Toxicological Study Of Cadmium Stress On Adult Onchidium Reevesii

Onchidium reevesii is a typical benthic shellfish belonging to the Mollusca.O.reevesii is a bare shell-free,hermaphroditic amphibious shellfish,often distributed in various coastal areas of China.Its survival temperature is wide,the distribution area is broad,and the wild resources are abundant.It is a kind of shellfish with important nutritional value and medicinal value.However,due to the widespread pollution of heavy metals in various sea areas in China in recent years,the destruction of aquatic ecosystems has intensified,which will inevitably affect the living environment of O.reevesii.Heavy metal stress,especially the time variation of non-essential metal stress,significantly affects the stress activity of the shellfish,the immune defense system,and the functional participation and expression of related genes.Therefore,exploring the response of shellfish stress activity to cadmium stress and its mechanism are of great significance for shellfish to prevent pollutants.In this paper,O.reevesii adult body was used as the research object,and the transcriptome changes of O.reevesii in response to cadmium stress time fluctuation were analyzed,and the relative expression of shellfish stress related genes was tested.The sequence,structure and function of O.reevesii metallothionein under cadmium stress and their relative expression changes in various tissues were studied.The effects of cadmium stress on key physiological indexes of shellfish were studied,and the trend of enzyme activity in hepatopancreas and the change trend of transmitter content in ganglion were studied.At the same time,the changes of cellular immune indexes of O.reevesii at different time of cadmium stress were determined.The aim is to provide a basis for O.reevesii to become a potential indicator for monitoring heavy metal pollution in coastal areas,and to provide a basis for the above-mentioned indicators as sensitive markers for early warning of environmental pollution,and to provide a basis for comprehensive evaluation of the potential impacts of pollutants on organisms.It provides a theoretical reference for the ecological toxicology research of shellfish.1.O.reevesii transcriptome changes in response to cadmium stressThe transcriptome analysis of O.reevesii ganglion at different times by cadmium treatment resulted in the assembly of 51,007 unigenes,of which 14,774 unigenes were annotated by NR,SWISSPROT,KOG,GO,KEGG databases,of which 4157(8.15 %)unigenes were in the intersection of the five major databases.Through GO and KOG functional classification analysis,3381 unigenes participated in the stimulation response process of O.reevesii;1425 unigenes participated in the signal transduction mechanism of O.reevesii;710 unigenes participated in the synaptic and synaptic part of O.reevesii;648 unigenes participate in the immune system process of O.reevesii.It indicated that cadmium stress at different times can activate the signaling pathway of O.reevesii and initiate stress response or immune process.Combined with Short Time-series Expression Miner cluster analysis,six groups of cadmium stress were compared between the experimental group and the control group.The responding transcripts were found to be clustered into sixteen statistically significant expression profiles,and there were 5666 unigenes intersections in the control group and six experimental groups,where metal ion binding,synaptic transmission,and oxidative stress were the main functions of the identified DEG.Expression analysis revealed genes important for invertebrate response under environmental stress.This is the first report using high-throughput sequencing to characterize the response of O.reevesii to cadmium exposure.This study provides O.reevesii with a comprehensive transcript sequence resource and provides a survey of gene expression for heavy metal exposure in non-model marine invertebrates via the Illumina sequencing platform.These results may help to clarify the molecular mechanisms involved in the response of shellfish to marine pollutants.2.Cloning and tissue expression analysis of O.reevesii metallothionein under cadmium stressThe transcriptome data of cadmium stress in response to cadmium stress was obtained,and the sequence of O.reevesii metallothionein(Or-MT)gene fragment was obtained and cloned.The full-length cDNA sequence was obtained by RACE.Or-MT is 1639 bp in length and includes a 195 bp open reading frame(ORF)encoding a total of 65 amino acids with a molecular weight of 6.48 kDa and a theoretical isoelectric point(PI)of 7.81.The Or-MT amino acid sequence is rich in cysteine residues(Cys),glycine(Gly),lysine(Lys),and serine(Ser),which are 27.7 %,16.9 %,13.8 %,and 12.3 %,respectively.Comparing with the domain of related proteins in the family,it was found that Or-MT has a highly conserved and high content Cys residue typical of metallothionein,and contains the Cys-X-Cys structure unique to this type of protein,and does not contain ?-helix peptides.Its spatial structure has two independent domains,each of which is spherical in shape,and is connected to a dumbbell shape by amino acid residues,so as to bond metal and have a strong conformation.From the constructed phylogenetic tree,Or-MT and the mollusk in invertebrates are clustered into one,and clustered with the MT of Lymnaea stagnalis as a branch.RT-qPCR showed that the relative expression of Or-MT in O.reevesii ganglion was lower than that in the control group,and the relative expression of the gene was the lowest when exposed to cadmium for 2 h.The relative expression of Or-MT in the dorsal tissue of O.reevesii showed a gradual increase and decreased.The relative expression of Or-MT was the highest at 48 h after cadmium exposure.The relative expression of Or-MT in each experimental group was higher than that in the control group.In the hepatopancreatic tissue of O.reevesii,the relative expression of Or-MT increased sharply at 12 h after cadmium treatment,and its expression was almost twice as high as that at each time point.At other time points,the relative expression of Or-MT tends to be stable.In the abdomen tissue of O.reevesii,the relative expression of Or-MT in the cadmium-treated experimental group was lower than that in the control group,and the relative expression of Or-MT in each experimental group increased gradually and then decreased.In conclusion,O.reevesii different tissues Or-MT have different responses to cadmium treatment time changes.Increasing MT can combine more metals and improve the adaptability of shellfish to the environment.However,long-term heavy metal stress will enrich the body,and when the accumulation reaches the toxicity threshold,it will cause a certain degree of damage to the body.3.Effects of cadmium stress on biochemical toxicological effects and cellular immunity of O.reevesiiShellfish,as invertebrates,have only a non-specific immune system.The blood cell activity and phagocytic activity of O.reevesii were measured by flow cytometry and microplate reader.The results showed that the phagocytic rate of blood cells in the O.reevesii experimental group was lower than that in the control group under cadmium stress,which was inhibited.This trend was related to the concentration of heavy metal stress.At 12 h after cadmium exposure,the phagocytosis rate of blood cells was significantly lower than that of the control group,and the inhibition rate of phagocytic activity was 31.62 %.The survival rate of O.reevesii blood cells was higher than that of the control group at different time of cadmium treatment.It was hypothesized that Cd accumulated slowly under cadmium stress,which accelerated the uptake and elimination rate of blood cells(or Cd transport).At 12 h and 48 h,cadmium-induced blood cell survival was significantly reduced.The hemolymph,hepatopancreatic tissue and ganglion of O.reevesii were studied,and the changes of cadmium effect on O.reevesii antioxidant enzyme(SOD),metabolic enzyme(AST)activity and neurotransmitter(ACh)content were studied.The results showed that under heavy metal treatment,the activity of SOD in O.reevesii hepatopancreas increased,which may be caused by cadmium stress leading to the accumulation of reactive oxygen species in the body,which enhanced the body's antioxidant enzyme activity,and the body activated the immune defense system.The activity of SOD in the experimental group was higher than that in the control group except for the cadmium treatment for 4 h,and the activity of SOD in the experimental group became stable after 24 h.AST is particularly associated with detoxification in hepatopancreas.Under the treatment of heavy metal cadmium,the activity of AST in O.reevesii hepatopancreas is wave-like.The AST activity was higher than that of the control group at 2 h and 24 h,and the AST activity of the other experimental groups was lower than that of the control group.Ach as an essential small molecule neurotransmitter,Ach content in O.reevesii ganglion under cadmium stress is lower than that in control group.At 2 h of stress,the Ach content in the ganglion decreased sharply,which was 20.57 % compared with the control group.After 24 h of stress,the Ach content in the ganglion of the experimental group decreased continuously.The above indicators are sensitive markers and can be used for early warning.These results have important implications for studying the immune mechanisms of shellfish treated at different times under cadmium stress.This will help to understand the response mechanism of cadmium stress to the physiological and biochemical activities of shellfish,and to assess the degree of damage and the adaptability of the body at different times of cadmium stress.