Jenny Rodríguez

Penaeidins, a New Family of Antimicrobial Peptides Isolated from the Shrimp Penaeus vannamei (Decapoda)

We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimpsPenaeus vannamei in which immune response has not been experimentally induced. The three molecules display antimicrobial activity against fungi and bacteria with a predominant activity against Gram-positive bacteria. The complete sequences of these peptides were determined by a combination of enzymatic cleavages, Edman degradation, mass spectrometry, and cDNA cloning using a hemocyte cDNA library. The mature molecules (50 and 62 residues) are characterized by an NH2-terminal domain rich in proline residues and a COOH-terminal domain containing three intramolecular disulfide bridges. One of these molecules is post-translationally modified by a pyroglutamic acid at the first position. Comparison of the data obtained from the cDNA clones and mass spectrometry showed that two of these peptides are probably COOH-terminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

Measurement of reactive oxygen intermediate production in haemocytes of the penaeid shrimp, Penaeus vannamei.

Ž. A spectrophotometric nitroblue tetrazolium NBT reduction assay was used to demonstrate the Ž y . production of superoxide anions O by haemocytes of the white shrimp Penaeus Oannamei .I t 2 was found that haemocytes, without receiving an experimental stimulant, showed a rather high Ž background activity. Therefore, optimal parameters number of haemocytes, type of incubation . medium, type and concentration of stimulants were first established, in order to obtain a reliable and reproducible quantitative assay. With this optimized assay, and using specific inhibitors, it was shown that it is indeed the production of O y that was measured. Activities varied strongly 2 among individual shrimp specimens. Live bacteria, among these Vibrio strains, induced O y production in the haemocytes, in a dose-dependent manner. Whereas Vibrio anguillarum and a probiotic strain of V. alginolyticus evoked clear reactions, a pathogenic strain of V. harOeyi failed to elicit O y production in the haemocytes. It is discussed that this may explain this strains 2

State of the art of immunological tools and health control of penaeid shrimp

Shrimp farming constitutes an important source of revenue and employment in many develop- ing countries. However, infectious diseases have affected the profitability of the shrimp industry. For this reason, disease prevention is a priority and shrimp immunology has become a prime area of research. In such a perspective, studies into the value of cellular and humoral parameters as indicators of shrimp condition are being carried out, with the intention of developing criteria for sanitary surveys, immunomodulation studies and selection programs for shrimp with high resis- tance to pathogens. Several quantitative, fast and easy procedures are being adapted to evaluate the expression of the immune response of shrimp. In regard to cellular parameters, the hemogram . and two cellular mechanisms, the radical oxygen intermediates ROIs generated during post- . phagocytic events and phenoloxidase PO activity have been considered as potential markers. Concerning humoral parameters, the antibacterial activity of plasma and the concentration of plasma proteins can be considered as criteria of health status. Information is presented about the immunological tools used to evaluate these health markers and the results concerning the clinical significance of response modifications. q 2000 Elsevier Science B.V. All rights reserved.

Knowledge and research prospects in marine mollusc and crustacean immunology

Abstract In the context of infectious diseases in mollusc and shrimp aquaculture, research must be focused on diagnosis for zoosanitary controls but also on obtaining resistant animals. This last strategy depends heavily on the development of knowledge about marine invertebrate immunology. With the establishment of purification protocols for the main invertebrate pathogens, progress has been made in the study of host-pathogen interactions at cellular and molecular levels and in identifying immune effectors involved in the destruction of pathogens. Recent information on molluscs and crustaceans is presented, concerning both hemocyte studies and cellular defence functions and humoral effectors, with special reference to their application to selection of pathogen-resistant animals. With this aim, research prospects will essentially be devoted to the identification and characterization of immune genes, either specific or heterologous, which could be candidates for mollusc and shrimp genetic transformation.

Terrazoanthines, 2-Aminoimidazole Alkaloids from the Tropical Eastern Pacific Zoantharian Terrazoanthus onoi

The first chemical study of the common species Terrazoanthus onoi, present off the coast of Ecuador, led to the identification of a new family of 2-aminoimidazole alkaloids named terrazoanthines A-C (1-3). Homologues 1 and 2 feature an unprecedented 6-(imidazol-5-yl)benzo[d]imidazole. Acyl substitution pattern and complete configurational assignments were deduced from comparison between experimental and theoretical 13C NMR and ECD data, respectively. These compounds may represent key derivatives in the biosynthesis of zoanthoxanthins.

Immunodetection of hemocytes, peneidins and α2-macroglobulin in the lymphoid organ of white spot syndrome virus infected shrimp.

Viral diseases restrict the development of the world shrimp industry and there are few studies on cell response to the presence of viral infections. We performed immunohistochemistry assays to characterize hemocytes subpopulations involved in the immune process occurring in the LO of Litopenaeus vannamei shrimp. Tissue sections of animals that increased their LO spheroids and hemocytes infiltration after WSSV induced infection, were used. Three MABs namely, 40E10 (recognizing small granule hemocytes), 40E2 (recognizing large granule hemocytes), and 41B12, which recognize α2‐macroglobulin were used. Additionally one polyclonal antibody was used against the penaeidins antimicrobial peptides, and to detect WSSV a commercial immunohistochemistry kit (DiagXotics) was used. Numerous small granule hemocytes were detected in the stromal matrix of LO tubules, whereas large granule hemocytes were less numerous and located mainly in hemal sinuses. The exocytosis of two molecules, which have been related to the phagocytosis process, i.e. penaeidins, and α2‐macroglobulin, was detected in the external stromal matrix and the outer tubule walls. α2‐macroglobulin inhibits phenoloxidase activity and its strong release in LO tissue may explain the absence of melanization in the immune processes occurring in it. The immunolabeling of vesicles within the LO spheroids with MABs 41B12 40E10 and antipenaedin antibody suggests that LOS are formed by phagocytic cells derived from small granule and hyaline hemocytes, with a possible role of peneidins and α2‐macroglobulin acting as opsonines.

Assessing the Zoantharian Diversity of the Tropical Eastern Pacific through an Integrative Approach

Zoantharians represent a group of marine invertebrates widely distributed from shallow waters to the deep sea. Despite a high diversity and abundance in the rocky reefs of the Pacific Ocean, very few studies have been reported on the diversity of this group in the Tropical Eastern Pacific coasts. While molecular techniques recently clarified some taxonomic relationships within the order, the taxonomy of zoantharians is still highly challenging due to a lack of clear morphological characters and confusing use of different data in previous studies. Our first insight into the zoantharian diversity at El Pelado Marine Protected Area - Ecuador led to the identification of six species: Terrazoanthus patagonichus; Terrazoanthus sp.; Antipathozoanthus hickmani; Parazoanthus darwini; Zoanthus cf. pulchellus; and Zoanthus cf. sociatus. A metabolomic approach using UHPLC-HRMS was proven to be very efficient as a complementary tool in the systematics of these species and specialized metabolites of the ecdysteroid and alkaloid families were identified as key biomarkers for interspecific discrimination. These results show good promise for an application of this integrative approach to other zoantharians.

Ecdysonelactones, Ecdysteroids from the Tropical Eastern Pacific Zoantharian Antipathozoanthus hickmani

Despite a large occurrence, especially over the Pacific Ocean, the chemical diversity of marine invertebrates belonging to the order Zoantharia is largely underexplored. For the two species of the genus Antipathozoanthus no chemical study has been reported so far. The first chemical investigation of Antipathozoanthus hickmani collected at the Marine Protected Area “El Pelado”, Santa Elena, Ecuador, led to the isolation of four new ecdysteroid derivatives named ecdysonelactones. The structures of ecdysonelactones A–D (1–4) were determined based on their spectroscopy data, including 1D and 2D NMR and HRMS. The four compounds of this family of ecdysteroids feature an unprecedented γ-lactone fused at the C-2/C-3 position of ring A. These derivatives exhibited neither antimicrobial nor cytotoxic activities.

A simple in vitro method to evaluate the toxicity of functional additives used in shrimp aquaculture

Graphical abstract

Zoanthamine Alkaloids from the Zoantharian Zoanthus cf. pulchellus and Their Effects in Neuroinflammation

Two new zoanthamine alkaloids, namely 3-acetoxynorzoanthamine (1) and 3-acetoxyzoanthamine (2), have been isolated from the zoantharian Zoanthus cf. pulchellus collected off the coast of the Santa Elena Peninsula, Ecuador, together with three known derivatives: zoanthamine, norzoanthamine, and 3-hydroxynorzoanthamine. The chemical structures of 1 and 2 were determined by interpretation of their 1D and 2D NMR data and comparison with literature data. This is the first report of zoanthamine-type alkaloids from Zoanthus cf. pulchellus collected in the Tropical Eastern Pacific. The neuroinflammatory activity of all the isolated compounds was evaluated in microglia BV-2 cells and high inhibitory effects were observed in reactive oxygen species (ROS) and nitric oxide (NO) generation.