Monitoring Of Zooplankton Community Responses To Typical Organophosphorus Chlorpyrifos

Chlorpyrifos is a typical broad-spectrum organophosphorus pesticide commonly used in agricultural pest control.A large number of chlorpyrifos residues in agriculture entered the water ecosystem along with surface runoff,causing damage to aquatic organisms.Zooplankton is an important part of the aquatic ecosystem,and it is one of the important indicator organisms for water ecological health.Studies have shown that zooplankton communities have significant differences in susceptibility to chlorpyrifos.Traditional ecotoxicology mostly based on laboratory single-species toxicity exposure,and more attention is paid to the impact of chlorpyrifos on individual and sub-individual levels of zooplankton,while there is less researches on chlorpyrifos on community level.With the development of high-throughput sequencing technologies,new eco-genomics technologies,represented by DNA metabarcoding,have provided highly efficient means for the identification of aquatic biodiversity,and provided opportunities and challenges for the response identification of toxic chemicals by aquatic organisms.The present research used DNA metabarcoding to analyze the species diversity of various ecological groups in the microcosm,and to analyze the toxic effects of chlorpyrifos on zooplankton at the community level,providing a methodological reference for the study of the ecological effects of toxic chemicals.The different toxicities of chlorpyrifos to five species of native zooplankton Daphnia magna,Daphnia similoides,Daphnia pulex,Simocephalus vetulus,Moina macrocopa were studied.Real-time fluorescent quantitative PCR(qPCR)was used as the quantitative standard to evaluate the accuracy of DNA metabarcoding.A zooplankton quantitative system based on DNA metabarcoding was established,which could further indicate the effect of chlorpyrifos on zooplankton,protozoa,algae,fungi and bacterial communities in the microcosm.At the same time,the differences between the results of DNA metabarcoding and RNA metabarcoding were compared,providing effective data support for metabarcoding in the application to monitor the effect of toxic chemicals on aquatic organisms.The main content and conclusions of this study:(1)There was no significant correlation between the lengths and the toxicity of chlorpyrifos of Daphnia magna,Daphnia similoides,Daphnia pulex,Simocephalus vetulus,Moina macrocopa.The five species had two orders of magnitude difference in toxicity sensitivity.The research for subsequent chlorpyrifos on toxic effect of zooplankton community study provides toxicity sensitivity reference.Moina macrocopa was the most sensitive and Daphnia similoides had the strongest tolerance,which were different from the existing research results.(2)Environmental DNA(eDNA)metabarcoding can be used on semi-quantitative monitoring of zooplankton species.There was no significant difference in DNA metabarcoding quantification by DNA extraction while PCR primers had a significant effect on species detection and quantification.Although 313 bp COI313 primer had high coverage of zooplankton community,the preference among species was large.COI116 primer based on a COI sequence redesign can detect five species simultaneously.Selecting suitable primers can identify different species in the ecological community.The copy number of qPCR were positively correlated with the relative proportion of species(R2 = 0.929,P<0.0001).similar correlation occurred between the species number of DNA metabacoding and the relative proportion of species(R2 = 0.885,P<0.0001).The number of reads of each species was highly consistent with the qPCR quantitative copies(R2 = 0.767,P<0.0001).(3)DNA metabarcoding has greatly improved the monitoring efficiency of chlorpyrifos on the zooplankton-dominated microcosm systems in various biological communities.The effects of chlorpyrifos on the experimentally-added animal Daphnia magna,Daphnia similoides,Simocephalus vetulus,Moina macrocopa was consistent with the laboratory single-species toxicity test.Chlorpyrifos had the strongest toxic effects on Cladocera species at the community level.Copepods and rotifers were more resistant to chlorpyrifos,and zooplankton communities had significantly different sensitivities to chlorpyrifos.The structure of protozoa,algae,fungi,and bacteria has undergone significant changes.DNA metabacoding monitoring has improved the efficiency of zooplankton communities identification,which provides reliable data support for ecological risk assessment of chlorpyrifos on aquatic organisms.(4)DNA metabarcoding and RNA metabarcoding showed that chlorpyrifos has a consistent effect on zooplankton community composition.The effects of chlorpyrifos on Daphnia magna,Daphnia similoides,Simocephalus vetulus,Moina macrocopa by RNA metabacoding was consistent with the laboratory single-species toxicity test,and the susceptibility to chlorpyrifos was in the order of Moina macrocopa,Simocephalus vetulus,Daphnia magna and Daphnia similoides.The detection of chlorpyrifos by RNA metabarcoding showed a significant negative effect on Cladocera.The tolerabilities of cockroach and rotifer were strong,which was consistent with the results of DNA metabarcoding detection.Conventional zooplankton diversity surveys can only use DNA metabacoding monitoring.DNA and RNA metabarcoding union methods should be used in studies that need to consider differences between living and dead organisms.