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

Influence of the application of juvenile hormone on the haemolymph total proteins and electrophoretic pattern of worker larvae of Apis mellifera

The aim of the present work was to verify the influence of the juvenile hormone (JH) applied on worker larvae of Apis mellifera 2 to 5 days old over the haemolymph total protein and electrophoretic pattern. Each larvae received topical applications of 1 ml of a solution of JH in hexane (1 μg/ml) on their 2 nd, 3 rd 4 th and 5 th day after hatching and had the amount and electrophoretic pattern of proteins from the haemolymph analyzed during the remaining days of their life. As a control, haemolymph of larvae of the same age that did not receive any kind of treatment was analyzed. The results show that the application of JH on larvae 3 or more days old affect the amount and electrophoretic pattern of the proteins, with this effect lasting through the subsequent days.

The prophenoloxidase system and in vitro interaction of Trypanosoma rangeli with Rhodnius prolixus and Triatoma infestans haemolymph

The presence of the prophenoloxidase (proPO) system in the haemolymph of Rhodnius prolixus and Triatoma infestans and the role played by Trypanosoma rangeli in the in vitro activation of proPO were tested. Both R. prolixus and T. infestans whole blood preparations showed a very active ProPO system. The proPO cascade of the two insect species were differentially activated by microbial-derived extracts: laminarin was a better activator of T. infestans haemolymph than of R. prolixus blood, and lipopolysaccharides from Shigella flexneri or Pseudomonas aeroginosa caused significant proPO activation of T. infestans haemolymph but not of R. prolixus preparations. For the two insect species, neither T. rangeli from culture nor parasite lysates were able to trigger proPO activation. The presence of the parasite in R. prolixus haemolymph/laminarin assays, however, significantly reduced the level of proPO activation to that of spontaneous activating controls. The immobilization of T. rangeli in vitro in haemolymph preparations occurred in both insect species and was dependent on the proPO activation intensity. Our results suggest that the susceptibility of R. prolixus to T. rangeli haemocoel infection may be explained, at least in part, by the suppression of the insect immune defence system i.e., inhibition of proPO in the presence of this protozoan parasite.

Dietary copper absorption and excretion in three semi-terrestrial grapsoid crabs with different levels of terrestrial adaptation

Three species of phylogenetically related semi-terrestrial crabs (Superfamily Grapsoidea - Sesarma rectum, Goniopsis cruentata and Neohelice granulata (formerly: Chasmagnathus granulatus) with different degrees of terrestriality were studied to quantify the accumulation of copper (Cu) in hemolymph, gills, hepatopancreas and antennal gland, and its excretion through the faeces. These crabs were fed for 15 days practical diets containing 0 (A), 0.5 (B), 1.0 (C), and 1.5% (D) of added CuCl2 (corresponding to 0, 0.2, 0.5 and 0.7% of Cu2+, respectively). The amount of food ingested was directly proportional to the degree of terrestriality: S. rectum, the most terrestrial species, ate around 2-3 times more than the other crabs, whereas G. cruentata ate 1.5-2 times more than N. granulata, the least terrestrial. The amount of Cu excreted in the feces was proportional to Cu ingestion, and was 76.8% and 64.2% higher for Sesarma fed diet D compared to G. cruentata and N. granulata, respectively. Sesarma also displayed higher Cu concentration in the haemolymph, gills and antennal glands, but not in the hepatopancreas. A detoxifying mechanism followed by elimination was probably present at this last organ, preventing Cu accumulation. More terrestrial crabs, such as Sesarma, may accumulate more Cu in hemolymph and tissues, showing a correlation between metal accumulation and increased terrestriality. In this aspect, contaminated feed sources with Cu may have more impact in conservation of terrestrial crabs. (C) 2008 Elsevier B.V. All rights reserved.

Changes in the morphology and ultrastructure of the Dufour's gland during the life cycle of the bumble bee queen, Bombus terrestris L-(Hymenoptera : Bombini)

The Dufour's gland is found closely associated with the sting apparatus of all female hymenopterans, playing multiple roles among bees. In some species of Bombus the gland may be involved in production of nestmate recognition pheromones, but in B. terrestris its function is not certain yet. The morphology of the :Dufour's gland of B. terrestris queens and the ultrastructural features of its cells were studied in different ages and behavioural stages using routine transmission electron microscopy. Measurements of the length and the diameter of the gland in the same conditions were also made. The Dufour's gland of the queen increases significantly in size (both in length and in diameter) with age and reproductive activity the ultrastructural features of the gland show electrondense material that comes from the haemolymph. This material is also present in the intercellular spaces, and is conducted to the subcuticular space, to be released directly into the glandular lumen. Hence at least part of the secretion is probably taken up directly from the haemolymph. The ultrastructural features indicate a more active phase of the gland corresponding to the period of egg-laying of the queen, and a decrease in activity when the queen is in hibernation as well as after the competition point. In conclusion, the gland is probably involved in reproduction, more specifically, in the marking off eggs.

Cytochemical localization of lipids in the venom glands of worker bees of Apis mellifera (Hymenoptera, Apidae)

The technique of osmium imidazol for the ultrastructural detection of lipids in the secretory cells of the venom gland of 14-days old worker bees of Apis mellifera L. demonstrated the presence of these components at various sites of the gland. These lipids were found mainly associated to the external region of the basal lamina and the microvilli, in the intercellular spaces, in the cuticle of the collecting canaliculi and in the secretion contained in the glandular lumen. Therefore, in addition to revealing the presence of lipids in the secretion, this technique also allowed us to attribute an exogenous origin to the lipids in the secretion; they are taken up from the haemolymph.

Morphological modifications induced by an artificial diet on the hypopharyngeal glands of Apis mellifera (Hymenoptera, apidae) during their degenerative process

The morphological aspects of the hypopharyngeal glands were analyzed in worker bees of Apis mellifera of 15 and 30 days of age. The individuals were kept in a room with controlled temperature at 32degreesC where they received water and either a protein or a high energy food. Nurse and foraging workers were used as a control for the experiment. The morphological results showed that the different diets modified the cell death characteristics and intensified its occurrence. Both diets caused precocious glandular degeneration. However, this anticipation of cell death was more pronounced in the glandular tissue of the workers who received the high energy diet when compared to the glands of the bees fed with the protein meal.The degenerative signs observed were an intense cytoplasmic vacuolization, with a loss of cytoplasm and of the cell boundaries, dilation or condensation of the cells and nuclei, and nuclear fragmentation. At the end of the degenerative process, we observed the extrusion of nuclei and, finally, the dissolution of the glands. The hypopharyngeal glands' remains were found in the haemolymph.

Lipid distribution in salivary glands of larvae and adult bees (Hymenoptera : Apidae)

Cytochemical studies were carried out to establish lipid distribution in the salivary glands of larvae and adult bees, using the imidazole buffer technique. In the duct cells of the larval salivary gland, the reaction was positive in the epicuticle and negative in the glandular lumen. The absence of smooth endoplasmic reticulum and the presence of lipids in the intercellular space suggest that lipids absorbed from the haemolymph could be used in the constitution of the epicuticle, after having been conveyed through the epithelium. In adult workers (new-emerged, nurse and forager workers), the head salivary glands presented a positive reaction in the secretion in glandular lumen, identifying its lipidic nature.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae)

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine, the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy. Tissue sections were subsequently stained with haematoxylin-eosin, bromophenol blue, silver, or a variant of the critical electrolyte concentration (CEC) method. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Glands were processed for the histochemical and cytochemical localization of acid phosphatase, as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed. The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and, additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen. The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed. The nuclei were pyknotic, with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Ocorrência, morfologia e ultra-estrutura da glândula de Dufour de Scaptotrigona postica Latreille (Hymenoptera: Apidae)

A glândula de Dufour é uma glândula acessória do aparelho reprodutivo feminino das abelhas. Nas abelhas neotropicais sem ferrão, tem sido pouca estudada sob todos os aspectos: morfológico, ontogenético e bioquímico. Na tentativa de colaborar com o conhecimento dessa glândula em abelhas sem ferrão, foi realizado um estudo da sua ocorrência, morfologia e desenvolvimento em Scaptotrigona postica Latreille. Os resultados mostraram que ela se encontra ausente nas operárias, como ocorre em muitas outras espécies desse grupo. Nas rainhas, as células glandulares parecem mais ativas nas virgens, possuindo uma desenvolvida rede de retículo endoplasmático liso tubular, grânulos de secreção e polirribossomos dispersos no citoplasma, além de apresentarem núcleos maiores do que os das células glandulares das fisogástricas. Nas rainhas fisogástricas há dois tipos de células glandulares, ambas aparentemente inativas sinteticamente. As glândulas das rainhas fisogástricas são claramente capazes de captar substâncias da hemolinfa, provavelmente lipídios, que não penetram nas células, mas passam pelos espaços intercelulares e, através da cutícula, chegam diretamente à luz da glândula. A bem desenvolvida dupla camada de lâmina basal ao redor da glândula pode atuar no processo de captação de substâncias da hemolinfa. A secreção, e conseqüentemente sua função, pode ser diferente nas duas classes de rainhas.

New morphological data on fat bodies of semi-engorged females of Amblyomma cajennense (Acari : Ixodidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morpho-physiological analysis of the insect fat body: A review

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

Fine structure of Mytella falcata (Bivalvia) gill filaments

Bivalve filter feeders are sessile animals that live in constant contact with water and its pollutants. Their gill is an organ highly exposed to these conditions due to its large surface and its involvement in gas exchanges and feeding. The bivalve Mytella falcata is found in estuaries of Latin America, on the Atlantic as well as the Pacific Coast. It is commonly consumed, and sometimes is the only source of protein of low-income communities. In this study, gill filaments of M. falcata were characterized using histology, histochemistry and transmission electron microscopy for future comparative studies among animals exposed to environmental pollutants. Gill filaments may be divided into abfrontal, intermediate and frontal zones. Filaments are interconnected by ciliary discs. In the center of filaments, haemocytes circulate through a haemolymph vessel internally lined by an endothelium and supported by an acellular connective tissue rich in polysaccharides and collagen. The abfrontal zone contains cuboidal cells, while the intermediate zone consists of a simple squamous epithelium. The frontal zone is composed of five columnar cell types: one absorptive, mainly characterized by the presence of pinocytic vesicles in the apical region of the cell; one secretory, rarely observed and three ciliated with abundant mitochondria. All cells lining the filament exhibit numerous microvilli and seem to absorb substances from the environment. PAS staining was observed in mucous cells in the frontal and abfrontal zones. Bromophenol blue allowed the distinction of haemocytes and detection of a glycoprotein secretion in the secretory cells of the frontal region. The characteristics of M. falcata gill filaments observed in this study were very similar to those of other bivalves, especially other Mytilidae, and are suitable for histopathological studies on the effect of water-soluble pollutants. (C) 2007 Elsevier Ltd. All rights reserved.

Aspectos da ultra-estrutura dos cecos gástricos da larva de Odontosciara sp. (Diptera: Sciaridae)

Ultrastructural features of the gastric caeca of Odontosciara sp. are reported. The species has 4 lateral caeca connected with the anterior midgut at the level of the foregut junction. The epithelial cell features indicate protein synthesis, digested material absorption from the lumen and haemolymph material absorption. Those functions, however, do not seem to be very intense.

Functional ultrastructure of the midgut of the fire ant Solenopsis saevissima Forel 1904 (Formicidae : Myrmicinae)

Solenopsis saevissima has a midgut composed of columnar, regenerative, and goblet cells. The midgut epithelium was covered by a basal lamina. Outside the basal lamina, layers of inner oblique, circular, and outer longitudinal muscles were present. Columnar cells showed a basal plasma membrane containing numerous folds, mitochondria, and the nucleus. Rough endoplasmic reticulum, Golgi bodies, membrane bounded vacuoles, and spherocrystals were found in this region. The apical plasma membrane was constituted by microvilli, which were above a region rich in mitochondria. Regenerative cells were found in groups lying by the basal lamina. Goblet cells were associated with an ion-transporting mechanism between the haemolymph and the midgut epithelium. These cells were lying by the midgut lumen and large microvilli were evident, but the cytoplasmic features were similar to the columnar cells.