Mitochondrial Genetic Diversity of the Freshwater Snail Melanoides tuberculata.

According to geological history, Peninsular Malaysia and Borneo formed at different times and were once connected during Quaternary glaciations. To determine how this history has influenced phylogeography, our study examined the population genetic structure of the tropical freshwater gastropod Melanoides tuberculata across Peninsular Malaysia and Borneo using the sequences from mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I genes (1168 bp). In total, 104 specimens were collected from seventeen populations. All mtDNA haplotypes were identified as belonging to two highly divergent lineages, and these lineages were almost allopatric in their distributions. Our study found that the freshwater fauna in Malaysia might be divided into four regions: northeast Peninsular Malaysia, northwest Peninsular Malaysia, south Peninsular Malaysia, and Borneo. The phylogeography of M. tuberculata in Malaysia was shaped by the landforms of Peninsular Malaysia and by the paleo-river systems in the Sunda continental shelf. In addition, our study found that these two lineages in Malaysia have invaded the globe. These results suggest that Malaysia is located in important shipping lanes throughout the world, and the populations of M. tuberculate might be widely distributed throughout the world by shipping.

Development and assays estradiol equivalent concentration from prawn (p-EEQ) in river prawn, Macrobrachium nipponense, in Taiwan.

The present study established a fast and convenient bioassay method for aqueous ecosystems using the prawn estradiol equivalent concentration (p-EEQ) of male Macrobrachium nipponense, which produce vitellogenin (VTG) after exposure to xeno-estrogens. This method was then used to determine the concentrations of xeno-estrogen pollutants in the rivers of Taiwan. To establish the calibration curve for the concentrations based on the p-EEQ, the induced VTG content was determined using the alkali-labile phosphate method after male M. nipponense were exposed to 0, 10, 100, 1,000 and 10,000ng/L of 17ß-estradiol for 1, 3, 5, 7, 10 and 14 days, respectively. The results of the experiments showed that the induced VTG content in all of the experimental groups stabilized after 10 days, except for the 10,000ng/L experimental group, in which the induced VTG content decreased after 10 days. A 17ß-estradiol-VTG10day response curve was then established based on the induced VTG content in the 0, 10, 100 and 1000ng/L experimental groups at day 10. After establishing the curve, male M. nipponense were captured from the upper, middle and lower reaches of the Chuo-shui River, the Beigang River, the Jishui River, the Agongdian River and the Sichong River in Taiwan, and the VTG content in these prawns was determined. In addition, the p-EEQ in the waters was determined based on the VTG content, and the estradiol equivalent concentration (EEQ) in the waters was also measured immediately after sampling using the solid-phase extraction-enzyme-linked immunosorbent assay (SPE-ELISA) method. The results showed that the p-EEQ in the middle and lower reaches of the rivers in certain parts of Taiwan ranged from 38 to 400ng/L, and the detection rate was 100%. Moreover, the EEQ ranged from 7.9 to 92.9ng/L, and the detection rate was 42.9%, indicating that most of the middle and lower reaches of the rivers in Taiwan were polluted by xeno-estrogens. The 17ß-estradiol concentrations determined based on the p-EEQ were all higher than those based on the EEQ (SPE-ELISA method). The results of the present study showed that the use of M. nipponense to determine the p-EEQ in environmental waters provided advantages that included a high detection rate, high sensitivity and convenience. However, the p-EEQ cannot be used in waters that do not contain M. nipponense.

Mitochondrial DNA phylogeography of Semisulcospira libertina (Gastropoda: Cerithioidea: Pleuroceridae): implications the history of landform changes in Taiwan.

The mitochondrial DNA cytochrome c oxidase subunit I sequences from 95 specimens of Semisulcospira libertina in Taiwan were identified as two major phylogroups, exhibiting a southern and northern distribution, north of Formosa Bank and south of Miaoli Plateau. The genetic distance between these two phylogroups was 12.20%, and the distances within-phylogroups were 4.97 and 5.56%. According to a molecular clock of 1.56% per lineage per million years, the divergence time between these two major phylogroups was estimated at 4.94 million years ago (mya), with the two phylogroups forming at 3.64 and 3.75 mya, respectively. Moreover, the geological events have suggested that Taiwan Island emerged above sea level at 4-5 mya, and became its present shape at 2 mya. These results suggested that these two phylogroups might originate from two independent ancestral populations or divergent before colonizing Taiwan. Within South phylogroup, the initial colonization was hypothesized to be in Kaoping River (WT), followed by its northward. The high divergence between south- and north of WT River was influenced by the formation of the Kaoping foreland basins. Within North phylogroup, the colonization was from central sub-region through paleo-Miaoli Plateau to northern and northeastern sub-regions. This study showed that the landform changes might have shaped the genetic structure of S. libertina in concert. Apparently, two cryptic species or five different genetic stocks of S. libertina could be identified; these results are useful for the evaluation and conservation of S. libertina in Taiwan.

Alterations of biochemical indicators in hepatopancreas of the golden apple snail, Pomacea canaliculata, from paddy fields in Taiwan.

The freshwater golden apple snail, Pomacea canaliculata, is one of the world's 100 worst invasive alien species. The snails' wide distribution, high abundance, and sensitivity to environmental pollution make them a potential bioindicator for environmental contamination. In this study, the biochemical status of golden apple snails collected from paddy fields throughout the island of Taiwan was examined. This study found that the biochemical status of apple snails collected from paddy fields differed from that of animals bred and maintained in the laboratory. Furthermore, certain biochemical endpoints of the snails collected from the paddy fields before and after agricultural activities were also different-hemolymphatic vitellogenin protein was induced in male snail after exposure to estrogen-like chemicals, the hepatic monooxygenase (1.97 +/- 0.50 deltaA(650mm) 30 min(-1) mg(-1) protein in control group) and glutathione S transferase (0.02 +/- 0.01 delta A(340mm) 30 min(-1) mg(-1) protein in control group) snails exposed to pesticides, as well as the hepatopancreatic levels of aspartate aminotransferase (450.00 +/- 59.40 U mg(-1) mg(-1) protein in control group) and alanine aminotransferase (233.27 +/- 42.09 U mg(-1) mg(-1) protein in control group) decreased the indicating that xenobiotics destroyed hepatopancreatic. The above findings reveal that apple snail could be used as a practical bioindicator to monitor anthropogenic environmental pollution.

Phylogeography and genetic differentiation among populations of the Moon Turban Snail Lunella granulata Gmelin, 1791 (Gastropoda: Turbinidae).

We examined the genetic variation and phylogeographic relationships among 10 populations of Lunella granulata from mainland China, Penghu Archipelago, Taiwan Island, and Japan using mitochondrial COI and 16S markers. A total of 45 haplotypes were obtained in 112 specimens, and relatively high levels of haplotype diversity (h = 0.903) and low levels of nucleotide diversity (π = 0.0046) were detected. Four major phylogenetic lineage clusters were revealed and were concordant with their geographic distribution, agreeing with the haplotype network. These results suggested that geographic barrier isolating effects were occurring among the populations. This hypothesis was also supported by a significant genetic differentiation index (FST = 0.709) and by a spatial analysis of molecular variance (SAMOVA) analysis. A mismatch distribution analysis, neutrality tests and Bayesian skyline plots found a single significant population expansion. This expansion occurred on the coast of mainland China before 20-17 ka. Consequently, although the dispersal ability of the planktonic stage and the circulation of ocean currents generally promote genetic exchanges among populations, L. granulata has tended to maintain distinct genetic groups that reflect the respective geographic origins of the constituent lineages. Although the circulation of ocean currents, in principle, may still play a role in determining the genetic composition of populations, long-distance migration between regions is difficult even at the planktonic stage.

Evaluation of the estrogenic effects of bisphenol-A on male freshwater prawn Macrobrachium asperulum.

Bisphenol-A(BPA), a known endocrine-disrupting chemical that is distributed world wide, is extensively employed in industry, dentistry and consumer household products. This study was conducted to explore the estrogenic effects of BA on the male freshwater prawn, Macrobrachium asperulum, by examining the daily survival rate, molting frequency, hepatosomatic index (HSI), and levels of vitellogenin-like protein. Results revealed that BPAcan elicitthe expression of vitellogenin-like protein (0.09 and 0.09 microg P x (mg protein))-1), respectively) in male organisms exposed to 0.1 and 1 ng l(-1) BPA at day 7. Moreover, prawns were perceived to be sensitive to estrogenic compounds. Hence, it can be concluded that M. asperulum can be used as a species for biomonitoring, and levels of vitellogenin-like protein in males are a useful biomarker for detecting estrogenic contaminations.

Population genetic diversity and structure of Rhinogobius candidianus (Gobiidae) in Taiwan: Translocation and release.

Rhinogobius candidianus is a freshwater goby distributed in north, northwest, west, and south Taiwan, but this species has been introduced to east Taiwan and became dominant. To investigate its native population genetic diversity and structure and evaluate the sources and diversity of translocated populations, the mitochondrial DNA control region and cytochrome b gene (1981 bp) from 220 specimens were analyzed. These results indicated that (1) the east populations originated from two sources in west Taiwan; (2) translocated populations exist in east Taiwan and south Taiwan; (3) many populations have likely been moved secondarily by human intervention; (4) the effective size of the populations had declined greatly; (5) within the native populations, the ancestral populations colonized Taiwan during the land bridge phase in the Pleistocene through north Taiwan; (6) the landform changes in Taiwan shaped the population structure; and (7) the landforms of the coastline during glaciation also shaped the native range. The low-level genetic diversity, high population differentiation, and population decline greatly suggest the need for resource management and conservation interventions. Four clades (α-δ) should be managed as four distinct evolutionarily significant units, while the translocated populations should be managed as separate management units. Moreover, the translocated populations in east Taiwan should be evaluated and monitored carefully.

Alterations of biochemical parameters in malformed Indian rice frogs, Rana limnocharis from Southern Taiwan.

The purpose of this study is to investigate the factors that cause malformed frogs in upstream Kaoping river (KP site) and Tungkang river (T site) of Southern Taiwan. In this experiment, the activities of monooxygenase (MO), glutathione-S-transferase (GST), acetylcholinesterase (AchE) as well as the concentration of vitellogenin (Vg) in the liver were measured. Results show that activities of MO, GST and AchE, and Vg levels in normal frogs (male/female) were 0.09 +/- 0.02/0.09+/-0.01 deltaA min(-1) mg(-1) protein, 0.12 +/- 0.04/0.13 +/- 0.04 deltaA min(-1) mg(-1) protein, 6.13 +/- 2.69/6.01 +/- 2.09 U mg(-1) protein and 0.87 +/- 0.42/2.18 +/- 0.50 microg mg(-1) protein, respectively. Activities of MO, GST and AchE, and Vg levels in malformed frogs (male/female) were 0.15 +/- 0.04/0.21 +/- 0.07 deltaA min(-1) mg(-1) protein, 0.27 +/- 0.08/0.30 +/- 0.12 deltaA min(-1) mg(-1) protein, 4.59 +/- 2.71/5.19 +/- 3.74 U mg(-1) protein and 1.46 +/- 0.61/3.15 +/- 0.88 microg mg(-1) protein, respectively in KP site, and were 0.16 +/- 0.69/0.1 +/- 80.07 deltaA min(-1) mg(-1) protein, 0.21 +/- 0.07/0.24 +/- 0.08 deltaA min(-1) mg(-1) protein, 5.13 +/- 4.58/3.94 +/- 1.33 U mg(-1) protein and 2.23 +/- 1.47/4.11 +/- 1.63 microg mg(-1) protein, respectively in T site. These results indicate that male and female malformed frogs in both rivers upstream are found with higher activities. No significant difference in AchE activity was found between normal and malformed frogs in this investigation. It is therefore reasonable to speculate that the organic chemicals released from agricultural activities are presumable the main factors that lead to the malformation of frogs.

Prevalence of malformed frogs in Kaoping and Tungkang river basins of southern Taiwan.

In this study we found many amphibians with bizarre appearances, known as malformations in Pingtung County southern Taiwan. For this investigation we collected frogs inhabiting the Kaoping and Tungkang river watersheds between February 2006 and June 2007. Among the total number of 10,909 normal frogs (i.e., anurans) collected during the investigation period, the Indian rice frogs (Rana limnocharis) account for the greatest number next is the Chinese bullfrog (Rana rugulosa). Of all the 244 captured malformed frogs, the Indian rice frog account for the greatest proportion. These malformed frogs have their main distribution in upstream areas of these two rivers. Our result indicates that the appearance rate of malformed frogs is 1.8% in the upstream reaches of the Kaoping River and 2.6%, and 0.8%, respectively in the upstream and midstream reaches of the Tungkang river. The most-commonly-found malformation is the lack of palms, followed by the lack of appendages, exostosis, and a malformed appendicular. It is, therefore, reasonable to speculate that the causes for the malformation may be related to the increased organic pollutants and agricultural chemicals used in the upstream reaches of these two rivers.

Neocaridinafonticulata, a new land-locked freshwater shrimp from Hengchun Peninsula, Taiwan (Decapoda, Caridea, Atyidae).

A new species of land-locked freshwater shrimp, Neocaridinafonticulata sp. n. (Atyidae), is described from Kenting, Hengchun Peninsula, Pingtung County, southern Taiwan. This new species can be distinguished from its congeners by rostrum structure, pereiopods, and male first and second pleopods. The molecular evidence of mitochondrial cytochrome oxidase subunit I (COI) also supports the establishment of a new species. This is the third endemic species of Neocaridina known from Taiwan.

Complete mitochondrial genome and the phylogenetic position of the pelagic octopus Tremoctopus violaceus (Mollusca: Tremoctopodidae).

Tremoctopus violaceus is a small species of pelagic octopods which inhabit in subtropical and tropical open oceans. The mitogenome of T. violaceus is 16,015 base pairs (bp) in length and contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. Sequence analysis showed that the overall base composition is 31.5% for A, 40.47% for T, 7.79% for C, and 20.24% for G. The newly characterized complete mitochondrial genome of T. violaceus will provide essential data for further studies of this endangered species.

Origins of Semisulcospira libertina (gastropoda: semisulcospiridae) in Taiwan.

The most accepted hypothesis has suggested that the fauna in Taiwan Island originated from South China, but some studies supported the Japan, Ryukyu Archipelago, and Taiwan Islands as a unique biogeographical district. This study examines whether the populations of freshwater snail Semisulcospira libertina in Taiwan are closer to those in Japan based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. Our study shows the populations in North Taiwan originated from Japan and the cyclic glacial caused the migrations among islands and continent repeatedly; the populations in South Taiwan might originate from South China or South Asia. Our results will not only affect the conclusions in phylogeography of freshwater species in Taiwan but also change the sampling plans in the future studies about evolutionary of freshwater species in East Asia.

Imposex in the golden apple snail Pomacea canaliculata in Taiwan.

The golden apple snail Pomacea canaliculata (Lamarck, 1822) was introduced into Taiwan intentionally in the early 1980s and has become a recurring pest that seriously threatens aquatic crops. In this study, a field description of imposex with a developed penis sheath and penis in female golden apple snails from crop/domestic wastewater drainage sites and a six-order river is presented for the first time. Based on the five field collections and the aquarium group, the vas deferens sequence (VDS) of P. canaliculata in imposex development was categorized into four stages, i.e., stage 0: without male genital system; stage 1: with rudimentary penis; stage 2: with rudimentary penis and penis sheath; and stage 3: the rudimentary penis developing into penis pouch and penis. The VDS indices varied between 1.07 and 2.82 and were lowest in the aquarium group and Yuanlin2. Regarding the severity of imposex, the aquarium group was less pronounced, as illustrated by the length of penis sheath and penis length, than the field collections (p<0.05). In respect of the penis length, males of the most imposex-affected site were up to 15% shorter than that of the aquarium group. Negative correlations between male penis length and female imposex characters (i.e., penis length and penis sheath length) were also observed.

Interspecific comparison of traffic noise effects on dove coo transmission in urban environments.

Most previous studies concerning avian adaptation to anthropogenic noise have focused on songbirds, but few have focused on non-songbirds commonly found in urban environments such as doves. We conducted field playback-recording experiments on the perch-coos of five dove species, including four native Taiwan species (the spotted dove, Spilopelia chinensis, the oriental turtle-dove, Streptopelia orientalis, the red collared-dove, Streptopelia tranquebarica, and the emerald dove, Chalcophaps indica) and one species not native to Taiwan (the zebra dove, Geopelia striata) to evaluate the detection and recognition of dove coos in habitats with differing levels of traffic noise. Our results suggest that traffic noise has selected dominant urban species such as the spotted dove to temporally and spatially adjust cooing to reduce the masking effects of traffic noise and rare urban species such as the emerald dove to avoid areas of high traffic noise. Additionally, although the zebra dove had the highest coo frequency among the study species, its coos showed the highest detection value but not the highest recognition value. We conclude that traffic noise is an important factor in shaping the distribution of rare and dominant dove species in urban environments through its significant effects on coo transmission.

Growth, Diet Composition and Reproductive Biology of the Invasive Freshwater Fish Chevron snakehead Channa striata on a Subtropical Island.

Kuan-chung Li, Bao-sen Shieh, Yuh-wen Chiu, Da-ji Huang, and Shih-hsiung Liang (2016) The Chevron snakehead (Channa striata) has been invading Taiwan for over 30 years, and it is currently widely distributed across diverse aquatic habitats within the island. Due to its strong environmental adaptability and carnivorous diet, C. striata has caused great negative impacts to the biodiversity of native fishes and aquatic organisms in Taiwan. To effectively restrain its spatial distribution and population, the objective of this study was to investigate the growing conditions, diet composition, and reproductive biology of C. striata in the field. In total, 294 individuals were collected from wetlands, irrigation canals, streams, and reservoirs in southern Taiwan from September 2008 to December 2010. Among 272 sex-identified individuals, more females (164) were collected than males (108). The morphological differences between the sexes could not be distinguished by the 10 body measurements recorded. Diverse food items, including snails, odonates, fishes, amphibians, and reptiles, were identified in the stomachs of 35 individuals. The minimum body length of sexually mature C. striata females exhibited at a standard length of 24.5 cm (total length 28 cm). The appearance of mature oocytes were mainly observed from July to November in 2009 and from April to October in 2010. Greater absolute fecundity (oocyte/individual) was estimated in Taiwan for C. striata than in its original distribution range possibly due to less water level fluctuation in the sampling habitats of Taiwan. The relative fecundity (oocyte/g) for C. striata was considered lower but within the documented range in Taiwan when compared with its original habitat in Malaysia. To effectively manage C. striata in Taiwan, regionally eradiating young and adult individuals, especially during the reproductive season and educating people to stop releasing it in the wild are possible ways to restrain and control the further spread of this exotic fish in Taiwan.

Oxytoxin-2, An Algal-Derived Molecule from a Cultured Mollusc Volvatella vigourouxi.

A natural caulerpenyne-derived sesquiterpene, oxytoxin-2 (1), was isolated from a cultured mollusc Volvatella vigourouxi. The structure of 1 was elucidated on the basis of spectroscopic analysis and this compound was suggested to be a diet-derived metabolite from the green alga Caulerpa sertularioides.-This is the first study on the chemical constituents of V. vigourouxi. Oxytoxin-2 (1) was found to inhibit significantly the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.

Acoustic and temporal partitioning of cicada assemblages in city and mountain environments.

Comparing adaptations to noisy city environments with those to natural mountain environments on the community level can provide significant insights that allow an understanding of the impact of anthropogenic noise on invertebrates that employ loud calling songs for mate attraction, especially when each species has its distinct song, as in the case of cicadas. In this study, we investigated the partitioning strategy of cicada assemblages in city and mountain environments by comparing the acoustic features and calling activity patterns of each species, recorded using automated digital recording systems. Our comparison of activity patterns of seasonal and diel calling revealed that there was no significant temporal partitioning of cicada assemblages in either environment. In addition, there was no correlation between the acoustic distance based on spectral features and temporal segregation. Heterospecific spectral overlap was low in both city and mountain environments, although city and mountain cicada assemblages were subject to significantly different levels of anthropogenic or interspecific noise. Furthermore, for the common species found in both environments, the calling activity patterns at both seasonal and diel time scales were significantly consistent across sites and across environments. We suggest that the temporal calling activity is constrained by endogenous factors for each species and is less flexible in response to external factors, such as anthropogenic noise. As a result, cicada assemblages in city environments with low species diversity do not demonstrate a more significant temporal partitioning than those in mountain environments with high species diversity.

Acute toxicity of mixture of acetaminophen and ibuprofen to Green Neon Shrimp, Neocaridina denticulate.

In recent years, numerous studies have indicated that various long-term use drugs, such as antibiotics or analgesics, not only cannot be completely decomposed via sewage treatment but also exhibit biological toxicity if they enter the environment; thus, the release of these drugs into the environment can damage ecological systems. This study sought to investigate the acute toxicity of two commonly utilized analgesics, ibuprofen (IBU) and acetaminophen (APAP), to aquatic organisms after these drugs have entered the water. To address this objective, the acute toxicity (median lethal concentration, LC50, for a 96-h exposure) of IBU alone, APAP alone, and mixtures containing different ratios of IBU and APAP in green neon shrimp (Neocaridina denticulata) were measured. The results of four tests revealed that the 96-h LC50 values for IBU and APAP alone were 6.07 mg/L and 6.60 mg/L, respectively. The 96-h LC50 for a 1:1 mixture of IBU and APAP was 6.23 mg/L, and the toxicity of this mixture did not significantly differ from the toxicity of either drug alone (p<0.05). The experimental results for mixtures containing unequal ratios of IBU and APAP indicated that mixtures with high APAP concentrations and low IBU concentrations exhibited markedly greater toxicity in N. denticulata (LC50=4.78 mg/L) than APAP or IBU alone. However, mixtures with high IBU concentrations and low APAP concentrations exhibited lower toxicity in N. denticulata (LC50=6.78 mg/L) than IBU or APAP alone. This study demonstrated that different mixtures of IBU and APAP were associated with different toxic effects in green neon shrimp.

The complete mitochondrial genome sequence of Onychostoma alticorpus (Cypriniformes, Cyprinidae).

We determined the complete mitochondrial genome (mitogenome) sequence of Onychostoma alticorpus, which is known as an endemic freshwater species in Taiwan, by using long polymerase chain reaction method. The total length of O. alticorpus mitogenome is 16,680 bp, consisting of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs genes and a noncoding control region. The overall base composition of O. alticorpus is 30.88% for A, 23.57% for T, 16.56% for G and 28.99% for C, with a slight AT bias of 54.45%. Gene location and specific usage of distinct termination codon types characterize typically the vertebrate mitochondrial genome. The determination of O. alticorpus mitogenome would play an important role not only in the delineation of phylogeographic history and population genetic structure, but reflection of conservation efforts on the genetic diversity as well as population vitality.

Cloning, expression, and characterization of cadmium-induced metallothionein-2 from the earthworms Metaphire posthuma and Polypheretima elongata.

In this study we report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Complementary (c)DNA was verified as the MT-2 gene by the characteristics of its predicted translation product, namely a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2 of both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The cDNAs corresponding to the two megascolecid MT-2 genes were expressed, and the MT-2 proteins were purified for biochemical characterization. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT and apo-MT were the most effective at scavenging free radicals, followed by Cd-MT. In conclusion, MT-2s from M. posthuma and P. elongata showed unique sequence features compared to those of lumbricid earthworms. These earthworms could be used to evaluate heavy-metal pollution in soil due to the inducible MT-2 by cadmium exposure.