Research On Diversity And Breeding Methods Of Tardigrades In Alpine Regions

Tardigrada,also known as water bears,are the one of the most resilient animals on earth.At present,there are more than 1000 clear species,almost all of which are distributed worldwide.Tardigrada are small in size,0.01 to 1.2 mm,which can be seen under a microscope.They mainly live in the sediment of fresh water and the water film of moss plants,and some species live in the intertidal zone of seawater.The biggest characteristic of tardigrada animals is their cryptic phenomenon,that is,when the living conditions are not suitable,tardigrada animals will show cryptic phenomena and show amazing resistance to stress.There are four types of cryptogenesis:dehydration(low humidity cryptogenesis),ultra-low temperature(hypogenetic cryptogenesis),oxygen deficiency(hypoxic cryptogenesis),and high salt concentration(variable osmosis).In addition to cryptogenesis,tardigrada animals can also survive in the adverse environment in the form of cysts.Foreign scholars have done a lot of researches on tardigrada animals,but in China,there are only a lilltle research on tardigrada animals,such as regional investigation,taxonomy,basic biology and life history,and other aspects of tardigrada animals(such as breeding and stress resistance mechanism,etc.)have not been reported.Generally speaking,the research on tardigrada animals is still few.The tardigrade animals in Liangshui National Nature Reserve,Yichun Fenglin National Nature Reserve,Normin Mountain,Inner Mongolia,Okridui Mountain and Changbai Mountain were selected as the research sites.Phase contrast microscope,differential interference microscope and scanning electron microscope were used for morphological observation,and combined with molecular biological detection method,i.e.,PCR amplification sequence alignment analysis of 18S rRNA,COI and 28S rRNA of individual tardigrade animals were used for species identification of tardigrade animals.Screen tardigrade animals suitable for survival and reproduction in laboratory conditions.The species diversity,genetic diversity and phylogenetic relationship of tardigrades in each sampling area were analyzed.This study is conducive to an in-depth understanding of the distribution structure and changes of tardigrades in northeast China,making the research methods of species classification of tardigrades more systematic and in line with international standards,and laying a foundation for further studies on the stress resistance of tardigrades,molecular systematic geography,and model species.The conclusions of this study are as follows:26 species,13 genera,6 families,3 orders,2 classes of tardigrades were found in these areas,among which two species were widely distributed,namely Adropion scoticum ommatophorum and Mesobiotus harmsworthi.Endemic to Fenglin reserve is Diphascon sp.Endemic to Norminda mountains is Pilatobius nuominensis sp.nov.Pilatobious recamieri is endemic to Oakley mound.Endemic to Changbai mountain is Murrayon dianeae.The Simpson index,Pielou index and Shannon-Wiener index of four areas of Liangshui reserve,Fenglin reserve,Normin mountain and Changbai mountain are similar,and the tardigrades in these four areas are at a medium level of similarity.Three new species,Diphascon wuyingensis,Pilatobius nuominensis sp.nov.and Macrobiotus fenglinensis sp.nov.were identified by the molecular biological method and morphological observation.New Chinese records include Murrayon dianeae,Macrobiotus paulinae,Hypsibius cf.Scabropygus,Pilatobious recamieri,Pilatobious iltisi.The constructed 18S rRNA and COI phylogenetic trees showed the phylogenetic relationship of local tardigrades.Haplotype diversity of the two sequences was higher and nucleotide diversity was lower.Through the cultivation of three kinds of tardigrades,Diphascon sp.,a tardigrades animal suitable for laboratory culture was selected,and its cultivation conditions were optimized.