Hsp70 Knockdown in the Brine Shrimp Artemia franciscana: Implication on Reproduction, Immune Response and Embryonic Cuticular Structure.

The potential functional role(s) of heat shock protein 70 (Hsp70) in the brine shrimp, Artemia franciscana, a crucial crustacean species for aquaculture and stress response studies, was investigated in this study. Though we have previously reported that Hsp70 knockdown may have little or no impact on Artemia development, the gestational survival and number of offspring released by adult females were impaired by obscuring Hsp70 synthesis. Transcriptomic analysis revealed that several cuticle and chitin synthetic genes were downregulated, and carbohydrate metabolic genes were differentially expressed in Hsp70-knockdown individuals. A more comprehensive microscopic examination performed in this study revealed exoskeleton structural destruction and abnormal eye lenses featured in Hsp70-deficient adult females 48 h after Hsp70 dsRNA injection. Cysts produced by these Hsp70-deficient broods, instead, had a defective shell and were smaller in size, whereas nauplii had shorter first antennae and a rougher body epicuticle surface. Changes in carbohydrate metabolism caused by Hsp70 knockdown affected glycogen levels in adult Artemia females, as well as trehalose in cysts released from these broods, indicating that Hsp70 may play a role in energy storage preservation. Outcomes from this work provided novel insights into the roles of Hsp70 in Artemia reproduction performance, cyst formation, and exoskeleton structure preservation. The findings also support our previous observation that Hsp70 knockdown reduced Artemia nauplius tolerance to bacterial pathogens, which could be explained by the fact that loss of Hsp70 downregulated several Toll receptor genes (NT1 and Spaetzle) and reduced the integrity of the exoskeleton, allowing pathogens to enter and cause infection, ultimately resulting in mortality.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.

Beneficial microbes to suppress Vibrio and improve the culture performance of copepod Tigriopus japonicus Mori.

The use of beneficial microbes, i.e., probiotics, to reduce pathogens and promote the performance of the target species is an important management strategy in mariculture. This study aimed to investigate the potential of four microbes, Debaryomyces hansenii, Ruegeria mobilis, Lactobacillus plantarum, and Bacillus subtilis, to suppress Vibrio and increase survival, population growth and digestive enzyme activity (protease, lipase, and amylase) in the harpacticoid copepod, Tigriopus japonicus. Copepod, T. japonicus stock culture with an initial mean density of 50 individual/mL (25 adult male and 25 adult female) was distributed into five treatments (i.e., four experimental and a control, each with four replicates; repeated twice) using 20 beakers (100 mL capacity each). The copepods were fed a mixture of the dinoflagellate Alexandrium tamarense and the diatom Phyaeodactylum tricornutum (3 × 104 cells/mL-1). Each microbe's concentration was adjusted at 108 CFU/mL-1 and applied to the culture condition. D. hansenii, L. plantarum, and B. subtilis all improved the copepods' survival and population growth, likely by including a higher lipase activity (P < 0.05). In contrast, using R. mobilis did not improve the copepod's culture performance compared to control. B. subtilis was the most effective in decreasing the copepod's external and internal Vibrio loading. The probiotic concentrations in the copepod decreased within days during starvation, suggesting that routine re-application of the probiotics would be needed to sustain the microbial populations and the benefits they provide. Our results demonstrated that D. hansenii and B. subtilis are promising probiotics for mass copepod culture as live food for mariculture purposes.

The effects of Pandanus tectorius leaf extract on the resistance of White-leg shrimp Penaeus vannamei towards pathogenic Vibrio parahaemolyticus.

Pandanus tectorius leaf extract effect on the White-leg shrimp Penaeus vannamei tolerance against Vibrio parahaemolyticus were investigated in this study. Thirty shrimp post-larvae measured at approximately 1 cm were exposed for 24 h to 0.5, 1, 2, 3, 4, 5 and 6 g/L leaf extract and subsequently observed for survival and immune-related genes expression (Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase), followed by determination of their tolerance and histological tissue profiles upon Vibrio challenge. Survival of shrimps treated with 6 g/L of leaf extract improved by up to 95% to controls. Hsp70, crustin, and prophenoloxidase mRNA levels were observed to be 8.5, 10.4, and 1.5-fold higher, respectively. Histopathological analysis of the hepatopancreas and the muscle tissues revealed major tissue degeneration in Vibrio-challenged shrimps but not in shrimps primed with P. tectorius leaf extract. Of all the dose examined, the best pathogen resistance results were obtained with a 24 h incubation of shrimp in 6 g/L P. tectorius methanolic leaf extract. The tolerance towards V. parahaemolyticus might be associated with the increased regulation of Hsp70, prophenoloxidase and crustin upon exposure to the extract, all immune-related proteins essential for pathogen elimination in Penaeid shrimp. The present study primarily demonstrated that P. tectorius leaf extract is a viable alternative for enhancing P. vannamei post-larvae resistance against V. parahaemolyticus, a major bacterial pathogen in aquaculture.

The genome of the extremophile Artemia provides insight into strategies to cope with extreme environments.

BACKGROUND: Brine shrimp Artemia have an unequalled ability to endure extreme salinity and complete anoxia. This study aims to elucidate its strategies to cope with these stressors. RESULTS AND DISCUSSION: Here, we present the genome of an inbred A. franciscana Kellogg, 1906. We identified 21,828 genes of which, under high salinity, 674 genes and under anoxia, 900 genes were differentially expressed (42%, respectively 30% were annotated). Under high salinity, relevant stress genes and pathways included several Heat Shock Protein and Leaf Embryogenesis Abundant genes, as well as the trehalose metabolism. In addition, based on differential gene expression analysis, it can be hypothesized that a high oxidative stress response and endocytosis/exocytosis are potential salt management strategies, in addition to the expression of major facilitator superfamily genes responsible for transmembrane ion transport. Under anoxia, genes involved in mitochondrial function, mTOR signalling and autophagy were differentially expressed. Both high salt and anoxia enhanced degradation of erroneous proteins and protein chaperoning. Compared with other branchiopod genomes, Artemia had 0.03% contracted and 6% expanded orthogroups, in which 14% of the genes were differentially expressed under high salinity or anoxia. One phospholipase D gene family, shown to be important in plant stress response, was uniquely present in both extremophiles Artemia and the tardigrade Hypsibius dujardini, yet not differentially expressed under the described experimental conditions. CONCLUSIONS: A relatively complete genome of Artemia was assembled, annotated and analysed, facilitating research on its extremophile features, and providing a reference sequence for crustacean research.

Probiotics: their action against pathogens can be turned around.

Probiotics when applied in complex evolving (micro-)ecosystems, might be selectively beneficial or detrimental to pathogens when their prophylactic efficacies are prone to ambient interactions. Here, we document a counter-intuitive phenomenon that probiotic-treated zebrafish (Danio rerio) were respectively healthy at higher but succumbed at lower level of challenge with a pathogenic Vibrio isolate. This was confirmed by prominent dissimilarities in fish survival and histology. Based upon the profiling of the zebrafish microbiome, and the probiotic and the pathogen shared gene orthogroups (genetic niche overlaps in genomes), this consequently might have modified the probiotic metabolome as well as the virulence of the pathogen. Although it did not reshuffle the architecture of the commensal microbiome of the vertebrate host, it might have altered the probiotic-pathogen inter-genus and intra-species communications. Such in-depth analyses are needed to avoid counteractive phenomena of probiotics and to optimise their efficacies to magnify human and animal well-being. Moreover, such studies will be valuable to improve the relevant guidelines published by organisations such as FAO, OIE and WHO.

Pandanus tectorius fruit extract promotes Hsp70 accumulation, immune-related genes expression and Vibrio parahaemolyticus tolerance in the white-leg shrimp Penaeus vannamei.

Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.

The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

Cyst viability and stress tolerance upon heat shock protein 70 knockdown in the brine shrimp Artemia franciscana.

Females of the brine shrimp Artemia franciscana produce either free-swimming nauplii via ovoviviparous pathway of reproduction or encysted embryos, known as cysts, via oviparous pathway, in which biological processes are arrested. While previous study has shown a crucial role of ATP-dependent molecular chaperone, heat shock protein 70 (Hsp70) in protecting A. franciscana nauplii against various abiotic and abiotic stressors, the function of this protein in diapausing embryos and cyst development, however, remains unknown. RNA interference (RNAi) was applied in this study to examine the role of Hsp70 in cyst development and stress tolerance, with the latter performed by desiccation and freezing, a common method used for diapause termination in Artemia cysts. Hsp70 knockdown was apparent in cysts released from females that were injected with Hsp70 dsRNA. The loss of Hsp70 affected neither the development nor morphology of the cysts. The time between fertilization and cyst release from Artemia females injected with Hsp70 dsRNA was delayed slightly, but the differences were not significant when compared to the controls. However, the hatching percentage of cysts which lacks Hsp70 were reduced following desiccation and freezing. Taken together, these results indicated that Hsp70 possibly plays a role in the stress tolerance but not in the development of diapause-destined embryos of Artemia. This research makes fundamental contributions to our understanding of the role molecular chaperone Hsp70 plays in Artemia, an excellent model organism for diapause studies of the crustaceans.

The blue mussel inside: 3D visualization and description of the vascular-related anatomy of Mytilus edulis to unravel hemolymph extraction.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.