Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae).

Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Negative impact of a cadmium concentration considered environmentally safe in Brazil on the cardiac performance of bullfrog tadpoles.

A drastic amphibian decline has been observed worldwide, which can be attributed (among other factors) to exposure to pollutants. Considering that cadmium corresponds to the most rapidly increasing trace metal in the environment, the aim of this work was to evaluate whether the exposure (2 and 16 days) of bullfrog tadpoles to this trace metal, at the concentration currently considered environmentally safe (at 1ppb) in class 1 and 2 waters by the Brazilian Environmental Council, can affect the cardiac performance of these animals. The acute exposure (2 days) of tadpoles to cadmium resulted in a marked bradycardic response, which was correlated with an incomplete cardiac relaxation, without any compensation by improved cardiac twitch force (Fc) or contraction velocity (TPT), nor even by cardiac hypertrophy. Indeed, after 16 days of exposure, the cardiac function of tadpoles became even more depressed due to a marked decrease in Fc, a prolongation of TPT, and also incomplete relaxation (i.e. increases in the ventricle resting tension), without changes in ventricle relative mass. Altogether, the cardiodepressive effects of cadmium (especially after more prolonged exposure periods) impose negative alterations on a tadpole׳s development and also impede adequate homeostatic adjustments to respond appropriately to the exposure to cadmium with increase in energetic demand to counteract the deleterious effects of the xenobiotic. These disturbances can impair tadpoles׳ growth, development and reproduction. It is a fact that allows us to strongly suggest that cadmium concentrations, which are currently considered environmentally safe in Brazil, should be revised.

Histochemistry of the mucus gland of Bombus morio Swedurus,1787 (Hymenoptera, Apidae)

The male mucus gland is present in all species of bees, exception for meliponines. In bees, the function of themucus glands is not certain so far and its contribution to form the female espermatecal fluid is not assured.With the aim to contribute to the knowledge of the mucus gland in bees we carried out a histochemical studyof the gland in mature, adult males of Bombus morio, focusing on the histochemical nature of the secretion.The males were collected around the forest fragments into UFSCar, Campus Sorocaba. The glands werefixed and analysed under routine microscopy and for histochemistry techniques: Periodic Acid Schiff (PAS),Bromophenol Blue (BB), Sudan Black (SB) and Critical Electrolyte Concentration (CEC) variant. The resultsshowed that the mucus gland is constituted by a pair of large, thick tubular structures, which presented theirdistal portion more dilated and corn shaped. The glandular cells are columnar and in the apical portion presentseveral apocrine vesicles being released into the gland lumen. The histochemistry showed that gland secretionis very complex and contains protein (BB), neutral polysaccharide (PAS), as well as lipid (SB). The luminalgland secretion presents a background of homogenous content, presenting little dark dots stained by alltechniques used for this work. The investigation of the nucleolar activity (CEC) showed that the mucus glandepithelium is evolved in protein synthesis, presenting nucleoli developed and much RNA in the cytoplasm.

An ancestral luciferase in the Malpighi tubules of a non-bioluminescent beetle!

The evolutionary origin of beetle bioluminescence is enigmatic. Previously, weak luciferase activity was found in the non-bioluminescent larvae of Tenebrio molitor (Coleoptera: Tenebrionidae), but the detailed tissular origin and identity of the luciferase-like enzyme remained unknown. Using a closely related giant mealworm, Zophobas morio, here we show that the luciferase-like enzyme is located in the Malpighi tubules. cDNA cloning of this luciferase like enzyme, showed that it is a short AMP-ligase with weak luciferase activity which diverged long ago from beetle luciferases. The results indicate that the potential for bioluminescence in AMP-ligases is very ancient and provide a first reasonable protoluciferase model to investigate the origin and evolution of beetle luciferases.

CCD imaging of basal bioluminescence in larval fireflies: clues on the anatomic origin and evolution of bioluminescence.

The anatomic and biochemical origin of beetle bioluminescence is still poorly understood. Through CCD imaging we report that larvae and pupae of the Brazilian fireflies Aspisoma lineatum and Cratomorphus sp emit a continuous weak glow throughout the entire body during all stages. This luminescence is especially developed after feeding, ecdysis and in the pupal stage, gradually disappearing as the cuticle becomes sclerotized and the adult emerges. This weak glow arises from the fat body, which consists of small lobes spread all over the body cavity. According to their pigmentation, these lobes can be divided in whitish and pinkish, and display different luciferase isozymes. Morphological studies suggest that the jelly-like ventral lanterns in the 8th abdominal segment evolved from these white lobes, providing a rationale for the widespread location of lanterns in larvae of different bioluminescent beetles. The biological and biochemical function of this weak diffuse bioluminescence is discussed in the context of the larval life-history.

Morphological changes of Sertoli cells during the male reproductive cycle of the teleost Piaractus mesopotamicus (Holmberg, 1887).

An investigation of the histological and ultrastructural changes of Sertoli cells during the male reproductive cycle in Piaractus mesopotamicus was made. The results showed that the Sertoli cell development is closely related with germ cell maturation. Therefore, these cells may have some role in germ cell maturation during the reproductive cycle of this species, whether in forming a tissue framework for the developing spermatogenic cysts, aiding in testes reorganization for a new reproductive cycle, in addition to other possible functions discussed in the text.

Morphological changes of Sertoli cells during the male reproductive cycle of the teleost Piaractus mesopotamicus (Holmberg, 1887)

Realizou-se uma investigação das mudanças histológicas e ultra-estruturais das células de Sertoli durante o ciclo reprodutivo de machos de Piaractus mesopotamicus. Os resultados mostraram que o desenvolvimento das células de Sertoli está estritamente relacionado à maturação das células gaméticas. Portanto, as células de Sertoli têm algum papel na maturação das células germinativas durante o ciclo reprodutivo dessa espécie, talvez formando um tecido de sustentação para os cistos espermatogênicos em desenvolvimento, ajudando a reorganização testicular para um novo ciclo reprodutivo, além de outras possíveis funções discutidas no texto.

Occurrence and ultrastructural characterization of "nuage" during oogenesis and early spermatogenesis of Piaractus mesopotamicus Holmberg, 1887 (Teleostei).

We investigated the occurrence and ultrastructurally characterized electrondense nuclear material (nuage) released from the nucleus during oogenesis and early spermatogenesis of Piaractus mesopotamicus, a fish from Pantanal Matogrossense (Brazil) having a seasonal reproductive cycle. The female germ cells presented two instances of nuclear material extrusion: in the oogonia and in the oocyte in the perinucleolar phase. In males, material with similar morphology and behavior occurred in the spematogonia. In all cases, this material was associated to mitochondria. The possible function of this material is discussed.

Occurrence and ultrastructural characterization of "nuage" during oogenesis and early spermatogenesis of Piaractus mesopotamicus Holmberg, 1887 (Teleostei)

Foi investigada a ocorrência e caracterizada ultra-estruturalmente a eliminação de material nuclear eletrondenso (nuage) para o citoplasma durante a ovogênese e durante os estágios iniciais da espermatogênese de Piaractus mesopotamicus, um peixe do Pantanal Matogrossense de ciclo reprodutivo sazonal. Constataram-se nas células germinativas femininas dois momentos de eliminação desse material, na ovogônia e no ovócito em fase perinucleolar. Nas células masculinas, material com morfologia e comportamento muito semelhante foi encontrado na espermatogônia. Em todos os casos, o material associou-se a mitocôndrias. A possível função desse material foi discutida.

Comparative study of the cuticular hydrocarbon composition of Melipona bicolor Lepeletier, 1836 (Hymenoptera, Meliponini) workers and queens

In social insects, cuticular hydrocarbons are involved in species, kin, caste and nestmate recognition. Gas chromatography and mass spectrometry were used to compare the cuticular hydrocarbon composition of workers, males and queens of Melipona bicolor. The cuticular hydrocarbon composition of this species was found to consist mainly of C23, C25:1, C25, C27:1, C27, C29:1 and C29, which are already present in imagoes that have not yet abandoned the brood cell. This composition varied quantitatively and qualitatively between and within the castes and sexes. The newly emerged workers and young queens (virgins) had similar cuticular hydrocarbon profiles, which were different from those of the males. When the females start executing their tasks in the colony, the cuticular hydrocarbon profile differences appear. The workers have less variety, while the queens conserve or increase the number of cuticular hydrocarbon compounds. The queens have more abdominal tegumentary glands than the workers, which apparently are the source of the new cuticular compounds

Dufour glands in the hymenopterans (Apidae, Formicidae, Vespidae): a review.

Associated to the sting apparatus of the aculeate hymenopterans is found the poison gland, originated from the glands associated to the ovipositor of the non-aculeate hymenopterans and the less derived Dufour gland, homologue of the coletterial gland of other insects, and found in all hymenopteran females. The Dufour gland functions is mostly uncertain in hymenopterans but in ants it is involved with communication and defense and in non social bees with the nest building and protection. In wasps possibly with kin-recognition. Differences in morphology and chemical composition of the gland secretion were observed among species, in the same species, between the castes in the social species and among individual of the same caste playing different tasks or belonging to different nest. Its original function of egg-protective substance producing, or favoring the oviposition, appear to have been replaced or complemented in hymenopterans by the production of semiochemicals with function in communication.

Dufour glands in the hymenopterans (Apidae, Formicidae, Vespidae): a review

Associated to the sting apparatus of the aculeate hymenopterans is found the poison gland, originated from the glands associated to the ovipositor of the non-aculeate hymenopterans and the less derived Dufour gland, homologue of the coletterial gland of other insects, and found in all hymenopteran females. The Dufour gland functions is mostly uncertain in hymenopterans but in ants it is involved with communication and defense and in non social bees with the nest building and protection. In wasps possibly with kin-recognition. Differences in morphology and chemical composition of the gland secretion were observed among species, in the same species, between the castes in the social species and among individual of the same caste playing different tasks or belonging to different nest. Its original function of egg-protective substance producing, or favoring the oviposition, appear to have been replaced or complemented in hymenopterans by the production of semiochemicals with function in communication