Anthropogenic nitrogen pollution inferred by stable isotope records of crustose coralline algae.

Since reef ecosystems can offer intricate habitats for various marine organisms, calcified reefs may contain valuable long-term environmental data. This study investigated stable isotopic composition of marine organisms from the Taoyuan and Linshanbi crustose coralline algae (CCA) reef ecosystems to understand sewage pollution. CCA samples from Taoyuan (Palaeo Xin A: ∼1000 years old and Palaeo G: ∼7000 years old) and Linshanbi (Palaeo L: ∼7000 years old and modern CCA) had significantly lower δ15N values (2.5-5.6 ‰) compared to modern CCA from Taoyuan (10.2 ± 1.2 ‰). Intertidal organisms from the Taoyuan CCA reef also showed higher δ15N values than those from Linshanbi CCA reef, indicating anthropogenic stress in both ecosystems. Long-term pollution monitoring and effective strategies to mitigate sewage pollution are recommended for these CCA reef ecosystems.

Evaluation of cooking effects on otolith stable carbon and oxygen isotope values of teleostean fish Pomadasys kaakan (Cuvier, 1830).

RATIONALE: For years, archaeologists, climatologists, and ecologists have used stable oxygen isotope values (δ13 C, δ18 O) in fish otoliths from archaeological sites to reconstruct the habitats, paleo-temperature, and seasonality of the fish captures. Otoliths from archaeological sites might have been heated when ancient people cooked the fish for food. Therefore, there are debates as to whether the cooking behaviors would cause further isotopic fractionations of the carbonate in the otoliths. METHODS: In this study, we have evaluated the effects of the cooking methods on the otolith δ13 C and δ18 O values by comparing the otoliths of the javelin grunter (Pomadasys kaakan) from the same individuals, with the left otoliths taken out before the different cooking processes. Otolith sections of the fish were then made and several subsamples were milled along the microstructures visible in the otolith pairs, mostly annual check rings, followed by the stable isotope analyses. RESULTS: There were no morphological changes between the cooked and uncooked otoliths. The δ13 C and δ18 O values were highly consistent for the otolith subsamples between the cooked and uncooked pairs, suggesting none or trivial effects of the cooking processes on the isotopic values of the otoliths. In addition, some javelin grunters showed lower δ13 C (-5‰ to -6‰) and δ18 O (-4‰ to -5‰) values deposited in the wide translucent zone of the otoliths, suggesting seasonal migration of the fish to estuaries during the summer. CONCLUSIONS: This study suggests that cooking processes do not change otolith stable isotopic compositions and will therefore allow for future research to use the otolith δ13 C and δ18 O values to better understand the life history and used habitats of the preserved fish remains in the middens.

Bluefin tuna reveal global patterns of mercury pollution and bioavailability in the world's oceans.

Bluefin tuna (BFT), highly prized among consumers, accumulate high levels of mercury (Hg) as neurotoxic methylmercury (MeHg). However, how Hg bioaccumulation varies among globally distributed BFT populations is not understood. Here, we show mercury accumulation rates (MARs) in BFT are highest in the Mediterranean Sea and decrease as North Pacific Ocean > Indian Ocean > North Atlantic Ocean. Moreover, MARs increase in proportion to the concentrations of MeHg in regional seawater and zooplankton, linking MeHg accumulation in BFT to MeHg bioavailability at the base of each subbasin's food web. Observed global patterns correspond to levels of Hg in each ocean subbasin; the Mediterranean, North Pacific, and Indian Oceans are subject to geogenic enrichment and anthropogenic contamination, while the North Atlantic Ocean is less so. MAR in BFT as a global pollution index reflects natural and human sources and global thermohaline circulation.

Natal origin and age-specific egress of Pacific bluefin tuna from coastal nurseries revealed with geochemical markers.

Geochemical chronologies were constructed from otoliths of adult Pacific bluefin tuna (PBT) to investigate the timing of age-specific egress of juveniles from coastal nurseries in the East China Sea or Sea of Japan to offshore waters of the Pacific Ocean. Element:Ca chronologies were developed for otolith Li, Mg, Mn, Zn, Sr, and Ba, and our assessment focused on the section of the otolith corresponding to the age-0 to age-1 + interval. Next, we applied a common time-series approach to geochemical profiles to identify divergences presumably linked to inshore-offshore migrations. Conspicuous geochemical shifts were detected during the juvenile interval for Mg:Ca, Mn:Ca, and Sr:Ca that were indicative of coastal-offshore transitions or egress generally occurring for individuals approximately 4-6 mo. old, with later departures (6 mo. or older) linked to overwintering being more limited. Changepoints in otolith Ba:Ca profiles were most common in the early age-1 period (ca. 12-16 mo.) and appear associated with entry into upwelling areas such as the California Current Large Marine Ecosystem following trans-Pacific migrations. Natal origin of PBT was also predicted using the early life portion of geochemical profile in relation to a baseline sample comprised of age-0 PBT from the two primary spawning areas in the East China Sea and Sea of Japan. Mixed-stock analysis indicated that the majority (66%) of adult PBT in our sample originated from the East China Sea, but individuals of Sea of Japan origin were also detected in the Ryukyu Archipelago.

Amphidromous but endemic: Population connectivity of Rhinogobius gigas (Teleostei: Gobioidei).

Rhinogobius gigas is an amphidromous fish endemic to eastern Taiwan. Fishes with the diadromous behavior are expected to have a broader distribution range and higher genetic homogeneity despite that some amphidromous fishes with limited distribution are observed and R. gigas is an additional exception with a limited distribution range. Rhinogobius gigas has been documented to be retained inshore near the river plume with a short pelagic larval duration of 30-40 days, which may account for the endemism of this species. The short marine larval stage of R. gigas may imply a population genetic structure and the aim of the present study is to test whether the population genetic structure is present in R. gigas. To test the population genetic structure, fragments of mitochondrial displacement loop and cytochrome c oxidase subunit I were sequenced to provide molecular inference for genetic structure among populations. Sixty-nine haplotypes were identified among 191 R. gigas from 10 populations of eastern Taiwan and the mean haplotype and nucleotide diversities for all samples were 0.956 and 0.0024, respectively, implying a bottleneck followed by a recent population expansion further supported by Fu's Fs (-26.6; p < 0.001) and Tajima's D (-1.5; p = 0.037) values. The phylogenetic analysis revealed lack of genetic structure and the bush-like median joining network without commonly shared haplotypes supports the same scenario. The genetic homogeneity is probably due to the amphidromous life history providing the opportunity for passive larval transportation among the rivers through coastal currents in eastern Taiwan. The endemism to eastern Taiwan may be a consequence of complicated interactions among short pelagic larval duration, interspecific competition and coastal currents.

Facultative amphidromy and pelagic larval duration plasticity of Rhinogobius formosanus (Teleostei, Gobioidei).

Rhinogobius formosanus Oshima, 1919 has long been considered an amphidromous goby. However, a landlocked population recently found in the Jingualiao Creek upstream of the Feitsui Reservoir in Taipei suggests that R. formosanus may complete its life in the river. This study aims to verify the habitat use of the landlocked population of R. formosanus collected from the Feitsui Reservoir and an amphidromous population collected in Malian Creek using otolith Sr:Ca ratio analysis. The hypothesis that early life history varies between the landlocked and migratory gobies was also tested. Genetic analyses show that the Feitsui Reservoir and Malian Creek populations are not genetically different. Rhinogobius formosanus from Malian Creek showed high-to-low otolith Sr:Ca ratios suggesting that these specimens spent a planktonic larval stage in the sea followed by a freshwater life at later stages. In contrast, R. formosanus from the Feitsui Reservoir showed constant lower otolith Sr:Ca ratios, implying a landlocked life history of fish in the creek upstream of the reservoir. In addition, the analysis of growth increments showed a longer pelagic larval duration for the fish in the Malian Creek (58.8 days) than those in the Feitsui Reservoir (38.8). Variation of pelagic larval duration in two genetically homogenous populations implies acclimatization to the reservoir by the landlocked gobies. This study shows that R. formosanus, like some other congeners, is capable of adapting to a freshwater landlocked environment in its early developmental stage and supports the hypothesis that landlocked populations may have a shorter pelagic larval duration.

Quantitative reconstruction of salinity history by otolith oxygen stable isotopes: An example of a euryhaline fish Lateolabrax japonicus.

RATIONALE: Otolith strontium:calcium (Sr:Ca) ratios have been extensively used to study fish migration across a river, estuary and ocean at each life stage. However, otolith Sr:Ca ratios only explain partial variations in salinity and quantitative reconstruction of salinity history of fishes remains a challenge. Using a euryhaline fish Lateolabrax japonicus as an example, this study demonstrated an isotopic method of quantitatively reconstructing the salinity histories and habitat uses of the fish. METHODS: Otolith oxygen stable isotopic ratios (δ18 Ooto values) were measured using a mass spectrometer for subsamples sequentially milled from the otolith core to the edge, and otolith Sr:Ca ratios were measured by an electron probe microanalyzer for the comparison. The mean water temperature within the studied area in the time period of each milled subsample was estimated from the Copernicus database. Based on an isotopic fractionation equation, each δ18 Ooto value and water temperature pair determined the water δ18 O value, which was then converted into salinity using published linear equations or an equation that was newly generated for this study. RESULTS: Individual fish clearly revealed different preferences in habitat use. The retrospectively reconstructed salinity history indicated that most L. japonicus inhabit and can spawn in seawater as well as in brackish estuaries. Few fish lived in freshwater during the young stage based on the analysis of δ18 Ooto profiles; however, otolith Sr:Ca ratios can only reveal freshwater residence and cannot distinguish residence in brackish water or seawater. CONCLUSIONS: This study demonstrated that otolith temporal microstructure and δ18 Ooto values are well-suited approaches for quantitative reconstruction of salinity histories of the fish. This method can improve the understanding of the habitat uses of other fishes inhabiting diverse habitats among the rivers, estuaries and oceans.

Fractionation of otolith nitrogen stable isotopes measured by peroxodisulfate oxidation-bacterial conversion and isotope ratio mass spectrometry.

RATIONALE: Otoliths are usually used to estimate the age of fish and the chemical composition such as nitrogen stable isotope ratios (δ15 N values) may record environmental information and ecological role of the fish. However, the isotopic fractionation of δ15 N values between diets and otoliths has rarely been investigated and remains unclear. METHODS: Nitrogen isotopic fractionation between five different diets (δ15 Ndiet values) and otoliths (δ15 Noto values) were elucidated in tilapia Oreochromis mossambica reared in controlled feeding experiments. The otoliths were dissolved with hydrogen chloride and peroxodisulfate was used to oxidize the total organic materials to nitrate, which was further converted into N2 O gas by denitrification bacteria before the measurement of δ15 Noto values by isotope ratio mass spectrometry. The δ15 N values of muscles, gills, scales and livers of the tilapias were also measured by isotope ratio mass spectrometry. RESULTS: The peroxodisulfate oxidation-bacterial conversion method reduced the minimum mass of the otoliths required for analysis to as low as 2 mg, unlike past methods, which have required masses of 8-155 mg. The δ15 Noto values were not significantly different from the δ15 Ndiet values of the five diets. Furthermore, the somatic growth rate had no effect on the δ15 Noto values. Nevertheless, the δ15 N values of metabolically active tissues were significantly different from each other and higher than the δ15 Ndiet values, due to the deamination of these tissues. CONCLUSIONS: These results suggest that diet was the main source of amino acids for the otolith organic matrix and there was no biochemical transamination during the assimilation of dietary amino acids to otoliths. The δ15 Noto value can be used as a proxy of nitrogen sources of fishes and may have potential application in ecological studies such as the detection of diet shift, migration, trophic levels and environmental changes experienced by the fish population.

Pelagic larval duration, growth rate, and population genetic structure of the tidepool snake moray Uropterygius micropterus around the southern Ryukyu Islands, Taiwan, and the central Philippines.

The relationships between pelagic larval duration (PLD) and geographic distribution patterns or population genetic structures of fishes remain obscure and highly variable among species. To further understand the early life history of the tidepool snake moray Uropterygius micropterus and the potential relationship between PLD and population genetic structure of this species, otolith microstructure and population genetics based on concatenated mtDNA sequence (cytochrome b and cytochrome oxidase subunit I, 1,336 bp) were analyzed for 195 specimens collected from eight locations around the southern Ryukyu Islands, Taiwan, and the central Philippines. Eels with longer PLD and lower otolith growth rates were observed at relatively higher latitudes with lower water temperatures (54.6 ± 7.7 days and 1.28 ± 0.16 µm day-1 on Ishigaki Island, Japan, vs. 43.9 ± 4.9 days and 1.60 ± 0.19 µm day-1 on Badian, the Philippines), suggesting that leptocephali grew faster and had shortened pelagic periods in warmer waters. Meanwhile, the eels along the southwest coast of Taiwan had relatively longer PLD (57.9 ± 10.5 days), which might be associated with the more complex ocean current systems compared to their counterparts collected along the east coast of Taiwan (52.6 ± 8.0 days). However, the southwestern and eastern Taiwan groups had similar otolith growth rates (1.33 ± 0.19 µm day-1 vs. 1.36 ± 0.16 µm day-1). Despite the intergroup variation in PLD, genetic analysis revealed fluent gene flow among the tidepool snake morays in the study regions, implying that intraspecies PLD variation had a weak effect on genetic structure. The leptocephalus stage might have ensured the widespread gene flow among the study areas and leptocephalus growth was likely influenced by regional water temperature.

Assimilation of nitrogen and carbon isotopes from fish diets to otoliths as measured by nanoscale secondary ion mass spectrometry.

RATIONALE: Nitrogen and carbon stable isotope ratios (δ15 N and δ13 C values) of carbonate-bound organic materials in otoliths can provide information to address the biological and ecological functions of fish. Correct interpretation of otolith δ15 N and δ13 C profiles requires knowledge of the metabolic routes of nitrogen and carbon isotopes. However, the isotopic assimilation of δ15 N and δ13 C compositions from diets to otoliths has rarely been investigated. METHODS: This study traced the daily nitrogen and carbon isotopic assimilation between diets and otoliths using nanoscale secondary ion mass spectrometry (NanoSIMS). Isotopically labeled algae (Tetraselmis chui) were fed to tilapia (Oreochromis niloticus) for 14-17 days. NanoSIMS and conventional isotope ratio mass spectrometry were used to measure δ15 N and δ13 C variations in the otoliths and fish muscle, respectively. RESULTS: Otolith δ15 N values abruptly surged from natural abundance levels by 1000-2300‰ after the fish ate 15 N-spiked algae with δ15 N values of approximately 2200‰. However, the δ15 N values of fish muscle increased to only approximately 500‰ at the end of the feeding experiment. Much higher δ15 N values (3700-14 000‰) and moderate δ13 C values (60-200‰) were detected in the otoliths after the tilapia ate 15 N- and 13 C-spiked algae with a δ15 N value of 36667‰ and a δ13 C value of 272‰. Mapping analysis showed sub-micrometer-scale distribution of 15 N embedded in the otolith growth increments with a low-to-high δ15 N signal after the tilapia shifted diets from non-spiked to 15 N-labeled algae. CONCLUSIONS: These results suggest that otolith nitrogen and carbon isotopes from food were directly assimilated on the same day. Food is the major and in some cases only source of otolith nitrogen isotopes but makes only a partial contribution to otolith carbon isotopes. Therefore, the δ15 N values recorded in the sclerochronological layers of the otoliths can be used to determine the trophic levels, food sources and diet changes of fish.

The Species and Origin of Shark Fins in Taiwan's Fishing Ports, Markets, and Customs Detention: A DNA Barcoding Analysis.

The increasing consumption of shark products, along with the shark's fishing vulnerabilities, has led to the decrease in certain shark populations. In this study we used a DNA barcoding method to identify the species of shark landings at fishing ports, shark fin products in retail stores, and shark fins detained by Taiwan customs. In total we identified 23, 24, and 14 species from 231 fishing landings, 316 fin products, and 113 detained shark fins, respectively. All the three sample sources were dominated by Prionace glauca, which accounted for more than 30% of the collected samples. Over 60% of the species identified in the fin products also appeared in the port landings, suggesting the domestic-dominance of shark fin products in Taiwan. However, international trade also contributes a certain proportion of the fin product markets, as four species identified from the shark fin products are not found in Taiwan's waters, and some domestic-available species were also found in the customs-detained sample. In addition to the species identification, we also found geographical differentiation in the cox1 gene of the common thresher sharks (Alopias vulpinus), the pelagic thresher shark (A. pelagicus), the smooth hammerhead shark (Sphyrna zygaena), and the scalloped hammerhead shark (S. lewini). This result might allow fishing authorities to more effectively trace the origins as well as enforce the management and conservation of these sharks.

Habitat Use and Migratory Life History of Salangid Icefish (Salangidae) Revealed by Otolith Sr/Ca Ratios.

Jen-Chieh Shiao, Chien-Yu Chen, Jie Zhang, and Yoshiyuki Iizuka (2016) Salangid icefish (Salangidae) are commercially important species and are widely distributed in lakes, rivers, estuaries, and coastal area of Asia. This study examined their habitat use and migratory patterns by analyzing otolith microstructure and Sr/ Ca ratios. Neosalanx tangkahkeii and Protosalanx chinesis collected in the isolated freshwater Taihu Lake in China showed consistently low otolith Sr/Ca ratios (< 5.0 × 10-3, mean + 2 SD), which were used to represent the freshwater residence. Another batch of P. chinesis collected from the Yangtze River estuary of China also showed low otolith Sr/Ca ratios (< 5.5 × 10-3, mean + 2 SD) throughout the life history, suggesting that these fish only use freshwater environments. A group of N. tangkahkeii collected in the Pearl River estuary of China showed otolith Sr/Ca ratios between 10.0 × 10-3 and 30.0 × 10-3, indicating habitat shifts between brackish and marine environments. Salanx ariakenesis collected in the Yangtze River estuary showed variable and higher otolith Sr/Ca ratios between 1.6 × 10-3 and 36.5 × 10-3, exhibiting the diverse migratory patterns between the river and the sea with the habitat shifts occurring at the juvenile, young, and adult stages. Neosalanx anderssoni collected in the Bohai Sea, China only used marine habitats based on their consistently high otolith Sr/Ca ratios with the mean values of each fish varying between 20.7 × 10-3 and 24.6 × 10-3. The habitat use by the icefish may differ within and among species. Different migratory patterns can coexist in the same species e.g., S. ariakenesis. The euryhaline icefish, even those living in the estuary or coastal water, do not necessarily migrate between the sea and rivers, suggesting their high plasticity of habitat use and facultative anadromous behaviors.

Toxin gene determination and evolution in scorpaenoid fish.

In this study, we determine the toxin genes from both cDNA and genomic DNA of four scorpaenoid fish and reconstruct their evolutionary relationship. The deduced protein sequences of the two toxin subunits in Sebastapistes strongia, Scorpaenopsis oxycephala, and Sebastiscus marmoratus are about 700 amino acid, similar to the sizes of the stonefish (Synanceia horrida, and Synanceia verrucosa) and lionfish (Pterois antennata and Pterois volitans) toxins previously published. The intron positions are highly conserved among these species, which indicate the applicability of gene finding by using genomic DNA template. The phylogenetic analysis shows that the two toxin subunits were duplicated prior to the speciation of Scorpaenoidei. The precedence of the gene duplication over speciation indicates that the toxin genes may be common to the whole family of Scorpaeniform. Furthermore, one additional toxin gene has been determined in the genomic DNA of Dendrochirus zebra. The phylogenetic analysis suggests that an additional gene duplication occurred before the speciation of the lionfish (Pteroinae) and a pseudogene may be generally present in the lineage of lionfish.

Acoustically evoked potentials in two cephalopods inferred using the auditory brainstem response (ABR) approach.

It is still a matter of debate whether cephalopods can detect sound frequencies above 400 Hz. So far there is no proof for the detection of underwater sound above 400 Hz via a physiological approach. The controversy of whether cephalopods have a sound detection ability above 400 Hz was tested using the auditory brainstem response (ABR) approach, which has been successfully applied in fish, crustaceans, amphibians, reptiles and birds. Using ABR we found that auditory evoked potentials can be obtained in the frequency range 400 to 1500 Hz (Sepiotheutis lessoniana) and 400 to 1000 Hz (Octopus vulgaris), respectively. The thresholds of S. lessoniana were generally lower than those of O. vulgaris.

Plasma membrane calcium ATPase required for semicircular canal formation and otolith growth in the zebrafish inner ear.

Fish otoliths consist of >90% calcium carbonate, the accretion of which depends on acellular endolymph. This study confirms the presence of plasma membrane calcium ATPase 1a isoform (Atp2b1a) in the auditory and vestibular system of a teleost fish. As shown by in situ hybridization, zebrafish atp2b1a is expressed mainly in larval otic placode and lateral-line neuromast as well as in the hair cells within the adult zebrafish inner ear chamber. Zebrafish atp2b1a knockdown by antisense morpholinos reduced the number of hair cells and produced malformation of semicircular canals and smaller otoliths. These defects coincide with unbalanced body orientation. The formation of smaller otoliths in atp2b1a morphants may stem from an impairment of calcium supply in the endolymph. However, otolith formation persists in most morphants, suggesting that other zebrafish Atp2b isoforms or paracellular pathways may also transport calcium into the endolymph. These results suggest that Atp2b1a plays an important role for normal development of the auditory and vestibular system as well as calcium transport in the inner ear of zebrafish.

Evaluation of thyroid-mediated otolith growth of larval and juvenile tilapia.

Thyroid-mediated otolith growth in tilapia was evaluated by the ontogenic triiodothyronine (T3) profile revealed by radioimmunoassay during the first month after hatching. Thyroid hormone receptor genes (TRalpha and TRbeta) were cloned and only the expression of TRalpha mRNA, quantified by real-time PCR, was similar to the T3 profile. Variations in otolith growth showed median correlation with the T3 profile and TRalpha mRNA expression pattern. Hypothyroidism and hyperthyroidism were induced in tilapia juveniles and larvae by administration of different concentrations of thiourea (TU) and T3, respectively, for 13 days. T3 and TU had little effect on otolith growth during the larval stage. However, T3 increased otolith growth and TU retarded, or stopped, otolith growth during the juvenile stage. Furthermore, TU treatment caused permanent changes in otolith shape in the ventral area. Otolith growth recovered slowly from hypothyroidism, requiring 2 days to form an increment during the first week. These results suggest that otolith growth, at least during the juvenile stage, is regulated by the thyroid hormones and the process may be mediated by TRalpha.

How can teleostean inner ear hair cells maintain the proper association with the accreting otolith?

The perception of equilibrium and sound in fish depends on the deflection of hair bundles of hair cell by the otolith. However, the accreting nature of teleostean otoliths poses a problem for maintenance of proper contact between the hair bundle and the otolith surface. Immunocytochemical staining localizes abundant proton-secreting H(+)-ATPase in the apical membrane of the hair cells. The H(+)-ATPase-mediated proton secretion into the endolymph causes an approximately 0.4-unit pH decrease, which was quantified by an H(+)-selective microelectrode. Thus, the hair cells maintain the proper distance from the otolith by neutralizing the alkaline endolymph to retard CaCO(3) deposition on the otolith opposite the sensory macula. Carbonic anhydrase, which hydrolyses CO(2) and produces HCO(3) (-) and H(+), was also localized in the hair cells. Ionocytes showed prominent immunostaining of carbonic anhydrase and Na(+)-K(+)-ATPase, indicating its role in transepithelial transport of HCO(3) (-) across the membranous labyrinth into the endolymph. Ionocytes form a ring closely surrounding the sensory macula. HCO(3) (-) secreted from the ionocytes may serve as a barrier to neutralize H(+) diffused from the sensory macula while keeping the endolymph alkaline outside the sensory macula. The ingenious arrangement of ionocytes and hair cells results in a unique sculptured groove, which is a common feature on the proximal surface of all teleostean otoliths.