José Roberto Machado Cunha da Silva

Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion

A large amount of heme is produced upon digestion of red cell hemoglobin in the midgut of mosquitoes. The interaction between heme and the peritrophic matrix (PM) was studied in Aedes aegypti. By light microscopy, the PM appeared as a light brownish layer between the intestinal epithelium and the alimentary bolus. This natural color can be attributed to the presence of heme bound to the matrix. In histochemical studies, a diffuse peroxidase activity of the heme molecules was clearly observed between the erythrocytes and the PM at 14 h after the blood meal. This activity tends to increase and concentrate in the PM reaching its maximum thickness at 24 h after feeding. Most of the heme of the PM was found associated to with enormous number of small electron-dense granules. The amount of heme bound to the PM increased in parallel with the progression of digestion, reaching a maximum at 48 h after feeding, when 18 nmol of heme were found in an individual matrix. The association of heme with PM from insects fed with plasma is saturable, suggesting the existence of specific binding sites for hemin in the PM. Taken all together, our data indicate that the PM performs a central role in heme detoxification in this insect.

Parotoid macroglands in toad (Rhinella jimi): Their structure and functioning in passive defence

When toads (Rhinella) are threatened they inflate their lungs and tilt the body towards the predator, exposing their parotoid macroglands. Venom discharge, however, needs a mechanical pressure onto the parotoids exerted by the bite of the predator. The structure of Rhinella jimi parotoids was described before and after manual compression onto the macroglands mimicking a predator attack. Parotoids are formed by honeycomb-like collagenous alveoli. Each alveolus contains a syncytial gland enveloped by a myoepithelium and is provided with a duct surrounded by differentiated glands. The epithelium lining the duct is very thick and practically obstructs the ductal lumen, leaving only a narrow slit in the centre. After mechanical compression the venom is expelled as a thin jet and the venom glands are entirely emptied. The force applied by a bite of a potential predator may increase alveolar pressure, forcing the venom to be expelled as a thin jet through the narrow ductal slit. We suggest that the mechanism for venom discharge within all bufonids is possibly similar to that described herein for Rhinella jimi and that parotoids should be considered as cutaneous organs separate from the rest of the skin specially evolved for an efficient passive defence.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus.

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors, antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens.

Induced in vitro phagocytosis of the Antarctic starfish Odontaster validus (Koehler 1906) at 0°C

Abstract Phagocytosis was studied in vitro using coelomic fluid of the Antarctic starfish Odontaster validus at 0°C. The number of coelomocytes present was determined and the phagocytic activity of the phagocytic amoebocytes (PA) was quantified with yeast during incubations of 1 and 2 h. The percentage of PA phagocytosing increased significantly from 42.29 ± 10.50% (SD) at 1 h to 52.57 ± 13.96% at 2 h. Numbers of yeast per PA also rose significantly from 2.27 to 2.45 cells per amoebocyte, indicating that phagocytic activity was maintained. In vitro phagocytosis of an Antarctic invertebrate at 0°C is shown for the first time, and the types of amoebocytes involved identified. Rates of phagocytosis were similar to, or higher than, reported data for temperate starfish, although this conclusion must be treated cautiously because of scarcity of data and differences in methods used. However, the data suggest that phagocytosis in O. validus is well adapted to low temperature.

Epidermal glands in Squamata: Microscopical examination of precloacal glands in Amphisbaena alba (Amphisbaenia, Amphisbaenidae)

The femoral or cloacal region of many species of lizards and amphisbaenians exhibits epidermal glands. The pores of these glands are plugged with holocrine solid secretions that serve as semiochemical sources. Many authors assume that these glands are mainly associated with reproduction and demarcation of territory. The structure of precloacal glands in Amphisbaena alba was previously studied by Antoniazzi et al. (Zoomorphology 113:199–203, 1993; J. Morphol. 221:101–109, 1994). These authors suggested that as the animal moves inside tunnels, the secretion plugs are abraded against the substrate, releasing a secretion trail. Some aspects of the plug were difficult to interpret in fine sections due to the dense and brittle nature of the plug. The morphology of the trail, and the manner of deposition on the substrate, have never been reported. This study presents a primarily scanning electron microscopic description of A. alba precloacal glands and of the secretion plugs. It also demonstrates experimentally the formation of the trail and its fine morphology. The results show that when the plugs scrape against the substrate, their constitution helps them to fragment into tiny pieces, which are spread on the ground, thus forming a trail. Each one of the fragments corresponds to a secretion granule of the precloacal glands secretory cells. In this way, the trail might have an extensive area for volatilization of semiochemicals, constituting an efficient means of intraspecific communication inside the tunnels. J. Morphol. 241:197–206, 1999.

New perspectives on the dispersal mechanisms of the Antarctic brooding bivalve Mysella charcoti (Lamy, 1906)

Abstract. Brooding is a widespread phenomenon among Antarctic bivalves. Although it should represent a handicap to dispersion, many brooding species have achieved a wide distribution in Antarctic and subantarctic waters, suggesting that they have alternative and effective methods of dispersal. Evidence of such an alternative method is presented here for the bivalve Mysella charcoti, unexpectedly found alive and healthy in feces expelled by Nototheniacoriiceps (Nototheniidae: Pisces). The finding indicates that the snug-fitting shell of Mysella functions as a barrier to digestive enzymes. Withstanding passage through the digestive tract of fish allows Mysella to be passively dispersed (within the home range of the fish) and colonize new habitats or re-colonize shallow-water substrates severely impacted by ice scours.

The impact of rising sea temperature on innate immune parameters in the tropical subtidal sea urchin Lytechinus variegatus and the intertidal sea urchin Echinometra lucunter

Ocean temperatures are rising throughout the world, making it necessary to evaluate the impact of these temperature changes on sea urchins, which are well-known bioindicators. This study evaluated the effect of an increase in temperature on the immune response of the subtidal Lytechinus variegatus and the intertidal Echinometra lucunter sea urchins. Both species were exposed to 20 (control), 25 and 30 °C temperatures for 24 h, 2, 7 and 14 days. Counting of coelomocytes and assays on the phagocytic response, adhesion and spreading of coelomocytes were performed. Red and colorless sphere cells were considered biomarkers for heat stress. Moreover, a significant decrease in the phagocytic indices and a decrease in both cell adhesion and cell spreading were observed at 25 and 30 °C for L. variegatus. For E. lucunter, the only alteration observed was for the cell proportions. This report shows how different species of sea urchins respond immunologically to rising temperatures.

Enhancement of liver size by stimulation of intact rat liver with exogenous hepatotrophic factors

In mammals, liver size is related to animal body weight at the 2.5 to 3% proportion, a ratio mediated by the afflux of hepatotrophic factors. Formulas capable of modifying this ratio have been developed in previous studies on the rat, with enhancement of liver size brought about by intraperitoneal (portal) infusion of exogenous factors such as glucose, amino acids, insulin, glucagon, vitamins, electrolytes, and triiodothyronine. However, the efficacy of these formulations was accompanied by increased animal mortality (PARRA et al.). The present study, which was carried out with small methodological modifications on a larger number of rats using daily intraperitoneal injections of a solution of exogenous hepatotrophic factors (40 ml/kg) for seven days, confirms the previous findings, with a 114.16 +/- 7.90% enhancement of liver size beyond the expected value for the body weight of the animal. However, the problem of animal mortality was not fully resolved.

CLONING AND EXPRESSION ANALYSIS OF ALLOGRAFT INFLAMMATORY FACTOR TYPE 1 IN COELOMOCYTES OF ANTARCTIC SEA URCHIN (STERECHINUS NEUMAYERI)

ABSTRACT We have cloned and characterized for the first time an allograft inflammatory factor 1 (Sn-AIF-1) from the Antarctic sea urchin. We report the cloning of Sn-AIF-1 cDNA and the characterization of its expression in coelomocytes after a bacterial challenge. The cDNA Sn-AIF-1 has a size of 608 bp and encodes a polypeptide of 151 aa. The deduced amino acid sequence has a putative size of 17.430 Da, an isoelectric point of 4.92, and shows 2 elongation factor handlike motifs that normally bind calcium ions. BLAST analysis revealed close matches with other known AIF-1. The deduced amino acid sequence of Sn-AIF-1 showed high homology with AIF-1 in vertebrates such as fish, mice, and humans; and in the case of invertebrates, the major degree of identity (55%) was with a predicted sequence of the purple sea urchin AIF-1, and 52% corresponded to a sponge. Expression of Sn-AIF-1 mRNA was analyzed by qPCR. Sn-AIF-1 mRNA expression was measured from coelomocytes after a bacterial challenge using RT-PCR and revealed that the gene was upregulated after 24 h. Sn-AIF-1 could participate in the inflammatory response, particularly in the activation of coelomocytes and their survival.

Induced inflammatory process in the Antarctic fish Notothenia neglecta

Abstract We studied the ability of the Antarctic fish Notothenia neglecta to conduct an induced inflammatory process at 0°C. Indian ink was injected and a cotton suture thread was implanted into muscle of different groups of fish. After 1–2 days of Indian ink injection, the ink was diffused in the perimysium and there was hemorrhage and cellular infiltrate composed mainly of macrophages A (with few and small lysosomes) and neutrophils; after 7–15 days, there were macrophages A and some macrophages B (cytoplasm clear, lamellar cytoplasmic system forming interdigitations); after 30 days, there was Indian ink in the interior of macrophages A. The suture thread process takes place in two phases: the first (up to 7 days) with predominance of macrophages A and few neutrophils, and the second (15–30 days) with predominance of macrophages B. It can be concluded that N. neglecta is responsive to irritant stimulus with inflammatory process indicating adaptation to the antarctic environment.