Analysis Of The Fish And Plankton Diversity In Chongde Lake At Southwest University Using Environmental DNA Technology

Environmental DNA?eDNA?refers to a mixed genetic material in environmental media such as water,soil,sediment,and air.The primary sources are skin,mucus,saliva,urine,feces,blood,sperm,eggs,roots,leaves,fruits,and rotting corpses of large organisms.In the case of microbes,it could be the whole organism.The eDNA technology refers to 1)capturing eDNA from environmental samples,2)extracting total eDNA,3)polymerase chain reaction?PCR?,and 4)sequencing and bioinformatics analysis.In recent years,eDNA metabarcoding technology,which is a novel,rapid,cost-effective,and noninvasive method,has been applied for monitoring aquatic species.Different research teams have developed different operating manuals.This study aimed to find the specific process of eDNA metabarcoding technology that would be suitable for our laboratory's current experimental conditions.In this study,we first tested the effectiveness of eDNA metabarcoding using aquarium trials.Then,eDNA metabarcoding technology was applied to the monitoring of fish and plankton diversity in Chongde Lake at Southwest University.Environmental parameters of the water in Chongde Lake were measured.The main results are as follows.?1?Detection of fish species composition using eDNA technology in aquarium.In this study,three fish species?Gambusia affinis,Hypophthalmichthys nobilis,and Pseudorasbora parva?were chosen as the subjects.We aimed to test the effectiveness of eDNA technology based on the MiFish-U primer using aquarium trials and to explore the correlations between valid sequence counts obtained from high-throughput sequencing and fish biomass.Water samples were collected daily in three replicates from day 1 to 4.After high-throughput sequencing,36 libraries yielded a total of37,022,428 valid sequences,which ranged from 661,708 to 1,882,980.The results showed that all target fishes were detected in each eDNA sample.Fish biomass and valid sequence count data from three tanks and three fish species were combined.Regardless of fish species and biomass changes after four days,the results of iteratively reweighted least squares regression analysis for four days?9 data points for each day?illustrated that there were consistent,significant positive relationships between valid sequence counts and fish biomass.Day 4 had the highest r²?0.95?.?2?Using eDNA technology to monitor fish and plankton diversity in Chongde Lake at Southwest University.Compared with the aquarium environment,the natural environment is more complex,and the water volume is very large.We chose the MiFish-U primer and3NDF/V4?euk?R2 primer to monitor fish and plankton diversity in the field—two small lakes?Lake?A and Lake?B?of Chongde Lake at Southwest University,respectively.We used 30 water samples for monitoring fish and 30 water samples for monitoring plankton in both summer and autumn.For monitoring fish,30 summer samples and 30autumn samples yielded 11,261,482 valid sequences and 11,465,587 valid sequences,respectively.For monitoring plankton,30 summer samples and 30 autumn samples yielded 1,822,757 valid sequences and 1,880,947 valid sequences,respectively.We detected 11 species-level fishes:Ctenopharyngodon idella,Cyprinus carpio,G.affinis,H.molitrix,H.nobilis,Monopterus albus,Mylopharyngodon piceus,P.parva,Rhinogobius cliffordpopei,Rhinogobius giurinus,and Rhodeus ocellatus;two genus-level fishes:Carassius and Oreochromis;and one family-level fish,Cobitidae.The results are roughly consistent with the results of historical records and traditional survey.Carassius,G.affinis,H.nobilis,H.molitrix,and P.parva have a high relative abundance.The plankton detected by eDNA analysis at the phylum level included members of Cryptophyta,Chlorophyta,Ochrophyta,Dinoflagellata,Arthropoda,Rotifera,Alveolata,Ciliophora,Cercozoa,Cryptomycota,and Chytridiomycota.At the class level,it comprised members of Cryptophyceae,Chlorophyceae,Trebouxiophyceae,Maxillopoda,and Monogononta.Cryptophyceae,Chlorophyceae,Trebouxiophyceae,Maxillopoda,and Monogononta members were highly abundant.Alpha diversity analysis showed that the abundance of fish communities in autumn was significantly higher than that in summer?P<0.001?;the diversity of plankton communities in summer was significantly higher than that in autumn?P<0.01?.Beta diversity analysis indicated that the plankton community assemblage structure differed significantly between summer and autumn.Beta diversity analysis in both summer and autumn showed that the plankton community assemblage structure differed significantly between Lake?A and Lake?B?P=0.001?.?3?Assessment of the water quality of Chongde Lake.Environmental parameters of the water were measured,including water temperature?WT?,pH,dissolved oxygen?DO?,total nitrogen?TN?,total phosphorus?TP?,chemical oxygen demand?COD?,phosphate?PO₄-P?,nitrate?NO₃-N?,and ammonia?NH₄-N?.According to the Environmental Quality Standard of Surface Water,the water quality rank of Lake?A belongs to?–?and the water quality rank of Lake?B belongs to III–V.According to The Standard Methods In Lake Eutrophication Investigation?second edition?,both Lake?A and Lake?B are eutrophic,and there is a high risk of algal bloom in summer because of the high relative abundance of Cryptophyta and Chlorophyta members.Shannon diversity index of plankton showed that both Lake?A and Lake?B possessed clean water in summer and lightly polluted water in autumn.The Gini-Simpson index of plankton showed that Lake?A and Lake?B possessed clean water in both summer and autumn.