Ming-Yih Leu

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 Taiwans 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, Taiwans 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 KNPs coral reefs.

Induced spawning and larval rearing of captive yellowfin porgy, Acanthopagrus latus (Houttuyn)

Females of yellowfin porgy (Acanthopagrus latus) were induced to spawn by a single injection of 1000 IU kg−1 human chorionic gonadotropin (hCG), 0.5 ml kg−1 Ovaprim (sGnRH-A in combination with domperidone) or a combination of hCG (500 IU kg−1) with Ovaprim (0.25 ml kg−1). Ovaprim, at a dosage of 0.5 ml kg−1, was the most effective. Fish injected with propylene glycol or 500 IU kg−1 body weight of hCG did not spawn at all. Fertilization rates ranged from 72 to 84% and hatching rates were between 60 and 88%. The hatched larvae were reared in 30–45 t cement tanks and fed initially on trochophore oyster larvae (Crassostrea gigas), followed by rotifers (Brachionus plicatilis), Artemia nauplii, copepods and finally weaned onto a zein micro-coated diet. Metamorphosis started when larvae reached 9.7 mm TL at 30 days of age, and was complete by Day 38 when larvae were 12.4 mm TL. From Day 20 after hatching, the larger larvae (7 mm TL) began to exhibit agonistic behavior. Intestinal swelling was frequently observed in larvae at 22–25 days after hatching (5–8 mm TL). p ]Two types of skeletal malformations, lordosis (10–21%) and brachyospondyliosis (1–4%), were frequently observed in the juvenile stage. Initial larval stocking densities were inversely correlated with survival (r = −0.80, P < 0.01). Optimal initial stocking density was 10–15 larvae 1−1. Larval rearing trials for growth and survival to 50– 56 days produced juveniles of 20 mm average total length, at a survival rate ranging from 0.7 to 27.5%.

Natural spawning and larval rearing of silver bream, Rhabdosargus sarba (Forsskål), in captivity

Abstract Silver bream, Rhabdosargus sarba , was spawned naturally in captivity without the use of hormones or other treatment. Spawning occurred continuously from 24 December 1989 to 29 March 1990 over a period of 96 days, with a water temperature range of 13.8–23.5°C. It is estimated that a female laid about 2 586 000 eggs during the spawning period. The hatched larvae were reared in 5-t rectangular concrete tanks and fed initially on rotifers, Brachionus plicatilis , followed by Artemia nauplii and finally weaned onto an artificial diet. Larval rearing trials for growth and survival to 45 days produced juveniles of 19.4 mm average total length, at a survival rate ranging from 7.3 to 43.2%. Few agonistic attacks were observed throughout the rearing period, despite high initial stocking densities. After 105 days the juveniles reached an average size of 61.6 mm fork length with a survival rate of 12.5%.

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.

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.

Characterising and predicting algal blooms in a subtropical coastal lagoon

Algal bloom is a major concern worldwide. In this study, we characterised the physical and biochemical parameters during an algal bloom event in a coastal lagoon in an attempt to predict local blooms in the future. Results showed that the highest concentrations of dissolved inorganic phosphorus (DIP), chlorophyll a (chl a) and phytoplankton abundance were found in the inner area, whereas the highest dissolved inorganic nitrogen (DIN) concentration occurred near the inlet-outlet channel. Chl a was correlated with DIP, and there was a significant exponential relationship between chl a and the nitrogen to phosphorus ratio (N/P ratio) across all sampling stations and times. A higher proportion of the variation in chl a was explained by the N/P ratio than either DIP or DIN. We found that a N/P ratio <2.38 will likely trigger an algal bloom (chl a ≥ 10 µgL–1) in the lagoon. Our results suggest that the N/P ratio could be used as an expedient and reliable measure of the potential eutrophic status of coastal lagoons.

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.

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.

TRANSPORTATION, HUSBANDARY, AND RELEASE OF A WHALE SHARK (RHINCODON TYPUS)

The National Museum of Marine Biology and Aquarium of Taiwan housed a whale shark in a 3.7 million liter indoor oval tank from June 2005 until July 2013, at which point it was released. This study reports the transport of the whale shark into and out of the Museum, its husbandry conditions, feeding regime, growth during captivity, and details on the release of the animal. During the eight years in captivity, the animal grew from 2.3 to 7.8 m in length and from 200 to 3,600 kg in weight when released, with estimated overall growth rates of 0.67 m yr^(-1) and 412 kg yr^(-1) in length and mass, respectively. The animal beached shortly after release, possibly due to having acclimated to life in captivity. Therefore, detailed behavioral knowledge is needed for future releases of animals that have been maintained in captivity for extended periods.