DNA barcoding of Antarctic marine zooplankton for species identification and recognition

Polar zooplankton are particularly sensitive to climate change, and have been used as rapid-responders to indicate climate-induced changes in the fragile Antarctic ecosystem. DNA barcoding provides an alternative approach for rapid zooplankton species identification. Ninety-four specimens belonging to 32 Antarctic zooplankton species were barcoded to construct a comprehensive reference library. An 830 to 1 050 base-pair region of the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was obtained as DNA barcodes. The intraspecific variation of the gene ranged from 0 to 2.6% (p-distance), with an average of 0.67% (SD=0.67%). The distance between species within the same genera ranged from 0.1% (Calanus) to 29.3%, with an average of 15.3% (SD=8.4%). The morphological and genetic similarities between Calanus propinquus and C. simillimus raise new questions about the taxonomic status of C. simillimus. With the exception of the two Calanus species, the intraspecific genetic divergence was much smaller than the interspecific divergence among congeneric species, confirming the existence of a barcode gap for Antarctic zooplankton. In addition, species other than Calanus sp. formed a monophyletic group. Therefore, we have confirmed DNA barcoding as an accurate and efficient approach for zooplankton identification in the Antarctic area (except for Hydromedusa, Tunicata, and other gelatinous zooplankton). Indicator vector analysis further confirmed this conclusion. The new primer sets issued here may facilitate the study of Antarctic marine zooplankton species composition by environmental metagenetic analysis

Variation in epibiotic bacteria on two squat lobster species of Munidopsidae

The relationships between epibiotic bacteria on deep-sea hosts and host lifestyle factors are of particular interest in the field of deep-sea chemoautotrophic environmental adaptations. The squat lobsters Shinkaia crosnieri and Munidopsis verrilli are both dominant species in cold-seep ecosystems, and they have different distributions and feeding behaviors. These species may have evolved to have distinct epibiotic microbiota. Here, we compared the epibiotic bacterial communities on the M. verrilli carapace (MVcarapace), S. crosnieri carapace (SCcarapace), and S. crosnieri ventral plumose setae (SCsetae). The epibiotic bacteria on SCsetae were dense and diverse and had a multi-layer configuration, while those on MVcarapace and SCcarapace were sparse and had a monolayer configuration. Chemoautotrophic bacteria had the highest relative abundance in all epibiotic bacterial communities. The relative abundance of amplicon sequence variant 3 (ASV3; unknown species in order Thiotrichales), which is associated with sulfide oxidation, was significantly higher in SCsetae than MVcarapace and SCcarapace. Thiotrichales species seemed to be specifically enriched on SCsetae, potentially due to the synthetic substrate supply, adhesion preference, and host behaviors. We hypothesize that the S. crosnieri episymbionts use chemical fluxes near cold seeps more efficiently, thereby supporting the host’s nutrient strategies, resulting in a different distribution of the two species of squat lobster

Confocal Raman microscopy for assessing effects of preservation methods on symbiotic deep-sea mussel gills

Confocal Raman microscopy (CRM) is a powerful tool for biological research, which can provide information regarding the composition and distribution of biomolecules in an in situ, label-free, non-destructive manner and with high spatial resolution. Sample preservation is often an unavoidable step, especially for symbiotic deep-sea samples. Moreover, protocols for the preservation of samples for CRM have not been established and specific effects of different preservation methods on biomolecules have not been studied for relevant samples. In this study, we used deep-sea mussel Gigantidas platifrons, an ideal model in the study of deep-sea symbiosis and investigated the effect of four common preservation methods on the results of CRM imaging and signals. The methods included snap-freeze (SF), SF followed by rapid fixation in methanol (SF-MeOH), 2.5% glutaraldehyde and 2% paraformaldehyde fixation (SF-GP), and 4% paraformaldehyde and alcohol fixation (PS-PA). The results of this study indicate that SF was the most effective method for the comprehensive analysis of the biomolecular composition although the sectioning success rate was relatively low. Moreover, SF-MeOH was found to be effective when SF is not sufficient in obtaining good morphology in sections, or when the effect of chemical bonding on the composition of biomolecules upon SF-MeOH can be neglected. Finally, SF-GP and PS-PA were found to be the most effective methods considering the overall morphological observation. However, they were less suitable for metabolic studies. We believe our results can provide guidance for further studies of Raman on symbiotic deep-sea biological samples. It is of great importance for the wide application of Raman technique

PO-178 Heat Treatment and Exercise Prevents Skeletal Muscle Insulin Resistance in Wistar Rats Fed High-Fat Diet

Objective  Insulin resistance (IR) is associated with many related health complications. Previous studies demonstrate that heat and exercise independently reduce IR. The purpose of this study was to test the hypothesis that combined exercise and heating is even more favorable in reducing IR. Methods Male Wistar rats were randomly divided into five groups: exercise (NE; n=10), heated (HC; n=10), exercise and heated (HE; n=10), sedentary (NC; n=10), and normal diet plus sedentary (CC; n=10). All but the latter group was fed a high-fat diet (60% calories from fat) for 10 weeks while receiving heat and/or exercise exposure for latter 8 weeks. Following this regimen, protein expression from the soleus and extensor digitorum longus muscles, serum, and brown fat were analyzed using Western blotting. Results Exercise combined with heating shifted the metabolic characteristics of rats on a high-fat diet toward that observed in the rats on a standard diet. Specifically, eight weeks of combined heat and endurance exercise increased PGC-1α, CnA, CaMKIV and p38 MAPK protein expression in the soleus (P < 0.05), insulin protein expression in the serum (P < 0.05), and UCP1 protein expression in the brown fat (P < 0.05), when compared to the high fat fed sedentary group. There were some significant differences in responses (i.e., body weight and Leptin & Adiponectin concentrations) between the combined exercise and heat group relative to the exercise alone group. Conclusions  Exercise combined with heat exposure mitigates the development of IR, presumably from the Irisin pathway. The study provides potential non-pharmaceutical methods for therapeutic treatment of IR

Toxicological effects of cadmium on deep-sea mussel Gigantidas platifrons revealed by a combined proteomic and metabolomic approach

IntroductionMarine metal contamination caused by deep-sea mining activities has elicited great concern from both social and scientific communities. Among the various metals deep-sea organisms might encounter, cadmium (Cd) is a widely detected metal that in very small amounts is nonetheless capable of severe toxicity. Yet due to both remoteness and technical challenges, insights into the effects of metal exposure resulting from mining activities upon deep-sea organisms are limited.MethodsHere, we investigated Cd’s toxicological effects on deep-sea mussels of Gigantidas platifrons exposed to 100 or 1000 g/L of Cd for 7 days; an integrated approach was used that incorporated proteomics and metabolomics along with traditional approaches (metal concentrations, metal subcellular distribution, and anti-oxidative and immune-related biochemical indexes).Results and DiscussionResults showed that Cd exposure caused significant Cd’s accumulation in mussel gills and redistribution of Cd among subcellular compartments, with cellular debris being the primary binding site. Although anti-oxidative enzymes activities (superoxide dismutase and catalase) were not significantly altered in mussel gills of both exposed groups, the markedly increased level of glutathione S-transferase detected via proteomic technique clearly evinced that deep-sea mussels suffered from oxidative stress under Cd exposure. Besides, altered activities of acid phosphatase and alkaline phosphatase assayed by traditional methods along with the predominant presence of largely altered immune-related proteins detected by proteomic data strongly revealed an immune response of deep-sea mussels elicited by Cd. In addition, results of proteomics combined with those of non-targeted metabolomics demonstrated that Cd could exert toxicity by disrupting cytoskeleton structure, ion homeostasis, and primary metabolisms of energy, lipid, and nucleotide in deep-sea mussels. As demonstrated in this study, proteomics and metabolomics can be used in tandem to provide valuable insights into the molecular mechanisms of deep-sea organisms’ response to Cd exposure and for helping to discover potential biomarkers for application during deep-sea mining assessments

PO-228 Effects of Different Cryotherapy Models on Timing Sequence Recovery of Exercise Induced Muscle Damage in Middle and Long Distance Runners

Objective Exercise induced muscle damage (EIMD) is one common physiological phenomenon in competitive sports and mass sports. Water immersion recovery and whole body cryotherapy (cryostimulation) (WBC) has become one of the fast recovery methods adopted by high level athletes in the world. The aim of this study was to compare the water immersion recovery and WBC from timing sequential recovery on EIMD, subjective scales, biochemical indicators, exercise performance indicators. Methods Twelve middle and long distance runners from Beijing Sport University were recruited in this study (exercise performance is secondary level in China). All participants performed four models in four weeks which included rest control (CON), cold water immersion(CWI), contrast water therapy (CWT) and whole body cryotherapy (cryostimulation) (WBC) separated by one week. The subjects needed to complete the EIMD exercise program, includes two parts: the treadmill running and the jump step. The individual speed of treadmill running was based on the individual VO2max. running including 5 sets and total time is about 90 min. Every set consist of 6 min flat running, 6 min uphill running and 6 min downhill running. The speed of the treadmill was stable in different stages. The slope of the uphill running is（＋6,＋5,＋4,＋3,＋2）, and the slope of downhill running is (-8, -7, -6, -5, -4). The jump was performed 20 times separated by 30 s via special step ( height is 40 cm) . After the EIMD exercise program, the subjects were treated with different recovery methods immediately after exercise, 24 h, 48 h, 72 h after exercise, while CON group received no intervention. The CWI group was immerged in 15 °C cold water for 12min, and the CWT groups was immerged in 15°C cold water for 1min and 38°C hot water for 1min with 6 cycles. The whole body cryotherapy (cryostimulation) group was immerged in special chamber (-110—-140℃）for 3 min. The three interventions were immerged body to the position of shoulders. The indexes including subjective scales (VAS scale, RPE scale, Borg scale, WHO deep sleep scale, PQSI scale), biochemical indicators (creatine kinase, lactate dehydrogenase, myoglobin, interleukin-6, C reactive protein, SICAM-1), exercise performance indicators ( vertical jump height, grip, running distance). The indexes was collected in different periods (i.e. Before exercise, immediately after exercise,Post1h, Post 24h, Post 48h, Post 72h, Post 96h after exercise etc.). Results  (1) Subjective scales. From results of VAS scale, RPE scale, and Borg scale, the WBC had best recovery effects than the other three interventions (P<0.05).  The recovery effect of the CWI group was similar with the CON group. From results of WHO deep sleep scale and PQSI scale, the WBC group had better recovery effects than CON group (P<0.05). (2) Biochemical indicators. From results of CK, Mb and SICAM-1, the WBC group had better recovery effect than the other three groups (P<0.05). In the LDH, the WBC group had similar effects with the CWT group (P>0.05). From results of CRP and IL-6, the WBC group had better recovery effects than the other three groups (P<0.05).（3）Exercise performance indicators. From results of vertical jump, WBC had lower decrease than the other three groups (P<0.05).  From results of the grip, of the WBC and CWT groups have significant retentive effects than CON group (P<0.05), but there was no significant difference between CON and CWI groups (P>0.05). Conclusions (1)Through combine the treadmill running and the jump step exercise program,Can effectively lead to human body appear the EIMD. (2)WBC has positive effects on the subjective scale, biochemical indicators, exercise performance indicators associated with EIMD.For middle and long distance runners EIMD,compared with CWI CWT,WBC effect is better.(3) CWI and CWT has a positive effect on some subjective scales, biochemical indicators, and exercise performance indicators related to EIMD. However, the positive effect of CWI and CWT is lower than WBC in the extent of action and timing sequence. (4)For the middle and long distance runners EIMD timing sequence recovery effects,WBC have better effects,Followed by CWT , CWI  effect is not significant