Metallothionein, hemocyte status and superoxide dismutase/aspartate aminotransferase activity are sensitive biomarkers of cadmium stress in Onchidium reevesii.

Metal pollution in the environment is a serious threat to the biological sustainability of coastal ecosystems. However, our current understanding of the biological effects of metals in these ecosystems is limited. Herein, we investigated the responses of the sea slug Onchidium reevesii to persistent sublethal Cd environmental stress. Dynamic expression was analyzed using various biomarkers. The full-length cDNA of O. reevesii metallothionein (MT) was cloned and consists of 1639 nucleotides encoding a 65 amino acid polypeptide. Phylogenetic analysis showed that Or-MT has conserved Cys residues typical of MTs, including a typical Cys-X-Cys motif, implying that it can function the same as the MT of other shellfish. Expression of Or-MT in response to Cd varied in different tissues, and was highest in gastropod tissues. Thus, regiotemporal expression of MT may be useful for assessing pollution in coastal areas. Cellular immunity (in the hemolymph) and enzyme activity (in the hepatopancreas) were investigated along with hemocyte viability, hemocyte phagocytosis, and superoxide dismutase (SOD) and aspartate aminotransferase (AST) activities. Hemocyte viability was elevated under continuous Cd exposure but hemocyte phagocytosis was decreased. SOD and AST activities in the hepatopancreas fluctuated considerably, and SOD activity was more sensitive. SOD activity was lowest at 4 h and highest at 12 h, while AST activity peaked at 2 h and was lowest at 48 h. Thus, changes in enzyme activity may reveal adaptation to stress. Furthermore, the response patterns of certain enzymes, cellular immunity, and MT expression in O. reevesii could serve as biomarkers of Cd pollution in aquatic environments.

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii.

Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

The Complete Mitochondrial Genome Sequences of the Philomycus bilineatus (Stylommatophora: Philomycidae) and Phylogenetic Analysis.

The mitochondrial genome (mitogenome) can provide information for phylogenetic analyses and evolutionary biology. We first sequenced, annotated, and characterized the mitogenome of Philomycus bilineatus in this study. The complete mitogenome was 14,347 bp in length, containing 13 protein-coding genes (PCGs), 23 transfer RNA genes, two ribosomal RNA genes, and two non-coding regions (A + T-rich region). There were 15 overlap locations and 18 intergenic spacer regions found throughout the mitogenome of P. bilineatus. The A + T content in the mitogenome was 72.11%. All PCGs used a standard ATN as a start codon, with the exception of cytochrome c oxidase 1 (cox1) and ATP synthase F0 subunit 8 (atp8) with TTG and GTG. Additionally, TAA or TAG was identified as the typical stop codon. All transfer RNA (tRNA) genes had a typical clover-leaf structure, except for trnS1 (AGC), trnS2 (TCA), and trnK (TTT). A phylogenetic analysis with another 37 species of gastropods was performed using Bayesian inference, based on the amino acid sequences of 13 mitochondrial PCGs. The results indicated that P. bilineatus shares a close ancestry with Meghimatium bilineatum. It seems more appropriate to reclassify it as Arionoidea rather than Limacoidea, as previously thought. Our research may provide a new meaningful insight into the evolution of P. bilineatus.