Neochloris aquatica induces larval mortality, molting defects, and unstable flightless adults in the Asian tiger mosquito.

The Asian tiger mosquito, Aedes albopictus, is a highly invasive and aggressive species capable of transmitting a large number of etiological agents of medical and veterinary importance, posing a high risk for the transmission of emerging viruses between animals and humans. In this work, we evaluated the mosquitocidal activity of Neochloris aquatica against A. albopictus throughout its development and analyzed whether this effect was potentiated when the microalga was cultivated under stress conditions due to nutrient deprivation. Our results suggest that N. aquatica produces metabolites that have negative effects on these insects, including larval mortality, interruption of pupal development, and incomplete emergence of adults when fed on microalgae in the larval stages. When microalgae were cultured under stress conditions, an increase in molting defects was recorded, and the number of healthy adults emerged drastically decreased. Histological studies revealed severe signs of total disintegration of different tissues and organs in the thorax and abdomen regions. The muscles and fat bodies in the midgut and foregut were severely distorted. In particular, larval intestinal tissue damage included vacuolization of the cytoplasm, destruction of brush border microvilli, and dilation of the intercellular space, which are distinctive morphological characteristics of apoptotic cells. Evidence suggests that N. aquatica produces metabolites with mosquitocidal effects that affect development and, therefore, the ability to vector etiological agents of medical and veterinary importance.

Wolbachia dominance influences the Culex quinquefasciatus microbiota.

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022.

Objectives: To implement and evaluate the use of wastewater sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods: In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sampler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription polymerase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results: In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrredon, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions: It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the usefulness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022

[ABSTRACT]. Objectives. To implement and evaluate the use of wastewater sampling for detection of severe acute respira- tory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods. In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sam- pler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription poly- merase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results. In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrre- don, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions. It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the useful- ness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

[RESUMEN]. Objetivos. Aplicar y evaluar la utilización de muestreos de aguas residuales como método para la detección del coronavirus del síndrome respiratorio agudo severo de tipo 2 (SARS-CoV-2) en dos distritos costeros de la Provincia de Buenos Aires, Argentina. Métodos. Se utilizó un dispositivo de muestreo automático para tomar muestras de 400 mL de las aguas residuales de 24 horas en el distrito de General Pueyrredon, mientras que en el distrito de Pinamar se tomaron muestras de 2,2 L a intervalos de 20 minutos hasta un volumen total de 20 L. Los muestreos se realizaron una vez por semana. Las muestras se concentraron mediante floculación con policloruro de aluminio. La purificación del ARN y la amplificación y detección del gen diana se llevaron a cabo mediante la prueba de reacción en cadena de la polimerasa con retrotranscripción para el diagnóstico clínico a partir de hisopados nasofaríngeos. Resultados. Se observó la presencia de SARS-CoV-2 en las aguas residuales de ambos distritos. En General Pueyrredon, el SARS-CoV-2 se halló en la semana epidemiológica 28 del 2020, es decir, 20 días antes del inicio del aumento de casos de enfermedad por coronavirus 2019 (COVID-19) registrado en la primera ola (semana epidemiológica 31) y nueve semanas antes de que se alcanzara el número máximo de casos de COVID-19 con confirmación de laboratorio. En el distrito de Pinamar se detectó el genoma viral en la semana epidemiológica 51 del 2020, pero solo se pudo volver a realizar el muestreo en la semana epidemiológica 4 del 2022, en la que se volvió a detectar la circulación del virus. Conclusiones. Se pudo detectar el genoma del virus SARS-CoV-2 en aguas residuales, lo que muestra la utilidad de la aplicación de la epidemiología de aguas residuales como método para la detección y el segui- miento del SARS-CoV-2 a largo plazo.

[RESUMO]. Objetivos. Implementar e avaliar o uso de amostragem de águas residuais na detecção do coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) em dois distritos costeiros da Província de Buenos Aires, Argentina. Métodos. No distrito de General Pueyrredon, amostras de 400 mL de águas residuais foram coletadas ao longo de 24 horas com um amostrador automático; já no distrito de Pinamar, foram coletados 20 L no total (2,2 L a intervalos de 20 minutos). As amostras foram coletadas uma vez por semana e concentradas por flocu- lação com cloreto de polialumínio. A purificação do RNA e a amplificação e detecção de genes-alvo foram realizadas por meio de reação em cadeia da polimerase com transcrição reversa para diagnóstico clínico de esfregaços nasofaríngeos humanos. Resultados. Detectou-se presença de SARS-CoV-2 em águas residuais dos dois distritos. Em General Puey- rredon, o SARS-CoV-2 foi detectado na semana epidemiológica 28 de 2020, ou seja, 20 dias antes do início de um aumento no número de casos da doença provocada pelo coronavírus de 2019 (COVID-19) na primeira onda (semana epidemiológica 31) e 9 semanas antes de se registrar o número máximo de casos de COVID-19 confirmados em laboratório. No distrito de Pinamar, o genoma viral foi detectado na semana epidemiológica 51 de 2020, mas não foi possível realizar a amostragem novamente até a semana epidemiológica 4 de 2022, quando a circulação do vírus foi novamente constatada. Conclusões. Foi possível detectar o genoma do vírus SARS-CoV-2 em águas residuais, demonstrando a uti- lidade da aplicação da epidemiologia baseada em águas residuais para detectar e monitorar o SARS-CoV-2 em longo prazo.

Hormone released by the microalgae Neochlorisaquatica and alkalinization influence growth of terrestrial and floating aquatic plants.

The microalgae Neochloris aquatica were previously evaluated as a potential biological control agent and source of bioactive compounds against immature stages of Culex quinquefasciatus. Larvae reared on microalgae suspension showed mortality or drastic effects with morphological alterations and damage in the midgut. N. aquatica have nutritional and toxic effects, resulting in delayed life cycle and incomplete adult development. Given the possibility of its use as a biological control agent, in this work we evaluate the effect of microalgae on other organisms of the environment, such as plants. Arabidopsis thaliana, a terrestrial plant, and Lemna sp., a floating aquatic plant, were selected as examples. Interaction assays and compound evaluations showed that the microalgae release auxins causing root inhibition, smaller epidermal cells and hairy root development. In Lemna sp., a slight decrease in growth rate was observed, with no deleterious effects on the fronds. On the other hand, we detected a detrimental effect on plants when interactions were performed in a closed environment, in a medium containing soluble carbonate, in which microalgae culture rapidly modifies the pH. The experiments showed that alkalinization of the medium inhibits plant growth, causing bleaching of leaves or fronds. This negative effect in plants was not observed when plants and microalgae were cultured in carbonate-free media. In conclusion, the results showed that N. aquatica can modify plant growth without being harmful, but the rapid alkalinization produced by carbon metabolism of microalgae under CO2-limiting conditions, could regulate the number of plants.

Presence of SARS-CoV-2 in urban effluents in south-east Buenos Aires, Argentina, May 2020 to March 2022 / Presencia del SARS-CoV-2 en aguas residuales urbanas del sudeste de Buenos Aires, Argentina, de mayo del 2020 a marzo del 2022 / Presença de SARS-CoV-2 em águas residuais urbanas no sudeste de Buenos Aires, Argentina, de maio de 2020 a março de 2022

ABSTRACT Objectives. To implement and evaluate the use of wastewater sampling for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in two coastal districts of Buenos Aires Province, Argentina. Methods. In General Pueyrredon district, 400 mL of wastewater samples were taken with an automatic sampler for 24 hours, while in Pinamar district, 20 L in total (2.2 L at 20-minute intervals) were taken. Samples were collected once a week. The samples were concentrated based on flocculation using polyaluminum chloride. RNA purification and target gene amplification and detection were performed using reverse transcription polymerase chain reaction for clinical diagnosis of human nasopharyngeal swabs. Results. In both districts, the presence of SARS-CoV-2 was detected in wastewater. In General Pueyrredon, SARS-CoV-2 was detected in epidemiological week 28, 2020, which was 20 days before the start of an increase in coronavirus virus disease 2019 (COVID-19) cases in the first wave (epidemiological week 31) and 9 weeks before the maximum number of laboratory-confirmed COVID-19 cases was recorded. In Pinamar district, the virus genome was detected in epidemiological week 51, 2020 but it was not possible to carry out the sampling again until epidemiological week 4, 2022, when viral circulation was again detected. Conclusions. It was possible to detect SARS-CoV-2 virus genome in wastewater, demonstrating the usefulness of the application of wastewater epidemiology for long-term SARS-CoV-2 detection and monitoring.

RESUMEN Objetivos. Aplicar y evaluar la utilización de muestreos de aguas residuales como método para la detección del coronavirus del síndrome respiratorio agudo severo de tipo 2 (SARS-CoV-2) en dos distritos costeros de la Provincia de Buenos Aires, Argentina. Métodos. Se utilizó un dispositivo de muestreo automático para tomar muestras de 400 mL de las aguas residuales de 24 horas en el distrito de General Pueyrredon, mientras que en el distrito de Pinamar se tomaron muestras de 2,2 L a intervalos de 20 minutos hasta un volumen total de 20 L. Los muestreos se realizaron una vez por semana. Las muestras se concentraron mediante floculación con policloruro de aluminio. La purificación del ARN y la amplificación y detección del gen diana se llevaron a cabo mediante la prueba de reacción en cadena de la polimerasa con retrotranscripción para el diagnóstico clínico a partir de hisopados nasofaríngeos. Resultados. Se observó la presencia de SARS-CoV-2 en las aguas residuales de ambos distritos. En General Pueyrredon, el SARS-CoV-2 se halló en la semana epidemiológica 28 del 2020, es decir, 20 días antes del inicio del aumento de casos de enfermedad por coronavirus 2019 (COVID-19) registrado en la primera ola (semana epidemiológica 31) y nueve semanas antes de que se alcanzara el número máximo de casos de COVID-19 con confirmación de laboratorio. En el distrito de Pinamar se detectó el genoma viral en la semana epidemiológica 51 del 2020, pero solo se pudo volver a realizar el muestreo en la semana epidemiológica 4 del 2022, en la que se volvió a detectar la circulación del virus. Conclusiones. Se pudo detectar el genoma del virus SARS-CoV-2 en aguas residuales, lo que muestra la utilidad de la aplicación de la epidemiología de aguas residuales como método para la detección y el seguimiento del SARS-CoV-2 a largo plazo.

RESUMO Objetivos. Implementar e avaliar o uso de amostragem de águas residuais na detecção do coronavírus da síndrome respiratória aguda grave 2 (SARS-CoV-2) em dois distritos costeiros da Província de Buenos Aires, Argentina. Métodos. No distrito de General Pueyrredon, amostras de 400 mL de águas residuais foram coletadas ao longo de 24 horas com um amostrador automático; já no distrito de Pinamar, foram coletados 20 L no total (2,2 L a intervalos de 20 minutos). As amostras foram coletadas uma vez por semana e concentradas por floculação com cloreto de polialumínio. A purificação do RNA e a amplificação e detecção de genes-alvo foram realizadas por meio de reação em cadeia da polimerase com transcrição reversa para diagnóstico clínico de esfregaços nasofaríngeos humanos. Resultados. Detectou-se presença de SARS-CoV-2 em águas residuais dos dois distritos. Em General Pueyrredon, o SARS-CoV-2 foi detectado na semana epidemiológica 28 de 2020, ou seja, 20 dias antes do início de um aumento no número de casos da doença provocada pelo coronavírus de 2019 (COVID-19) na primeira onda (semana epidemiológica 31) e 9 semanas antes de se registrar o número máximo de casos de COVID-19 confirmados em laboratório. No distrito de Pinamar, o genoma viral foi detectado na semana epidemiológica 51 de 2020, mas não foi possível realizar a amostragem novamente até a semana epidemiológica 4 de 2022, quando a circulação do vírus foi novamente constatada. Conclusões. Foi possível detectar o genoma do vírus SARS-CoV-2 em águas residuais, demonstrando a utilidade da aplicação da epidemiologia baseada em águas residuais para detectar e monitorar o SARS-CoV-2 em longo prazo.

Culex quinquefasciatus Holobiont: A Fungal Metagenomic Approach.

Microorganisms associated with mosquitoes have fundamental roles, not only in their nutrition, but also in physiological and immunological processes, and in their adaptation to the environment as well. Studies on mosquito hologenomes have increased significantly during the last years, achieving important advances in the characterization of the "core bacteriome" of some species of health importance. However, the fungal mycobiome has not been exhaustively researched, especially throughout the life cycle of some hematophagous mosquito species. In this work, the diversity and composition of fungal communities in different developmental stages, sexes, and adult nutrition of Culex quinquefasciatus reared on laboratory conditions were characterized, using internal transcribed spacer high throughput amplicon sequencing. Larvae presented a higher fungal richness, while sucrose-fed males and females showed a similar diversity between them. Blood-fed females presented few operational taxonomic units with an even distribution. Results are consistent with the reduction of larval microbiota after molting, observed for the bacterial microbiome in other mosquito species. The filamentous Ascomycota Penicillium polonicum and Cladosporium sp. were present in all stages of the mosquitoes; in addition, the presence of yeasts in the insects or their subsequent colonization associated with their diet is also discussed. These results suggest that some species of fungi could be essential for the nutrition and development of mosquitoes throughout their life cycle.

Culex quinquefasciatus larvae development arrested when fed on Neochloris aquatica.

Culex quinquefasciatus is a cosmopolitan species widely distributed in the tropical and subtropical areas of the world. Due to its long history of close association with humans, the transmission of arboviruses and parasites have an important role in veterinary and public health. Adult females feed mainly on birds although they can also feed on humans and other mammals. On the other hand, larvae are able to feed on a great diversity of microorganisms, including microalgae, present in natural or artificial breeding sites with a high organic load. These two particularities, mentioned above, are some of the reasons why this mosquito is so successful in the environment. In this work, we report the identification of a microalga found during field sampling in artificial breeding sites, in a group of discarded tires with accumulated rainwater. Surprisingly, only one of them had a bright green culture without mosquito larvae while the other surrounding tires contained a large number of mosquito larvae. We isolated and identified this microorganism as Neochloris aquatica, and it was evaluated as a potential biological control agent against Cx. quinquefasciatus. The oviposition site preference in the presence of the alga by gravid females, and the effects on larval development were analyzed. Additionally, microalga effect on Cx. quinquefasciatus wild type, naturally infected with the endosymbiotic bacterium Wolbachia (w+) and Wolbachia free (w-) laboratory lines was explored. According to our results, even though it is chosen by gravid females to lay their eggs, the microalga had a negative effect on the development of larvae from both populations. Additionally, when the larvae were fed with a culture of alga supplemented with balanced fish food used as control diet, they were not able to reverse its effect, and were unable to complete development until adulthood. Here, N. aquatica is described as a biological agent, and as a potential source of bioactive compounds for the control of mosquito populations important in veterinary and human health.

Molecular characterization of a Bacillus thuringiensis strain from Argentina, toxic against Lepidoptera and Coleoptera, based on its whole-genome and Cry protein analysis.

The insecticidal proteins of Bacillus thuringiensis are used in formulations of spore-crystal complexes and their genes have been incorporated into several crops, providing a model for genetic engineering in agriculture. Despite the variability of the Cry proteins described so far, it is still necessary to look for toxins with a broad spectrum of action, since a significant number of pests are not controlled with the available Cry proteins. It is also important to provide alternatives to address the problem of insect resistance, which has already appeared with the use of formulations and with transgenic plants that express cry genes that code for insecticidal proteins. The FCC 7 strain was characterized by the ultrastructural parasporal body under optical and electronic microscopy, and for the detection of Cry8-type proteins by genomic and proteomic approaches. The identity of the strain and the presence of putative toxin encoding genes, and virulence factors analyzed by Illumina Miseq 1500 platform genomic sequencing, was confirmed. The identity of the two Cry8 proteins that make up the parasporal body was confirmed by MALDI-TOF/TOF. To expand knowledge about the insecticidal activity of this strain, we conducted preliminary tests against the cotton boll weevil, Anthonomus grandis. Here we report the characterization of a novel B. thuringiensis isolate native to Argentina (FCC 7) toxic against A. grandis. The strain shows a rounded parasporal body harboring mainly a protein of about 140 kDa and two different types of Cry8 proteins. Through whole-genome sequencing, we identified the presence of two cry8-like crystal protein genes, one vpa-like and two vpb-like genes, and multiple virulence factors, deepening the knowledge of a strain that had already been described as toxic against some lepidopterans and coleopterans, including Spodoptera frugiperda, Anticarsia gemmatalis, Tenebrio molitor and Diabrotica speciosa.

Culex quinquefasciatus carrying Wolbachia is less susceptible to entomopathogenic bacteria.

In an attempt to evaluate the susceptibility of the mosquito Culex quinquefasciatus to bacterial agents, a population naturally infected with a Wolbachia pipientis wPipSJ native strain was tested against the action of three bacterial mosquitocides, Bacillus thuringiensis subsp. israelensis, Bacillus wiedmannii biovar thuringiensis and Lysinibacillus sphaericus. Tests were carried out on mosquito larvae with and without Wolbachia (controls). Cx. quinquefasciatus naturally infected with the native wPipSJ strain proved to be more resistant to the pathogenic action of the three mosquitocidal bacterial strains. Additionally, wPipSJ was fully characterised using metagenome-assembled genomics, PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and MLST (MultiLocus Sequence Typing) analyses. This Wolbachia strain wPipSJ belongs to haplotype I, group wPip-III and supergroup B, clustering with other mosquito wPip strains, such as wPip PEL, wPip JHB, wPip Mol, and wAlbB; showing the southernmost distribution in America. The cytoplasmic incompatibility phenotype of this strain was revealed via crosses between wildtype (Wolbachia+) and antibiotic treated mosquito populations. The results of the tests with the bacterial agents suggest that Cx. quinquefasciatus naturally infected with wPipSJ is less susceptible to the pathogenic action of mosquitocidal bacterial strains when compared with the antibiotic-treated mosquito isoline, and is more susceptible to B. thuringiensis subsp. israelensis than to the other two mosquitocidal agents.

Molecular tool for monitoring the safety of Aedes (Stegomyia) aegypti Rockefeller rearing in arthropod containment facilities

Abstract The interest in and use of Aedes (Stegomyia) aegypti (Linnaeus) (Diptera: Culicidae) insectary lines increased in most laboratories around the world since the recognition of the transmission of human and animal pathogens by this mosquito species, resulting in further scientific research on tropical diseases and vectors, and the development of chemical and biological products for mosquito populations control. In recent years, approaches to mosquito populations reduction have focused on new technologies that include the release of Wolbachia-infected lines, genetically modified vector and insects subjected to radiation in the Sterile Insect Technique. In order to evaluate some of these techniques, it is essential to count with wild A. aegypti populations and the reference strain, accurately identified, maintained under laboratory conditions. This work proposes a new tool to monitor possible exchanges between reference mosquito strain and wild native populations of A. aegypti in neighboring areas, or between different lines in the same insectary. We aligned and compared ND5 gene fragments of A. aegypti from diverse sources, finding a region with putative Single Nucleotide Polymorphisms between individuals of Rockefeller (Rock) strain and different wild A. aegypti populations. These polymorphic sites in the molecular marker, allowed us to discriminate Rock reference strain from the wild A. aegypti haplotypes found in the southeast of Argentina and bordering areas with Brazil, Uruguay and Paraguay, and it can be useful as a tool for regulatory entities of mosquito insectaries at different Arthropod Containment Levels.

Bacillus wiedmannii biovar thuringiensis: A Specialized Mosquitocidal Pathogen with Plasmids from Diverse Origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous efforts have been undertaken to allow presumptive differentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490 kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.

Culex pipiens Development Is Greatly Influenced by Native Bacteria and Exogenous Yeast.

Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage.

Distribution of mosquitoes in the south east of Argentina and first report on the analysis based on 18S rDNA and COI sequences.

Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors.

Molecular systematic of three species of Oithona (Copepoda, Cyclopoida) from the Atlantic Ocean: comparative analysis using 28S rDNA.

Species of Oithona (Copepoda, Cyclopoida) are highly abundant, ecologically important, and widely distributed throughout the world oceans. Although there are valid and detailed descriptions of the species, routine species identifications remain challenging due to their small size, subtle morphological diagnostic traits, and the description of geographic forms or varieties. This study examined three species of Oithona (O. similis, O. atlantica and O. nana) occurring in the Argentine sector of the South Atlantic Ocean based on DNA sequence variation of a 575 base-pair region of 28S rDNA, with comparative analysis of these species from other North and South Atlantic regions. DNA sequence variation clearly resolved and discriminated the species, and revealed low levels of intraspecific variation among North and South Atlantic populations of each species. The 28S rDNA region was thus shown to provide an accurate and reliable means of identifying the species throughout the sampled domain. Analysis of 28S rDNA variation for additional species collected throughout the global ocean will be useful to accurately characterize biogeographical distributions of the species and to examine phylogenetic relationships among them.

Genetic diversity of cry gene sequences of Bacillus thuringiensis strains analyzed by denaturing gradient gel electrophoresis.

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.

Cloning and characterization of a novel crystal protein from a native Bacillus thuringiensis isolate highly active against Aedes aegypti.

We characterized a novel Bacillus thuringiensis isolate native to Argentina (FCC 41) that exhibits a mosquitocidal activity higher than the reference B. thuringiensis subsp. israelensis. This isolate shows a rounded crystal harboring two major proteins of about 70-80 kDa. Moreover, we cloned and sequenced the encoding gene of one of the crystal proteins (Cry) consisting of an open reading frame of 2061 pb that encodes a protein of 687 amino acid residues. The deduced amino acid sequence has a predicted relative molecular mass of 78 kDa and is 52% and 45% identical to those of the reported Cry24Aa and Cry24Ba sequences, respectively. The novel Cry protein was designated as Cry24Ca, which also exhibited larvicidal activity against Aedes aegypti when its encoding gene was expressed in an Escherichia coli host strain.

New strategy for identification of novel Cry-type genes from Bacillus thuringiensis strains.

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.