Interactive Effects Of Ocean Acidification And Hypoxia On Gonad Function And Larval Development Of Mussels

With the development of the industrialization,a large amount of carbon dioxide（CO₂）is discharged into the air,which causes the increase of seawater dissolved carbon dioxide.The p H value of the seawater decreases as the seawater partial pressure of carbon dioxide（p CO₂）increases,resulting in ocean acidification（OA）.At the same time,algae reproduce vigorously and microbial respiration consumes a large number of oxygen and increases CO₂ in some coastal eutrophic areas,resulting in hypoxia as well as acidification in the local seawater.Marine benthic filter-feeding bivalves are the important part of marine ecosystems,and may experience seasonal fluctuations in the marine environment for a long time so they reproduce through broadcast-type spawning and ovulation.Their reproduction is extremely impacted by the interference of seawater acidification and hypoxia.To investigate the interactive effects of OA and hypoxia on mussels’reproductive capacity,this study takes typical marine economic mussels including the thick-shelled mussel Mytilus coruscus and the blue mussel Mytilus edulis as experimental objects and tests the combined effects of OA and hypoxia on mussel gonad function and larval development.It is revealed for the first time that thick-shelled mussels may modulate the development of gonads by regulating the wnt/β-catenin signaling pathway.It also shows the transgenerational effects of OA and hypoxia on mussel larval development.This study provides a basis for exploring the adaption of mussels under future marine environmental changes.Based on these backgrounds,to study the combined effects of OA and hypoxia on the gonadal development and the related gene expression in thick-shelled mussel,mussels were exposed to three p H levels（8.1,7.7,7.3）and two dissolved oxygen concentration level(6 mgO₂ L^-1,2 mgO₂ L^-1)in a factorial-design experimental conditions.The gonad index and gonad area as gonadal characterization parameters as well as the relative expression levels of Wnt4,Wnt7b andβ-catenin genes in the gonad of the thick-shelled mussels were tested in six treatments.The experiment lasted for 30days,including the first 20 days of exposure period and the subsequent 10 days of recovery period.The male and female gonads of thick-shelled mussels were sampled on days 1,20,and 30 respectively.The results showed that at the first day,there was no significant difference between the gonad index and gonad area of all individuals.However,after 20 days of exposure,gonad index of male showed a significant decline in the p H 7.3 groups,while the female gonad index only significantly decreased in the 6mgO₂ L^-1×p H 7.3 treatment by OA.As for the gonad area,a significant decrease of both male and female individuals was observed in the 6 mgO₂ L^-1×low p H treatments.However,results also showed that ten-day recovery period can only partially restore the gonad area and gonad index of male individuals,while female individuals have no significant recovery.In addition,gene expressions ofβ-catenin and Wnt7b decreased but gene expression of Wnt4 increased under OA and hypoxia.This study showed that OA and hypoxia have adverse and combined effects on the development of thick-shelled mussel gonads.Also,Wnt7b andβ-catenin genes were involved in regulating the mussel gonads development.In order to study the combined effects of ocean acidification and hypoxia on the dynamic changes of gonadal hormone contents in thick-shelled mussels,three types of male and female typical sex hormone levels including estradiol,testosterone and progesterone were measured after individuals were exposed to six treatments with the same conditions described above.The experiment lasted for a total of 30 days,including the first 20 days of exposure and the subsequent 10 days of recovery.The male and female gonads of thick-shelled mussels were sampled on days 1,20,and 30,respectively.The content of the three sex hormones showed an overall downward trend under acidification,while a significant decrease in testosterone concentrations in male and female individuals at p H 8.1 treatment group under hypoxia.In addition,estradiol,testosterone and progesterone concentrations in the male gonads remained low after the ten-day recovery period,while female gonads were relatively less affected by experimental exposure.This experiment showed that thick-shelled mussels may regulate gonadal function by adjusting the concentrations of estradiol,testosterone and progesterone in the gonads under OA and hypoxic stress.In order to figure out whether transgenerational effects can enhance mussel larval tolerance to adverse environmental stress such as OA and hypoxia,and thereby alleviate the effects of OA and hypoxia on the early development of larvae.The impacts of OA and hypoxia on the early development of mussel larvae across generations were tested.The experiment was divided into two groups.The first group of parental mussels was exposed to six different acidification and hypoxia treatments(DO 6 mgO₂ L^-1,2 mgO₂L^-1×p H 8.1,7.7,7.3),and the parental mussels in the second group was exposed to 6mgO₂ L^-1×p H 8.1 conditions（ambient conditions）.Then the parental mussels in two groups were artificially propagated after 30 days of exposure period.The fertilization rate,embryo deformity rate,and D-type larval shell length were measured.Experiments have shown that OA had a negative impact on fertilization rate,embryo deformity rate and larval shell length,while hypoxia increases embryo deformity rate at p H 8.1.Results showed that the offspring of the experimental group were less affected by acidification and hypoxia than the control group.However,compared with the normal seawater conditions treatment,OA and hypoxia still had significant adverse effects on the offspring in experimental group,indicating that OA and hypoxia would have a negative impact on the early development of mussel larvae,and the pre-adaptation of parental mussels could only slightly alleviate this effect.In summary,ocean acidification and hypoxia have negative effects on male and female mussel gonads by regulating the expression of genes related to gonadal development and the concentration of sex hormones in gonads as well as the short-term carry-over effects have been observed.The adverse effects of ocean acidification and hypoxia on the early development of mussel larvae were observed,while the transgenerational effect of parental mussels from acidification and hypoxia exposure could only partially alleviate these negative effects.