Calnexin is a 90-kDa integral membrane protein of the endoplasmic reticulum (ER). Calnexin binds Ca2+ and may function as a chaperone in the transition of proteins from the ER to the outer cellular membrane. We have purified human calnexin in association with the human interferon-gamma receptor and cloned calnexin cDNA from placenta. Fragments of calnexin have been prepared as glutathione S-transferase fusion proteins and analyzed for their abilities to bind 45Ca2+ and ruthenium red. A subdomain containing four internal repeats binds Ca2+ with the highest affinity. This sequence is highly conserved when compared to calreticulin (a luminal ER protein), an Onchocerca surface antigen, and yeast and plant calnexin homologues. Consequently, this sequence represents a conserved motif for the high-affinity binding of Ca2+, which is clearly distinct from the "E-F hand" motif. An adjacent subdomain, also highly conserved and containing four internal repeats, fails to bind Ca2+. The carboxyl-terminal, cytosolic domain is highly charged and binds Ca2+ with moderate affinity, presumably by electrostatic interactions. The calnexin amino-terminal domain (residues 1-253) also binds Ca2+, in contrast to the amino-terminal domain of calreticulin, which is relatively less acidic. We have also determined the cDNA sequences of mouse and rat calnexins. Comparison of the known mammalian calnexin sequences reveals very high conservation of sequence identity (93-98%), suggesting that calnexin performs important cellular functions. The gene for human calnexin is located on the distal end of the long arm of human chromosome 5, at 5q35.

Wild Litopenaeus Õannamei females in different stages of sexual maturation were sampled, Ž . including spent females and their nauplii, for determination of the lipid content, lipid class LC Ž . composition, fatty acid FA composition, vitamin C content and vitamin E content. Free FA Ž . Ž . Ž . Ž . FFA , triacylglycerol TAG , phosphatidylcholine PC and sterol esters SE were the dominant Ž . LC in the midgut gland. TAG and phospholipids PL , mainly PC and phosphatidylethanolamine Ž .Ž . Ž . PE , were the dominant ovarian LC. Neutral lipids NL prevailed over polar lipids POL in midgut gland lipids, while ovarian lipids displayed an inverse relationship. An increase in ovarian Ž . Ž . TL was observed from stage 0 immature to stage 1 early maturing . Later, from stage 1 to stage Ž . 2 mid maturing , a decrease in midgut gland TL was observed. TAG was most responsible for these changes in TL. Lipids were preferentially transferred to the nauplii, which contained relatively high TAG and PC levels. In both midgut gland and ovaries, 16:00, 18:00, 16:1n y 7, Ž . Ž . 18:1n y 9, arachidonic acid ARA; 20:4n y 6 , eicosapentaenoic acid EPA; 20:5n y 3 and Ž . docosahexaenoic acid DHA; 22:6n y 3 were the principal FA. All tissues and nauplii displayed n y 3 ) n y 6 and EPA ) DHA relationships, and contained high proportions of n y 3 highly Ž . unsaturated FA n y 3 HUFA . During sexual maturation, the sum of poly-unsaturated FA Ž . PUFA decreased in the ovaries due to the decrease in n y 6 PUFA such as ARA. The sum of Ž . mono-unsaturated FA MUFA , on the other hand, increased in the ovaries. AA levels were high ) Corresponding author. Laboratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, B-9000 Gent, Belgium. Tel.: q32-9-264-37-54; fax: q32-9-264-41-93.Ž . E-mail address: r.wouters@inve.be R. Wouters . Wouters et al.r Aquaculture 198 2001 307-323 308 in immature, maturing and mature ovaries. They were low in the ovaries of spent females and nauplii. Vitamin E levels were low in immature ovaries, increased substantially during ovarian maturation, and then decreased again upon spawning. High vitamin E levels were retained in the nauplii. The findings of this study, combined with those reported in related studies, suggest the importance of n y 3 HUFA for larval development, of vitamin C for egg development and hatching, and of vitamin E for ovarian maturation and larval development. These nutrients cannot be synthesised de novo by shrimp, and should be included at high levels in the broodstock diet. q

ABSTRACT. A culture of Vibrio harveyi, isolated from diseased Penaeus vannamei, was pathogenic in penaeid shrimp larvae when used in a bath at 105 cells n~l-' for 2 h. The resultant disease had characteristics of Bolitas negricans, as observed in Ecuadorian hatcheries, namely the development of bioluminescence, reduced feeding and retarded development, sluggish swimming, reduced escape mechanisms, degeneration of hepatopancreatic tissue with resultant formation of necrotic bundles, and increased mortality. Koch's Postulates were confirmed by reisolation and identification of the organism. Histopathology showed the presence of distlnctive melanotic tissue aggregates within the hepatopancreas, with irnmunohistochemistry confirming the presence of large numbers of v harveyi in the intestine and hepatopancreas.These results indicate a suitable infection protocol, which can be used to test the pathogenicity of putative pathogens of penaeid shrimp larvae.

Several bacteriological surveys were performed from 1994 to 1996 at different Litopenaeus vannamei hatcheries (in Ecuador) and shrimp farms (in Mexico). Samples were taken from routine productions of healthy and diseased L. vannamei larvae, postlarvae, and their culture environment and from healthy and diseased juveniles and broodstock. In Ecuador, the dominant bacterial flora associated with shrimp larvae showing symptoms of zoea 2 syndrome, mysis mold syndrome, and bolitas syndrome has been determined. Strains were characterized by Biolog metabolic fingerprinting and identified by comparison to a database of 850 Vibrio type and reference strains. A selection of strains was further genotypically fine typed by AFLP. Vibrio alginolyticus is predominantly present in all larval stages and is associated with healthy nauplius and zoea stages. AFLP genetic fingerprinting shows high genetic heterogeneity amongV. alginolyticus strains, and the results suggest that putative probiotic and pathogenic strains each have specific genotypes. V. alginolyticus was found to be associated with larvae with the zoea 2 syndrome and the mysis mold syndrome, while different Vibrio species (V. alginolyticus and V. harveyi) are associated with the bolitas syndrome. V. harveyi is associated with diseased postlarvae, juveniles, and broodstock. The identities of the strains identified as V. harveyi by the Biolog system could not be unambiguously confirmed by AFLP genomic fingerprinting.Vibrio strain STD3-988 and one unidentified strain (STD3-959) are suspected pathogens of only juvenile and adult stages. V. parahaemolyticus, Photobacterium damselae, and V. mimicus are associated with juvenile and adult stages.

We report on the data mining of publicly available Litopenaeus vannamei expressed sequence tags (ESTs) to generate simple sequence repeat (SSRs) markers and on their transferability between related Penaeid shrimp species. Repeat motifs were found in 3.8% of the evaluated ESTs at a frequency of one repeat every 7.8 kb of sequence data. A total of 206 primer pairs were designed, and 112 loci were amplified with the highest success in L. vannamei. A high percentage (69%) of EST-SSRs were transferable within the genus Litopenaeus. More than half of the amplified products were polymorphic in a small testing panel of L. vannamei. Evaluation of those primers in a larger testing panel showed that 72% of the markers fit Hardy-Weinberg equilibrium, which shows their utility for population genetic analysis. Additionally, a set of 26 of the EST-SSRs were evaluated for Mendelian segregation. A high percentage of monomorphic markers (46%) proved to be polymorphic by singles-stranded conformational polymorphism analysis. Because of the high number of ESTs available in public databases, a data mining approach similar to the one outlined here might yield high numbers of SSR markers in many animal taxa.

Culture fluids of peritoneal exudate cells rich in macrophages stimulated DNA synthesis of thymocytes and, to lesser extent, of spleen cells. We also investigated the effects of culture fluids from macrophages on the in vitro response to a hapten-carrier protein (fluorescein-menocyanin) using spleen cells from immune mice. Macrophage culture fluids contained an activity that increased the plaque-forming cell response of both IgG and IgM class. This increase was observed in the absence of any added hapten protein to the culture. The helper function of T lymphocytes (as evidenced by challenging with the hapten on the homologous carrier) was also increased by the macrophage culture fluid. However, this enhancement was best observed in conditions of relatively low T-cell activity. Also, the macrophage fluid allowed spleen cells of nude athymic mice to make a plaque-forming cell response to sheep red blood cells of both the IgM and IgG class. The macrophage was the cell source of the stimulatory molecule since it was generated only in cultures of macrophages devoid of significant number of lymphocytes. Stimulatory activity was not found in cultures of lymphocytes, mouse embryo cells, or 3T3 cells. The thymocyte stimulatory molecule did not contain H-2 antigens, was resistant to diisopropylfluorophosphate treatment, eluted from Sephadex with a size ranging from 15,000 to 21,000 daltons, and was sensitive to chymotrypsin and pepsin.

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.

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.