Molecular characterization of Nosema bombi (Microsporidia : Nosematidae) and a note on its sites of infection in Bombus terrestris (Hymenoptera : Apoidea)

Investigations of queen, worker and male bumble bees (Bombus terrestris) showed that all individuals became infected with Nosema bombi. Infections were found in Malpighian tubules, thorax muscles, fat body tissue and nerve tissue, including the brain. Ultrastructural studies revealed thin walled emptied spores in host cell cytoplasm interpreted as autoinfective spores, besides normal spores (environmental spores) intended for parasite transmission between hosts. The nucleotide sequence of the gene coding for the small subunit rRNA (SSU-rRNA) from Microsporidia isolated from B. terrestris, B. lucorum, and B. hortorum were identical, providing evidence that N. bombi infects multiple hosts. The sequence presented here (GenBank Accession no AY008373) is different from an earlier submission to GenBank (Accession no U26158) of a partial sequence of the same gene based on material collected from B. terrestris. It still remains to be investigated if there is species diversity among Microsporidia found in bumble bees.

Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects. (C) 2010 Elsevier Inc. All rights reserved.

Susceptibility of mosquito vectors to Dirofilaria immitis on Madeira Island, Portugal

Dirofilaria immitis (Leidy, 1856), an agent of heartworm disease, is an important parasite from both the veterinary standpoint and as a model to study human filariasis. It is a mosquito-borne filarial nematode which inhabits the right ventricle and pulmonary arteries of dogs. D. immitis is an important disease agent on Madeira Island with about 30% of dogs testing positive for this worm. Nevertheless, the vectors of this parasite in Madeira have never been studied, nor has the interaction between pathogen and vector, or the environmental variables that might influence heartworm transmission. Innate susceptibility to infection is only one component of vector competence, and field isolation of naturally infected mosquitoes has shown the capability of D. immitis to exploit a great diversity of vector species under natural conditions. The purpose of this work was to determine which mosquitoes are vectors of heartworm disease, the relation between population density and environment, and the association between immune response of the vector to the filarial parasite. Seasonal abundance of Culex theileri and Culex pipiens molestus was studied. Correlation and canonical correspondence analysis were performed using abundance data of these two species with selected weather variables, including mean temperature, relative humidity and accumulated precipitation. The most important factor determining Cx. theileri abundance was accumulated precipitation, while Cx. pipiens molestus abundance did not have any relationship with weather variables. Field studies were performed to verify whether Cx. theileri Theobald functions as a natural vector of D. immitis on Madeira Island, Portugal. Cx. theileri tested positive for D. immitis for the first time. The same study was made regarding Cx. p. molestus. Two abnormal L2 stage filarial worms were found in Malpighian tubules in field caught Cx. p. molestus. In the laboratory, two strains of Cx. p. molestus were studied for their susceptibility to D. immitis. None presented infective-stage larvae. Finally, because Cx. p. molestus is an autogenous mosquito, we evaluated the reproductive costs when this mosquito mounts an immune response against D. immitis in the absence of a blood meal. This mosquito showed an active immune response when inoculated intrathoracically with microfilariae (mf) of the heartworm. The ovaries from mosquitoes undergoing melanotic encapsulation developed more eggs than those which could not melanize the mf. This fact is contradictory with some previous studies of reproductive costs in Armigeres subalbatus and Ochlerotatus trivittatus, and it was the first time that an autogenous mosquito was used to study this subject.

Morphological study of the hindgut in larvae of Anticarsia gemmatalis Hübner(Lepidoptera: Noctuidae)

A lagarta da soja (Anticarsia gemmatalis Hübner) tem grande interesse econômico, pois afeta significativamente a cultura da soja em todo o mundo. Este trabalho descreve a morfologia do intestino posterior de larvas de A. gemmatalis, com ênfase nos seus aspectos histológicos. O intestino posterior é constituído por regiões morfologicamente distintas, identificadas como piloro, íleo, cólon e reto. Independente da região, a parede do intestino posterior é constituída por fina cutícula, epitélio simples e camada muscular. A íntima cuticular apresenta espículas no anel intersticial posterior, entre o intestino médio e o posterior, e na região posterior do piloro. A musculatura do reto é formada por camada única de largas fibras circulares, diferindo das demais regiões do intestino posterior que apresentam duas camadas de fibras musculares. As extremidades distais dos túbulos de Malpighi atravessam as paredes do reto, constituindo o sistema criptonefridial característico de Lepidoptera.

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrateerythrocytes and cultured epimatigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titres of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus

Ultramorphology of the Digestive Tract of Paratrechina longicornis (Hymenoptera, Formicidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tissue-specific expression of esterases in Triatoma infestans (Triatominae, Heteroptera)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Post-embrionic development of the digestive tube of Dermatobia hominis (Linnaeus) (Diptera, Cuterebridae)

The digestive tube of 2nd and 3rd instar larvae, pupae and newly emerged adults of Dermatobia hominis (Linnaeus, 1781) was studied anatomically. The specimens were dissected in buffer saline under a stereomicroscope, and the digestive tubes were placed on slides and fixed in 10% buffered formalin. Each tube was measured using a micrometric eye piece, and drawings were made with camera lucida. The results showed that the midgut, the hindgut and the Malpighian tubules with their ducts grow gradually during the larval development. The oesophagus and the salivary glands with their ducts grow only during the moult from the 2nd to the 3rd instar. In the pupal period, salivary glands grow gradually but disappeared after the 20th day. After metamorphosis the digestive tube regressed. This is expected since adult D. hominis lives about nine days without feeding. This fly, similar to other calyptratae muscoid flies shows no vestige of a crop during all post-embrionic development, and the adult has no salivary glands.

CROSS-REACTIVITY BETWEEN HARD TICK ANTIGENS

1. The present study was carried out to determine the target cells and tissues for anti-tick immunoglobulins using an indirect immunohistochemical technique.2. Sections in triplicate prepared from unfed ticks Rhipicephalus appendiculatus, R. evertsi and Amblyomma variegatum were used to assess the cross-reactivity of serum from guineapigs naturally infested with these tick species or immunized against them.3. The sections showed slight (+) to strong (++++) labelling of several structures in the tick body, e.g. salivary gland, gut lumen and malpighian tubules, depending on the serum used.4. The immune serum resulting from the immunization of guinea pigs with an extract of unfed nymphs of R. appendiculatus ticks showed the most intense cross-reactivity with the sections examined.

Histopathology of Rhipicephalus sanguineus (Acari: Ixodidae) ticks fed on resistant hosts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analysis of chromatin and nuclear area alterations in triatomines during starvation

In the present study changes in nuclear area and chromatin distribution in the Malpighian tubules of a blood-feeding insect, the vector of Chagas' disease were analysed. Male and female adult insects were dissected, after a starving period of up to 28 days. The Malpighian tubules were fixed and stained by orcein lacto-acetic. The nuclei were analyzed by photomicrography, karyometry and a statistical analysis was performed. It was observed that with the increase of the starvation period there were alterations in the nuclear phenotype, that is, changes in the nuclear area and chromatin extenuation. In males, starting from the second week of starvation, chromatin extenuation was accentuated, and the beginning of nuclear coalition occurred more frequently in the third and fourth starvation weeks. In females, the accentuated chromatin extenuation was observed after the third starvation week, and irregularities in nuclear borders were more frequent. The descriptive statistical analysis of the male and temale nuclear areas revealed that the nuclear areas continued to increase during starvation reaching highest values in the third week. Another outstanding aspect is that in the third starvation week karyometry presented larger heterogeneity with higher standard deviation values for both genders. Therefore, nuclear alterations related to the distribution and extenuation chromatin and increase in nuclear area were observed. These aspects suggest the presence of nuclear metabolic mechanisms that lead to synthesis of specific proteins necessary for the protection and maintenance of the cell during physiologic stress.

Toxicological and histopathological effects of boric acid on atta sexdens rubropilosa (Hymenoptera: Formicidae) workers

The current study compared the toxicity of different concentrations of boric acid in adult workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae), with toxicological bioassays, and examining the dose-dependent and time-dependent histopathological changes, of the midgut, Malpighian tubules, and postpharyngeal glands. Our results revealed the importance of conducting toxicological bioassays combined with morphological analyses of the organs of ants chronically exposed to insecticides used in commercial ant baits. In vitro bioassays showed that boric acid significantly decreases the survivorship of workers regardless of concentration, whereas the morphological data suggested progressive dose-dependent and time-dependent changes in the organs examined, which were evident in the midgut. The midgut is the first organ to be affected, followed by the postpharyngeal gland and Malpighian tubules. This sequence is in agreement with the absorption pathway of this chemical compound in the midgut, its transference to the hemolymph, possibly reaching the postpharyngeal glands, and excretion by the Malpighian tubules. These progressive changes might be due to the cumulative and delayed effect of boric acid. Our findings provide important information for the understanding of the action of boric acid in ant baits in direct and indirect target organs. © 2010 Entomological Society of America.

Toxicological and histopathological effects of hydramethylnon on Atta sexdens rubropilosa (Hymenoptera: Formicidae) workers

The leaf-cut ants are important agricultural pest, because they can cause intense defoliation in plants and destroy large areas cultivated. Although there are several works for the control of these insects by examining the toxicity of natural chemical compounds on various species of ants, few are focused on analyses of morphological changes caused in the affected organs. The aim of this study was to evaluate the effects of hydramethylnon on Atta sexdens rubropilosa workers through toxicological bioassays and morphological analysis of the post-pharyngeal glands, midgut, and Malpighian tubules of these ants. Hydramethylnon dissolved either in acetone (HA) or in a mixture of acetone and soy oil (HAO) was added to the artificial diet at a concentration of 200 μg/mL. The workers fed daily with the diet containing hydramethylnon showed higher mortality than the controls, especially when HAO was used. Moreover, light and electron microscopy revealed morphological alterations in the midgut and Malpighian tubules of workers treated with HA, whereas alterations of the post-pharyngeal glands were observed in the HAO-treated group. These results indicated that the presence of soy oil provided an alternate route for the ingestion of the formicide's active ingredient and corroborated previous studies that suggested a role for the post-pharyngeal glands in lipid metabolism. Our findings suggest that the oil may carry hydramethylnon to the gland lumen, resulting in lower quantity of the active ingredient in the intestinal lumen and Malpighian tubules that explains the lower degree of morphological alterations in these structures in the workers treated with HAO. These results may provide insight into the toxicological effects of hydramethylnon on leaf-cutting ants and the use of vegetable oil as an adjuvant in baits to control ants. © 2012 Elsevier Ltd.

Aspectos morfológicos, ultra-estruturais e distribuição dos túbulos de Malpighi do diplópodo Urostreptus atrobrunneus Pierozzi & Fontanetti, 2006

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Efeitos de doses subletais do imidaclopride no cérebro, ventrículo e túbulo de Malpighi de Apis melifera africanizada

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)