larimichthys is a genus of ray-finned saltwater fishes commonly known as yellow croakers .

pseudosciaena is a commonly used but invalid synonym for larimichthys .

the genus includes major fishery species in the northwest pacific , principally by china : with a catch of 438 thousand tonnes in 2012 , small yellow croaker larimichthys polyactis is 24th among the 70 " principal " capture species , and also the annual catches of large yellow croaker larimichthys crocea are significant at 70 thousand tonnes .

larimichthys

genome sequencing of the perciform fish larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation .
the complete mitochondrial genome of the large yellow croaker , larimichthys crocea ( perciformes , sciaenidae ) : unusual features of its control region and the phylogenetic position of the sciaenidae .
( of larimichthys croceus ( richardson , 1846 ) ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of larimichthys rathbunae jordan & starks , 1905 ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
citation : ao j , mu y , xiang l - x , fan d , feng m , zhang s , et al . ( 2015 ) genome sequencing of the perciform fish larimichthys crocea provides insights into molecular and genetic mechanisms of stress adaptation . plos genet 11 ( 4 ) : e1005118 . urltoken
to detect variations in the l . crocea genome , we chose nine species ( larimichthys crocea , gasterosteus aculeatus , takifugu rubripes , tetraodon nigroviridis , oryzias latipes , gadus morhua , danio rerio , gallus gallus , and homo sapiens ) . proteins that were greater than 50 amino acids in size were aligned by blast ( - p blastp - e 1e - 7 ) , and treefam [ 70 ] was used to construct gene families for comparison .
amino acid sequences from six representative teleosts ( larimichthys crocea , gasterosteus aculeatus , danio rerio , oryzias latipes , takifugu rubripes , and tetraodon nigroviridis ) were aligned by blast ( - p blastp - e 1e - 5 - m 8 ) , and reciprocal - best - blast - hit methods were used to define orthologous genes in six teleost fish . because alignment errors are an important concern in molecular data analysis , we made alignments of codon sequences , which are nucleotide sequences that code for proteins , using the prank aligner [ 75 ] . positive selection was inferred , based on the branch - site k a / k s test by codeml in the paml 3 . 0 package [ 19 ] .
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marine ; benthopelagic ; oceanodromous ( ref . 51243 ) ; depth range ? - 120 m ( ref . 11230 ) . subtropical ; 41°n - 22°n , 117°e - 141°e ( ref . 54561 )
maturity : l m 18 . 1 range ? - ? cm max length : 40 . 0 cm sl male / unsexed ; ( ref . 559 )
dorsal spines ( total ) : 10 - 11 ; dorsal soft rays ( total ) : 31 - 36 ; anal spines : 2 ; anal soft rays : 9 - 10 ; vertebrae : 28 - 30 . body gold when fresh . soft - rayed portions of dorsal and anal fins mostly covered with scales . posterior part of pectoral fin not black . anal soft rays usually 9 ( ref . 41299 ) .
found in the sublittoral zone above 120 m in sandy mud bottoms ( ref . 11230 ) . used in chinese medicine ( ref . 12166 ) .
trewavas , e . , 1977 . the sciaenid fishes ( croakers or drums ) of the indo - west pacific . trans . zool . soc . lond . 33 : 253 - 541 . ( ref . 5369 )
) : 9 . 4 - 24 . 2 , mean 16 . 7 ( based on 107 cells ) .
phylogenetic diversity index ( ref . 82805 ) : pd 50 = 0 . 6250 [ uniqueness , from 0 . 5 = low to 2 . 0 = high ] .
bayesian length - weight : a = 0 . 00891 ( 0 . 00798 - 0 . 00995 ) , b = 3 . 08 ( 3 . 05 - 3 . 11 ) , in cm total length , based on lwr estimates for this species ( ref . 93245 ) .
trophic level ( ref . 69278 ) : 3 . 6 ±0 . 63 se ; based on food items .
resilience ( ref . 69278 ) : medium , minimum population doubling time 1 . 4 - 4 . 4 years ( tmax = 3 - 23 ; k = 0 . 2 - 0 . 7 ) .
vulnerability ( ref . 59153 ) : low to moderate vulnerability ( 32 of 100 ) .
marine ; brackish ; benthopelagic ; depth range 1 - 50 m ( ref . 9772 ) . tropical
western central pacific : along coasts of northwestern australia and southern papua new guinea .
maturity : l m ? range ? - ? cm max length : 13 . 5 cm sl male / unsexed ; ( ref . 9772 )
dorsal spines ( total ) : 10 ; dorsal soft rays ( total ) : 33 - 36 ; anal spines : 2 ; anal soft rays : 7 . upper rostral pore median only ; mental pores in 3 pairs , the first open at front of chin , separated by symphysis . gill rakers long , slender , about 1 . 5 times longer than gill filaments at angle of arch , 13 to 17 on lower limb of first arch . dorsal fin with ix spines , the second part of fin with i spine and 33 to 36 soft rays . second spine of anal fin short , slender , 23 to 33 % of head length . pectoral fin long , 25 to 27 % of standard length . scales on head and body cycloid ( smooth ) , except the belly and under side of caudal peduncle which are covered with weak ctenoid scales ( slightly rough to touch ) ; small scales present on basal quarter of soft parts of dorsal and anal fins . swim bladder carrot - shaped ; with about 26 pairs of arborescent appendages along its sides ; each appendage with a well - developed dorsal and ventral limbs , the first pair entering head beyond transverse septum .
inhabits shallow coastal waters and estuaries to depths of 50 m ( ref . 9772 ) .
sasaki , k . , 2001 . sciaenidae . croakers ( drums ) . p . 3117 - 3174 . in k . e . carpenter and v . h . niem ( eds . ) fao species identification guide for fishery purposes . the living marine resources of the western central pacific . volume 5 . bony fishes part 3 ( menidae to pomacentridae ) . rome , fao . pp . 2791 - 3380 . ( ref . 9772 )
) : 24 . 6 - 28 . 8 , mean 27 . 9 ( based on 198 cells ) .
bayesian length - weight : a = 0 . 00871 ( 0 . 00425 - 0 . 01785 ) , b = 3 . 08 ( 2 . 91 - 3 . 25 ) , in cm total length , based on lwr estimates for this ( sub ) family - body shape ( ref . 93245 ) .
trophic level ( ref . 69278 ) : 3 . 4 ±0 . 6 se ; based on size and trophs of closest relatives
resilience ( ref . 69278 ) : high , minimum population doubling time less than 15 months ( preliminary k or fecundity . ) .
vulnerability ( ref . 59153 ) : low vulnerability ( 14 of 100 ) .
en - large yellow croaker , fr - tambour à gros yeux , sp - corvina japonesa .
body elongate , compressed . scales cycloid ( smooth ) on head and above pectoral fin , elsewhere ctenoid ( rough to touch ) . lateral line with 51 - 57 scales reaching to tip of caudal fin . mouth large , maxillary extending to backward beyond eye . teeth in double series on both jaws , enlarged in outer series ; posterior margin of preoperculum with dermal serration . gillrakers on first gill arch 8 - 9 + 16 - 18 . dorsal fin with 8 - 10 spines and 30 - 35 soft rays . anal fin with 2 spines and 7 - 9 soft rays ; second anal espine weak , longer than eye . pectoral with 15 - 17 . caudal fin pointed . colour body yellow , darker above and golden sheen below ; ventral scales yellow ( luminous organs ) ; fins all yellow ; lips sometimes red ; lining of body cavity light dusky grey .
northwest pacific ocean ( yellow and east china seas ) . shallow waters , along the chinese coast ( canton , hong kong ) .
benthopelagic . inhabits coastal waters and estuaries . a seasonal migratory species . feeds on crustaceans and fishes . spawning in spring and autum in turbid waters , close to river estuaries .
caught with bottom trawls , longlines and handlines . marketed fresh ; also dried - salted ; swimbladder dried . the total catch reported for this species to fao for 1999 was 66 076 t . the countries with the largest catches were china ( 65 806 t ) and korea , republic of ( 270 t ) .
chan , w . & u . bathia . - 1974sciaenidae . in : w . fischer ; p . j . p . whithead ( eds . ) fao species identification sheets for fishery purposes . estern indian ocean ( fishing area 57 ) and western central pacific ( fishing area 71 ) . volume . i , rome fao , pag . var .
froese , r . and d . pauly . editors . 2003 . fishbase . world wide web electronic publication . urltoken
okamura , o . - 1984 sciaenidae . in : h . masuda ; k . amaoka ; c . araga ; t . uyeno ; t . yoshino ( eds . ) . the fishes of the japanese archipelago . tokai . univ . press . 161 - 163 . species 2000 dynamic ( checklist query ) .
vera , j . - 1992diccionario multilingüe de especies marinas para el mundo hispano . ministerio de agricultura , pesca y alimentación . secretaria general técnica . 1282 pp .
marine ; brackish ; benthopelagic ; oceanodromous ( ref . 51243 ) ; depth range 0 - 120 m ( ref . 43239 ) . temperate ; 38°n - 13°n , 106°e - 141°e
northwest pacific : from central vietnam to south korea and japan , yellow and east china seas .
maturity : l m ? , range 17 - ? cm max length : 80 . 0 cm tl male / unsexed ; ( ref . 4537 )
dorsal spines ( total ) : 9 - 10 ; dorsal soft rays ( total ) : 30 - 35 ; anal spines : 2 ; anal soft rays : 7 - 9 ; vertebrae : 25 - 26 . body gold when fresh . soft - rayed portions of dorsal and anal fins mostly covered with scales . posterior part of pectoral fin not black . anal soft rays usually 8 ( ref . 41299 ) .
inhabit coastal waters and estuaries ( ref . 2113 ) . found in muddy or muddy - sandy bottoms shallower than 120 m depth ( ref . 43239 ) . feed on crustaceans and fishes .
masuda , h . , k . amaoka , c . araga , t . uyeno and t . yoshino , 1984 . the fishes of the japanese archipelago . vol . 1 . tokai university press , tokyo , japan . 437 p . ( text ) . ( ref . 559 )
) : 20 . 8 - 24 . 7 , mean 22 . 8 ( based on 54 cells ) .
bayesian length - weight : a = 0 . 00832 ( 0 . 00484 - 0 . 01430 ) , b = 3 . 06 ( 2 . 92 - 3 . 20 ) , in cm total length , based on lwr estimates for this species & ( sub ) family - body ( ref . 93245 ) .
trophic level ( ref . 69278 ) : 3 . 7 ±0 . 56 se ; based on food items .
resilience ( ref . 69278 ) : medium , minimum population doubling time 1 . 4 - 4 . 4 years ( assuming tm = 2 - 4 ) .
vulnerability ( ref . 59153 ) : moderate vulnerability ( 42 of 100 ) .
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disclaimer : itis taxonomy is based on the latest scientific consensus available , and is provided as a general reference source for interested parties . however , it is not a legal authority for statutory or regulatory purposes . while every effort has been made to provide the most reliable and up - to - date information available , ultimate legal requirements with respect to species are contained in provisions of treaties to which the united states is a party , wildlife statutes , regulations , and any applicable notices that have been published in the federal register . for further information on u . s . legal requirements with respect to protected taxa , please contact the u . s . fish and wildlife service .
the webpage text is licensed under a creative commons attribution - noncommercial 4 . 0 license
froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
liu j . y . [ ruiyu ] ( ed . ) . ( 2008 ) . checklist of marine biota of china seas . china science press . 1267 pp . ( look up in imis ) [ details ] available for editors [ request ]
( of sciaena crocea richardson , 1846 ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of collichthys chinensis steindachner , 1866 ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of collichthys croceus ( richardson , 1846 ) ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of pseudosciaena amblyceps bleeker , 1863 ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of pseudosciaena crocea ( richardson , 1846 ) ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
( of pseudosciaena undovittata jordan & seale , 1905 ) froese , r . & d . pauly ( editors ) . ( 2018 ) . fishbase . world wide web electronic publication . , available online at urltoken [ details ]
ang , p . o . ; wong , c . k . ; lin , t . p . ; ma , w . c . ; hung , s . ( 2005 ) . biological monitoring in sha chau and lung kwu chau marine park . submitted to agriculture , fisheries and conservation department , the hong kong sar government . [ details ]
key laboratory of marine biogenetics and resources , third institute of oceanography , state oceanic administration , fujian collaborative innovation center for exploitation and utilization of marine biological resources , key laboratory of marine genetic resources of fujian province , xiamen , p . r . china
college of life sciences , zhejiang university , key laboratory for cell and gene engineering of zhejiang province , hangzhou , zhejiang , p . r . china
bgi - tech , bgi - shenzhen , shenzhen , guangdong , p . r . china
college of life sciences , shenzhen university , shenzhen , guangdong , p . r . china
l . crocea is a temperate - water migratory fish that belongs to the order perciformes and the family sciaenidae . in china , the annual yield from l . crocea aquaculture exceeds that of any other net - cage - farmed marine fish species . l . crocea also exhibits peculiar behavioral and physiological characteristics and is especially sensitive to various environmental stresses . to understand the molecular and genetic mechanisms underlying the adaptation and response of l . crocea to environmental stress , we sequenced and assembled the genome of l . crocea . further genomic analyses showed the significant expansion of several gene families , such as vision - related crystallins , olfactory receptors , and auditory sense - related genes , and provided a genetic basis for the peculiar physiological characteristics of l . crocea . transcriptome analyses of the hypoxia - exposed l . crocea brain revealed new aspects of neuro - endocrine - immune / metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia . proteomics data demonstrate that skin mucus of the air - exposed l . crocea had a complex composition , suggesting its multiple protective mechanisms involved in antioxidant functions , oxygen transport , immune defence , and osmotic and ionic regulation . these findings provide novel insights into the mechanisms of fish stress adaptation .
copyright : © 2015 ao et al . this is an open access article distributed under the terms of the creative commons attribution license , which permits unrestricted use , distribution , and reproduction in any medium , provided the original author and source are credited
data availability : the whole - genome sequences of l . crocea have been deposited in the dna data bank of japan ( ddbj ) , the european molecular biology laboratory ( embl ) nucleotide sequencing database and genbank under the accession jrpu00000000 . the version described in this paper is the first version jrpu01000000 . all short - read data of wgs and bac have been deposited in the short read archive ( sra ) under accession sra159210 and sra159209 respectively . raw sequencing data of transcriptomes have been deposited in the gene expression omnibus under the accession number gse57608 . the mass spectrometry proteomics data have been deposited to the proteomexchange consortium ( urltoken ) with the dataset identifier pxd001218 .
funding : the work is supported by grants from the nation ‘863’ project ( 2012aa092202 ) , national science fund for distinguished young scholars ( 31125027 ) , national basic research program of china ( 2012cb114402 ) , national natural science foundation of china ( 31372556 ) , and fund of xiamen south ocean research center ( 13gzp002nf08 ) . the funders had no role in study design , data collection and analysis , decision to publish , or preparation of the manuscript .
teleost fish , nearly half of all living vertebrates , display an amazing level of diversity in body forms , behaviors , physiologies , and environments that they occupy . strategies for coping with diverse environmental stresses have evolved in different teleost species . therefore , teleost fish are considered to be good models for investigating the adaptation and response to many natural and anthropogenic environmental stressors [ 1 – 3 ] . recent genome - sequencing projects in several fish have provided insights into the molecular and genetic mechanisms underlying their responses to some environmental stressors [ 4 – 6 ] . however , to better clarify the conserved and differentiated features of the adaptive response to specific stresses and to trace the evolutionary process of environmental adaptation and response in teleost fish , insight from more teleost species with different evolutionary positions , such as perciformes , is required . perciformes are by far the largest and most diverse order of vertebrates , and thus offer a large number of models of adaptation and response to various environmental stresses .
to understand the molecular and genetic mechanisms underlying the responses of l . crocea to environmental stress , we sequenced its whole genome . furthermore , we sequenced the transcriptome of the hypoxia - exposed l . crocea brain and profiled the proteome of its skin mucus under exposure to air . our results revealed the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure .
we applied a bacterial artificial chromosome ( bac ) and whole - genome shotgun ( wgs ) hierarchical assembly strategy for the l . crocea genome to overcome the high levels of genome heterozygosity ( table 1 and s1 – s2 fig ) . the 42 , 528 bacs were sequenced by the hiseq 2000 platform and each bac was assembled by soapdenovo [ 18 ] ( s1 table ) . the total length of all combined bacs was 3 , 006 megabases ( mb ) , which corresponded to approximately 4 . 3 - fold genome coverage ( s2 – s3 tables ) . all bac assemblies were then merged into super - contigs and oriented to super - scaffolds with large mate - paired libraries ( 2–40 kb ) . gap filling was made with reads from short insert - sized libraries ( 170–500 bp ) ( s3 – s4 tables ) . in total , we sequenced 563 - fold coverage bases of the estimated 691 mb genome size . the final assembly was 679 mb , with a contig n50 of 63 . 11 kb and a scaffold n50 of 1 . 03 mb ( table 1 ) . the 672 longest scaffolds ( 11 . 2 % of all scaffolds ) covered more than 90 % of the assembly ( s5 table ) . to assess the completeness of the l . crocea assembly , 52 - fold coverage paired - end high - quality reads were aligned against the assembly ( s3 fig ) . more than 95 . 63 % of the generated reads could be mapped to the assembly by burrows - wheeler aligner and 98 . 99 % of assembled sequences could be covered by at least 5 reads . furthermore , the integrity of the assembly was validated by the successful mapping of 95 . 80 % of 18 , 184 transcripts ( greater than 1000 bp ) with an identity cutoff of 80 % ( s6 table ) . these results indicate that the genome assembly of l . crocea has high coverage and is of high quality ( s7 table ) .
the repetitive elements comprise 18 . 1 % of the l . crocea genome ( s8 table ) , which is a relatively low percentage when compared with other fish species , such as danio rerio ( 52 . 2 % ) , gadus morhua ( 25 . 4 % ) , and gasterosteus aculeatus ( 25 . 2 % ) . this suggests that l . crocea may have a more compact genome ( s9 – s10 tables ) . we identified 25 , 401 protein - coding genes based on ab initio gene prediction and evidence - based searches from the reference proteomes of six other teleost fish and humans ( s4 fig and s11 table ) , in which 24 , 941 genes ( 98 . 20 % of the whole gene set ) were supported by homology or rnaseq evidence ( s5 fig ) . over 97 . 35 % of the inferred proteins matched entries in the interpro , swiss - prot , kegg or trembl database ( s12 table ) .
the phylogenetic relationships of l . crocea to seven other sequenced teleost species were estimated based on 2 , 257 one - to - one high - quality orthologues , using the maximum likelihood method . according to the phylogeny and the fossil record of teleosts , we dated the divergence of l . crocea from the other teleost species to approximately 64 . 7 million years ago ( fig . 1a ) . we also detected 19 , 283 orthologous gene families ( s3 table ) , of which 14 , 698 families were found in l . crocea . the gene components of l . crocea were similar to those of d . rerio ( fig . 1b ) . the gene contents in four representative teleost species and l . crocea genomes were also analysed , and 11 , 205 ( 76 . 23 % ) gene families were found to be shared by five teleosts ( fig . 1c ) . we confirmed that the one - to - one orthologous genes of g . aculeatus and l . crocea have higher sequence identities from the distribution of the percent identity of proteins ( fig . 1d ) , which indicates that sciaenidae has a closer affinity to gasterosteiformes and coincides with our genome - level phylogeny position .
( a ) the phylogenetic tree was constructed from 2 , 257 single - copy genes with 3 . 18 m reliable sites by maximum likelihood methods . the red points on six of the internal nodes indicate fossil calibration times in the analysis . blue numbers indicate the divergence time ( myr , million years ago ) , and the green and red numbers represent the expanded and extracted gene families , respectively , in l . crocea . ( b ) the different types of orthologous relationships are shown . “1 : 1 : 1” = universal single - copy genes ; “n : n : n” = orthologues exist in all genomes ; “fish” = fish - specific genes ; “sd” = genes that have undergone species - specific duplication ; “homology” = genes with an e - value less than 1e - 5 by blast but do not cluster to a gene family ; “nd” = species - specific genes ; and “others” = orthologues that do not fit into the other categories . ( c ) the shared and unique gene families in five teleost fish are shown in the venn diagram . ( d ) distribution of the identity values of orthologous genes is compared among l . crocea and other teleosts .
furthermore , 121 significantly expanded and 27 contracted gene families ( p < 0 . 01 ) were identified by comparing the family size of l . crocea with that of the other vertebrates used in the phylogenetic analysis ( s14 – s15 tables ) . based on the ratios of the number of nonsynonymous substitutions per nonsynonymous site ( k a ) to the number of synonymous substitutions per synonymous site ( k s ; k a / k s ratios ) in a branch - site model of paml [ 19 ] , 92 genes were found to be positively selected in l . crocea compared with their orthologues in the other six teleost species ( p < 0 . 001 , s16 table ) .
l . crocea is a migratory fish with good photosensitivity , olfactory detection , and sound perception , and it contains high levels of selenium [ 8 ] . our genomic analyses provide genetic basis for these physiological characteristics . several crystallin genes ( crygm2b , cryba1 , and crybb3 ) , which encode proteins that maintain the transparency and refractive index of the lens [ 4 ] , were significantly expanded in the genome of l . crocea relative to those of other sequenced teleosts ( s17 table ) . phylogenetic analysis showed that the crystallin genes from l . crocea cluster together , indicating that these genes were specifically duplicated in l . crocea lineage ( s6 fig ) . the specific expansion of these crystallin genes may be helpful for improving photosensitivity by increasing lens transparency , thereby enabling the fish to easily find food and avoid predation underwater .
we also identified 112 olfactory receptor ( or ) - like genes from the l . crocea genome ( s18 table and s7 fig . ) , and almost all of them ( 111 ) have been reported to be expressed in the olfactory epithelial tissues of l . crocea [ 14 ] . the majority of these genes ( 66 ) were classified into the “delta” group , which is important for the perception of water - borne odorants [ 20 ] . l . crocea also possessed the highest number of genes that were classified into the “eta” group ( 30 , p < 0 . 001 ) , and these genes may contribute to the olfactory detection abilities , which could be useful for feeding and migration [ 21 ] .
l . crocea is named for its ability to generate strong repetitive drumming sounds , especially during reproduction [ 8 ] . for good communication , fish have developed high sensitivities to environmental sound . three important auditory genes , otoferlin ( otof ) , claudinj , and otolin 1 ( otol1 ) , were significantly expanded in the l . crocea genome ( p < 0 . 01 , s19 table ) . these expansions may contribute to the detection of sound signaling during communication , and thus to reproduction and survival [ 22 ] .
selenium is highly enriched in l . crocea [ 8 ] , and it is mainly present as selenoproteins . we used the selgenamic - based selenoprotein prediction method [ 23 ] to analyse the l . crocea genome and identified 40 selenoprotein genes , which is the highest number among all examined vertebrates ( s20 table ) . interestingly , five copies of msrb1 , which encodes methionine sulfoxide reductase , were found in l . crocea ( msrb1a , msrb1b , msrb1c , msrb1d , and msrb1e ) , whereas only two copies ( msrb1a and msrb1b ) were found in other fish , thus suggesting its broader specificity to reduce all possible substrates [ 24 ] .
( a ) a schematic diagram summarising genes related to different t - cell lineages in l . crocea is shown . the inducible factors , the main regulatory transcriptional factors , and the immune effectors of t cells are present in green , blue , and orange backgrounds , respectively . the genes that have been annotated by genome survey are shown in black , and the unannotated genes are shown in red . il - 2r represents all three subunit genes of l . crocea il - 2 receptor , il - 2rb , il - 2rg and il - 2ra / il - 15ra genes . the majority of the cd4 + t - helper type 1 ( th1 ) , cd4 + t - helper type 2 ( th2 ) and cd8 + t cell - related genes have been found in the l . crocea genome . the genes related to th17 cell - and γδ - t cell - mediated mucosal immunity are also conserved in l . crocea . ( b ) several key genes are expanded in the antiviral immunity pathways in l . crocea . the genes that have been identified in the l . crocea genome are shown in orange boxes , and the lost gene ( rig - i ) is shown in the grey box . lgp2 is able to bind to double - stranded rna ( dsrna ) to trigger interferon production , but the adaptor molecule of lgp2 is still unknown in fish . the red boxes indicate gene families ( trim25 , cgas , ddx41 , and nlrc3 ) that are expanded in l . crocea . the arrow represents induction , and the interrupted line represents inhibition .
the brain allows rapid and coordinated responses to the environmental stress by driving the secretion of hormones . therefore , we studied the response of the l . crocea brain to hypoxia . we sequenced seven transcriptomes of the brains at different times of hypoxia exposure and found that 8 , 402 genes were differentially expressed at one or more time points ( fold change ≥ 2 , false discovery rate [ fdr ] ≤ 0 . 001 ; s10 fig ) . hypoxia stress induced a response with the largest number of genes ( 4 , 535 genes ) at 6 h ( s11 fig ) , indicating that genes with regulated expression at 6 h may be critical for the response .
under hypoxia , the potential neuro - endocrine - immune / metabolism networks contribute to the regulation of moderate inflammation and the maintenance of energy balance . hypoxia may inhibit the hypothalamic - pituitary - adrenal ( hpa ) axis and induce the expression of et - 1 / adm , and the latter then promote the expression of il - 6 / tnf - α and form a positive feedback loop with them . on the other hand , the et - 1 / adm and il - 6 / tnf - α may in turn activate the hpa axis , and the latter subsequently induces glucocorticoids and generates a negative feedback to inhibit et - 1 / adm and il - 6 / tnf - α expression to reduce the inflammatory responses in brain . therefore a hpa axis - et - 1 / adm - il - 6 / tnf - α feedback regulatory loop is outlined here . besides , both socs - 1 and socs - 3 may have complementary roles in down - regulating il - 6 and tnf - α , and both il - 6 and tnf - α have reciprocal functions to induce socs - 1 and socs - 3 expression , thus constituting a socs - 1 / 3 - dependent feedback regulation to modify cerebral inflammation . the hypothalamic - pituitary - thyroid ( hpt ) axis was inhibited in l . crocea brains during the early period of hypoxia , thus leading to a decrease in thyroid hormone ( t3 and t4 ) production . decrease of hpt axis - thyroid hormones subsequently may inhibit protein synthesis by the pi3k - akt - mtor - s6k signaling pathway , which contributes to the reduction in energy consumption during hypoxia stress . meanwhile , down - regulation of hpt axis - thyroid hormones also represses the tricarboxylic acid ( tca ) cycle and accelerates the anaerobic glycolytic pathway , which facilitates o 2 - independent atp production under hypoxia . therefore the hpt axis - mediated effects may play roles in response to hypoxia by reorganizing energy consumption and generation . genes related to the neuro - endocrine system ( orange ) , immunity ( red ) , and metabolic system and protein synthesis ( blue ) are indicated . the outer border indicates the brain of l . crocea . the arrow represents promotion , and the interrupted line represents inhibition . solid lines indicate direct relationships between genes . dashed lines indicate that more than one step is involved in the process .
energy maintenance and homeostasis under hypoxia is another critical event in brain . however , the mechanisms underlying this issue remain limited . here , we found that the major hypothalamic - pituitary - thyroid ( hpt ) axis - related genes ( thyrotropin - releasing hormone [ trh ] , trh receptor [ trhr ] , thyroid - stimulating hormone β [ tshβ ] , and thyroid hormone receptor α [ trα ] ) were significantly down - regulated in the l . crocea brain at 1 h to 6 h under hypoxia ( s14 fig ) . down - regulation of these gene expressions during hypoxia was also confirmed by real - time pcr ( s15 fig ) . these findings therefore indicate that the hpt axis may be inhibited during the early period of hypoxia , which agreed with the previous observations that hypoxia could influence the hpt axis [ 43 ] .
the skin mucus is considered as the first defensive barrier between fish and its aquatic environment , and it plays a role in a number of functions , including locomotion , antioxidant responses , respiration , disease resistance , communication , ionic and osmotic regulation [ 49 ] . however , the exact mechanisms underlying these functions remain unknown . mucus is composed mainly of the gel - forming macromolecule mucins and inorganic salts suspended in water [ 50 ] . we identified 159 genes that are implicated in mucin biosynthesis and mucus production in the l . crocea genome ( s23 table ) , based on previous studies in mammals [ 51 ] . this indicates that the mucin synthetic pathway is conserved between fish and mammals . among these gene families , galnt , which encodes n - acetylgalactosaminyl transferases [ 52 ] , was significantly expanded in l . crocea ( 27 copies versus 15–20 copies in other fish ; s17 fig ) . syntaxin - 11 was also expanded . additionally , genes encoding syntaxin - binding protein 1 and syntaxin - binding protein 5 , which are related to mucus secretion , were positively selected in the l . crocea genome ( s16 table ) . the expansion and positive selection of these genes may explain why the l . crocea secretes more mucus than other fish under stress .
( a ) the distribution of mucus proteins in the molecular function class of gene ontology is shown . the over - represented functional categories are indicated in the pie chart , of which oxidoreductase activity - , peroxidase activity - , oxygen binding - , and ion binding - related proteins are enriched . ( b ) a representation of the functional mechanisms of the mucus barrier is shown . the continuously replenished thick mucus layer can retain a large number of antioxidant , immune , oxygen - binding , and ion - binding molecules , which are involved in antioxidant functions , immune defence , oxygen transport , and osmotic and ionic regulation , respectively . antioxidant , immune , oxygen - binding , and ion - binding molecules are indicated in red , green , blue , and gray , respectively .
we sequenced and assembled the genome of the large yellow croakerr ( l . crocea ) using bacs and the wgs hierarchical assembly strategy . this methodology is effective for high - polymorphism genomes and produces a high quality genome assembly , with the 63 . 11 kb contig n50 and 1 . 03 mb scaffold n50 ( table 1 ) . support from the 563 - fold coverage of genome yields high single - base resolution and 95 . 80 % completeness of the coding region ( s6 table ) . further genomic analyses showed the significant expansion of several gene families , such as vision - related crystallins , olfactory receptors , and auditory sense - related genes , and provided a genetic basis for the peculiar physiological characteristics of l . crocea .
during the early stages of hypoxia stress , the induction of et - 1 / adm and il - 6 / tnf - α generates the primary protective effect to increase blood pressure , enhance vascular permeability and trigger inflammatory response [ 54 , 55 ] . these mechanisms maintain the brain oxygen supply and resist pathogen infection when the blood brain barrier is disrupted by hypoxia [ 56 ] . as the stress response progresses , several natural brakes , including hpa axis - glucocorticoids and socs family members , exhibit secondary protection effects to avoid excessive inflammatory responses in the brain . our transcriptome results show that a novel hpa axis - et - 1 / adm - il - 6 / tnf - α feedback regulatory loop in neuro - endocrine - immune networks contributed to the protective effect and regulated moderate inflammation under hypoxia stress ( fig . 3 ) . on the other hand , the hypoxia - induced down - regulation of the hpt axis may lead to the inhibition of protein synthesis and the activation of anaerobic metabolism ( fig . 3 and s14 – s16 fig ) . inhibition of protein synthesis principally contributes to the reduction in cellular energy consumption during hypoxia [ 3 , 57 ] . activation of anaerobic metabolism facilitates o 2 - independent atp production under hypoxia , albeit with low atp yield [ 57 ] . therefore the reduction in atp consumption through the hpt axis - mediated inhibition of protein synthesis matched the lower atp yield by the hpt axis - activated anaerobic metabolism , which may aid to maintain cellular energy balance under hypoxia , thus extending fish survival . hence , our results reveal new aspects of neuro - endocrine - immune / metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia stress . these discoveries will help to improve current understanding of neuro - endocrine - immune / metabolism regulatory networks and protective mechanisms against hypoxia - induced cerebral injury in vertebrates , providing clues for research on the pathogenesis and treatment of hypoxia - induced cerebral diseases .
amazingly , 3 , 209 different proteins were identified in the l . crocea skin mucus under air exposure . of these , oxidoreductase activity - , oxygen binding - , immunity - , and ion binding - related proteins were enriched ( fig . 4a and s18 fig ) . the increase in secretion of the skin mucus of l . crocea under air exposure may reflect a physiological adjustment of the fish to cope with environmental changes , and the complex components suggest that the skin mucus exerts multiple protective mechanisms , which are involved in antioxidant functions , oxygen transport , immune defence , and osmotic and ionic regulation ( fig . 4b ) . these results expand our knowledge of skin mucus secretion and function in fish , highlighting its importance in response to stress . in addition , the mucus proteome shares many proteins with the mucus from humans and other animals [ 58 , 59 ] . these characteristics thus make l . crocea a pertinent model for studying mucus biology .
in summary , our sequencing of the genome of the large yellow croaker provided the genetic basis for its peculiar behavioral and physiological characteristics . results from transcriptome analyses revealed new aspects of neuro - endocrine - immune / metabolism regulatory networks that may help the fish to avoid cerebral inflammatory injury and maintain energy balance under hypoxia stress . proteomic profiling suggested that the skin mucus of the fish exhibits multiple protective mechanisms in response to air - exposure stress . overall , our results revealed the molecular and genetic basis of fish adaptation and response to hypoxia and air exposure . in addition , the data generated by this study will facilitate the genetic dissection of aquaculture traits in this species and provide valuable resources for the genetic improvement of stress resistance and yield potential in l . crocea .
the studies were carried out in strict accordance with the regulations of the administration of affairs concerning experimental animals established by the fujian provincial department of science and technology . animal experiments were approved by the animal care and use committee of the third institute of oceanography , state oceanic administration . all surgery was performed under tricaine - s anesthesia , and all efforts were made to minimize suffering .
the wild l . crocea individuals were collected from the sanduao sea area in ningde , fujian , china . genomic dna was isolated from the blood of a female fish by using standard molecular biology techniques for bac library construction and sequencing by the hiseq 2000 sequencing system in bgi ( beijing genomics institute , shenzhen , china ) . subsequently , low - quality and duplicated reads were filtered out , and sequencing errors were removed . the bacs of l . crocea were assembled by using soapdenovo2 [ 60 ] ( urltoken ) with k - mers that ranged from 25 to 63 in size . then , we selected the assembly with the longest scaffold n50 for gap filling . the bacs were merged together based on the overlap found by blat , using the custom script : rabbit ( urltoken ) . the redundant sequences that were produced by high polymorphisms were removed by sequence depth and shared k - mer percentage . assembly was performed by scaffolding with mate - paired libraries ( 2–40 kb ) using sspace v2 [ 61 ] , and gap filling was made by gapcloser ( urltoken ) with small - insert libraries ( 170–500 bp ) .
the whole - genome sequences of l . crocea have been deposited in the dna data bank of japan ( ddbj ) , the european molecular biology laboratory ( embl ) nucleotide sequencing database and genbank under the accession jrpu00000000 . the version described in this paper is the first version jrpu01000000 . all short - read data of wgs and bac have been deposited in the short read archive ( sra ) under accession sra159210 and sra159209 respectively .
for the annotation of repetitive elements , we used a combination of homology - based and ab initio predictions . repeatmasker [ 62 ] and protein - based repeatmasking [ 62 ] were used to search repbase , which contains a vast amount of known transcriptional elements at the dna and protein levels . during the process of ab initio prediction , repeatscout [ 63 ] was used to build the ab initio repeat library based on k - mer , using the fit - preferred alignment score on the l . crocea genome . contamination and multi - copy genes in the library were filtered out before the repeatscout library was used to find homologs in the genome and to categorise the found repeats by repeatmasker [ 62 ] .
gene models were integrated based on ab initio predictions , homologue prediction , and transcription evidence .
the protein sequences of seven species ( danio rerio , gasterosteus aculeatus , oreochromis niloticus , oryzias latipes , takifugu rubripes , tetraodon nigroviridis , and homo sapiens ) were aligned to the l . crocea assembly using blast ( e - value ≤ 1e - 5 ) , and the matches with length coverage > 30 % of the homologous proteins were considered as gene - model candidates . the corresponding homologous genome sequences were then aligned against the matching proteins by using genewise [ 64 ] to improve gene models .
augustus [ 65 ] , snap [ 66 ] , and genescan [ 67 ] were used for the ab initio predictions of gene structures on the repeat - masked assembly .
rnaseq reads from the transcriptomes of the mixed tissues of a female and a male ( eleven tissues each ) were aligned to the genome assembly by tophat [ 68 ] , which can identify splice junctions between exons . cufflinks [ 69 ] was used to obtain transcript structures .
homology - based , ab initio derived and transcript gene sets were integrated to form a comprehensive and non - redundant gene set . the overlap length of each gene was verified by different methods , and genes showing 50 % overlap by at least one method were selected . to eliminate false positives ( genes only supported by ab initio methods ) , novel genes with the reads per kilobase of gene model per million mapped reads ( rpkm ) ≤ 1 were removed . a method based on gene synteny and fgenesh program ( urltoken ) was used for the prediction of interleukin genes .
the 2 , 257 single - copy genes from the gene family analysis were aligned using muscle [ 71 ] , and alignments were concatenated as a single data set . to reduce the error topology of phylogeny by alignment inaccuracies , we used gblock [ 72 ] ( codon model ) to remove unreliably aligned sites and gaps in the alignments . the phylogenetic tree and divergence time were calculated using the paml 3 . 0 package [ 19 ] .
gene family expansion and contraction analyses were performed by cafe [ 73 ] . for optical , olfactory receptor , and auditory system - related genes , we downloaded the genes from swissprot or genebank and predicted their candidates using blast and genewise to determinate copy numbers . pseudogenes produced by frame shift were removed . phylogenetic analysis of the expanded gene families was based on maximum likelihood methods by paml 3 . 0 [ 19 ] , and the phylogenetic tree was represented by evolview [ 74 ] .
l . crocea ( 90–100 g ) individuals were purchased from a mariculture farm in ningde , fujian , china . the fish were maintained at 25°c in aerated water tanks ( dissolved oxygen [ do ] concentration : 7 . 8±0 . 5 mg / l ) with a flow - through seawater supply . after 7 days of acclimation , hypoxia - exposure experiments were conducted at 25°c using published methods [ 3 ] by bubbling nitrogen gas into an aquarium . the desired concentration of do was detected by using a do meter ( ysi , canada ) . l . crocea can not maintain the aerobic pathway at do levels below 2 . 0 mg / l , and it resorts to anaerobic metabolism [ 15 ] . therefore , at the onset of hypoxia , the oxygen content in the tank was lowered from 7 . 8±0 . 5 mg / l to 1 . 6±0 . 2 mg / l over a 10 - min period . brains were harvested from six fish at the 0 , 1 , 3 , 6 , 12 , 24 , and 48 h time points and frozen immediately in liquid nitrogen until rna extraction and transcriptome analyses were performed . to investigate whether the gene expression alterations under hypoxia come from changes in the baseline expression levels , brain tissues were collected from five fish with no hypoxia treatment at several corresponding time points ( 0 , 3 , 6 , 12 , and 24 h ) and subjected to real - time pcr analysis of selected genes ( s19 fig ) .
total rna was extracted from the brain tissues harvested at 0 , 1 , 3 , 6 , 12 , 24 , and 48 h after hypoxia exposure using the guanidinium thiocyanate - phenol - chloroform extraction method ( trizol , invitrogen , usa ) . the rna samples ( 5 μg each ) were used to construct rna - seq libraries using the illumina mrna - seq prep kit . the libraries were sequenced by using the illumina hiseq 2000 sequencing platform with the paired - end sequencing module [ 76 ] . more than 4 gigabase ( gb ) of clean data were generated in each library ( s29 table ) . clean reads were picked out from the raw reads of each library following removal of reads containing adaptor sequences , reads with an n ( unknown bases in read ) percentage higher than 5 % and low quality reads ( > 30 % of bases with a quantity score q - value ≤ 10 ) using an in - house c + + program .
clean reads were aligned to the l . crocea genome and its coding sequences ( cds ) which were produced from genome - wide prediction of protein - coding genes ( s29 table ) , with soapaligner / soap2 [ 60 ] . the alignment to the cds of l . crocea genome was then utilised to calculate the distribution of reads on reference genes and to perform coverage analysis . if an alignment result passed quality control ( alignment ratio > 70 % ) , we proceeded to gene expression calculations and differential expression comparisons . the gene expression levels were calculated based on rpkm values [ 69 ] , and comparisons of gene expression difference between control ( 0 h ) and each time point after hypoxia exposure ( 1 , 3 , 6 , 12 , 24 , and 48 h ) were performed using the method described by audic and claverie [ 77 ] . genes with fold change ≥ 2 and fdr ≤ 0 . 001 were defined as differentially expressed genes ( degs ) [ 78 ] . gene expression patterns were analyzed by the open source clustering software cluster 3 . 0 and presented using the graphpad prism 5 software or java treeview . raw sequencing data for the transcriptome have been deposited in the gene expression omnibus ( geo ) under accession gse57608 .
real - time pcr analysis was performed using the mastercycler epgradient realplex4 ( eppendorf , germany ) with sybr green as the fluorescent dye , according to the manufacturer’s protocol ( takara , china ) . primer set was designed based on the coding sequence of each identified gene in l . crocea genome ( s30 table ) . total rna was extracted from brain tissues of five fish sampled at 0 , 1 , 3 , 6 , 12 , 24 , and 48 h after hypoxia induction . first - strand cdna was synthesized from 2 μg total rna and used as a template for real - time pcr . real - time pcr was performed in a total volume of 20 μl , and cycling conditions were 95°c for 1 min , followed by 40 cycles of 94°c for 5 s , 57°c for 15 s , and 72°c for 20 s . the expression levels of each gene were expressed relative to those of β - actin in each sample using the 2 - δδct method [ 79 ] . each real - time pcr assay was repeated three times . the data of real - time pcr were expressed as the standard error of the mean ( sem ) . two - tailed student’s t test was used for the significance test of the gene expression levels in brain tissues between 0 h and each time point after hypoxia exposure . a p - value < 0 . 05 was considered to be statistically significant .
skin mucus was collected from six healthy l . crocea individuals under air exposure as previously described [ 50 ] . briefly , the fish were anesthetised with a sub - lethal dose of tricaine - s ( 100 mg / l ) , and transferred gently to a sterile plastic bag for 3 min to slough off the mucus under air exposure . to exclude the cell contamination , mucus was diluted in fresh , cold phosphate - buffered saline and drop - splashed onto slides , which were then air - dried . after staining with 10 % giemsa dye ( sigma , st louis , mo , usa ) for 20 min , the mucus was observed under a nikon microscope with a 20 × objective . no cell was observed .