Study On The Immune Characteristics Of Triplophysa Rosa And The Effect Of Starvation

Caves are peculiar environments.An important hallmark of cave environments is an overall decrease in biodiversity.Due to the lack of sunlight,no photosynthesis-driven primary producers exist in caves.Food scarcity is a major challenge for cave residents.Consequently,cave dwellers rely entirely on limited and unstable external energy sources.Therefore,nutri-tional factors may represent important selective pressures in the adaptive evolution of cave-dwelling animals.In addition,parasite diversity is relatively low,and bacterial diversity is high but relatively fixed in composition.As one of the most successful groups of vertebrates surviving in caves,cavefish are limited by the darkness of the cave environment and usually face severe survival challenges.During the long process of evolution,the morphology,behav-ior,and physiology of cavefish have undergone alteration,such as visual degradation,loss of pigment,decreased metabolic rate,and loss of circadian rhythm to conserve energy,improved metabolic efficiency,and reduced weight loss during nutritional deficiency to resist starvation,all of which have improved the adaptability of cavefish to such extreme environments.Resistance to pathogen invasion is also important for survival during periods of energy deprivation.Limited energy or resources must be allocated among multiple physiological functions,resulting in trade-offs between immune and life-history components,such as growth and reproduction,where increased investment in one process reduces investment in another.The vertebrate immune system is comprised of two main systems:innate and ac-quired immunity.The former is necessary for an initial response to pathogens.Because of the short lifespan and high complexity of innate immune cells,the innate immune system is very costly to the host.However,adaptive immunity is defined as long-term protection against pathogens by the production of pathogen-specific antibodies.This is considered less costly for the host because its cells,such as B and T cells,have low complexity and long lifespans.It has been suggested that the vertebrate immune system is capable of adjusting the immune in-vestment strategies.Given the differences in immune costs,how did the immune system of the cavefish T.rosa evolve adaptively in this cave environment in which antigens(e.g.,para-sites and bacteria)are limited and more species-specific?How did T.rosa allocate energy in its investment in immunity under energy-deprived conditions?To address these questions,we conducted histological,genomic,and transcriptomic analyses.1.Structural characteristics of the major immune organs(tissues)in T.rosa.The histological structures and cellular characteristics of the major immune organs(spleen,head kidney,and thymus)and mucosal-associated lymphoid tissues(gills,skin,and intestine)of T.rosa were systematically investigated based on anatomy,serial paraffin sections,and electron microscopy.The main results and conclusions are as follows.(1)The spleen of T.rosa presented as an elongated,thin,small organ located at the back of the intestine.It was composed of a capsule,red pulp,and white pulp.There was no obvious boundary between the red and white pulps.The red pulp,which occupied most of the organs,was filled with erythrocytes.The staining of the white pulp was relatively intense.These cells were distributed as dense lymphocytes.Ellipsoids and melano-macrophage centres were visi-ble in the spleen.Transmission electron microscopy revealed the presence of granulocytes,monocytes,and an abundance of lymphocytes.(2)The head kidney of T.rosa was small,slender,and located at the forefront of the kid-ney.It consisted of lymphoid tissue and blood sinuses supported by the reticuloendothelial system.The cells in the head kidney were dispersed and there were numerous lymphocytes.The distribution of lymphocyte aggregates was determined using TEM.Eosinophils and mac-rophages were also observed in the head kidney.(3)The thymus of T.rosa was located in the gill chamber,at the superior edge.No clear distinction between the cortical and medullary areas was observed in the thymus.The cellular components were demarcated into outer and inner zones,with dense thymocytes in the outer zone and sparse thymocytes in the inner zone.Type II mucous cells lined the epithelial sur-face.Hassall’s corpuscles,lymphocytes,thrombocytes,and macrophages were also observed.The blood-thymus barrier,which is a biological barrier,could be seen in the thymus.Age-related thymic degeneration was not observed in sexually mature T.rosa.However,the thy-mus of sexually mature individuals of the surface fish T.bleekeri was significantly degraded and filled with adipose.The undegraded thymus of T.rosa may be an adaptation to the cave environment.(4)Mucous cells were abundant in the gills,skin,and intestinal epithelium of T.rosa.Many immune-related cells such as lymphocytes,granulocytes,and macrophages were dis-tributed in these tissues,providing the first line of defense against pathogen invasion.Histological studies showed that T.rosa had intact immune organs(tissues).It is possible that T.rosa developed an efficient adaptive immune system owing to the non-degradation of its thymus,which may be a sign of adaptation to the cave environment.A well-developed adaptive immunity is advantageous for cave fish to survive in the extreme cave environments with limited and fixed antigens.2.Comparative genomics analysis of important immune genes in T.rosa.The TLR gene family,which is important for innate immunity,was identified based on the T.rosa ge-nome.To understand how the innate immune mechanism of T.rosa acts against pathogen in-fection,its expression patterns in response to Aeromonas hydrophila infection were investi-gated.Furthermore,a comparative genomic analysis of the cavefish T.rosa and surface fishes was carried out to explore the adaptive evolutionary characteristics of important immune genes from a broader perspective.The main results and conclusions are as follows.(1)In total,16 TLR genes were identified in the T.rosa genome.A comprehensive analy-sis of the TLR gene family in T.rosa was performed in the present study based on phylogenet-ic relationships,structural characteristics,chromosomal locations,and expression profiles.Phylogenetic analysis and domain architecture prediction of TLRs have revealed the evolu-tionary conservation of these genes.The expression profiles of these genes were quantified,showing that they were expressed ubiquitously in the tested tissues,although in distinct pat-terns.Similar to other fishes,most TLRs in T.rosa respond significantly to A.hydrophila(concentration:1×10~4 CFU/m L)infections in mucosal-associated lymphoid tissues.(2)Comparative genomic analysis showed that the expansion gene families of T.rosa were mostly related to T cells,such as activation of the membrane attack complex,natural killer cell cytokine production,gamma-delta T-cell differentiation and activation,and alpha-beta T-cell differentiation and activation.The contraction gene families,on the other hand,were mostly related to innate immunity,such as mucosal immunity and interleukin production,suggesting that T.rosa possesses a well-developed adaptive immune system owing to the ex-pansion of its genes related to T-cell activation and differentiation.The results of genomic analysis revealed that T.rosa has owned a well-developed adap-tive immune system owing to the expansion of its genes related to T-cell activation and dif-ferentiation,which is consistent with the histological results.It was speculated that the expan-sion of gene families related to T-cell activation and differentiation may be related to the fact that the thymus of T.rosa did not undergo age-related degeneration.3.Effects of starvation on T.rosa.A rigorous starvation experiment(starvation for 30d,60 d and 90 d)was conducted to investigate the effects of starvation on the growth,me-tabolism,and immunity of T.rosa by performing histological and transcriptomic analyses of the liver and spleen.The main results and conclusions are as follows.(1)The growth of T.rosa was significantly inhibited after starvation.Increasing energy storage by ingestion when food is available and a decrease in the speed of weight loss during starvation improved the adaptability of T.rosa to nutrient-deficient environments.(2)Energy allocation in the liver of T.rosa was altered after starvation.By inhibiting the expression of genes related to fatty acid biosynthesis,cholesterol metabolism,protein diges-tion and absorption,and the cell cycle,the relative energy-consuming cell activities and met-abolic levels were reduced in response to starvation.Increased autophagy activity in the liver may also be one of the strategies used by T.rosa to resist starvation.(3)Energy allocation in the spleen of T.rosa was altered after starvation.Energy-consuming activities in the spleen were reduced to conserve energy by inhibiting protein translation,DNA replication,and gene expression related to organ growth in the spleen.Many differentially expressed genes in antigen processing and presentation,T-cell receptor signal-ing pathway,and FcεRI signaling pathway were upregulated in the early stages of starvation(<30 days),and their expression was downregulated during prolonged starvation(>30 days).It has been suggested that during the early stages of starvation,T.rosa may allocate some of its conserved energy to immunity by upregulating its immune levels,particularly the already well-developed adaptive immunity,to defend against pathogen invasion in caves.However,in case of prolonged starvation,the overall energy supply is insufficient,leading to a decrease in the overall level of immunity of T.rosa.In conclusion,we investigated the adaptive evolutionary characteristics of the immune system of T.rosa and the effects of starvation on T.rosa.It was found that the thymus of 8.5-year-old T.rosa was not degraded,and the genes related to T-cell activation and differentia-tion were expanded,suggesting that T.rosa may possess well-developed adaptive immunity.In addition,T.rosa coped with food deprivation by altering energy distribution,reducing en-ergy-consuming cellular activities and metabolic levels,and enhancing autophagy.Simulta-neously,energy was partially allocated to immunity during short periods of starvation;how-ever,prolonged starvation led to decreased levels of immunity.This study fills a gap in the histological analysis of immune organs(tissues)of cavefish,resulting in new insights into the understanding of how cavefish adapt to the extreme environment of caves and providing new ideas for further research on immunity in cavefish.