We report here the isolation of three members of a new family of antimicrobial peptides from the hemolymph of shrimps Penaeus 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 NH 2 -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 COOHterminally amidated by elimination of a glycine residue. These molecules with no evident homology to other hitherto described antimicrobial peptides were named penaeidins.

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 Õannamei. It 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 2 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. harÕeyi failed to elicit O y production in the haemocytes. It is discussed that this may explain this strain's 2 capability of evading the host's oxidative microbicidal activity, which would be a virulence factor in these bacteria. Heat-killed bacteria hardly induced NBT reduction in the cells. The fungicide propiconazole or Tilt w , found as a pollutant in the aquatic environment where the shrimp are reared, was tested for its effect on NBT reduction by the haemocytes. In haemocytes that did not receive an experimental stimulant, Tilt w induced the reduction of NBT in a dose-dependent manner. In experimentally stimulated haemocytes, however, Tilt w strongly reduced the reaction upon the stimulant PMA. Probable explanations for these seemingly controversial effects of Tilt w are discussed, as are possible consequences of this sort of pollutants for shrimp aquaculture. This easy to perform and relatively cheap and simple quantitative assay for measuring the activity of an oxidative microbicidal mechanism in shrimp haemocytes, appears quite reliable and may therefore prove to be a valuable tool for monitoring shrimp health and immunologic status. q

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Choice of forage species and harvest management may influence protein degradability in cattle diets. We measured forage yield, undegradable intake protein (UIP), and forage quality at three maturities in two cuttings (spring, regrowth) of alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), and birdsfoot trefoil (Lotus corniculatus L.) in the seeding and two subsequent years. At equivalent harvest stage, trefoil and clover were usually lower in neutral detergent fiber (NDF) and acid detergent fiber (ADF) than alfalfa. Crude protein (CP) was lower in red clover than trefoil or alfalfa for three of six cuttings, but red clover was equal to or higher in UIP than alfalfa or trefoil in all cuttings. Trefoil never had higher UIP concentrations than alfalfa. Trefoil tannin concentrations increased with maturity and were positively correlated with UIP concentration. For all species, UIP was positively correlated with NDF and ADF and negatively correlated with in situ dry matter disappearance (ISDMD). In alfalfa and trefoil, UIP was negatively correlated with CP. In all legumes, dry matter yield (DMY), NDF, and ADF increased with maturity, while CP and ISDMD decreased. Changes in NDF, ADF, CP, and ISDMD with maturity were usually most rapid in trefoil. The proportion of UIP increased with maturity for all species in all cuttings, but it never comprised more than 240 g kg−1 CP. Small increases in UIP as legumes matured were gained at the expense of other forage quality measures.

Mortalities of cultured shrimp, Penaeus vannamei (Boone), induced by white spot syndrome virus (WSSV) have occurred in Ecuador since May 1999. Three epidemiological surveys in Ecuadorian farms were carried out and showed an apparent association between lower temperature and increased mortality rates in commercial ponds. Infected animals showed a reddish discolouration and lethargy and occasionally, white spots in the exoskeleton. Histopathological studies revealed that infected cells presented nuclear hypertrophy with eosinophilic to basophilic inclusions. In some cases, two other pathologies were observed: (a) lymphoid organ spheroids and (b) cells with pyknotic and karyorrhectic nuclei in the lymphoid organ, haematopoietic tissue, connective tissue, heart and antennal gland. Occasionally pyknotic cells were encapsulated without apparent injury to the adjacent tissue and without melanization. Transmission electron microscopy showed the presence of WSSV particles in the cytoplasm of cells with pyknotic nuclei in the stomach hypodermis. Viral structure and morphogenesis agreed with previous descriptions by other authors in WSSV-infected shrimp. Occasionally, two nucleocapsids within one envelope were present amongst single enveloped nucleocapsids. A long rod-shaped structure that could reach 2.4 lm in length was present in the nuclei of some infected cells.

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