| Ozobranchus jantseanus Oka,1912,is a freshwater blood sucking leech that lives in the folds of the neck and limbs in Mauremys reevesii.It has only been reported in China and Japan.In 2014,Suzuki et.al first reported that the survival rate of O.jantseanus could reach 100%after direct injection into liquid nitrogen for 24 hours.It is the largest multicellular animal known to survive at ultra-low temperatures without any pretreatment.However,the mechanism of its resistance to ultra-low temperature has not been reported.In this paper,the ultra-low temperature tolerance,long-term survival and desiccation tolerance of O.jantseanus were studied.Following,the morphological characteristics,the distribution of glycogen and lipid,and low temperature adaptive morphological changes were studied by light microscopy and electron microscopy,tissue section staining analysis and other techniques.To further explore the mechanism of its resistance to ultra-low temperature,a comparative study of transcriptome and proteomics was performed between O.jantseanus and Placobdelloides siamensis Oka,1917,a parasite belonging to Rhynchobdellida,too.The purpose of this study is to reveal the mechanism of morphology and molecular biology of ultralow temperature tolerance.The main research contents and results are as follows:1.Detection the capacity of tolerate to ultra-low temperature and desiccation in Ozobranchus jantseanusWe continuously observed the recovery of O.jantseanus in 600 minutes after 96 hours of freezing with liquid nitrogen and-80 ?.The results indicated the early recovery process of liquid nitrogen group after freezing was slower than that of-80 °C group,and 13.33±5.77% of individuals with original gill filaments activity died in about 10 hours.Long-term survival after frozen experiment showed that the longevity of O.jantseanus,which survived 96 hours of liquid nitrogen and freezing at-80 °C,can survive for up to 41 and 45 days,respectively,and the unfrozen group(20?)was 43 days,all groups were fed with anticoagulant blood during the experimental process.There is still the existence of spawning during long-term survival after freezing,which suggesting that ultra-low temperature freezing has little effect on the survival and reproductive capacity of O.jantseanus.Survival rate were 88.33±2.89%,100±0%,95±5.00% after recovery for 24 h from frozen in liquid nitrogen,-80 ? and 4 ?for 30 days,respectively.The survival rate of frozen O.jantseanus at 4? was slightly lower than that at-80?,the reason of this phenomenon maybe that the O.jantseanus are still active.Freeze-thaw test showed that O.jantseanus can undergo freeze-thaw cycles of liquid nitrogen and-80?(freezing for 12 hours,room temperature for 12 hours)twice and thirteen times,respectively.The water content of O.jantseanus was 74.92±2.95%.In the desiccation tolerant experiment,the survival rate of O.jantseanus after rehydration was still 100% when the water loss rate was below 84.78%.2.Morphological observation of Ozobranchus jantseanusThe morphological characteristics of appearance and dissecting digestive system and reproductive system of O.jantseanus were described in detail,the surface of O.jantseanus was observed by scanning electron microscopy,and HE,glycogen and lipid staining of frozen sections were analyzed.The results showed that there are 3 pairs of testes and multiple branches of ovarian sac which is different from other reported close species.O.jantseanus has an adaptive posture when it is in environments of alcohol,freezing,and desiccation,the change,that is,the formation of a hemispherical state by shrinkage.And the apparent vitrification change of the body surface is observed during the desiccation process.Scanning electron microscopy revealed that the posterior sucker had multiple holes like sponge,the ventral surface was completely folded and the mouth contracted significantly,and more mucopolysaccharides were seen on the dorsal side.HE staining revealed a large number of Acid cells with abundant basophilic granules and collagen fibers exist in spaces between the epidermis,visceral and interstitial spaces.Glycogen staining shows that the body surface has a large amount of secreted mucopolysaccharide,and the Acid cells and collagen fibers in the body also contain a large amount of glycogen.Lipid staining showed that only oocytes within ovarian tissue contained lipid droplets that can be stained by Sudan ?.When O.jantseanus encounters cold,the body contracts strongly in a hemispherical state.It may be that the body's free water is discharged through the holes on the body surface and the posterior sucker to produce rapid dehydration,then vitrification of their body to tolerate freeze.In addition,body shrinkage,as well as a large amount of mucopolysaccharide can protect cells from freezing damage.3.The comparative study of transcriptomics of Ozobranchus jantseanus and Placobdelloides siamensisIn this study,one group of Ozobranchus jantseanus(O.J.)with untreated(20 ?)and treated(liquid nitrogen and-80?)samples,and another group of Placobdelloides siamensis(P.S.)with untreated(20 ?),who is parasitic on Orlitia borneensis and lacks the ability of freeze tolerance,were respectively collected to sequence the transcriptome by High-throughout sequencing technology to explore the molecular mechanisms of freeze tolerance of O.jantseanus.Totally,43218976 and 42700616 raw reads were respectively obtained from O.jantseanus and P.siamensis,then 19781 and 13601 unigenes were generated respectively from results de novo assembly.According to the results of analysis of the gene expression in O.J.and P.S.,200 candidate highly expressed genes in each group were selected to further screen out the genes related freeze tolerance.Finally,seven genes,respectively encoded the proteins of BAG domain-containing protein,Stress-associated endoplasmic reticulum protein 2,Aquaporin-4,Superoxide dismutase [Mn/Fe],Microsomal glutathione S-transferase 3,Peroxiredoxin 1 and Heat shock protein HSP 90-beta,were significantly higher expressed in O.J.compared with P.S.,but genes related to the enzymes involved in glucose metabolism and cytoskeleton showed a contrary result.Accompanied by the analysis of KEGG,several genes(included HMCN1,col1a2,col11a2,col2a1,THBS1,etc.)involved in ECM-receptor interaction pathway were showed significantly higher expressed in O.J.,which demonstrated ECM-receptor interaction pathway might be the key factor in tolerating ultra-low.4.The comparative study of proteomics of O.jantseanus and P.siamensisThe groups of O.jantseanus with untreated(20 ?)and treated(liquid nitrogen and-80?)samples,and another group(P.S.)of the untreated P.siamensis(20 ?),were respectively collected to sequence the proteome for further analysis.The four groups were named Pctr1,PN2,P80 and Pctr12,respectively.In total,2955 protein were identified in iTRAQ proteome.Compared with Pctr1(interspecific comparison),51 and 461 proteins were up-regulated in PN2 and P80,295 and 131 proteins were down-regulated;Compared with Pctrl2(intra-specific comparison),1022,1264 and 1058 proteins were respectively up-regulated in PN2,P80 and Pctr1,198,118 and 178 were respectively down-regulated.In addition,with comparison between PN2 and P80,21 proteins were up-regulated and 1106 were down-regulated.And 15 proteins were up-regulated in both inter-specific and intra-specific comparison.The GO protein enrichment and KEGG pathway enrichment analysis of the treatment group up-regulated proteins showed that the proteins were mainly related to extracellular matrix structure and collagen fibers,located in the pathways of ECM-receptor interaction and Focal adhesion in PN2;the majority of proteins in P80 were related to the composition of the membrane,the structural components of the ribosome,the ribosome,the ATP synthesis,the extracellular matrix structural components,the synthesis of carbohydrates,and lipids,and located in the pathways of the ribosome,N-glycine biosynthesis,peroxisomes,ECM-receptor interactions,complement and coagulation cascades.There were 15 up-regulation proteins in both intra-specific and inter-specific comparison,including 9 collagen proteins(3 Collagen ? and each one of Collagen?,?,?,?,?,?),2cysteine-rich secretory proteins,von Willebrand factor,Glycosyl transferase family,Semaphorin-5A and Cystathionine beta-synthase.Combined with transcriptome data,the expression levels of the transcriptome genes corresponding to these 15 up-regulation proteins were analyzed,and it was found that the corresponding proteins of these genes with high expression levels(TPM>300)were 3 Collagen ? and 2 cysteine-rich secretory proteins,as well as Collagen ?,?,? and Semaphorin-5A.Anti-freeze proteins(AFP)were searched among the 15 proteins,6 of them were found to be anti-freeze proteins,including 3 Collagen ?,and each one of Collagen ?,?,?.Those 15 proteins were all up-regulated in intra-species and inter-species comparision,and more significantly in the later.Analysis of pathway showed that10 proteins were located in the ECM-receptor interaction pathway,including 9 collagen proteins and Von willebrand factor.The remaining proteins,containing 2 cysteine-rich secretory proteins,Glycosyl transferase family 90 and Cystathionine beta-synthase,were all related to the s-glycosylation of proteins,which may play a vital role in the glycosylation of collagen.The comparative analysis of iTRAQ proteomics demonstrated that there were several upregulated proteins of glycoproteins and glycosylases.Furthermore,glycoproteins are present in the form of polysaccharides in O.jantseanus,especially,the observation of Acid cells and epidermal components of mucopolysaccharides.Thus,O.jantseanus may vitrify through these glycoproteins and the glyco-binding collagen during ultra-low temperature freeze and desiccation,filling tissues and protecting cell membranes.This study further verified the ability of O.jantseanus to tolerate ultra-low temperature and desiccation,which was reported in the previous study;morphological observation showed that the body was contracted into a hemispherical state when it was freezed,which was conducive to the massive elimination of free water and the formation of vitrification,thus causing anhydrobiosis.HE and glycogen staining showed that the content of glycogen was high in vivo,which was mainly composed of a large number of Acid cells containing mucopolysaccharides distributed in the area between epidermis and interstitial tissues,as well as collagen fibers composed of extracellular matrix.Combined with transcriptome and proteomic data,it was further proved that acid mucopolysaccharide and glycogen binded collagen components with antifreeze protien(AFP)effects,especially type ? collagen,which played a vitrification role in the freezing process,and plays an important role in changing the shape of ice,filling tissues and protecting cell membranes. |
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