Guangxing Liu

Prevalent Ciliate Symbiosis on Copepods: High Genetic Diversity and Wide Distribution Detected Using Small Subunit Ribosomal RNA Gene

Toward understanding the genetic diversity and distribution of copepod-associated symbiotic ciliates and the evolutionary relationships with their hosts in the marine environment, we developed a small subunit ribosomal RNA gene (18S rDNA)-based molecular method and investigated the genetic diversity and genotype distribution of the symbiotic ciliates on copepods. Of the 10 copepod species representing six families collected from six locations of Pacific and Atlantic Oceans, 9 were found to harbor ciliate symbionts. Phylogenetic analysis of the 391 ciliate 18S rDNA sequences obtained revealed seven groups (ribogroups), six (containing 99% of all the sequences) belonging to subclass Apostomatida, the other clustered with peritrich ciliate Vorticella gracilis. Among the Apostomatida groups, Group III were essentially identical to Vampyrophrya pelagica, and the other five groups represented the undocumented ciliates that were close to Vampyrophrya/Gymnodinioides/Hyalophysa. Group VI ciliates were found in all copepod species but one (Calanus sinicus), and were most abundant among all ciliate sequences obtained, indicating that they are the dominant symbiotic ciliates universally associated with copepods. In contrast, some ciliate sequences were found only in some of the copepods examined, suggesting the host selectivity and geographic differentiation of ciliates, which requires further verification by more extensive sampling. Our results reveal the wide occurrence and high genetic diversity of symbiotic ciliates on marine copepods and highlight the need to systematically investigate the host- and geography-based genetic differentiation and ecological roles of these ciliates globally.

In situ diet of the copepod Calanus sinicus in coastal waters of the South Yellow Sea and the Bohai Sea

Copepods are a key trophic link between primary producers and predatory animals at higher trophic levels in the marine ecosystem. Knowledge of the in situ composition of the copepod diet is critical for the accurate evaluation of trophic relationships and energy transfer in marine food webs. In this study, we applied a PCR-based cloning technique developed previously to investigate the in situ diet of Calanus sinicus, an ecologically important largesized calanoid copepod that dominates in the shelf waters around China, Japan and Korea. Analyses of the 18S rDNA sequences obtained from the copepod diet revealed the diverse food composition of C. sinicus from two stations (Y19 in the South Yellow Sea and B49 in the Bohai Sea). A total of 43 operational taxonomic units (OTUs) were detected, which belonged to 13 diverse lineages: Bacillariophyta, Dinoflagellata, Dictyochophyceae, Chrysophyta, Katablepharidophyta, Pelagophyceae, Apusozoa, Hydrozoa, Ctenophora, Echinodermata, Tunicata, Chaetognatha and marine fungi. The results indicate that during an algae bloom, C. sinicus can graze on the bloom causative species. When the abundance of phytoplankton in ambient water is relatively low, C. sinicus can choose eggs, larvae, or organic particles/detritus of various metazoans, especially hydrozoans and ctenophores, as alternative food sources. Our result suggests that C. sinicus is an omnivorous species, and its prey choice may depend on the food availability in the ambient waters.

Spliced leader RNA trans-splicing discovered in copepods

Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

Toxicity of five phenolic compounds to brine shrimp Artemia sinica (Crustacea: Artemiidae)

The acute toxicity of five phenolic compounds each to 15 d old Artemia sinica was determined in this study. The brine shrimp A. sinica was hatched from the encysted dry eggs (Bohai Bay Brand) produced by Dongying Ocean Artemia Co., Ltd., China at 27°C ± 1°C in pre-filtered (through pores of 0.45 μm in diameter) and autoclaved seawater (salinity 31, pH 7.5–8.0) in a cilindroconical glass beaker (2000 mL in volume) under continuous illumination (provided by a side set 20 W fluorescent lamp) with slight aeration. Ten Artemia individuals from the same batch of the hatched were cultured in 10 mL toxicant solution prepared with seawater (salinity 31, pH 7.5–8.0) at room temperature (about 20°C) to determine 24 h, 48 h and 72 h medium lethal concentration (LC50) of 5 phenolic compounds each. It was found that the toxicity of n-heptylphenol was the highest followed by nonylphenol, t-butylphenol, 2,4-dichlorophenol and bisphenol A in order. The LC50 values of the 5 compounds were calculated with regression analysis. The real concentration (in μg L−1) of 5 phenolic compounds each in toxicant solutions was measured with GC/MS analysis. Significant loss of phenolic compounds caused by either adsorption or desorption was not found. The significant difference of LC50 values was found among the five compounds 3 exposure times each. The range between the highest no-observed-effect concentration (NOEC) and 100% death causing concentration of five phenolic compounds each was determined. The toxicity in term of 24 h LC50 value of n-HP was 9.10 times higher than that of BPA, 1.71 times higher than t-BP, 1.53 times higher than 2,4-DCP and 1.36 times higher than NP, respectively.

Diagnostic model construction and example analysis of habitat degradation in enclosed bay: II. spatiotemporal variations in habitat degradation in Sansha Bay

The process of habitat degradation varies in habitat type and driving force which shows certain spatial and temporal heterogeneity on regional scales. In the present study, a new diagnostic model for enclosed bay habitat degradation was established, with which the spatial and temporal variation patterns of habitat degradation during 1991–2012 in Sansha Bay, Fujian, China was investigated. The results show that anthropogenic disturbance is the major controlling factor for the habitat degradation in large temporal heterogeneity in the bay. On the other hand, the habitat degradation experienced significant spatial variations among six sub-bays. Under the joint action of temporal and spatial heterogeneity, the degradation trend in growing scale shows a more significant correlation with the distribution of local leading industries along shorelines. Therefore, we quantified the main characters of habitat degradation in Sansha Bay, and have understood the relationship between the status of habitats spatio-temporal variation value and the main controlling factor leading to the changes. However, a deficiency of this research is the lack of or inaccessible to the detailed data, which shall be better solved in the future study for accessing more data from more sources.

Distribution and abundance of pelagic tunicates in the North Yellow Sea

In this paper, the distribution patterns and abundance of pelagic tunicates in the North Yellow Sea of China during the period 2006–2007 were analyzed. Zooplankton samples were obtained with vertical towing from bottom to surface using a WP2 plankton net (200 μm mesh size; mouth area: 0.25 m2). Five species belonging to two classes were identified: Oikopleura dioica, O. longicauda and Fritillaria borealis belonging to class Appendicularia; Salpa fusiformis and Doliolum denticulatum of class Thaliacea. O. dioica and O. longicauda were the dominant species, occurring in the samples of all four seasons, with different distribution patterns. Their maximum abundance were 1664.7 ind. m−3 (spring) and 1031.7 ind. m−3 (spring) respectively. Following Oikopleura spp. were D. denticulatum, which was found only in autumn with an average abundance of 149.6 ind. m−3, and S. fusiformis, which was detected all the year long except for autumn with low abundance (max. abundance 289.4 ind. m−3 in summer). Only a very small amount of F. borealis was detected in summer samples, with an average abundance of 2.7 ind. m−3. The relationship between tunicates abundances and the environmental factors was analyzed using the stepwise regression model for each species. The variation of appendicularian abundance showed a significant correlation with the surface water temperature and with the concentration of Chl-a. No relationship was found between tunicates abundance and salinity, likely due to the slight changes in surface salinity of the studied area during the four seasons. Salps abundance and that of doliolids were significantly correlated to bottom water temperature, indicating that these two species (S. fusiformis and D. denticulatum) migrate vertically in the water column. In particular D. denticulatum, known to be a warm water species, showed not only an important correlation with water temperature, but also a spatial distribution connected to the warm currents in the North Yellow Sea. The occurrence of D. denticulatum represents an interesting result never found in past research work. Water temperature, algal distribution and currents were the most relevant environmental factors influencing the tunicate abundance and distribution in the North Yellow Sea. Further research is needed in order to get more information on the ecology of these organisms and to better understand their role in the ecosystem including the oceanic food web.

Diagnostic model construction and example analysis of habitat degradation in enclosed bay: I. diagnostic model construction

Presently, research is lacking regarding the diagnosis and evaluation of habitat degradation in enclosed bay systems. We established a diagnostic model for enclosed bay habitat degradation (EBHD model) using a multi-approach integrated diagnostic method in consideration of driving force-pressure-state-influence-response. The model optimizes the indicator standardization with annual average change rate of habitat degradation as the basic element, to reflect accurately the impact of the change and speed of degradation on the diagnostic results, to quantify reasonably the contribution of individual diagnostic indicator to habitat degradation, and to solve the issue regarding the influence of subjective factors on the evaluation results during indicator scoring. We then applied the EBHD model for the Sansha Bay in Fujian Province, China, evaluated comprehensively the situation of habitat degradation in the bay, and screened out the major controlling factors in the study area. Results show that the diagnostic results are consistent in overall with the real situation of the study area. Therefore, the EBHD model is advantageous in terms of objectivity and accuracy, making a breakthrough in diagnosis and evaluation for habitat degradation in enclosed bay systems.

Molecular phylogeography and population genetic structure of the planktonic copepod Calanus sinicus Brodsky in the coastal waters of China

Planktonic copepod Calanus sinicus is the dominant meso-zooplankton in the Northwest Pacific Ocean. To better understand its population dynamics and phylogeographic patterns, 243 C. sinicus individuals were collected from seven locations across the shelf waters of China and its population genetics was studied by mitochondrial DNA cytochrome oxidase I (mtCOI) sequences analyses. Thirty-nine different sequences, or haplotypes, were detected with moderate haplotype diversity (h=0.749) and low nucleotide diversity (π=0.003) for all populations. The evolutionary divergence between geographic populations varied from 0.24% to 0.37%, indicative of very limited genetic differentiation. Visualized minimum spanning network (MSN) and phylogenetic analysis of all the detected haplotypes did not reveal any clear phylogeographic pattern. Furthermore, AMOVA data showed no significant spatial population differentiation existed among the individuals collected across China shelf waters. Pairwise FST values showed that population collected from northwest of the East China Sea (ECS) displayed a low difference to other populations. Mismatch distribution analyses and neutrality tests indicated that C. sinicus might undergo a demographic/population expansion. No significant population genetic structuring was detected, indicating an extensive gene flow among the C. sinicus populations. Our results provide molecular evidence for the hypothesis that C. sinicus in the northwestern South China Sea in winter is transported from the East China Sea and the Yellow Sea by the China Coastal Current during the northeast monsoon period.

Primers to block the amplification of symbiotic apostome ciliate 18S rRNA gene in a PCR-based copepod diet study

Pelagic copepods play an important role in the marine food web. However, a full understanding of the ecological status of this zooplankton group depends on the careful study of their natural diets. In previous PCR-based copepod diet studies, we found many apostome ciliates that live symbiotically under the exoskeleton of the copepods, and their sequences were often over-represented in the 18S rRNA gene (18S rDNA) libraries. As a first step to address this issue, we designed three apostome ciliate 18S rDNA blocking primers, and tested their blocking efficiency against apostome ciliate 18s rDNA under various PCR conditions. Using a semi-quantitative PCR method, we optimized the conditions to efficiently amplify the 18S rDNA of the prey while simultaneously excluding the symbiotic apostome ciliates. This technique will facilitate PCR-based diet studies of copepods and other zooplankton in their natural environments.

Effects of elevated seawater pCO2 on early development of scallop Argopecten irradias (Lamarck, 1819)

To investigate the effects of elevated seawater pCO2 on the early developmental stages of marine benthic calcifying organisms, we exposed the eggs and larvae of Argopecten irradias, an important bivalve species in Chinese aquaculture, in seawater equilibrated with CO2-enriched (1000 ppm) gas mixtures. We demonstrated that elevated seawater pCO2 significantly interfered with fertilization and larval development and resulted in an increased aberration rate. Fertilization in the treatment (pH 7.6) was 74.3% ± 3.8%, which was 9.7% lower than that in the control (pH 8.3) (84.0% ±3.0%). Hatching success decreased by 23.7%, and aberration rate increased by 30.3% under acidic condition. Larvae in acidified seawater still developed a shell during the post-embryonic phase. However, the shell length and height in the treatment were smaller than those in the control. The development of embryos differed significantly at 12 h after fertilization between the two experimental groups. Embryos developed slower in acidified seawater. Nearly half of the embryos in the control developed into D-shaped larvae at 48 h after fertilization, which were considerably more than those in the treatment (11.7%). Results suggest that future ocean acidification (OA) would cause detrimental effects on the early development of A. irradias.