Feeding Preferences Of Soil Bacterial-feeding Nematodes And Its Molecular Mechanism

Bacterial-feeding nematodes(BFNs),which are regarded as one of the main soil microfauna predators,play an essential role in soil food webs via linking primary consumers of bacteria to higher trophic levels.Soil bacteria,serving as food for BFNs,could influence the growth,reproduction,lifespan and behavior of BFNs.On the contrary,BFNs could not only affect the soil microbial populations and activity,but also affect the structure of soil microbial communities by consuming bacteria.BFNs have significant and practical importance for agriculture and forestry as well as soil ecosystem by influencing bacteria,such as enhancing the rate of nitrogen mineralization and improving the growth of plants.However,the relationships between BFNs and bacteria were not known very well.Feeding preference is an important feeding behavior in animals.Generally,feeding choices and preferences are thought to be the instinct of all animals,and this natural phenomenon is not only suitable for higher organisms,but also for low protists,such as nematodes and protozoan.BFNs could change the competition and distribution of bacteria in soils by selectively grazing bacteria.The feeding preferences of BFNs could explain the energy flow in soil food web and nutrition transformation.The molecular mechanism of the BFNs feeding preferences plays a key role in explaining how BFNs regulate the microbial populations,activities and the structure of soil microbial communities.This paper contained three main researches:(1)we employed five bacteria(B.amyloliquefaciens JX1,Variovorax sp.JX14,B.megaterium JX15,P.fluorescens Y1,E.coli OP50)and the typical model BFN C.elegans to evaluate the feeding preferences,life traits and feeding related functional genes of C.elegans;(2)we isolated two soil native BFNs(Mesorhabditis sp.NJ and Acrobeloides sp.NJ)from soil,and we used Mesorhabditis sp.NJ and Acrobeloides sp.NJ with the above five bacteria to explore the feeding preferences and life traits of Mesorhabditis sp.NJ and Acrobeloides sp.NJ;we successfully isolated 5 feeding related functional genes from Mesorhabditis sp.NJ and Acrobeloides sp.NJ,respectively,and obtained all the full length cDNAs of these genes by the methods of RT-PCR and RACE,then we used the methods of qRT-PCR and RNAi to detect the functions of these genes in Mesorhabditis sp.NJ and Acrobeloides sp.NJ;(3)we analyzed the transcriptomes of C.elegans feeding on different bacteria using RNA sequencing to explore the genetic mechanisms of BFNs feeding preferences deeply.The main results were as follows:1.The feeding preferences experiment was conducted to evaluate the preferences of C.elegans for the five bacteria,and C.elegans showed pronounced feeding preferences in this experiment:C.elegans preferred the active bacteria P.fluorescens Y1 and E.coli OP50 significantly more than the inactive bacteria B.amyloliquefaciens JX1 and B.megaterium JX15.Moreover,C.elegans feeding on the active and preferred bacteria produced more offspring but had shorter lifespan,while the inactive and less preferred bacteria increased the worms lifespan but decreased the brood size.The bacteria that most increased the lifespan of C.elegans was B.amyloliquefaciens JX1,increasing it up to 80%.2.C.elegans changed the feeding preferences when they continued feeding on the five tested bacteria respectively.When C.elegans fed on P.fluorescens Y1 for a long time,the nematodes would decrease the ability to distinguish the five bacteria and they showed the same preferences for the tested bacteria.When C.elegans fed on their preferred bacteria P.fluorescens Y1,they felt comfortable and became satiety quiescence,so their feeding related functional genes(egl-4,tax-2,tax-4,gcy-28,odr-3,tyra-3)were down regulated and the expressions of these genes were significantly lower than nematodes fed on E.coli OP50.3.Two species of soil native BFNs were isolated from soil,and they were identified as Mesorhabditis and Acrobeloides based on morphology and 18S rRNA sequencing,named as Mesorhabditis sp.NJ and Acrobeloides sp.NJ,respectively.The feeding preferences experiment was conducted to research the feeding preferences of Mesorhabditis sp.NJ and Acrobeloides sp.NJ for the five tested bacteria,and these two soil native BFNs exhibited marked feeding preferences:they both significantly preferred the active bacteria P.fluorescens Y1 and E.coli OP50,and the Mesorhabditis sp.NJ on the P.fluorescens Y1 and E.coli OP50 lawns was 80%of the total Mesorhabditis sp.NJ on the five bacteial lawns,meanwhile,the Acrobeloides sp.NJ on the P.fluorescens Y1 and E.coli OP50 lawns was less than 60%of the total Acrobeloides sp.NJ on the five bacteial lawns.Furthermore,these two soil native BFNs feeding on the active and preferred bacteria produced more offspring but had shorter lifespan,while the inactive and less preferred bacteria increased the worms lifespan but decreased the brood size.4.Based on the full length cDNAs of feeding related functional genes in C.elegans and other nematodes or worms,we successfully cloned 5 genes from Mesorhabditis sp.NJ and Acrobeloides sp.NJ,respectively,and we isolated the full length cDNAs of these genes.The results of multiple sequences alignment demonstrated that the gene we cloned from Mesorhabditis sp.NJ and Acrobeloides sp.NJ were homologous with the corresponding feeding related functional genes in C.elegans and other nematodes or worms.It was convenient to research the molecular mechanism of soil native BFNs feeding preferences by detecting the functions of these cloned genes from soil native BFNs.5.Mesorhabditis sp.NJ and Acrobeloides sp.NJ changed their feeding preferences when they continued feeding on the five tested bacteria respectively.The soil bacteria promoted the preferences of Mesorhabditis sp.NJ for P.fluorescens Y1 at 8 h compared to Mesorhabditis sp.NJ feeding on E.coli OP50.Since most of the feeding related functional genes were up-regulated after Mesorhabditis sp.NJ fed on soil bacteria.While B.megaterium JX15 and P.fluorescens Y1 decreased the ability of Acrobeloides sp.NJ to distinguish the food,Acrobeloides sp.NJ feeding on P.fluorescens Y1 or B.megaterium JX15,showed the same preferences for P.fluorescens Y1 and E.coli OP50.Because most of the feeding related functional genes were down-regulated after Acrobeloides sp.NJ fed on P.fluorescens Y1 or B.megaterium JX15.6.The plasmid L4440,which contained the feeding related functional gene(Asp-egl-4,Asp-tax-4,Asp-gcy-28,Asp-odr-3)from Acrobeloides sp.NJ,was transformed into E.coli HT115,respectively.Acrobeloides sp.NJ fed on these E.coli HT115,which were cultured with IPTG to produce and deliver dsRNA,respectively.Therefore,we got five types of gene silencing Acrobeloides sp.NJ,and Acrobeloides sp.NJ feeding on E.coli HT115 with empty L4440 were as control(RNAi-CK).Acrobeloides sp.NJ changed their feeding preferences when their feeding related functional gene was silencing.The control Acrobeloides sp.NJ(RNAi-CK)showed no different preferences between P.fluorescens Y1 and E.coli OP50,but Acrobeloides sp.NJ(gene silencing)all significantly preferred P.fluorescens Y1 than E.coli OP50.The qRT-PCR results demonstrated that when one of the feeding related functional genes was silencing in Acrobeloides sp.NJ,most of the rest four genes were up regulated to improve the distinguish food ability of Acrobeloides sp.NJ.When the gene egl-4 or gcy-28 of Acrobeloides sp.NJ were silencing,Acrobeloides sp.NJ decreased preferences differences between E.coli OP50 and inactive bacteria.7.We analyzed the transcriptomes of C.elegans feeding on B.megaterium JX15,P.fluorescens Y1 and E.coli OP50 respectively,using RNA sequencing,and the transcriptome of C.elegans feeding on E.coli OP50 was as control.C.elegans feeding on B.megaterium JX15,116 genes were changed significantly compared to C.elegans feeding on E.coli OP50,and 70 genes were down regulated,46 genes were up regulated.However,C.elegans feeding on P.fluorescens Y1,1404 genes were changed significantly compared to C.elegans feeding on E.coli OP50,and 409 genes were down regulated,995 genes were up regulated.The rusults of Gene ontology(GO)and KEGG pathway analysis demonstrated that:when C.elegans fed on B.megaterium JX15,the growth,reproduction,aging,response to stress and some other GO terms were changed,and the nutrition digestion and absorption,MAPK signaling pathway,metabolic pathway were also changed;however,after C.elegans feeding on P.fluorescens Y1,not only these GO terms and KEGG pathways were changed,the chemotaxis,learning or memory,and lots of other GO terms were also changed,moreover the olfactory transduction,taste transduction,TGF-?signaling pathway,insulin signaling pathway,serotonergic synapse and many other KEGG pathways were also changed.These changed genes,GO terms and KEGG pathways provide the opportunities to explore the genetic mechanisms of BFNs feeding preferences deeply.