Intermittent hypoxia leads to functional reorganization of mitochondria and affects cellular bioenergetics in marine molluscs.

Fluctuations in oxygen (O2) concentrations represent a major challenge to aerobic organisms and can be extremely damaging to their mitochondria. Marine intertidal molluscs are well-adapted to frequent O2 fluctuations, yet it remains unknown how their mitochondrial functions are regulated to sustain energy metabolism and prevent cellular damage during hypoxia and reoxygenation (H/R). We used metabolic control analysis to investigate the mechanisms of mitochondrial responses to H/R stress (18 h at <0.1% O2 followed by 1 h of reoxygenation) using hypoxia-tolerant intertidal clams Mercenaria mercenaria and hypoxia-sensitive subtidal scallops Argopecten irradians as models. We also assessed H/R-induced changes in cellular energy balance, oxidative damage and unfolded protein response to determine the potential links between mitochondrial dysfunction and cellular injury. Mitochondrial responses to H/R in scallops strongly resembled those in other hypoxia-sensitive organisms. Exposure to hypoxia followed by reoxygenation led to a strong decrease in the substrate oxidation (SOX) and phosphorylation (PHOS) capacities as well as partial depolarization of mitochondria of scallops. Elevated mRNA expression of a reactive oxygen species-sensitive enzyme aconitase and Lon protease (responsible for degradation of oxidized mitochondrial proteins) during H/R stress was consistent with elevated levels of oxidative stress in mitochondria of scallops. In hypoxia-tolerant clams, mitochondrial SOX capacity was enhanced during hypoxia and continued rising during the first hour of reoxygenation. In both species, the mitochondrial PHOS capacity was suppressed during hypoxia, likely to prevent ATP wastage by the reverse action of FO,F1-ATPase. The PHOS capacity recovered after 1 h of reoxygenation in clams but not in scallops. Compared with scallops, clams showed a greater suppression of energy-consuming processes (such as protein turnover and ion transport) during hypoxia, indicated by inactivation of the translation initiation factor EIF-2α, suppression of 26S proteasome activity and a dramatic decrease in the activity of Na(+)/K(+)-ATPase. The steady-state levels of adenylates were preserved during H/R exposure and AMP-dependent protein kinase was not activated in either species, indicating that the H/R exposure did not lead to severe energy deficiency. Taken together, our findings suggest that mitochondrial reorganizations sustaining high oxidative phosphorylation flux during recovery, combined with the ability to suppress ATP-demanding cellular functions during hypoxia, may contribute to high resilience of clams to H/R stress and help maintain energy homeostasis during frequent H/R cycles in the intertidal zone.

Expression of fructose 1,6-bisphosphatase and phosphofructokinase is induced in hepatopancreas of the white shrimp Litopenaeus vannamei by hypoxia.

Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.

Bacteriostatic suppression in Norway lobster (Nephrops norvegicus) exposed to manganese or hypoxia under pressure of ocean acidification.

Future ocean acidification (OA) and warming following climate change elicit pervasive stressors to the inhabitants of the sea. Previous experimental exposure to OA for 16 weeks at pH levels predicted for 2100 has shown to result in serious immune suppression of the Norway lobster, Nephrops norvegicus. The lobsters are currently affected by stressors such as periodical hypoxia inducing high levels of bioavailable manganese (Mn). Here, we aimed to investigate possible effects of interactions between OA and these stressors on total hemocyte counts (THCs) and on recovery of inoculated bacteria in the lobsters, measured as a proxy for bacteriostatic response. The effects were judged by following numbers of culturable Vibrio parahaemolyticus in hepatopancreas, 4 and 24h post inoculation in lobsters kept in replicate tanks with six different treatments: either ambient (pCO2∼500 µatm/pH∼8.1 U) or CO2-manipulated seawater (OA; pCO2∼1550 µatm/pH∼7.6 U) for 8 weeks. During the last 2 weeks, additional stress of either hypoxia (∼23% oxygen saturation) or Mn (∼9 mg L(-1)) was added except in control treatments. Our results showed clear effect on bacteriostatic response in Norway lobsters exposed to these stressors. In lobsters kept in ambient seawater without additional stressors, the number of culturable bacteria in hepatopancreas was reduced by ∼34%. In combined treatment of ambient seawater and hypoxia, the reduction was ∼23%, while in the Mn-exposed animals, there was no reduction at all. This was also the case in all OA treatments where mean numbers of culturable V. parahaemolyticus tended to increase. In lobsters from ambient seawater with or without hypoxia, the THC was not significantly different as was also the case in OA without additional stressors. However, in OA treatments combined with either hypoxia or Mn, THC was reduced by ∼35%. While the reduction of culturable V. parahaemolyticus in lobsters was clearly affected by these stressors, we found no notable effects on growth, survival or hemolytic properties of the bacteria itself. Thus, we conclude that this predicted stress scenario is beneficial for the pathogen in its interaction with the host. As OA proceeds, it may force the health of the ecologically and economically important N. norvegicus to a tipping point if exposed to more short-term stressors such as the periodical events of hypoxia and Mn. This could impact lobster condition and biomass and may as well increase the risk for bacterial transmission to consumers.

Transcriptome analysis of Pacific white shrimp (Litopenaeus vannamei) hepatopancreas in response to Taura syndrome Virus (TSV) experimental infection.

BACKGROUND: The Pacific white shrimp, Litopenaeus vannamei, is a worldwide cultured crustacean species with important commercial value. Over the last two decades, Taura syndrome virus (TSV) has seriously threatened the shrimp aquaculture industry in the Western Hemisphere. To better understand the interaction between shrimp immune and TSV, we performed a transcriptome analysis in the hepatopancreas of L. vannamei challenged with TSV, using the 454 pyrosequencing (Roche) technology. METHODOLOGY/PRINCIPAL FINDINGS: We obtained 126919 and 102181 high-quality reads from TSV-infected and non-infected (control) L. vannamei cDNA libraries, respectively. The overall de novo assembly of cDNA sequence data generated 15004 unigenes, with an average length of 507 bp. Based on BLASTX search (E-value <10-5) against NR, Swissprot, GO, COG and KEGG databases, 10425 unigenes (69.50% of all unigenes) were annotated with gene descriptions, gene ontology terms, or metabolic pathways. In addition, we identified 770 microsatellites and designed 497 sets of primers. Comparative genomic analysis revealed that 1311 genes differentially expressed in the infected shrimp compared to the controls, including 559 up- and 752 down- regulated genes. Among the differentially expressed genes, several are involved in various animal immune functions, such as antiviral, antimicrobial, proteases, protease inhibitors, signal transduction, transcriptional control, cell death and cell adhesion. CONCLUSIONS/SIGNIFICANCE: This study provides valuable information on shrimp gene activities against TSV infection. Results can contribute to the in-depth study of candidate genes in shrimp immunity, and improves our current understanding of this host-virus interaction. In addition, the large amount of transcripts reported in this study provide a rich source for identification of novel genes in shrimp.

Development of monoclonal antibodies for detection of necrotizing hepatopancreatitis in penaeid shrimp.

Monoclonal antibodies (MAbs) were produced against necrotizing hepatopancreatitis bacteria (NHP-B) of penaeid shrimp. The MAbs tested in dot-immunoblot (D-IB) assays were capable of detecting the NHP-B in hepatopancreas samples collected from moribund juvenile Litopenaeus vannamei during an experimentally induced NHP-B infection. The MAbs were also screened by immunohistochemistry (IHC) using case submissions that were determined to be infected not only by histology, but also polymerase chain reaction (PCR) and in situ hybridization (ISH) assays using specific digoxigenin (DIG)-labeled probes on histological sections prepared from naturally infected shrimp. Two of the MAbs were chosen for development of detection methods for NHP. The MAbs were tested using IHC methods on Davidson's alcohol-formalin-acetic acid (AFA) fixed tissue sections and identified NHP-B infected cells and tissues in a pattern similar to that seen with DIG-labeled NHP-specific gene probes. None of the MAbs reacted with tissue from specific pathogen-free (SPF) shrimp or with shrimp tissues infected with a rickettsia-like bacteria, Vibrio sp., Campylobacter sp., and Spiroplasma sp. The MAbs were found to be negative against these other organisms, demonstrating that they are species specific and useful for rapid diagnostic detection of NHP-B.