DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.

Performance evaluation of fixed-station sampling design for a fishery-independent survey with multiple objectives

Fixed-station sampling design was widely used in fishery-independent surveys because of its characteristics of convenient sampling station setting,but the non-probabilistic(fixed)nature made it more uncertainty of drawing inferences on population.The performance of fixed-station sampling design for multispecies survey has not been evaluated,and we are uncertain if the design could detect the temporal trends of different populations in multispecies fishery-independent survey.In this study,spatial distribution of abundance indices for three species with different spatial distribution patterns including small yellow croaker(Larimichthys polyactis),whitespotted conger(Conger myriaster)and Fang’s blenny(Enedrias fangi)were simulated using ordinary kriging interpolation as the“true”population distribution.The performance of fixed-station sampling design was compared with simple random sampling design by resampling the simulated“true”populations in this simulation study.The results showed that the fixed-station sampling design had the power to detect the seasonal trends of species abundance.The effectiveness of fixed-station sampling design were different in different species distribution patterns.When the species had even distribution,fixed-station sampling design could get high quality abundance data;when the distribution was uneven with heterogeneity or patchiness,fixed-station sampling design tended to underestimate or overestimate the abundance.Evidently,the estimates of abundance index based on the fixedstation sampling design must be calibrated cautiously while applying them for fisheries stock assessment and management.This study suggested that fixed-station sampling design could catch the temporal dynamics of population abundance,but the abundance estimates from the fixed-station sampling design could not be treated as the absolute estimates of populations.

Identification of navigation characteristics of single otter trawl vessel using four machine learning models

Fishing logbook records the fishing behaviors and other information of fishing vessels.However,the accuracy of the recorded information is often difficult to guarantee due to the misreport and concealment.The fishing vessel monitoring system(VMS)can monitor and record the navigation information of fishing vessels in real time,and it may be used to improve the accuracy of identifying the state of fishing vessels.If the VMS data and fishing logbook are combined to establish their relationships,then the navigation characteristics and fishing behavior of fishing vessels can be more accurately identified.Therefore,first,a method for determining the state of VMS data points using fishing log data was proposed.Secondly,the relationship between VMS data and the different states of fishing vessels was further explored.Thirdly,the state of the fishing vessel was predicted using VMS data by building machine learning models.The speed,heading,longitude,latitude,and time as features from the VMS data were extracted by matching the VMS and logbook data of three single otter trawl vessels from September 2012 to January 2013,and four machine learning models were established,i.e.,Random Forest(RF),Adaptive Boosting(AdaBoost),K-Nearest Neighbor(KNN),and Gradient Boosting Decision Tree(GBDT)to predict the behavior of fishing vessels.The prediction performances of the models were evaluated by using normalized confusion matrix and receiver operator characteristic curve.Results show that the importance rankings of spatial(longitude and latitude)and time features were higher than those of speed and heading.The prediction performances of the RF and AdaBoost models were higher than those of the KNN and GBDT models.RF model showed the highest prediction performance for fishing state.Meanwhile,AdaBoost model exhibited the highest prediction performance for non-fishing state.This study offered a technical basis for judging the navigation characteristics of fishing vessels,which improved the algorithm for judging the behavior of fishing vessels based on VMS data,enhanced the prediction accuracy,and upgraded the fishery management being more scientific and efficient.

Analyses of Trawling Track and Fishing Activity Based on the Data of Vessel Monitoring System(VMS): A Case Study of the Single Otter Trawl Vessels in the Zhoushan Fishing Ground

The original purpose of Vessel Monitoring System(VMS) is for enforcement and control of vessel sailing. With the application of VMS in fishing vessels, more and more population dynamic studies have used VMS data to improve the accuracy of fisheries stock assessment. In this paper, we simulated the trawl trajectory under different time intervals using the cubic Hermite spline(c Hs) interpolation method based on the VMS data of 8 single otter trawl vessels(totally 36000 data items) fishing in Zhoushan fishing ground from September 2012 to December 2012, and selected the appropriate time interval. We then determined vessels' activities(fishing or non-fishing) by comparing VMS speed data with the corresponding speeds from logbooks. The results showed that the error of simulated trajectory greatly increased with the increase of time intervals of VMS data when they were longer than 30 minutes. Comparing the speeds from VMS with those from the corresponding logbooks, we found that the vessels' speeds were between 2.5 kn and 5.0 kn in fishing. The c Hs interpolation method is a new choice for improving the accuracy of estimation of sailing trajectory, and the VMS can be used to determine the vessels' activities with the analysis of their trajectories and speeds. Therefore, when the fishery information is limited, VMS can be one of the important data sources for fisheries stock assessment, and more attention should be paid to its construction and application to fisheries stock assessment and management.

Effects of different dietary lipid sources on growth performance and tissue fatty acid composition of juvenile swimming crab Portunus trituberculatus

This study was conducted to evaluate the effects of dietary lipid sources on the growth performance and fatty acid composition of the swimming crab, P ortunus trituberculatus. Four isonitrogenous and isoenergetic experimental diets were formulated to contain four separate lipid sources, including fish, soybean, rapeseed, and linseed oils(FO, SO, RO, and LO, respectively). With three replicates of 18 crabs each for each diet, crabs(initial body weight, 17.00 ±0.09 g) were fed twice daily for 8 weeks. There were no significant differences among these groups in terms of weight gain, specific growth rate, and hepatosomatic index. However, the RO groups' survival rate was significantly lower than FO groups. The feed conversion and protein efficiency ratios of RO groups were poorer than other groups. The proximate compositions of whole body and hepatopancreas were significantly affected by these dietary treatments. Tissue fatty acid composition mainly reflected dietary fatty acid compositions. Crabs fed FO diets exhibited significantly higher arachidonic, eicosapentaenoic, and docosahexaenoic acid contents in muscle and hepatopancreas compared with VO crabs. Linoleic, oleic, and linolenic acids in muscle and hepatopancreas were the highest in the SO, RO, and LO groups, respectively. The present study suggested that SO and LO could substitute for FO in fishmeal-based diets for swimming crabs, without affecting growth performance and survival.

Complete Mitochondrial Genome of Myra affinis(Decapoda:Brachyura:Leucosiidae)and Its Phylogenetic Implications for Brachyura

Knowledge of complete mitochondrial genomes(mitogenomes)can help understand the molecular evolution and phylogenetic relationships of various species.To date,the phylogenetic status of Leucosiidae within Brachyura remains unresolved because of the limited number of mitogenomes available.In the present study,the complete mitogenome of Myra affinis was sequenced using next-generation sequencing to supplement the limited mitogenome information of Leucosiidae.The 15349 bp-long mitogenome includes 13 protein-coding genes(PCGs),22 transfer RNAs(tRNAs),2 ribosomal RNAs(rRNAs),and a putative control region.The overall base composition is 34.5%A,36.1%T,11.5%G,and 17.9%C,with a high AT bias(70.6%).All 13 PCGs start with the standard ATN codon and stop with the TAN codon or incomplete T.Except for tRNA-Ser,all other tRNAs have a typical cloverleaf-like secondary structure.Phylogenetic analyses based on the maximum likelihood and Bayesian inference methods are employed to generate identical phylogenetic topologies,thereby supporting the sister relationship between Leucosiidae and Matutidae for the first time.The monophyly of Eubrachyura is well established,and its sister relationship with Raninoida is strongly supported.The results of this work will not only help achieve a better understanding of the characteristics of the M.affinis mitogenome and the phylogenetic position of Leucosiidae,but also provide relevant information for further studies on the phylogeny of Brachyura.

Influences of environmental factors on the spawning stock-recruitment relationship of Portunus trituberculatus in the northern East China Sea

Based on the Ricker-type models,the spawning stock-recruitment(S-R)relationship of Portunus trituberculatus was analysed under the impacts of environmental factors(including red tide area(AORT),sea level height(SLH),sea surface salinity(SSS)and typhoon landing times(TYP))in the northern East China Sea in 2001 and 2014.Besides the traditional Ricker model,two other Ricker-type S-R models were built:Ricker model with ln-linear environmental impact(Ricker-type 2)and Ricker model with ln-quadratic polynomial environmental impact(Ricker-type 3).Results showed that AORT,SLH,SSS and TYP had great influences on the recruitment of P.trituberculatus.When SSS reached 29 and 31,recruitment decreased from 20.7×10^(3) million to 8.3×10^(3) million individuals.In this case,recruitment declined,whereas AORT and TYP increased.Analysis of the S-R model showed that the Akaike information criterion(AIC)value of the traditional Ricker model was 14.619,which remarkably decreased after addition of the environmental factors.Different numbers of environmental factors were added to the Ricker model,and the best result was obtained when four factors were added to the model together.Moreover,Ricker-type 2 model,with the AIC value of-5.307,was better than Ricker-type 3 model(add above four environmental factors at the same time).The findings indicated that the mechanisms by which various environmental factors affect the S-R relationship are different.

A New Record of Ponyfish Deveximentum megalolepis(Perciformes: Leiognathidae) in Beibu Gulf of China

A new record ponyfish, Deveximentum megalolepis Mochizuki and Hayashi, 1989, was documented based on its morphological characteristics and DNA barcode. Fifty specimens were collected from Beibu Gulf of China and identified as D. megalolepis by morphological characterization. The coloration, meristic traits, and morphometric measurements were consistent with previously published records. In general, it is a silver-white, laterally compressed and deep bodied ponyfish with 6–9 rows of scales on cheek; scale rows above lateral line 6–8; scale rows below lateral line 14–17. Mitochondrial cytochrome oxidase I subunit(COI) gene fragment was sequenced for phylogenetic analysis. There is no sequence variation of COI gene between the specimens collected in this study. The genetic distances between D. megalolepis and other congeneric species range from 3.6% to 14.0%, which were greater than the threshold for fish species delimitation. The COI sequence analysis also supported the validity of D. megalolepis at genetic level. However, the genetic distance between Chinese and Philippine individuals was about 1.2% and they formed two lineages in gene tree, which may be caused by the geographical distance.

Comparative mitochondrial genome analysis of Sesarmidae and its phylogenetic implications

Here,we sequenced the complete mitogenome of Parasesarma eumolpe(Brachyura:Grapsoidea:Sesarmidae)for the first time.The characteristics of this newly sequenced mitogenome were described and compared with other Sesarmidae species.The 15646-bp mitogenome contains 13 protein-coding genes(PCGs),two ribosomal RNA genes(r RNAs),22 transfer RNA genes(t RNAs),and an A-T rich region.All of the PCGs are initiated by the start codon ATN and terminated by the standard TAN codon or an incomplete T.The pairwise Ka/Ks ratio analysis shows that all 13 PCGs are under purifying selection,whereas the ATP8 gene is an outlier,with pairwise comparison values ranging from neutral selection(0.000)to positive selection(1.039).The gene arrangement of P.eumolpe compared with ancestral Decapoda shows the translocation of two t RNAs(t RNA-His and t RNA-Gln),which is identical to other Sesarmidae species.Phylogenetic analyses show that all Sesarmidae species are placed into one group,and the polyphyly of Eriphioidea,Ocypodoidea,and Grapsoidea is well supported.The relationship between gaps in the QIM region and the phylogeny of Sesarmidae is analyzed.It is obvious that both the G5(the gap between Q and I)and G6(the gap between I and M)decrease progressively with the evolution process.These results will help to better understand the genomic evolution within Sesarmidae and provide insights into the phylogeny of Brachyura.

Characterization of ghrelin mRNA expression in fasting Larimichthys crocea juveniles

Larimichthys crocea is a marine fish species cultured in China.Short-term starvation is often applied to improve the quality of cultured L.crocea,and the expression of ghrelin in tissues of stomach,muscle,brain,intestines,liver,and kidney,involved in starvation response,under starvation conditions were studied to understand the effect of starvation on the expression of ghrelin in L.crocea juveniles.The ghrelin expression was tissue-specific,and expression was significantly higher in the stomach compared to other tissues(P<0.01).Additionally,ghrelin expression in different tissues changed along with prolongation of fasting.In the stomach,ghrelin expression levels increased gradually at the beginning of the fast,and then declined after eight days of fasting.Gene expression in the brain and intestines increased at the beginning of the fast,and then decreased with longer fasting time.Interestingly,ghrelin expression declined at the beginning of the fast,then increased with longer fasting in the kidneys and muscles.These results suggest that ghrelin is involved in starvation response in L.crocea juveniles.This study provids insights into ghrelin function and an important reference for the development of reasonable feeding strategies for L.crocea juveniles.

Evaluation of Nutrient Components and Nutritive Quality of Larimichthys crocea(Big Yellow Croaker)in Different Aquaculture Models

To ascertain the nutrient components and nutritive quality of the flesh of big yellow croaker in three culture conditions （ traditional cage, offshore cage and cage-free）, basic nutritional components,amino acid,fatty acid and mineral elements were determined. The results indicated that crude protein in flesh of the big yellow croaker in cage-free culture was higher than that in offshore cage and much higher than that in traditional cage （ P 〈0.05）. Crude fat of the croaker cul- tured in the traditional cage was twice as high as that in cage-free culture, while that in the offshore cage was in the middle. Proline content in the cage-free culture was much higher than that in the offshore cage, and also than that in the traditional cage （P 〈 0.05 ）. There was no significant difference in the content of alanine, methionine and tryptophan （ P 〈 0.05）. Contents of other amino acids had no significant difference between the cage-free culture and offshore cage, but were much lower in the traditional cage （P 〈 0.05 ）. Top six fatty acids were 9-Hexadecenoic acid, palmitic acid,9-Octadecenoic acid, Octadecauoic acid, DHA and EPA. The palmitic acid content was the highest in cage-free culture and in traditional cage, 9-Octadeeenoic acid content was the highest in offshore cage. Content of unsaturat- ed fatty acids in cage-free culture, offshore cage and traditional cage was 63.60,66.32,57.67, respectively, and polyunsaturated fatty acid was 29.10, 28.57, and 24.40. Content of DHA in cage-free culture was significantly higher than that in the offshore and traditional cage. Content of zinc had no significant difference in three culture models. Content of phosphorus had no significant difference between that in cage-free culture and offshore cage, was lower in the traditional cage. The cage-free cultured croakers had the highest content of calcium and phosphorus. Content of selenium was about the same between the offshore cage and the traditional cage stocking, higher in the cage-free culture. This research has considerable application value for identifying quality and sources of the big yellow croakers.

High genetic diversity and strong differentiation in dramatically fluctuating populations of Zostera japonica(Zosteraceae):implication for conservation

Aims Seagrasses provide a variety of ecosystem goods and services,but they are subjected to frequently anthropogenic disturbances.In this study,we genotyped samples collected from Zostera japonica mead-ows with dramatic fluctuations in the area in order to understand the distribution of genetic variation within and among populations.Methods We collected samples from eight extant populations along coastal areas of southern China.Ten polymorphic microsatellites were adopted to genotype the samples.Parameters of genetic diversity and differentiation were calculated with general software.Important Findings High levels of genetic diversity were found in the studied populations,suggesting that the effective population size has not decreased signifi-cantly,which was supported by no signs of recent bottlenecks.High genetic diversity reflects an important role of sexual seedling recruit-ment in Z.japonica populations.We found a significant relationship between genetic differentiation and the shortest sea surface distance of populations,suggesting that ocean currents play a critical role in shap-ing the genetic structure of Z.japonica populations.STRUCTURE soft-ware analysis clustered the eight populations into two groups:western and eastern populations separated by the Qiongzhou Strait/Leizhou Peninsula,hinting that there was very limited gene flow through the narrow strait in this marine plant.Four populations had high contribu-tion diversity and,thus,high priority for in situ conservation.