Hongsheng Yang

Growth characters and photosynthetic capacity of Gracilaria lemaneiformis as a biofilter in a shellfish farming area in Sanggou Bay, China

Experiments on growth characters and ecological functions of the macroalgae Gracilaria lemaneiformis, collected from south China, were conducted in polyculture areas of kelp and filter-feeding bivalve in Sanggou Bay in Weihai City, Shandong, in north China from May 2002 to May 2003. The results of 116 days cultivation showed that the average wet weight of alga increased 89 times from 0.1 to 8.9 kg rope−1, with an average specific growth rate (based on wet weight) of 3.95% per day. The most favorable water layer for its growth was 1.0–1.8 m below the surface in July and August, with an average specific growth rate of 8.2% per day in 30-day experiments. Photosynthetic activity changed seasonally, with an average of 7.3 mg O2 g dw−1 h−1. The maximum rate (14.4 mg O2 g dw−1 h−1) was recorded in July, or 19.3 mg CO2 g dw−1 h−1, while the minimum (0.40 mg CO2 g dw−1 h−1) was in April. This study indicated that the culture of G. lemaneiformis is an effective way to improve water quality where scallops are cultivated intensively.

Effects of aestivation on the energy budget of sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea)

Abstract Apostichopus japonicus is a kind of temperate sea cucumbers, known to aestivate when water temperature rises above 20°C to 24.5°C. In this study, we measured the effects of aestivation on the energy utilization (i.e., energy allocation in growth, feces discharge, respiration and excretion) of A. japonicus with two different body weights (134.0 g ± 3.5 g and 73.6 g ± 2.2 g) at water temperature from 10°C to 30°C with an interval of 5°C. Noticeable variation in the energy utilization of sea cucumbers was observed in this study. During the non-aestivation period, energy deposit in growth was lower and the energy loss in feces accounted for the majority of the feeding energy. Under the threshold temperature, the feeding energy reduced and the proportion of energy deposit in growth became negative. During aestivation, sea cucumbers discontinued feeding energy, resulting in weight loss. Our study suggested that the ecological implication of aestivation in this species could lead to a model of energy saving during the long-term hot period.

RNA-Seq Reveals Dynamic Changes of Gene Expression in Key Stages of Intestine Regeneration in the Sea Cucumber Apostichopus japonicas

Background Sea cucumbers (Holothuroidea; Echinodermata) have the capacity to regenerate lost tissues and organs. Although the histological and cytological aspects of intestine regeneration have been extensively studied, little is known of the genetic mechanisms involved. There has, however, been a renewed effort to develop a database of Expressed Sequence Tags (ESTs) in Apostichopus japonicus, an economically-important species that occurs in China. This is important for studies on genetic breeding, molecular markers and special physiological phenomena. We have also constructed a library of ESTs obtained from the regenerative body wall and intestine of A. japonicus. The database has increased to ∼30000 ESTs. Results We used RNA-Seq to determine gene expression profiles associated with intestinal regeneration in A. japonicus at 3, 7, 14 and 21 days post evisceration (dpe). This was compared to profiles obtained from a normally-functioning intestine. Approximately 5 million (M) reads were sequenced in every library. Over 2400 up-regulated genes (>10%) and over 1000 down-regulated genes (∼5%) were observed at 3 and 7dpe (log2Ratio≥1, FDR≤0.001). Specific “Go terms” revealed that the DEGs (Differentially Expressed Genes) performed an important function at every regeneration stage. Besides some expected pathways (for example, Ribosome and Spliceosome pathway term), the “Notch signaling pathway,” the “ECM-receptor interaction” and the “Cytokine-cytokine receptor interaction” were significantly enriched. We also investigated the expression profiles of developmental genes, ECM-associated genes and Cytoskeletal genes. Twenty of the most important differentially expressed genes (DEGs) were verified by Real-time PCR, which resulted in a trend concordance of almost 100% between the two techniques. Conclusion Our studies demonstrated dynamic changes in global gene expression during intestine regeneration and presented a series of candidate genes and enriched pathways that contribute to intestine regeneration in sea cucumbers. This provides a foundation for future studies on the genetics/molecular mechanisms associated with intestine regeneration.

Catecholaminergic responses to environmental stress in the hemolymph of Zhikong scallop Chlamys farreri.

Catecholamines have long been known to play essential roles in several physiological processes in invertebrates. In this study, enzyme immunoassays were used to examine the levels of catecholamines (including adrenaline (AD), noradrenaline (NA) and dopamine (DA)) in the hemolymph of the scallop, Chlamys farreri, in responses to selected environmental stress. These stressors include high temperature (acute transfer from 17 to 28 degrees C), low salinity (acute transfer from salinity 31 to 20 psu) and air exposure, which are commonly encountered in aquaculture practices or in their natural habitat. The results showed that both AD and NA concentrations increased significantly in response to all the stressors. The levels of these catecholamines peaked 72 hr after acute temperature or salinity stresses, and then decreased gradually. For scallops subjected to air exposure, the duration of AD concentration elevation depended on the exposure temperature with a higher air temperature resulting in a shorter time to return to control level. In contrast, the NA responses were long lasting and no obvious decreases were observed after reaching the peak value. Interestingly, the DA concentration showed an inverse trend in response to stresses compared with AD or NA. These results suggested that the catecholaminergic responses in bivalve hemolymph will provide useful references in predicting the success of aquaculture practices and monitoring the environmental stresses.

Histological, ultrastructural and heat shock protein 70 (HSP70) responses to heat stress in the sea cucumber Apostichopus japonicus.

The aquaculture industry for Apostichopus japonicus has suffered severe economic and resource losses due to high temperature in recent summers. There is increasing concern about the effect of high temperature on this species. Histological, ultrastructural and HSP70 responses to heat stress were investigated in the intestine of A. japonicus. Tissue degradation was observed in muscular, submucosal and mucosal layers, with significant decrease in plicae circulares of the mucosal layer. Ultrastructural damage intensified with increasing stress time, and indicators of cell apoptosis were evident after 192 h heat stress. Immunostaining showed HSP70 mainly in mucosa and serosa, with faint staining in non-stressed individuals (the control group) and denser staining under stress (the 6, 48 and 192 h groups). Western blot detection confirmed ocurrence of HSP70 in all groups and significant up-regulation under stress. The rapid and persistent response of HSP70 implies its critical role in the heat shock response of A. japonicus.

Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis

The response of the defense components lysozyme (LYZ), metallothionein (MT), and superoxide dismutase (SOD) to combined exposure to heavy metals and bacteria was assessed at transcriptional level in the surf clam Mactra veneriformis. First, the full-length LYZ cDNA containing 808 nucleotides and encoding 194 deduced amino acids was identified from the clam. Multiple alignments revealed that MvLYZ had a high identity with invertebrate-type LYZs from other mollusks. Next, clams were exposed to Vibrio parahaemolyticus and a mixture of cadmium and mercury, alone or in combination, for 7 days. Cumulative mortality of clams and mRNA expressions of the three defense components were analyzed. The highest cumulative mortality took place in the combined treatment on day 7. The expression of the three genes was up-regulated in response to treatments compared to the control with different response times and transcriptional levels; the response to combined exposure occurred earlier than to single exposure. Among the experimental groups, MvLYZ expression and MvSOD expression peaked in the combined treatment on day 3, whereas MvMT expression peaked in heavy metals treatment on day 5. Furthermore, interactive effects of heavy metals and Vibrio on transcriptional response changed over the exposure time. Therefore, transcriptional regulation of the three genes under combined exposure was more complex than under single exposure.

Restoring Eelgrass (Zostera marina L.) Habitats Using a Simple and Effective Transplanting Technique

Eelgrass beds in coastal waters of China have declined substantially over the past 30 years. In this study, a simple new transplanting technique was developed for eelgrass (Zostera marina L.) restoration. To assist in anchoring single shoots, several rhizomes of rooted shoots were bound to a small elongate stone (50–150 g) with biodegradable thread (cotton or hemp), and then the bound packet was buried at an angle in the sediments at a depth of 2–4 cm. This stone anchoring method was used to transplant eelgrass in early November 2009 and late May 2010 in Huiquan Bay, Qingdao. The method led to high success. Three month survivorship of the transplanted shoots at the two transplant sites was >95%. From April 20 to November 19, 2012, the following characteristics of the 2009 and 2010 transplanted eelgrass beds were monitored: morphological changes, shoot density, shoot height, leaf biomass, and sediment particle size. Results showed that the sexual reproduction period of the planted eelgrass was from April to August, and vegetative reproduction reached its peak in autumn. Maximum shoot height and biomass were observed in June and July. After becoming established, the transplanted eelgrass beds were statistically equal to natural eelgrass beds nearby in terms of shoot height, biomass, and seasonal variations. This indicates that the transplant technique is effective for eelgrass restoration in coastal waters.

Analysis of metallotionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure.

To investigate the possible role of metallothioneins (MTs) and antioxidant enzymes in cadmium (Cd) tolerance in Meretrix meretrix larvae, a new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full-length cDNA of MmMT consisted of an open reading frame (ORF) of 231bp encoding a protein of 76 amino acids, with 21 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57-84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was used to analyze the expression level of MmMT mRNA at different M. meretrix larval stages under Cd exposure (25μgL⁻¹). Results showed that Cd could induce the expression of MmMT mRNA in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers, respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all of the soft parts at the trochophore and postlarva stages, whereas it was only synthesized in the velum and epidermis at the D-shaped larva and pediveliger stages. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), also were measured in larvae at different developmental stages. Increased enzymatic activities were detected mainly in D-shaped larvae and pediveligers under Cd stress, suggesting that these enzymes respond synchronously with MT. Our results indicate that MmMT and antioxidant enzymes played important roles in counteracting Cd stress in M. meretrix larvae.

Understanding the Heat Shock Response in the Sea Cucumber Apostichopus japonicus, Using iTRAQ-Based Proteomics

The sea cucumber Apostichopus japonicus is exploited as a commercial species owing to their high nutritive and medicinal value. Recent high summer temperatures have caused high mortality rates in A. japonicus. In this study, we applied the isobaric tag for relative and absolute quantitation (iTRAQ) technique to investigate the global protein expression profile under an acute short-term (48 h) heat stress. In total, 3432 proteins were identified, and 127 proteins showed significant heat stress responses, with 61 upregulated proteins and 66 downregulated proteins. Our results suggest that heat stress influenced the expression of proteins involved in various biological processes, such as tissue protection and detoxification, lipid and amino acid metabolism, energy production and usage, transcription and translation, cell apoptosis, and cell proliferation. These findings provide a better understanding about the response and thermo-tolerance mechanisms of A. japonicus under heat stress.

Temporal pattern in the bloom-forming macroalgae Chaetomorpha linum and Ulva pertusa in seagrass beds, Swan Lake lagoon, North China

Seagrasses that are distributed over a large area of the Swan Lake, Weihai, China, support a productive ecosystem. In recent years, however, frequent macroalgal blooms have changed the ecosystem structure and threatened the seagrasses. To understand the bloom-forming macroalgae we conducted a yearly field survey of Swan Lake. Results indicated that the macroalgae Chaetomorpha linum and Ulva pertusa both exhibited a much higher productivity and attained a greater maximum biomass (of 1712±780gDWm(-)(2) and 1511 ± 555 gDW m(-2), respectively) than was the case for the seagrasses. The mean annual atomic ratios of C/N, C/P and N/P in C. linum were 14.31 ± 4.45, 402.82 ± 130.25, and 28.12±2.08, respectively. The δ(15)N values (11.09 ± 0.91‰ for C. linum; 9.27 ± 2.83‰ for U. pertusa) indicated a land-based source of N enrichment to the macroalgal blooms. High concentrations of nitrogen and phosphorus in the lagoon, particularly near the river mouth, supported the blooms.