Trypanocidal and leishmanicidal activities of different antimicrobial peptides (AMPs) isolated from aquatic animals.

Most of the available animal antimicrobial peptides (AMPs) have been tested against bacteria and fungi, but very few against protozoan parasites. In the present study, we investigated the antiparasitic activity of different AMPs isolated from aquatic animals: tachyplesin (Tach, from Tachypleus tridentatus), magainin (Mag, from Xenopus laevis), clavanin (Clav, from Styela clava), penaeidin (Pen, from Litopenaeus vannamei), mytilin (Myt, from Mytilus edulis) and anti-lipopolysaccharide factor (ALF, from Penaeus monodon). The antiparasitic activity was evaluated against the promastigote form of Leishmania braziliensis and epi and trypomastigote forms of Trypanosoma cruzi, through the MTT method. Tach was the most potent peptide, killing completely L. braziliensis and trypomastigote T. cruzi from 12.5microM, whereas Pen and Clav were weakly active against trypomastigotes and Myt against L. braziliensis, only at a high concentration (100microM). Tach and Mag were markedly hemolytic at high concentrations, whereas the other peptides caused only a slight hemolysis (<10% up to 50microM). Our results point to Tach as the only potential candidate for further investigation and potential application as a therapeutic agent.

Hemocytes of the palaemonids Macrobrachium rosenbergii and M. acanthurus, and of the penaeid Penaeus paulensis.

The hemocytes of two palaemonids and one penaeid were characterized using light and transmission electron microscopy (TEM). The blood cells in all three species were classified as hyaline hemocytes (HH), small granule hemocytes (SGH), and large granule hemocytes (LGH). The HH are unstable hemocytes with a characteristic high nucleo-cytoplasmic ratio. Their cytoplasm appears particularly dense and has from few to numerous granules that often exhibit a typical striated substructure. In both palaemonids, the great majority of the HH contain numerous granules, whereas in Penaeus paulensis, a small number of these cells have few or no granules. The cytoplasm of some HH of the penaeid exhibits typical electron-dense deposits. The granulocytes, LGH and SGH, contain abundant electron-dense granules that are usually smaller in the SGH. In both hemocyte types, the cytosol, but not the granules, is rich in carbohydrates (PAS positive) and numerous vesicles contain acid phosphatase (Gomori reactive). In all studied shrimps, the SGH and LGH were actively phagocytic when examined on blood cell monolayers incubated with the yeast Saccharomyces cerevisiae. A few mitotic figures (less than 1%) were observed in the granulocytes of P. paulensis, but not in the palaemonids. SGH is the main circulating blood cell type in both palaemonids, whereas HH is predominant in the penaeid. Based on morphological and functional features, it appears that the hyaline and the granular hemocytes of the three shrimp species represent different cell lineages.

The prophenoloxidase activating system of the shrimp Penaeus paulensis and associated factors.

In the present study we investigated the proPO activating system of the penaeid Penaeus paulensis, focusing on its role in the shrimp immune system. The great majority of PO activity (more than 90%) was found in the shrimp hemocytes. The enzyme activity was greatly enhanced by components of microorganism cell walls, such as LPS and beta-1,3-glucans, suggesting its involvement in non-self recognition. PO activity was also found in the shrimp serum and trypsin, and LPS were able to increase the enzyme activity. Thus, serum can be used as an alternative for the study of the shrimp proPO activating system, as it is much more readily obtained than HLS. PO activity was cation-dependent, and 5 mM of calcium and 10 mM of magnesium were the optimal concentrations for the enzyme activity. An immune factor was found in the shrimp HLS, capable of inducing cell-adhesion and degranulation of the penaeid hemocytes.

Effect of fixed epimastigote forms of Trypanosoma cruzi on the hemocytes and the prophenoloxidase-activating system of the crayfish Pacifastacus leniusculus.

The effect of the parasite Trypanosoma cruzi on the hemocytes and the prophenoloxidase (proPO)-activating system of the crayfish Pacifastacus leniusculus was studied. Incubation of the crayfish hemocyte lysate with fixed epimastigote forms of the parasites (4 x 10(5) cells/ml) induced a marked activation of the crayfish proPO system, measured as phenoloxidase activity. The activation of proPO by the parasite was much stronger (7-fold) than that induced by beta-1, 3-glucans (1 mg/ml) which are known to be efficient elicitors of the proPO system. The fixed parasites promoted the spreading and degranulation of different populations of the crayfish hemocytes isolated by Percoll gradients, and were often observed to be attached to the crayfish hemocytes in rosette-like fashion. The attachment of the epimastigote forms of T. cruzi to the crayfish blood cell surface was not dependent on the adhesive 76-kDa protein released by the crayfish hemocytes, since the exocytotic inhibitor SITS and monospecific antibodies to the 76-kDa protein did not prevent parasite adhesion. The crayfish hemocytes apparently are able to phagocytose the fixed epimastigote forms of T. cruzi in vitro.

Effect of fixed epimastigote forms of Trypanosoma cruzi on the hemocytes and the prophenoloxidase-activating system of the crayfish Pacifastacus leniusculus

The effect of the parasite Trypanosoma cruzi on the hemocystes and the prophenoloxidase (proPO)-activating system of the crayfish Pacifastacus leniusculus was studied. Incubation of the crayfish hemocyte lysate with fixed epimastigote forms of the parasites (4 x 10(5) cells/ml) induced a marked activation of the crayfish proPO system, measured as phenoloxidase activity. The activation of proPO by the parasite was much stronger (7-fold) than that induced by beta-1,3-glucans (1 mg/ml) which are known to be efficient elicitors of the proPO system. The fixed parasites promoted the spreading and degranulation of different populations of the crayfish hemocytes isolated by Percoll gradients, and were often observed to be attached to the crayfish hemocytes in rosette-like fashion. The attachment of the epimastigote forms of T. cruzi to the crayfish blood cell surface was not dependent on the adhesive 76-kDa protein released by the crayfish hemocytes, since the exocytotic inhibitor SITS and monospecific antibodies to the 76-kDa protein did not prevent parasite adhesion. The crayfish hemocytes apparently are able to phagocytose the fixed epimastigote forms of T. cruzi in vitro.

Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila.

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmitter of the trematode Schistosoma mansoni in Brazil, were examined by light and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.

Ultrastructural studies of the hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae).

Ultrastructural analyses revealed the presence of six hemocyte types in the hemolymph of Panstrongylus megistus, partially confirming our previous results obtained through light microscopy. Prohemocytes: small, round hemocytes with a thin cytoplasm layer, especially rich in free ribosomes and poor in membranous systems. Plasmatocytes: polymorphic cells, whose cytoplasm contains many lysosomes and a well developed rough endoplasmic reticulum (RER). They are extremely phagocytic. Sometimes, they show a large vacuolation. Granulocytes: granular hemocytes whose granules show different degrees of electrodensity. Most of them, have an internal structuration. Coagulocytes: oval or elongated hemocytes, which show pronounced perinuclear cisternae as normally observed in coagulocytes. The cytoplasm is usually electrodense, poor in membranous systems and contains many labile granules. Oenocytoids: large and very stable hemocytes, whose homogeneous cytoplasm is rich in loose ribosomes and poor in membranous systems. Adipohemocytes: large cells, containing several characteristic lipid droplets. The cytoplasm is also rich in glycogen, RER and large mitochondria. The total and differential hemocyte count (THC and DHC) were also calculated for this reduviid. THC increases from 2,900 hemocytes/mm3 of hemolymph in the 4th instar to 4,350 in the 5th and then, decreases to 1,950 in the adults. Plasmatocytes and coagulocytes are the predominant hemocyte types.

The hemocytes of Panstrongylus megistus (Hemiptera: Reduviidae).

Five hemocyte types were identified in the hemolymph of Panstrongylus megistus by phase contrast and common light microscopy using some histochemical methods. These are: Prohemocytes, small cells presenting a great nucleus/cytoplasm ratio; Plasmatocytes, the most numerous hemocytes, are polymorphic cells mainly characterized by a large amount of lysosomes; Granulocytes, hemocytes very similar to plasmatocytes which contain cytoplasmic granules and are especially rich in polysaccharides; Oenocytoids, cells presenting a small nucleus and a thick cytoplasm; they show many small round vacuoles when observed in Giemsa smears and many cytoplasmic granules under phase microscopy; Adipohemocytes, very large hemocytes, presenting many fat droplet inclusions which could correspond to free fat bodies which entered the hemolymph. Only prohemocytes and plasmatocytes can be clearly classified; all the other hemocyte types have a more ambiguous classification.