A field and laboratory study of the responses of cytoprotection and osmoregulation to salinity stress in mosquitofish (Gambusia affinis).

The mosquitofish (Gambusia affinis) naturally inhabits freshwater (FW; 1-3‰) and seawater (SW; 28-33‰) ponds in constructed wetland. To explore the physiological status and molecular mechanisms for salinity adaptation of the mosquitofish, cytoprotective responses and osmoregulation were examined. In the field study, activation of protein quality control (PQC) mechanism through upregulation of the abundance of heat shock protein (HSP) 90 and 70 and ubiquitin-conjugated proteins was found in the mosquitofish gills from SW pond compared to the individuals of FW pond. The levels of aggregated proteins in mosquitofish gills had no significant difference between FW and SW ponds. Furthermore, the osmoregulatory responses revealed that the body fluid osmolality and muscle water contents of the mosquitofish from two ponds were maintained within a physiological range while branchial Na+/K+-ATPase (NKA) expression was higher in the individuals from SW than FW ponds. Subsequently, to further clarify whether the cellular stress responses and osmoregulation were mainly induced by hypertonicity, a laboratory salinity acclimation experiment was conducted. The results from the laboratory experiment were similar to the field study. Branchial PQC as well as NKA responses were induced by SW acclimation compared to FW-acclimated individuals. Taken together, induction of gill PQC and NKA responses implied that SW represents an osmotic stress for mosquitofish. Activation of PQC was suggested to provide an osmoprotection to prevent the accumulation of aggregated proteins. Moreover, an increase in branchial NKA responses for osmoregulatory adjustment was required for the physiological homeostasis of body fluid osmolality and muscle water content.

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas).

Reverse transcription quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct results. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from four beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have the potential to serve as an important approach to evaluating cetacean health influenced by environmental insults.

Exploration of the mechanisms of protein quality control and osmoregulation in gills of Chromis viridis in response to reduced salinity.

Fish gills are the vital multifunctional organ in direct contact with external environment. Therefore, activation of the cytoprotective mechanisms to maintain branchial cell viability is important for fish upon stresses. Salinity is one of the major factors strongly affecting cellular and organismal functions. Reduction of ambient salinity may occur in coral reef and leads to osmotic stress for reef-associated stenohaline fish. However, the physiological responses to salinity stress in reef-associated fish were not examined substantially. With this regard, the physiological parameters and the responses of protein quality control (PQC) and osmoregulatory mechanisms in gills of seawater (SW; 33-35 ‰)- and brackish water (BW; 20 ‰)-acclimated blue-green damselfish (Chromis viridis) were explored. The results showed that the examined physiological parameters were maintained within certain physiological ranges in C. viridis acclimated to different salinities. In PQC mechanism, expression of heat-shock protein (HSP) 90, 70, and 60 elevated in response to BW acclimation while the levels of ubiquitin-conjugated proteins were similar between the two groups. Thus, it was presumed that upregulation of HSPs was sufficient to prevent the accumulation of aggregated proteins for maintaining the protein quality and viability of gill cells when C. viridis were acclimated to BW. Moreover, gill Na(+)/K(+)-ATPase expression and protein amounts of basolaterally located Na(+)/K(+)/2Cl(-) cotransporter were higher in SW fish than in BW fish. Taken together, this study showed that the cytoprotective and osmoregulatory mechanisms of blue-green damselfish were functionally activated and modulated to withstand the challenge of reduction in salinity for maintaining physiological homeostasis.

A continuous, real-time water quality monitoring system for the coral reef ecosystems of Nanwan Bay, Southern Taiwan.

The coral reef ecosystems of Nanwan Bay, Southern Taiwan are undergoing degradation due to anthropogenic impacts, and as such have resulted in a decline in coral cover. As a first step in preventing the continual degradation of these coral reef environments, it is important to understand how changes in water quality affect these ecosystems on a fine-tuned timescale. To this end, a real-time water quality monitoring system was implemented in Nanwan Bay in 2010. We found that natural events, such as cold water intrusion due to upwelling, tended to elicit temporal shifts in coral spawning between 2010 and 2011. In addition, Degree Heating Weeks (DHWs), a commonly utilized predictor of coral bleaching, were 0.92 and 0.59 in summer 2010 and 2011, respectively. Though this quantity of DHW was below the presumed stress-inducing value for these reefs, a rise in DHWs in the future may stress the resident corals.

Evaluating the accuracy of morphological identification of larval fishes by applying DNA barcoding.

Due to insufficient morphological diagnostic characters in larval fishes, it is easy to misidentify them and difficult to key to the genus or species level. The identification results from different laboratories are often inconsistent. This experiment aims to find out, by applying DNA barcoding, how inconsistent the identifications can be among larval fish taxonomists. One hundred morphotypes of larval fishes were chosen as test specimens. The fishes were collected with either larval fish nets or light traps in the northern, southern and northwestern waters of Taiwan. After their body lengths (SL) were measured and specimen photos were taken, all specimens were delivered, in turn, to five laboratories (A-E) in Taiwan to be identified independently. When all the results were collected, these specimens were then identified using COI barcoding. Out of a total of 100 specimens, 87 were identified to the family level, 79 to the genus level and 69 to the species level, based on the COI database currently available. The average accuracy rates of the five laboratories were quite low: 80.1% for the family level, 41.1% for the genus level, and 13.5% for the species level. If the results marked as "unidentified" were excluded from calculations, the rates went up to 75.4% and 43.7% for the genus and species levels, respectively. Thus, we suggest that larval fish identification should be more conservative; i.e., when in doubt, it is better to key only to the family and not to the genus or species level. As to the most misidentified families in our experiment, they were Sparidae, Scorpaenidae, Scombridae, Serranidae and Malacanthidae. On the other hand, Mene maculata and Microcanthus strigatus were all correctly identified to the species level because their larvae have distinct morphology. Nevertheless, barcoding remains one of the best methods to confirm species identification.

Impacts of human activities on coral reef ecosystems of southern Taiwan: a long-term study.

In July 2001, the National Museum of Marine Biology and Aquarium, co-sponsored by the Kenting National Park Headquarters and Taiwan's National Science Council, launched a Long-Term Ecological Research (LTER) program to monitor anthropogenic impacts on the ecosystems of southern Taiwan, specifically the coral reefs of Kenting National Park (KNP), which are facing an increasing amount of anthropogenic pressure. We found that the seawater of the reef flats along Nanwan Bay, Taiwan's southernmost embayment, was polluted by sewage discharge at certain monitoring stations. Furthermore, the consequently higher nutrient and suspended sediment levels had led to algal blooms and sediment smothering of shallow water corals at some sampling sites. Finally, our results show that, in addition to this influx of anthropogenically-derived sewage, increasing tourist numbers are correlated with decreasing shallow water coral cover, highlighting the urgency of a more proactive management plan for KNP's coral reefs.

Distribution and accumulation of organotin species in seawater, sediments and organisms collected from a Taiwan mariculture area.

The present study was undertaken to evaluate the distribution and accumulation of tributyltin (TBT) and triphenyltin (TPhT) in seawater, sediments and selected organisms from a cage mariculture area in southern Taiwan, Hsiao Liouchiou Island. Our results show that ΣOTs were found in concentrations as high as 196 ng/L in seawater collected from the sites in Pai-Sa harbor, and up 1040 ng/g dry wt. in sediments dredged from sites within Da-Fu harbor. Also, ΣOTs concentrations of 859 ng/g dry wt. were observed in the liver of cobia (Rachycentron canadum) from mariculture cages. As most published studies have focused on the acute toxicity and bioaccumulation of organotins in mussels, the effects of organotins on cobia and other marine fauna are still poorly understood. This study highlights the significance of ΣBTs accumulation in cobia, as well as in the sediments and seawater surrounding their culture facilities.