Fishery Biology Of Loliginidae In China Seas Based On Hard Tissues

Uroteuthis chinensis and U.edulis are important target species in East China Sea and South China Sea.Species and stock identification of these two species are the most important issue need to be solved by scientist because of their similar appearance which is hard to tell by naked eyes.Nowadays,studies on age and growth are limited to statoltih for these two species.Therefore,it is necessary to use other hard tissues of squid to study their age and growth.On the other hand,studies about trace elements of hard tissues of squid are rare.Hence,there are several aims need to be conduct in the present study,i.e.combining DNA based species identification with their traditional morphology and geomorphology to identify these species;selecting best hard tissues for age determination from statolith,beak and eye lens;analyzing age and growth of two species based on best tissue;comparing trace element composition and content and their tendency in different life stages.In the present study,we used U.chinensis and U.edulis,sampled in northern South China Sea from 2015 to 2016,to identify species based on morphology and DNA information;then compare the beak morphology of two species to analyze the difference of beak characters in different mantle length for two species;use outline method and stepwise discriminant method to identify statolith and beak of three species for identification;compare microstructure of statoltih,beak and eye lens,and select best tissue for age determination,then establish relationship between mantle and age,and back-calculate hatching date;describe the trace element composition and content of three hard tissues of two species,analyze the difference of element/Ca ratio among different life stages,use element/Ca ratio to identify two species;predict the migratory routes of U.chinensis and U.edulis based on Sr/Ca and Ba/Ca of statolith and previous studies.The main results of the present study are as follows:?1?Species identification of U.chinensis and U.edulis based on morphology and DNA methods.In order to distinguish these two species,two methods?teeth shape of arm sucker ring and DNA sequence?were used to identify them.In present study,341 individuals?210 U.chinensis and 131 U.edulis?from different locations of China Seas were used for analyses.There were 14%of specimens had discrepancy in species identification between two identification methods?i.e.teeth shape of arm sucker ring and DNA?.Teeth shape of arm sucker ring identification is an ideal method after considering the high match rate to DNA results.The ratio between Mantle Width?MW?and Mantle length?ML?was not suitable for identification because of the overlap in two species.The ratio between MW and ML changed sharply among different ML groups indicating the allometric growth in both species.The Principal Component Analysis?PCA?and Step-wise Discriminant Analysis?SDA?results showed that the morphological similarity of two species in the same area is greater than that of one species in different areas.SDA results also showed that validation rate for three groups?U.edulis in East China Sea,U.edulis in South China Sea and U.chinensis in South China Sea?were above 75%.Phylogenetic analysis based on COI showed that U.edulis in present study formed two clades while U.chinensis just formed one clade.U.chinensis was only found in the south of 25°N which needs to be confirmed through amounts of sampling in East China Sea.?2?The traditional morphology difference of beaks in U.chinensis and U.edulis.The results indicated that morphological beak values were greater for U.edulis,compared to U.chinensis,for upper hood length?UHL?,upper crest length?UCL?,upper lateral wall length?ULWL?,lower crest length?LCL?,and lower lateral wall length?LLWL?.According to principal component analysis,UHL/ML,UCL/ML,ULWL/ML,LCL/ML,LLWL/ML and LWL/ML could represent the characteristics of beaks for U.chinensis,while UHL/ML,UCL/ML,ULWL/ML,LHL/ML,LCL/ML and LLWL/ML could represent it for U.edulis.According to Akaike's information criterion?AIC?values,a power function was the most suitable model for U.chinensis,while a linear function was the most suitable model for U.edulis.The beak variable-mantle length ratio?beak variable/mantle length?declined with the increasing of mantle length and declined sharply at the early stage of growth in both beaks and species.The ratio changed quickly after achieving the mantle length of140 mm for U.chinensis,while the ratio changed quickly after 170 mm for U.edulis.Beaks in both species experienced sharper changes through maturity stage?to?than other maturity stages.This study gives us basic beak morphology information for U.chinensis and U.edulis in the East and South China Seas.?3?Species identification based on geometric morphologyical method.In the present study,we used statolith and beak to identify three squid species including U.duvaucelii,Loliolus beka,and U.edulis in the South China Sea.Because of the highly overlapping habitat and similar body morphology of the three squid species,we explored four different ways to identify them,by using statolith,upper beak,lower beak and a combination of statolith and beak using two hard tissues,statolith and beaks.An outline geometric morphometric method and stepwise discriminant analysis were used to evaluate the most suitable method for the identification.We found that the combination of statolith and beak had the highest cross validation rate that was75.0%,87.5%and 88.7%for U.duvaucelii,L.beka and U.edulis,respectively.Using two beaks had similar results and the lowest cross validation rate was 60.0%,50.0%,and 73.7%for the upper beak,46.9%,58.5%and 75.3%for the lower beak of U.duvaucelii,L.beka and U.edulis,respectively.Analyzing with the statolith had moderate cross validation which was 72.2%,80.0%,and 87.7%for U.duvaucelii,L.beka and U.edulis,respectively.From the results it is suggested when the entire body of a squid is available,a combination of statolith and beak should be used for the identification.When only one hard tissue is available,species identification can be subjected to large errors.?4?Comparative study of hard tissue microstructures in U.chinensis.To explore microstructure and growth increments of hard tissues of U.chinensis,we use statoliths,beaks and eye lenses to reveal details of microstructure of three hard tissues,and to interpret their relationships with body size and periodicity validation of increments.In this study,we observed growth increments in three domes[dorsal dome?SDD?,lateral dome?SLD?and rostrum]of statolith.Growth increments of SLD had the most stable increment width among all domes.Growth increments of rostrum sagittal section?RSS?,especially hood of RSS,were seen in upper beak.Eye lens had concentric growth increments.The number of growth increments of hard tissues differed from each other.SLD and SDD had similar growth increment number while that of beak were lower than them.The number of growth increments of eye lens was multiple time larger than that of statolith and beak.The relationship between mantle length and growth increments of beak were best fitting,following with SLD and eye lens.Also,the relationship between mantle length and growth increments of SLD had two separate equations,which might result from two different cohorts.Considering the validated growth increments of statolith for age determination,growth increments of SLD were finally used for ageing.Hence,the maximum age of U.chinensis was 169 days in the present study.The SLD-beak equation had a linear slope of 0.97 that suggests beak might be potential age determination material for U.chinensis.?5?Age and growth of U.chinensis and U.edulis based on statolith.To understand age and growth of U.chinensis and U.edulis,growth increments of lateral dome of statolith were used in the present study.The specimens of two species were caught in the South China Sea?SCS?and East China Sea?ECS?at different seasons and were used to compare age and growth differences between two species.In the present study,we found two species have similar lifespan less than 200 days.The difference of mantle length and body weight relationship between species were greater than that between seasons of the same species.The back-calculated hatching date for two species were similar,i.e.October to January for specimens caught in spring of two species and March to June for specimens caught in summer?U.edulis?or fall?U.chinensis?.The exponential and logistic growth curves were fitted to build age and mantle length relationship.Both growth curves showed U.chinensis has larger mantle length than U.edulis at the same age.The results of growth rates also showed U.chinensis grows faster than U.edulis.However,there are discrepancies between previous studies and the present study on the lifespan of the two species.Also,the sample size of the present studies were small especially for U.edulis.Hence,future studies should be conduct to validate daily periodicity of growth increment in domes of statolith?except lateral dome which has been validated?and sample size should be increased for age and growth study.?6?Comparative study of hard tissue trace elements composition.In the present study,we used 83 squid individuals for trace element analysis.The results indicated that,there are nine trace elements whose average is over 10.00 ppm and have detection rate over 80%in statolith,i.e.¹¹B,²³Na,²⁵Mg,³¹P,³⁹K,⁴²Ca,⁵⁷Fe,⁸⁸Sr and¹³⁷Ba.There are thirteen trace elements whose average is over 10.00 ppm and have detection rate over 80%in beak,i.e.¹¹B,²³Na,²⁵Mg,³¹P,³⁹K,⁴²Ca,⁶³Cu,⁶⁶Zn,⁸⁵Rb,⁸⁸Sr,⁹⁷Mo,²⁰⁷Pb and ²³⁸U.There are eight trace elements whose average is over 10.00 ppm and have detection rate over 80%in eye lens,²³Na,²⁵Mg,³¹P,³⁹K,⁴⁹Ti,⁶³Cu,⁶⁶Zn and ⁸⁸Sr.According to t-test,there are no significant difference between U.chinensis and U.edulis for all trace elements?P>0.05?,which can be interpreted by severe overlapping of habitat.ANOVA showed that,element/Ca ratios of life history of two species have different tendency,i.e.Na/Ca,Mg/Ca,P/Ca,K/Ca,Sr/Ca and Ba/Ca of statolith of two species have similar tendency,B/Ca,Mg/Ca,K/Ca,Cu/Ca,Sr/Ca and Pb/Ca of beak of two species have similar tendency,Na/Ca,Mg/Ca,K/Ca and Sr/Ca of eye lens of two species have similar tendency,while other element/Ca ratios have different tendency for three hard tissues.Discriminant analysis showed that,adult stage has the highest cross-validation rate when using statolith trace element for species identification,sub-adult stage has the highest cross-validation rate when using beak,and adult stage has the highest cross-validation rate when using eye lens.?7?Migratory routes inferences based on Sr/Ca and Ba/Ca of statolith in U.chinensis and U.edulis.For the samples that measured trace elements,the hatching months of U.chinensis were October,2015 to January,2016,and the hatching months of U.edulis were November,2015 to February,2016.For Sr/Ca and Ba/Ca of each life stage of U.chinensis and U.edulis in different hatching months,their trends were similar,i.e.Sr/Ca of hatching had higher values while juvenile or sub-adult had lower value;Ba/Ca of hatching or larvae had higher value while sub-adult had lower value.Based on the hypothetical living temperature,U.chinensis and U.edulis hatched from October to February lived in the waters that had the temperature between 21?and27??Sr/Ca of statolith of U.chinensis hatched in January had negative relationship with hypothetical living temperature,while those of U.edulis hatched from November to February had had negative relationship with hypothetical living temperature.For U.chinensis hatched in January and U.edulis hatched between November and February,the inferred migration routes were as follows:from October to February,they hatched at the bottom of water depth between 30 to 100 meters;from hatchling to March,they migrated to south where bottom depth were around 200 meters for wintering;from the end of March to June,they migrated to north to the coast of China for feeding;from July to September,they migrated to south again for mating with water depth between 100 and 200 meters;after mating,female squid moved to north at the bottom with depth between 30 and 100 meters for spawning.In the present study,we conclude that,in order to distinguish U.chinensis and U.edulis accurately,teeth shape of arm sucker ring was used for species identification.According to the number of increment of statolith lateral dome,U.chinensis and U.edulis have life span about 200 days.The growth rates of U.edulis in different waters were significant different,while those of U.chinensis and U.edulis in the same water were similar.Composition and content of trace element in statolith,beak and eye lens were different,which might be related to the formation mechanism in different tissues.According to the specimens in the present study,the migration routes of U.chinensis hatched in January and U.edulis hatched between November and February were inferred:they migrate between China coast and waters with depth of 200 meters,i.e.south-north migration,and shallow and deeper water migration.