| As a soil macro fauna,earthworms are known as "soil ecosystem engineers".Their activities in the soil,such as movement,feeding,and excretion,can significantly affect the physical,chemical,and biological properties of the soil.They strongly affect soil inhabitants-micro-fauna.Soil nematodes are metazoan(also micro-or meso-fauna)with the largest number and taxa in the soil ecosystem.Among them,bacterivores are the most abundant,accounting for 60 to 90%of the total number of nematodes.The bacterivores regulate the quantity,activity and community structure of soil microorganisms by feeding on bacteria.Thereby it affect the ecological functions of soil such as carbon,nitrogen and phosphorus transformation and circulation.Many studies have confirmed that earthworms can significantly affect the number and community structure of nematodes in soil.The mechanism of action is mainly three kinds of viewpoints:1.The nematode habitat is changed by earthworm’s phagocytosis and defecation,which affects the number and community structure of nematodes;2.The earthworm feeds on nematodes is capable of reducing the number of nematodes in the soil and changing their community structure;3.The competition between earthworm and nematodes for food resources leads to changes in the number and the community structure of nematodes.We believe that earthworm can also directly affect the quantity,activity and community structure of soil nematodes through secretory action,thereby regulating the structure and function of microbial communities.The main research contents of this dissertation include the following aspects:1.The purpose of preventing earthworm from swallowing soil is achieved by closing the earthworm prostomium(hereinafter referred to sutural earthworm),and comparing with normal earthworms and soil without earthworms to study the secretion of earthworms impact on soil nematode communities.Using the go after profits and advoid disadvantages of C.elegans,Mesorhabditis sp.NJ,Mesorhabditis sp.NJ,and Protorhabditis sp.,the phobotaxis nature of the earthworm mucus indirectly inferred whether the earthworm mucus were "advantaged","neutral" or "harmful" to bacterivores.2.The C.elegans,Mesorhabditis sp.NJ,Rhabdits sp.,and Protorhabditis sp.were exposed in the earthworm mucus,and their mortality,reproductive rate,motility,feeding inhibition rate,and feeding preference were measured to examin the physiological and ecological effects of earthworm mucus on bacterivores.3.By analyzing the secretions of earthworm mucus by proteinase K,ethyl acetate extraction and other treatments,determined the types of active substances that affect the feeding habit of bacterivore nematodes in earthworm mucus,and then used ultrafiltration and column chromatography to purify the active substances.The structure was finally identified by UPLC-MS and synthesized artificially.4.Determination of the feeding preferences of C.elegans after exposure to hexacyclic peptides(synthetic the active substance in earthworm mucus altering the feeding habits of bacteria-feeding nematodes)to verify that hexacyclic peptides is active substance which change the bacterivores feeding preference;explores the effect of hexacycline on the reproduction and longevity of C.elegans;uses RNA-seq technology to perform transcriptome sequencing of C.elegans exposed to cyclic peptides,and attempts to explore the mechanism of hexacyclic peptides effect C.elegans in combination with other information.5.Cyclic peptides were added to the soil and the effect of cyclic peptides on the nematode community in the soil ecosystem and the effect on the microbial community were explored through a soil culture test;the inhibition zone test on the bacteria by the cyclic peptide and the contact killing test on the fungi.To determine whether cyclic peptides have a direct inhibitory effect on bacteria and fungi;use Biolog culture method to explore whether the cyclic peptide has a direct impact on the soil microbial communities.The main findings are as follows:1.The total number of soil nematodes after inoculation with sutural earthworm was significantly higher than that of non-inoculated earthworm and normal earhtworm.The percentage of bacterivores that took up the entire nematode community increased in turn,which was 72.58%,77.41%,and 91.94%,respectively,in comparison with control,normal earhtworm and sutural earthworm.In contrast,the fungal-feeding nematodes was 24.93%,18.95%and 7.05%.Inoculation of earthworms caused a significant reduction in the number of Protorhabditis sp.,which was 37.10%.However,the inoculation of sutural earthworm did not change the number of Protorhabditis sp.;the Mesorhabditis sp.were found only in the control,and were treated in both the larvae and the seal locusts.It was not found;inoculation of normal earthworm reduced the number of Cephalobus sp.,whereas inoculation on sutural earthworm increased the number of Cephalobus sp.;the number of Rhabditis sp.was increased in the control,inoculated normal earthworm and inoculated sutural earthworm.2.Explore the reluctance of C.elegans,Mesorhabditis sp.NJ,Protorhabditis sp.,and Rhabditis sp.to extract crude extracts of earthworm mucus.The results showed that:C.elegans will escape from the crude extracts of earthworm mucus;Mesorhabditis sp.NJ and Protorhabditis sp.also show a tendency to escape crude extracts of earthworm;Rhabditis sp.to produce crude extracts of earthworm.3.To explore the effects of earthworm excrement on the physiological indices of C.elegans,Mesorhabditis sp.NJ,Protorhabditis sp.,and Rhabditis sp.The results showed that the crude extracts of earthworm mucus reduced the number of offspring of C.elegans by 63.41%;the crude extracts of earthworm mucus increased the mortality rate of C.elegans and-Mesorhabditis sp.NJ(increased by 3.30 and 3.26 times compared with the control,respectively),but does not change the mortality of the Protorhabditis sp.and Rhabditis sp.;the crude extracts of earthworm mucus suppresses the motility of C.elegans,but does not inhibit the motility of Mesorhabditis sp.NJ;crude extracts of earthworm mucus inhibits C.elegans,Protorhabditis sp.,and Rhabditis sp.feed.The highest feeding inhibition rates were:89.91%,12.42%,and 16.72%,respectively.The crude extracts of earthworm mucus alters feeding preferences of C.elegans,Mesorhabditis sp.NJ,Protorhabditis sp.,and Rhabditis sp.:increase C.elegans feeding on B.megaterium JX15(JX15),reduce feeding on P.fluorescens Y1(Pseudomonas fluorescens,abbreviated as Y1);reduce Mesorhabditis sp.NJ feeding on Y1 and E.coli OP50;reduce Protorhabditis sp.feeding on E.coli OP50;reduce Rhabditis sp.feeding on Y1 and increases its feeding on JX15.4.In order to determine the type of active substance in the earthworm mucus that changes the feed preference of nematodes,ethyl acetate extraction,proteinase K hydrolysis,proteinase K hydrolysis and ultrafiltration were used to treat blister mucus.The results showed that the ethyl acetate extract could not change the feeding preferences of bacteria-feeding nematodes,proteinase K hydrolysate and proteinase K hydrolysis and ultrafiltration products could change the feed preference of bacteriafeeding nematode,and the initial judgment was that the active substance was small peptides.Then the earthworm mucus were sequentially separated by ultrafiltration,ion exchange chromatography,gel filtration and RP-HPLC.The purified product was analyzed by UPLC-MS.A series of novel cyclic peptides that are homologs or isomers have been found:Cyclo(L(I)-L(I)-L(I)-L(I)-L(I));Cyclo(L(I)-L(I)-L(I)L(I)-L(I)-L(I);Cyclo(L(I)-L(I)-L(I)-L(I))-L(I)-L(I)-L(I));Cyclo(L(I)-L(I)-L(I)L(I)-L(I)-L(I)--L(I)-L(I)).The company also synthesized its representative hexacyclic peptide Cyclo(L-L-L-I-I-I).5.Hexapeptide reduced the reproduction rate of C.elegans(up to 43.80%)and affected its feeding preference(the FPI of feed preference index had the highest reduction of 28.83%),but had no effect on its life span.It was also found that C.elegans,which interacts with cyclic peptides,like to feed on JX1(which can increase the life span of C.elegans,but does not inhibit its reproduction).Hexapeptide also changed the feeding preference of the Protorhabditis sp.(falling 29.21%in FPI).The results of C.elegans RNA-seq after cyclic peptide showed that the number of inDel of nematodes increased significantly;the KEGG pathways such as growth,development,reproduction and DNA repair of nematodes significantly changed,but the olfactory and learning signaling pathways related to feeding did not changed..These phenomena indicate that the DNA of C.elegans is damaged.In order to combat this damage,C.elegans actively reduces its energy for reproduction and transfers this energy to growth.However,because DNA damage repair takes a lot of energy,so the nematode did not live longer because of its reduced reproduction.Therefore,C.elegans favors this survival strategy by preferring foods(JX1)that can extend their lifespan without excessively reducing their reproduction.6.It was found through inhibition zone and contact test that hexacycline had no direct inhibitory effect on bacteria and fungi;Biolog test showed that there was no difference in AWCD values between the two concentrations of cyclic peptides and the control,and hexacycline did not change the microorganisms community structure.7.After addition the hexacyclic peptide to the soil,the number and community structure of bacterial-feeding nematode did not change(one of the dominant species is the Protorhabditis sp.).The results of high-throughput sequencing showed that the abundance of 11 genera in the soil bacteria significantly changed after the addition of hexacyclic peptide for 5 days,and the bacterial community structure significantly changed after 21 days(PCoA analysis results),among which the abundance of Nitrospira spp.With a decrease of 12.63%,the concentration of soil nitrate decreased by 12.54%.After the addition of hexacyclic peptide,the community structure of the fungi changed on both the 5th and 21st days(PCoA analysis results).The addition of hexacyclic peptide had no significant effect on soil basal respiration,soil ammonium,available potassium,available phosphorus and pH.Changes in the above indicators show that:Hexacyclic peptides can regulate the soil microbial structure by affecting feeding preferences of bacteria-feeding nematodes,and thus lead to changes in soil environment(changes in nitrate nitrogen). |
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