water level is 15 mm above the bottom rim of the cup and 5 mm above the hole. There was significant correlation between 24-h samples of Aedes albopictus and Culex quinquefasciatus 4th-instar larvae in the larval sampling device and populations in tires. Greater numbers of immature mosquitoes were found per unit surface area of the sampling device than the tire as a whole, demonstrating that immature mosquitoes were trapped by the sampler.

Mosquito host range and specificity of Edhazardia aedis (Microspora: Culicosporidae).

Edhazardia aedis was transmitted horizontally to its natural host, Aedes aegypti, and to 6 alternate hosts: Ae. albopictus, Ae. triseriatus, Ae. taeniorhynchus, Anopheles quadrimaculatus, Orthopodomyia signifera and Toxorhynchites rutilus rutilus. The microsporidium produced both binucleate and uninucleate spores in all susceptible hosts. Transovarial transmission, however, was only successful in Ae. aegypti. Therefore, while E. aedis can infect a variety of mosquito species from diverse genera, it is specific for its natural host, Ae. aegypti. Five other mosquito species were not susceptible to E. aedis.

Seasonality, prevalence and pathogenicity of the gregarine Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in mosquitoes from Florida.

Aedes albopictus larvae collected in Gainesville, FL, were infected with the gregarine Ascogregarina taiwanensis. Natural prevalence varied from 68 to 100%. Eight mosquito species were tested in the laboratory for susceptibility to A. taiwanensis isolated from field-collected Ae. albopictus. Aedes aegypti, Aedes albopictus, and Aedes taeniorhynchus became 100% infected in the larval stage, whereas Aedes triseriatus was less susceptible; Culex quinquefasciatus, Culex nigripalpus, Culex territans, and Anopheles quadrimaculatus were not susceptible. Viable A. taiwanensis oocysts from adults were recovered from Ae. taeniorhynchus (30%) and Ae. albopictus (100%); no oocysts were produced in the other exposed hosts. Mortality induced by A. taiwanensis infection was low in all mosquitoes except Ae. taeniorhynchus. We conclude that A. taiwanensis has little short-term impact on the mortality of the 3 most common container-inhabiting mosquito species in Florida; however, the long-term impact on overall host population regulation has yet to be determined.

Effects of three larvicides on the production of Aedes albopictus based on removal of pupal exuviae.

The production of adult Aedes albopictus from tires in northcentral Florida was monitored for 169 days by the daily removal of pupal exuviae. More than twice as many adults emerged from tires located in the shade (1.74 adults/tire/day) compared to tires in the sun (0.64 adults/tire/day). The effect of 3 larvicides on the production of adult Ae albopictus was evaluated. The fungal pathogen Lagenidium giganteum was ineffective. A liquid formulation of Bacillus thuringiensis israelensis (Acrobe) provided significant control for 47 days, whereas a slow-release pellet formulation of the insect growth regulator methoprene (Altosid) provided almost complete control for 116 days.

Effect of per os Edhazardia aedis (Microsporida: Amblyosporidae) infection on Aedes aegypti mortality and body size.

Infection with Edhazardia aedis uninucleate spores had less effect on Aedes aegypti larval mortality and adult body size than did larval diet. Larval mortality averaged 60-81% in starved larvae and 2-16% in well-fed larvae. No significant amounts of larval mortality could consistently be attributed to exposure to the parasite at dosages of 1.5 x 10(3) or 1.5 x 10(5) spores/ml. Infection rates in adults surviving exposure to the parasites as larvae ranged from 30 to 59%. Infected adults had significantly smaller body sizes than uninfected adults or controls. Storage of spores in water reduced infectivity gradually over the course of 36 h. By 48 h, the spores were not able to infect mosquito larvae. Spore infectivity was eliminated by drying.

Pathogenicity Tests on Nine Mosquito Species and Several Non-target Organisms with Strelkovimermis spiculatus (Nemata Mermithidae).

Nine species of mosquitoes and several species of non-target aquatic organisms were tested for susceptibility to the mernaithid nematode, Strelkovimermis spiculatus. All species of Anopheles, Aedes, Culex, and Toxorhynchites exposed to S. spiculatus were susceptible. Of the nine mosquito species tested, C. pipiens quinquefasciatus had the greatest tolerance to initial invasion and the highest percent infection of those that survived. High levels of infection were also achieved with Aedes taeniorhynchus and A. albopictus, but these mosquitoes were significantly less tolerant to parasitism than C. pipiens quinquefasciatus. Strelkovimermis spiculatus did not infect or develop in any of the non-target hosts tested.

Induction of reaper ortholog mx in mosquito midgut cells following baculovirus infection.

Many vertebrate and insect viruses possess antiapoptotic genes that are required for their infectivity. This led to the hypothesis that apoptosis is an innate immunoresponse important for limiting virus infections. The role of apoptosis may be especially important in insect antiviral defense because of the lack of adaptive immunity. However, the cellular mechanism that elicits apoptosis in response to viral infection in insects has not been determined. Using an in vivo infection system with the mosquito baculovirus CuniNPV (Culex nigripalpus nucleopolyhedrovirus), we demonstrated that michelob_x (mx), the mosquito ortholog of Drosophila proapoptotic gene reaper, is specifically induced in larval midgut cells following viral infection. Interestingly, the dynamics of mx induction corresponds with the outcome of the infection. In the permissive mosquito C. quinquefasciatus, a slow induction of mx failed to induce prompt apoptosis, and the infected cells eventually undergo necrosis with heavy loads of encapsulated viruses. In contrast, in the refractory mosquito Aedes aegypti, a rapid induction of mx within 30 min p.i. is followed by apoptosis within 2-6 h p.i., suggesting a possible role for apoptosis in limiting viral infection. When the execution of apoptosis was delayed by caspase inhibitors, viral gene expression became detectable in the A. aegypti larvae.

Epizootiological studies of Amblyospora camposi (Microsporidia: Amblyosporidae) in Culex renatoi (Diptera: Culicidae) and Paracyclops fimbriatus fimbriatus (Copepoda: Cyclopidae) in a bromeliad habitat.

The epizootiology of Amblyospora camposi was studied in a natural population of Culex renatoi, a bromeliad-inhabiting mosquito, and its intermediate host, Paracyclops fimbriatus fimbriatus, over a 2-year period. Twenty Eryngium cabrerae plants were sampled monthly from January 2003 to January 2005 and the prevalence of A. camposi in P.f. fimbriatus and Cx. renatoi populations was determined. The monthly prevalence rates of meiospore infections in Cx. renatoi larvae never exceeded 5.5% and was detected in 50% of the monthly samples. Meiospores were available in plants over the course of the study at a mean concentration of 2 x 10(4) meiospores/ml. Within each plant the parasite was maintained by horizontal transmission. P.f. fimbriatus with vegetative stages and mature spores were found regularly in bromeliads suggesting efficient meiospore infectivity to field copepod populations. The mean concentration of spores from copepods found in plants was 8 x 10(2) spores/ml. Infections in copepods were detected in 54% of the monthly samples with a prevalence rate ranging from 0.55 to 17.4% and an overall average of 5.1%. Vegetative stages in fourth instar mosquito larvae (probably derived from the horizontal pathway via spores formed in copepods) were detected in 12.5% of the monthly samples with an overall prevalence rate of 1.1%. Infections in female and male adults were detected in 20.8% of the monthly samples with an overall average of 4.1% and 6.8%, respectively.

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae.

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.

Potential of microsporidia for the biological control of mosquitoes.

The use of larvicides for the control of disease vector mosquitoes is not always practical. Tony Sweeney and Jimmy Becnel discuss prospects for the use of microsporidio, particularly Edhazardia aedis, as biocontrol agents.

Influence of temperature on developmental parameters of the parasite/host system Edhazardia aedis (Microsporida: Amblyosporidae) and Aedes aegypti (Diptera: Culicidae).

Larvae of Aedes aegypti, transovarially infected with Edhazardia aedis, were reared between 20 and 36 degrees C to determine the influence of temperature on the development of the parasite and the infected host. Development of the parasite was evaluated based on spore yield and size. The predicted optimum temperature for maximum spore production of E. aedis in A. aegypti was 30.8 degrees C. The results demonstrate that the E. aedis-A. aegypti system has a wide temperature tolerance; whereas spore yield will be lower at unfavorable temperatures, the host will remain infected. Additionally, spores were significantly smaller from individual reared at 34 degrees C than those reared at either 20 or 27 degrees C. Development of the infected host was evaluated based on pupal weight and time of pupation. Infected pupae were significantly larger than uninfected pupae. There was also a significant difference in the pupation rate between controls and infected A. aegypti larvae. Controls had a 50% cumulative pupation time (CPT50) of 65.7 degree days and infected individuals a CPT50 of 76.6 degree days.