First report of natural Wolbachia infections in mosquitoes from Cuba.

Mosquitoes are extensively responsible for the transmission of pathogens. Novel strategies using Wolbachia could transform that scenario, since these bacteria manipulate mosquito reproduction, and can confer a pathogen transmission-blocking phenotype in culicids. Here, we screened the Wolbachia surface protein region by PCR in eight Cuban mosquito species. We confirmed the natural infections by sequencing and assessed the phylogenetic relationships among the Wolbachia strains detected. We identified four Wolbachia hosts: Aedes albopictus, Culex quinquefasciatus, Mansonia titillans, and Aedes mediovittatus (first report worldwide). Knowledge of Wolbachia strains and their natural hosts is essential for future operationalization of this vector control strategy in Cuba.

Sterile Insect Technique: Successful Suppression of an Aedes aegypti Field Population in Cuba.

Dengue virus infections are a serious public health problem worldwide. Aedes aegypti is the primary vector of dengue in Cuba. As there is no vaccine or specific treatment, the control efforts are directed to the reduction of mosquito populations. The indiscriminate use of insecticides can lead to adverse effects on ecosystems, including human health. The sterile insect technique is a species-specific and environment-friendly method of insect population control based on the release of large numbers of sterile insects, ideally males only. The success of this technique for the sustainable management of agricultural pests has encouraged its evaluation for the population suppression of mosquito vector species. Here, we describe an open field trial to evaluate the effect of the release of irradiated male Ae. aegypti on a wild population. The pilot trial was carried out in a suburb of Havana and compared the mosquito population density before and after the intervention, in both untreated control and release areas. The wild population was monitored by an ovitrap network, recording frequency and density of eggs as well as their hatch rate. A significant amount of sterility was induced in the field population of the release area, as compared with the untreated control area. The ovitrap index and the mean number of eggs/trap declined dramatically after 12 and 5 weeks of releases, respectively. For the last 3 weeks, no eggs were collected in the treatment area, clearly indicating a significant suppression of the wild target population. We conclude that the sterile males released competed successfully and induced enough sterility to suppress the local Ae. aegypti population.

Effect of Chlorine and Temperature on Larvicidal Activity of Cuban Bacillus thuringiensis Isolates.

BACKGROUND: The efficacy of biolarvicides may be influenced by species of mosquito, larval age and density, temperature, water quality, bacterial formulation, and others. The aim of this study was to evaluate the influence of temperature and chlorine on larvicidal activity of Bacillus thuringiensis Cuban isolates against Aedes aegypti. METHODS: The influence of temperature (25, 30, 35 °C) and chlorine (2.25mg/L) on the larvicidal activity of eleven B. thuringiensis Cuban isolates (collected between 2007 and 2009) were tested under laboratory conditions following WHO protocols. Bioassay data were analyzed by Probit program. The effect of chlorine and temperature (25, 30, 35 and 40 °C) on the Cry and Cyt proteins of these isolates was determined by SDS-PAGE polyacrylamide gel electrophoresis. RESULTS: The pathogenicity of the isolates U81, X48 was affected at 35 °C. However, A21, A51, L910, and R89 isolates increase their entomopathogen activity at 35 °C. No differences were observed in toxicity of M29, R84, R85 and R87 isolates at different temperatures. The Cry 4, Cry 10 and Cry 11 proteins were reduced in A21, X48, R85 isolates at 35 and 40 °C. The Cyt proteins were reduced at 35 and 40 °C in A21, X48, R85, and A51 isolates. In L910 and R84 isolates, the Cyt toxin was degraded only at 40 °C. In chlorinated water, the lethal concentrations 50 and 90 in A21, A51, M29, R84, U81, and X48 isolates were increase. CONCLUSION: A21, A51, L910, R85, and X48 isolates have a strong larvicidal activity for the treatment of Ae. aegypti breeding's sites exposed to high temperature and chlorine.

Infección por Angiostrongylus cantonensis (Nematoda: Metastrongylidae) en Rattus rattus (Rodentia: Muridae) en una localidad de La Habana, Cuba / Infection by Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Rattus rattus (Rodentia: Muridae) in a locality of Havana, Cuba

Introducción: La meningoencefalitis eosinofílica producida por Angiostrongylus cantonensis (Chen, 1935), es una zoonosis emergente presente en Cuba. En el país existen escasos estudios de prevalencia de infección en los hospederos definitivos, que puedan determinar sitios de riesgo para el establecimiento de medidas efectivas en la prevención de la enfermedad. Objetivo: Determinar la prevalencia de infección por A. cantonensis en una población natural de R. rattus, su relación con la estructura poblacional y la época del año en una finca que pertenece al Programa de la Agricultura Urbana y Suburbana de Cuba. Material y Métodos: Se realizó un estudio descriptivo en el municipio La Lisa, La Habana. Se hicieron capturas de roedores mensuales durante los períodos lluvioso y poco lluvioso. Se examinaron las arterias pulmonares de los roedores capturados en búsqueda de parásitos adultos. El índice parasitológico de prevalencia fue calculado y comparadas las frecuencias entre los períodos del año y la edad ecológica. Resultados: Se capturaron 63 roedores identificados como Rattus rattus, de ellos 46,03 por ciento estuvo infectado con A. cantonensis, con un incremento de la infección en los individuos adultos (59,46 por ciento) y en el período lluvioso (55,88 por ciento). Conclusiones: Se demuestra por primera vez en Cuba la infección de A. cantonensis en R. rattus. La presencia de individuos adultos, la alta abundancia de roedores y la temporada lluviosa son los factores que incidieron en una mayor infección de A. cantonensis, y que aumentan la probabilidad de transmisión a los hospederos intermediarios y, por ende, el riesgo de transmisión al humano(AU)

Introduction: Eosinophilic meningitis caused by Angiostrongylus cantonensis (Chen, 1935) is an emergent zoonosis present in Cuba. In the country, few studies about the prevalence of infection in definitive hosts, which can determine risk sites for the establishment of effective measures to prevent the disease, have not been carried out so far. Objective: To determine the prevalence of infection with Angiostrongylus cantonensis in a natural population of Rattus rattus, its relationship with the population structure, and the season of the year in an urban farm field belonging to the Programme of Urban and Suburban Agriculture of Cuba. Material and Methods: A descriptive study was conducted in La Lisa Municipality, Havana, Cuba. The rats were collected monthly during the rainy and dry seasons. The pulmonary arteries of the captured rodents were examined for adult worms. The prevalence of parasitological indices was calculated. The frequencies of infection were compared between the seasons of the year and ecological ages. Results: A total of 63 rodents identified as Rattus rattus were captured. Of the total of individuals captured, 46.03 percent were infected with A. cantonensis, with an increase of infection in adult individuals (59.46 percent) and during the rainy season (55.88 percent). Conclusions: The infection of A. cantonensis in its definitive host R. rattus, and its circulation in the study area in the analyzed period was demonstrated for the first time in Cuba. The presence of adult rats, the high quantity of rodents and the rainy season were the factors that contributed to a greater infection of A. cantonensis, which increase the probability of transmission to intermediate hosts, and consequently, the risk of transmission to humans(AU)

Large indoor cage study of the suppression of stable Aedes aegypti populations by the release of thiotepa-sterilised males

The sterile insect technique (SIT) is a promising pest control method in terms of efficacy and environmental compatibility. In this study, we determined the efficacy of thiotepa-sterilised males in reducing the target Aedes aegypti populations. Treated male pupae were released weekly into large laboratory cages at a constant ratio of either 5:1 or 2:1 sterile-to-fertile males. A two-to-one release ratio reduced the hatch rate of eggs laid in the cage by approximately a third and reduced the adult catch rate by approximately a quarter, but a 5:1 release drove the population to elimination after 15 weeks of release. These results indicate that thiotepa exposure is an effective means of sterilising Ae. aegypti and males thus treated are able to reduce the reproductive capacity of a stable population under laboratory conditions. Further testing of the method in semi-field enclosures is required to evaluate the mating competitiveness of sterile males when exposed to natural environmental conditions. If proven effective, SIT using thiotepa-sterilised males may be incorporated into an integrated programme of vector control to combat dengue in Cuba.

Large indoor cage study of the suppression of stable Aedes aegypti populations by the release of thiotepa-sterilised males.

The sterile insect technique (SIT) is a promising pest control method in terms of efficacy and environmental compatibility. In this study, we determined the efficacy of thiotepa-sterilised males in reducing the target Aedes aegypti populations. Treated male pupae were released weekly into large laboratory cages at a constant ratio of either 5:1 or 2:1 sterile-to-fertile males. A two-to-one release ratio reduced the hatch rate of eggs laid in the cage by approximately a third and reduced the adult catch rate by approximately a quarter, but a 5:1 release drove the population to elimination after 15 weeks of release. These results indicate that thiotepa exposure is an effective means of sterilising Ae. aegypti and males thus treated are able to reduce the reproductive capacity of a stable population under laboratory conditions. Further testing of the method in semi-field enclosures is required to evaluate the mating competitiveness of sterile males when exposed to natural environmental conditions. If proven effective, SIT using thiotepa-sterilised males may be incorporated into an integrated programme of vector control to combat dengue in Cuba.

Release of thiotepa sterilized males into caged populations of Aedes aegypti: life table analysis.

Successful SIT trials against mosquitoes in the 1960-70s were achieved by sterilizing male mosquitoes using chemosterilants. Their use was discontinued after concerns were raised about the effect of residues on non-target organisms, although scant evidence has been published. Irradiation is an expensive process; chemosterilization could be an affordable option for implementing SIT programs in developing countries. We compare life table parameters of three Aedes aegypti populations comprising different ratios of thiotepa-treated and non-treated males in order to identify the impact on reproductive potential of the presence of sterile males. No difference was observed in the survival of the treated and untreated males. The release of thiotepa sterilized males into caged Ae. aegypti populations had no effect on death or survival probability of the individuals in the cages but the fecundity of females was significantly reduced, as evaluated by hatch rate and stable age structure parameters. The significant decreases in net reproduction rate, finite rate of natural increase and intrinsic rate of natural increase in populations including sterile males are sufficient to indicate that such populations would not be able to proliferate in natural conditions. This suggests that release of Ae. aegypti thiotepa-treated males could be effective in reducing the reproductive capability of the target population and consequently contribute to vector control.

Isolation and characterization of entomopathogenic bacteria from soil samples from the western region of Cuba.

The use of insect pathogens is a viable alternative for insect control because of their relative specificity and lower environmental impact. The search for wild strains against dipterans could have an impact on mosquito control programs. We have made an extensive screening of soil in western Cuba to find bacteria with larvicidal activity against mosquitoes. A total of 150 soil samples were collected and isolates were identifying using the API 50 CHB gallery. Phenotypic characteristics were analyzed by hierarchical ascending classification. Quantitative bioassays were conducted under laboratory conditions following the World Health Organization protocol in order to ascertain the toxicity and efficacy of isolates. The protein profiles of the crystal components were determined by SDS-PAGE. Eight hundred and eighty-one bacterial isolates were obtained, and 13 isolates with entomopathogenic activity were isolated from nine samples. Nine isolates displayed higher entomopathogenic activity against both Cx. quinquefasciatus and Ae. aegypti compared with the reference strain 266/2. All toxic isolates showed higher biological potency than the 266/2 strain. These isolates with high entomopathogenic activity displayed a protein pattern similar to the B. thuringiensis var. israelensis IPS-82 and 266/2 strains. These results are a valuable tool for the control of Diptera of medical importance.

Efecto patogénico de 3 nematodos parásitos en larvas de Aedes aegypti en condiciones de laboratorio, en Cuba / Pathogenic effect of 3 parasitic nematodes in Aedes aegypti larvae under laboratory conditions in Cuba

Se evaluó el efecto patogénico de 3 nematodos parásitos Romanomermis culicivorax Ross y Smith, 1976, Romanomermis iyengari Welch, 1964 y Strelkovimermis spiculatus Poinar y Camino, 1986, a diferentes dosis de aplicación, en larvas de Aedes aegypti Linnaeus, 1762. Para cada unidad experimental se utilizaron 100 larvas de II estadio de este mosquito y se infestaron con nematodos preparasíticos de las 3 especies, en proporciones de 3:1, 5:1, 10:1, 15:1 y 20:1. Las medias de infestación y las tasas de parasitismo aumentaron en la medida en que se incrementaron las dosis. A bajas densidades de nemátodos (3 y 5 nemátodos/larva) R culicivorax resultó ser más efectivo al compararla con las otras especies, sin embargo con una proporción de 10:1 todos los nemátodos rovocaron 100 por ciento de mortalidad, siendo S spiculatus el nemátodo que provocó las medias de infestación más altas. Por esta razón, una proporción de 10:1 pudiera ser recomendada para que estos nematodos sean evaluados en condiciones naturales en recipientes artificiales no útiles como una alternativa biológica para el control de Aedes aegypti

[Pathogenic effect of 3 parasitic nematodes in Aedes aegypti larvae under laboratory conditions in Cuba]. / Efecto patogénico de 3 nematodos parásitos en larvas de Aedes aegypti en condiciones de laboratorio, en Cuba.

The pathogenic effect of three parasitic nematodes, Romanomermis culicivorax Ross y Smith, 1976, Romanomermis iyengari Welch, 1964, and Strelkovimermis spiculatus Poinar and Camino, 1986, was evaluated at different application doses in larvae of Aedes aegypti. For each experimental unit, one hundred second instar larvae of this mosquitoe were infested with preparasites of the three nematode species in the following proportions: 3:1, 5:1, 10:1, 15:1 and 20:1. Both infestation mean and parasitism rate increased as the doses augmented. Romanomermis culicivorax proved to be more effective at low doses when compared to other species; however, with the 10:1 proportion, all the nematodes caused 100% of mortality. S. spiculatus showed the highest infestation rate. For this reason, a proportion of 10:1 could be recommended to evaluate these nematodes under natural conditions in useless artificial containers as a biological alternative for Aedes aegypti control.

Laboratory and field studies of Macrocyclops albidus (Crustacea: Copepoda) for biological control of mosquitoes in artificial containers in a subtropical environment.

The cyclopoid copepod Macrocyclops albidus (Jurine) was tested as a potential biological control agent of mosquitoes in laboratory microcosms, in controlled field conditions, and in a 22-mo field experiment using discarded tires. The predator was highly efficient in controlling mosquitoes in all three settings, reaching close to 90% reduction in larval survival under field conditions and exceeding the recommended predation rates for effective mosquito control in laboratory experiments. The predator was most effective on 1-4-d-old larvae. Alternate food and habitat structure significantly influenced the predation rates on mosquito larvae. Once established, the copepod was able to maintain reproducing populations in the field for the duration of the experiments. However, the predator failed to establish populations at four of the experimental field sites. Two of the failures can be attributed to characteristics of the individual tires, such as leaching chemicals, whereas the other two were probably due to site-specific factors. This copepod species is a promising candidate for control of mosquito larvae because it is a widespread and highly effective predator that is capable of establishing and maintaining populations under a wide variety of field conditions. Additionally, M. albidus is relatively easy to culture, maintain, and deliver to the target areas.