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.

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging bacterial disease of cultured shrimp caused mainly by Vibrio parahaemolyticus, which harbors the lethal PirAB toxin genes. Although Penaeus vannamei (P. vannamei) postlarvae are susceptible to AHPND, the changes in the bacterial communities through the larval stages affected by the disease are unknown. We characterized, through high-throughput sequencing, the microbiome of P. vannamei larvae infected with AHPND-causing bacteria through the larval stages and compared the microbiome of larvae collected from high- and low-survival tanks. A total of 64 tanks from a commercial hatchery were sampled at mysis 3, postlarvae 4, postlarvae 7, and postlarvae 10 stages. PirAB toxin genes were detected by PCR and confirmed by histopathology analysis in 58 tanks. Seven from the 58 AHPND-positive tanks exhibited a survival rate higher than 60% at harvest, despite the AHPND affectation, being selected for further analysis, whereas 51 tanks exhibited survival rates lower than 60%. A random sample of 7 out of these 51 AHPND-positive tanks was also selected. Samples collected from the selected tanks were processed for the microbiome analysis. The V3–V4 hypervariable regions of the 16S ribosomal RNA (rRNA) gene of the samples collected from both the groups were sequenced. The Shannon diversity index was significantly lower at the low-survival tanks. The microbiomes were significantly different between high- and low-survival tanks at M3, PL4, PL7, but not at PL10. Differential abundance analysis determined that biomarkers associated with high and low survival in shrimp hatchery tanks affected with AHPND. The genera Bacillus, Vibrio, Yangia, Roseobacter, Tenacibaculum, Bdellovibrio, Mameliella, and Cognatishimia, among others, were enriched in the high-survival tanks. On the other hand, Gilvibacter, Marinibacterium, Spongiimonas, Catenococcus, and Sneathiella, among others, were enriched in the low-survival tanks. The results can be used to develop applications to prevent losses in shrimp hatchery tanks affected by AHPND.

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