Intelligent monitoring of Aedes aegypti in a rural area of Rio de Janeiro State, Brazil

The aim of this research was to monitor the presence of females of Aedes aegypti (Linnaeus, 1762) on the Seropédica municipality, Rio de Janeiro State, from 2010 to 2013. For this purpose, the Intelligent Dengue Monitoring (IM-Dengue) and Intelligent Virus Monitoring (IM-Virus) developed by Universidade Federal de Minas Gerais (Ecovec – Minas Gerais, Brazil), were used. IM-Dengue is a tool that allows achieving a weekly overview of A. aegypti infestation, while IM-Virus is another tool that allows detecting dengue virus directly from the mosquito, by Real Time-PCR. Both tools were developed for diagnosis in a prepathogenesis period of the disease, before infection occurrence. Traps were distributed in 19 locations inside the municipality and the bugs were collected weekly during the years of the research. As a result, the presence of 163 females of A. aegypti was recorded over the period; there was no circulation of the virus in the municipality. In one of the 19 study sites, a high degree of disease transmission risk was verified. The study concluded that the municipality, as a whole, showed no risk of disease transmission throughout the field research period

Ultrastructural and Cytotoxic Effects of Metarhizium robertsii Infection on Rhipicephalus microplus Hemocytes

Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 108 conidia mL−1). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control

Comparação entre as técnicas Gordon & Whitlock modificada e Centrífugo Flutuação para diagnóstico de helmintose em bovinos

O presente estudo objetivou verificar o grau de correlação existente entre as técnicas Gordon & Whitlock modificada (GW) e Centrifugo Flutuação (CF) no diagnóstico de endoparasitoses gastrintestinais em bovinos, e elaborar uma tabela com o grau de infecção de helmintos da Superfamília Strongyloidea, medido pelo número de ovos por grama de fezes (OPG) através das duas técnicas. Foram coletadas amostras de fezes, diretamente da ampola retal, de 12 fêmeas, ¾ holandês/zebu, a partir de seis meses de idade, pesando entre 100 a 150 kg, naturalmente parasitadas por nematóides gastrointestinais, na Fazenda Santa Mônica, da Embrapa Gado de Leite, em Valença, RJ, Brasil. As amostras foram coletadas a cada 21 dias, no período de 28 meses. As contagens de OPG foram realizadas no Laboratório de Parasitologia da Embrapa, em Juiz de Fora, MG. A técnica CF detectou 80.81% de amostras positivas, contra 15.17% da técnica GW, demonstrando que a técnica CF é mais sensível que a GW para contagem de OPG, em bovinos. A técnica GW apresentou maior Coeficiente de Variação (CV = 237%) que a CF (CV = 68%). Entretanto, verificou-se concordância significativa entre elas (rho = 0,118), confirmando a existência de uma correlação entre as mesmas. Esta confirmação permitiu a elaboração de uma tabela do grau de infecção por helmintos da Superfamília Strongyloidea baseada nas duas técnicas, que auxiliará na determinação do limiar de tratamento. Dessa forma, a ausência de parâmetros na tomada de decisão para tratamento de helmintose em bovinos, a partir do grau de infecção, considerada a grande desvantagem da técnica CF, foi eliminada

Effects of fluazuron on the biological parameters of engorged females of Rhipicephalus microplus

The objective of this study was to evaluate the biological parameters of Rhipicephalus microplus engorged females exposed to the arthropod growth regulator fluazuron. A commercial formulation based on fluazuron (Acatak®, Novartis) was administered topically to stabled cattle artificially infested with R. microplus larvae. The infestations occurred on four alternate days (days -21, -17, -13 and -6) taking as day 0 (zero) the day of treatment with a single dose of fluazuron for the treated group. Fifteen engorged females from each group were collected from the floor of the stalls at the end of the engorgement period (21 days) on pre-established days: before (day 0) and after treatment (days 4, 8 and 15), respectively. After cleaning and preparation of the engorged females in the laboratory, the following biological parameters were analyzed: weight of the engorged female, weight of the female after laying (spent female), laying period, weight of the posture, egg production index, reproductive efficiency index, hatching percentage, and nutritional index. The average weight of engorged females showed a significant reduction on all days after treatment in the exposed group. Aside from the weight after laying, all other biological parameters showed a significant reduction on the eighth post-treatment day due to exposure to fluazuron. Knowledge of the effects of an active principle on biological parameters in a population of ticks is essential for appropriate implementation of integrated management practices for parasite control

Alterations in the oxidative metabolism of Rhipicephalus (Boophilus) microplus ticks in response to exposure to the insect growth regulator fluazuron

Abstract Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick’s fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects

PATOGENICIDADE IN VITRO DE Metarhizium anisopliae S.L. SOBRE LARVAS E NINFAS DE Ornithodoros mimon (ACARI: ARGASIDAE)

Ornithodoros mimon é um carrapato argasídeo comum a Chiroptera na região Neotropical, mas bastante agressivos aos humanos, pois a espécie é frequentemente encontrada em habitações humanas. Atualmente, não foram estabelecidos métodos de controle. Porém, trabalhos prévios já demonstraram a susceptibilidade de outros argasí- deos a fungos entomopatogênicos. Dessa forma, o presente estudo teve como objetivo verificar a patogenicidade in vitro de Metarhizium anisopliae s.l. sobre larvas e ninfas de primeiro ínstar ingurgitadas e não ingurgitadas de O. mimon. O parâmetro avaliado foi o percentual de mortalidade das larvas e ninfas. Como resultados, observaram-se 80% de mortalidade do estágio larval na concentração de 107 conídios/mL e 91,4% para larvas submetidas à concentração de 108 conídios/mL. As ninfas ingurgitadas imersas na concentração 107 conídios/ mL foram mais susceptíveis ao fungo que as ninfas não ingurgitadas. Com relação à concentração 108 conídios/mL não foi observada diferença significativa, uma vez que se obteve um percentual médio de mortalidade acima de 95% tanto para as ninfas alimentadas como para as não alimentadas. Com base nos resultados obtidos, pode-se concluir que os estágios de O. mimon estudados são susceptíveis a M. anisopliae

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity

Abstract Background Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. Methods Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. Results Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. Conclusion By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae. Graphical Abstrac

Estudo morfológico de isolados de Beauveria bassiana antes e após reisolamento em Rhipicephalus microplus

Beauveria bassiana é um fungo artropodopatogênico cosmopolita que possui o carrapato dos bovinos Rhipicephalus microplus como hospedeiro. Neste cenário, estudos morfológicos e de produtividade conidial em meio de cultura podem indicar isolados que possuam potencial para produção em larga escala. O presente trabalho avaliou a macromorfologia e micromorfologia, além da produção conidial de dez isolados de B. bassiana antes e após uma passagem (ciclo de desenvolvimento) sobre R. microplus. Macroscopicamente, variações de diâmetro, aparência, aspecto de colônia, exsudação e cor de reverso foram observadas após o reisolamento. Por outro lado, o processo não alterou parâmetros como desenvolvimento dos conidióforos, formato e tamanho de conídios. Em relação à produção conidial, seis isolados aumentaram significativamente o número de conídios contados por área. Como destaque, o isolado Bb 02, que possui o carrapato como hospedeiro original de isolamento, apresentou aumento da pulverulência e produção conidial após apenas uma única passagem. Assim, acredita-se que para avaliações futuras com cunho comercial, estudos que envolvam mais passagens em hospedeiros em potencial, bem como sua associação a análises de patogenicidade e atividade enzimática, podem expressar resultados mais relevantes para o controle biológico

Asssociação de Metarhizium anisopliae sensu lato e cipermetrina para controle de Rhipicephalus microplus

Rhipicephalus microplus é responsável por grandes perdas econômicas na pecuária brasileira. Com o intuito de controlar esse artrópode, o uso indiscriminado de produtos químicos é amplamente empregado, acarretando em prejuízos ao meio ambiente, aos animais e humanos, além de contribuir para o aparecimento de cepas de carrapatos resistentes. Assim, o emprego de produtos químicos associados a entomopatógenos surge como uma alternativa para aumentar a eficácia do controle de carrapato e reduzir os danos causados pelo uso inapropriado de produtos químicos. Desta forma, o objetivo do presente estudo foi de avaliar a eficiência da associação de Metarhizium anisopliae e cipermetrina sobre estágios imaturos de R. microplus. O tratamento dos grupos foi realizado através da imersão de ovos e larvas em 1mL de suspensão aquosa do fungo, solução de cipermetrina, suspenção fúngica associada à cipermetrina e um grupo controle. Os parâmetros biológicos avaliados foram o percentual de eclosão e o percentual de mortalidade das larvas. Observou se que o fungo quando utilizado sozinho em menor concentração (106 conídios mL-1) ou a cipermetrina também em menor concentração (25 ppm) não foram capazes de alterar os parâmetros biológicos quando comparados ao grupo controle. No entanto, a associação do fungo a 106 conídios mL-1 com cipermetrina em menor concentração, foi capaz de reduzir o percentual de eclosão pela metade e causar o dobro de mortalidade quando comparado ao uso do fungo e cipermetrina isoladamente. Desta forma, conclui se que a associação de M. anisopliae com cipermetrina é eficaz no controle de estágios imaturos de R. microplus

Oil-based formulations containing arthropodpathogenic fungi to control Rhipicephalus microplus

ABSTRACT. Angelo I.C., Diniz-Neto H.C., Santos S.V., Perinotto W.M.S., Quinelato S., Santos H.A., Gôlo P.S., Camargo M.G., Marciano A.F. & Bittencourt V.R.E.P. [Oil-based formulations containing arthropodpathogenic fungi to control Rhipicephalus microplus.] Formulações oleosas contendo fungos artropodopatogênicos para o controle de Rhipicephalus microplus. Revista Brasileira de Medicina Veterinária, 37(Supl.1):18-24, 2015. Departamento de Epidemiologia e Saúde Pública, Universidade Federal Rural do Estado do Rio de Janeiro, BR 465, Km 7, Seropédica, RJ 23897-970, Brasil. E-mail: [email protected] Rhipicephalus microplus ticks are responsible for economical losses estimated in U$ 3.24 billions per year in Brazil. Arthropodpathogenic fungi are an important alternative to the chemical control of this arthropod; however to be efficient in the field, formulations must be developed to protect conidia against environmental stresses. The present study compared the efficacy of mineral or vegetal oil-based formulations containing Metarhizium anisopliae s.l. or Beauveria bassiana s.l. as well as their association to control R. microplus engorged females. M. anisopliae and B. bassiana isolates were cultivated in Potato Dextrose Agar medium and maintained under optimal conditions. Single fungus oil-based formulations were prepared with the following proportions: 84% aqueous fungal suspension; 15% sterile oil; 1% Tween 80. Associated fungal formulations were prepared as follows: 42% aqueous M. anisopliae suspension; 42% aqueous B. bassiana suspension; 15% sterile oil and 1% Tween. Engorged females were immersed in 1 mL oil formulations. The following biological parameters were evaluated: initial weigh of engorged female; weight of egg mass; egg production index; nutritional index and percentage of tick control. There was no difference in the percentage of tick control when M. anisopliae s.l. and/or B. bassiana s.l. were tested in mineral oil. Although, when vegetable oil-based formulations were tested, only M. anisopliae s.l. formulations (both single and associated) had satisfactory control percentage (41.97% and 40. 13%) while single fungus B. bassiana s.l. formulation yield a very low percentage of tick control (10.88%). Accordingly, the use of different oils (mineral or vegetable oils) may affect in different ways the arthropodpathogenic fungi efficacy to control R. microplus. Therefore, different oils should be tested in formulations to control ticks, as well as their interactions with different fungal isolates what will contribute to the development of a promising alternative to control this important parasite