| Since the 21st century,noise generated by marine activities such as ships,port construction,wind farms,oil and geological exploration has continued to grow and has become a non-negligible source of pollution in the ocean.However,most studies have focused only on marine mammals and fish,and very few have studied mollusks,which does not reflect their importance in marine ecosystems.Many mollusks have relatively small sizes,short life cycles,are easy to study under laboratory and field conditions,and are more sensitive to low-frequency sound than vertebrates,making them the best material for exploring the effects of low-frequency noise.Therefore,in this paper,we used sea slug（Onchidium reevesii）as the research object,and studied the changes of sea slug behavior,biochemical responses,antioxidant indexes and immune indexes under the dual exposure of low-frequency noise and other environmental stressors（pathogens and heavy metals）with the technical methods of behavior,physiology and molecular biology to reveal the effects of low-frequency noise on sea slug behavior,oxidative stress and immune defense.At the same time,the molecular mechanism of the potential stress response caused by low-frequency noise on sea slug was comprehensively described in conjunction with transcriptomics,providing a theoretical basis for the study of marine noise pollution in mollusks.Expt.1 Behavioral and physiological responses of sea slug to low-frequency noiseA large amount of marine noise pollution from coastal industrial construction and the water transportation industry affects the growth and development of marine life,which is a big issue threatening marine organisms.However,most studies have focused on vertebrates,such as marine mammals and fishes,and little is known about the effects of noise on invertebrates.Therefore,the impacts of low-frequency noise（100,300,and500 Hz）on behavioral and physiological responsesof the sea slug（Onchidium reevesii）were investigated.Under laboratory conditions,sea slugs were stimulated with low-frequency noise at 100,300,and 500 Hz for 1 h.Then,hemolymph enzyme activities（glucose,albumin,triglycerides,superoxide dismutase,catalase,and malondialdehyde）were measured,and m RNA expression of the hsp70 gene was detected in hemolymph and the nervous system by quantitative real-time polymerase chain reaction（q RT-PCR）analysis,while expression of the hsp70 protein was analyzed by immunohistochemistry.The results demonstrated that sea slugs were away from the stimulus source under the influence of low-frequency noise compared to the control group.Enzyme activities,as well as hsp70 gene m RNA and protein expression levels,were significantly higher in the noise-exposed groups than those in the control group.Overall,these changes indicate that low-frequency noise caused oxidative stress in sea slugs in vivo,and the oxidative damage gradually increased when the noise frequency was increased from 100to 500 Hz.Expt.2 Transcriptome analysis of the central nervous system of sea slug exposed to low-frequency noiseLow-frequency noise has become a marine pollutant that cannot be ignored,but most studies have focused on the behavioral and physiological effects on marine vertebrates,with few studies in marine mollusks.Therefore,sea slug was used in this study to investigate the effect of low-frequency noise on its physiological aspects.This experiment was designed with different low-frequency noise（0,100,300,and 500 Hz）and different stimulation times（0,6,and 12 h）to measure superoxide dismutase（SOD）,malondialdehyde（MDA）,and catalase（CAT）activities in hemolymph and transcriptomics in the control（C）and 6 and 12 h groups（L1 and L2）with 500 Hz noise.The results showed a positive correlation between antioxidant enzyme activity and low-frequency noise frequency（P<0.05）and no correlation with time（P>0.05）.In central nervous system（CNS）transcriptomics,2,460 and 3,268 genes had upregulated expression and 2,765 and 2,783 genes had downregulated expression in the L1 and L2 groups,respectively,compared to the C group.According to Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis,low-frequency noise mainly affects signaling pathways such as cytokine-cytokine receptor interaction,the Fox O signaling pathway,natural killer cell-mediated cytotoxicity,apoptosis immune-related pathways,and energy metabolic pathways such as glycolysis,the TCA cycle,and glycerophospholipid metabolism,as well as neurological pathways such as GABAergic synapses,the synaptic vesicle cycle,amyotrophic lateral sclerosis（ALS）and other neurological pathways.This study would provide valuable reference information on the potential response of mollusks to low-frequency noise stress.Expt.3 Effect of low-frequency noise on immune indexes of sea slug hemocytes and hepatopancreasHemocytes and hepatopancreas play an important role in shellfish non-specific immunity.However,there are few studies on the effects of low frequency noise on the immune system of marine animals.Therefore,in this study,in order to investigate the effect of low frequency noise on the immune system of sea slug.We conducted 1,3,6and 12 h exposure experiments with sea slug in low frequency noise at 1000 Hz.Then we analyzed the ROS production and cell viability of blood cells as well as antioxidant enzymes such as superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-PX）and glutathione-S transferase（GST）and immune-related genes human nuclear factorκB inhibitor proteinα（IκBα）,transcription factor p65（p65）,Bcl-2-related protein x（Bax）,B-cell lymphoma 2（bcl-2）,interleukin-8（IL-8）,interleukin-1β（IL-1β）and Toll-like receptor 4（TLR4）m RNA expressions.The results showed that compared with the unexposed group,ROS production in sea slug blood cells was significantly higher（P<0.05）and cell viability was significantly lower（P<0.05）;and there was a different degree of increase in SOD,CAT,GSH-PX and GST antioxidant enzyme activities in hepatopancreas（P<0.05）,and immune-related genes IκBα,p65,Bax,bcl-2 and m RNA expression of IL-8,IL-1βand TLR4 genes were also increased to different degrees（P<0.05）.Moreover,the changes of the above indicators were positively correlated with the exposure time（P<0.05）.Taken together,these results suggest that low-frequency noise can cause oxidative stress and immune response in sea slug.Expt.4 Effect of low-frequency noise on the survival rate and immunity of infected Vibrio parahaemolyticus sea slugMarine noise has become an important environmental pollutant that affects the behavior,physiology and immunity of marine animals.However,the resistance of marine animals to pathogens while under the influence of noise is a topic that has received little attention.To assess the immune defense response of sea slugs against pathogens when exposed to low frequency noise,we performed 120 h exposure experiments on sea slugs after a Vibrio parahaemolyticus application in low frequency noise at 500 Hz and 1000 Hz.The results showed that after the V.parahaemolyticus attack,the survival rate of the sea slugs decreased,the apoptosis rate and ROS production of hemocytes increased significantly,the proliferation of hemocytes accelerated,the activities of enzymes such as SOD,CAT,AKP,ALT,and LZM in the hepatopancreas increased significantly,and the expression of TNF signaling pathway-related genes（TNF-α,FADD,Caspase 8,Caspase 3）and Hsp70 genes were generally upregulated.In addition,after exposing sea slugs that were infected with V.parahaemolyticus to low frequency noise at 500 Hz and 1000 Hz,all of the above indicators were significantly higher and positively correlated with frequency.The results of a two-way ANOVA on low-frequency noise frequency and exposure time showed that noise frequency and exposure time had an interactive effect on the above indicators.In summary,low frequency noise exposure increases the risk of pathogenic infections in sea slugs and exacerbates the negative effects on the antioxidant capacity and immune metabolism of the organism.Expt.5 Low-frequency noise aggravates the enrichment and toxicity of cadmium on sea slugCadmium has become an important environmental pollutant affecting the behavior,physiology and immunity of marine animals.In the context of increasing cadmium pollution,an increasing amount of noise pollution has also been generated in the ocean.However,the physiological responses to marine organisms when anthropogenic noise and cadmium pollution are present simultaneously are unknown.To examine the immune defense response of sea slug exposed to both low-frequency noise and cadmium,we subjected sea slug in a cadmium environment to low frequency noise at500 Hz and 1000 Hz for 120 h exposure experiments.The results showed that after exposure to cadmium alone,the concentration of Cd2+in the hepatopancreas of sea slug increased,the apoptosis rate and ROS production of hemocytes increased significantly,hemocyte proliferation was accelerated,TAC activity in the hepatopancreas decreased,GSH,LPO and ALT enzyme activities increased significantly,and p53 signaling pathway-related genes（p53,Cyt C,caspase 3 and caspase 9）.The expression of p53signaling pathway-related genes（p53,Cyt C,caspase 3 and caspase 9）,MT and Hsp70genes were also generally upregulated.In addition,after exposing sea slug to both Cd and low-frequency noise at 500 Hz and 1000 Hz,all of the above indicators were significantly increased and positively correlated with frequency.The results of two-way ANOVA on low-frequency noise frequency and exposure time showed that noise frequency and exposure time had an interactive effect on the above indices.In conclusion,low-frequency noise exposure increased the enrichment of Cd2+by sea slug,which exacerbated the negative effects on the antioxidant capacity and immune metabolism of the organism.Expt.6 Cloning,characterization and correlation of transcription factor HSF1 with Hsp70 expression in sea slugHeat shock transcription factor 1（HSF1）is thought to activate Hsp70 transcription for cell protection under environmental disturbances as well as proteotoxic stress.HSF1has been extensively studied in marine vertebrates,however,very few studies have been conducted on mollusks,especially under the stress of environmental factors,and the regulatory relationship between HSF1 and Hsp70 is still unclear.Therefore,in this study,we explored the functional role played by HSF1 in Hsp70 expression during sea slug exposure to low frequency noise.We obtained the first 1572 bp sea slug Or HSF1 gene sequence,of which the coding region is 819 bp long and encodes 273 amino acids.The phylogenetic tree results showed that sea slug has high homology with Aplysia californica,Biomphalaria glabrata and Bulinus truncatus.After RNA interference of the Or HSF1 gene,the gene silencing effect was found to be systematic in the central nervous system,hepatopancreas,intestine,gonads,muscles,gastropods and hemolymph of sea slug.The m RNA expression of Or HSF1 gene showed a decreasing and then increasing trend within 5 d of the RNA interference experiment,indicating that the gene silencing effect was persistent and that the expression pattern of Or HSF1 gene and Hsp70 were uniform.After exposing the sea slug to low frequency noise at 500 Hz for 6h after RNA interference,the m RNA expression and protein expression of Hsp70 in the CNS were significantly decreased relative to the noise group,but higher than the control group,indicating that HSF1 has a regulatory effect on the expression of sea slug Hsp70,and it tentatively revealed that the HSF1-Hsp70 regulatory axis involved in the stress response of sea slug. |
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