Recovery of gonadal development in tiger puffer Takifugu rubripes after exposure to 17β-estradiol during early life stages

The aim of the present study was to investigate the long-term effects of 17β-estradiol(E2) exposure on gonadal development in the tiger puffer( Takifugu rubripe s), which has a genetic sex determination system of male homogametic XY-XX. Tiger puffer larvae were exposed to 1, 10 and 100 μg/L E2 from 15 to 100 days post-hatch(dph) and then maintained in clean seawater until 400 dph. Changes in sex ratio, gonadal structure and gonadosomatic index(GSI) were monitored at 100, 160, 270 and 400 dph. Sex-associated single nucleotide polymorphism(SNP) markers were used to analyze the genetic sex of samples, except those at 100 dph. Exposure had a positive effect on the conversion of genetically male gonads into phenotypically female gonads at 100 dph. However, gonads from 60% of genetic XY males in the 1-μg/L E2 group and 100% in the 10-μg/L E2 group developed intersexual gonads at 160 dph; gonads of all genetic XY males in the two treatment groups reverted to testis by 270 dph. While 38%, 57% and 44% of gonads of XY fish in the 100-μg/L E2 group reverted to intersexual gonads at 160, 270 and 400 dph, respectively, none reverted to testis after E2 treatment. In addition, E2 exposure inhibited gonadal growth of both genetic sexes, as indicated by the clear dose-dependent decrease in GSI at 270 and 400 dph. The results showed that exposure to E2 during the early life stages of tiger puffer disrupted gonadal development, but that fish recovered after migration to clean seawater. The study suggests the potential use of tiger puffer as a valuable indicator species to evaluate the effects of environmental estrogens on marine fish, thereby protecting valuable fishery resources.     

Temperature-control-induced masculinization in tiger puffer Takifugu rubripes

This study used tiger puffer Takifugu rubripes to explore new, simple methods for lowtemperature-induced masculinization in a cultured fish without the use of ultraviolet irradiation or sex hormones. An orthogonal test L_9(3~4) design was used to consider three factors at three levels: treatment starting times(days post-hatch, dph: factor A) of 20, 50 and 80 dph; treatment temperatures(factor B)of 13℃, 15℃ and 17℃; and treatment durations(factor C) of 30, 45 and 60 days. A control group was reared at 21±1℃. The experiments were repeated twice. At 230 dph, the gonads were removed from thirty randomly sampled fish in each group. Histological observations and analysis of single nucleotide polymorphisms(SNP) were used to identified pseudo males, which biological sex was male and genetic sex were female(XX). Treatment group 4(A2B1C2) resulted in the highest proportion of males(75%).According to the intuitive analysis of the orthogonal-array experiments, the optimal combination of lowtemperature-induced masculinization of T. rubripes was A2B1C2. The population sex ratio depended on the three factors in the sequence B→A→C. A comparison of the daily increases in length and weight during and after the low-temperature conditions showed that the absolute daily increases in weight and length were significantly less during treatment than after treatment. Daily increases in weight and length did not significantly differ between the treatment groups and the controls(P0.05), demonstrating that the growth rate could return to normal after the low-temperature conditions. This study establishes a low-temperatureinduced masculinization technology for T. rubripes and demonstrates that although the growth rate(length and weight) decreased in an array of nine treatment groups during the processing time, it returned to a normal level after processing. The results should serve as a guide for achieving the masculinization of T.rubripes in production.     

Reproductive cycle of Ophiopholis mirabilis (Echinodermata: Ophiuroidea) in Zhangzi Island area,northern Yellow Sea

Ophiopholis mirabilis is a common species with a high population density on the western coasts of the northern Pacific Ocean.The number of O.mirabilis has been increasing recently in the scallop aquaculture zone(the Zhangzi Island area,northern Yellow Sea) in China.To explore the mechanism of its population variation,the reproductive cycle of O.mirabilis was investigated in this area(39°04'N;122°51'E) from February 2017 through January 2018 and determined by the monthly gonad index(GI),histological examinations of the gonads and the oocyte size-frequency distribution.O.mirabilis had a clear annual reproductive cycle that was synchronous between males and females.Sea temperature and food availability played important roles in O.mirabilis reproduction.The GI value was less reliable for determining reproductive activity in O.mirabilis because the nutritive tissues within the gonads may be utilized to synthesize gametes,leading to a decrease in GI during maturation.The histological results also show that abundant nutritive phagocytes were present in the gonads of O.mirabilis,which,together with the germ cells,affected the weight of the gonads.In addition,the mature oocytes of O.mirabilis were relatively small(75-150 μm),indicating that the larval development was planktotrophic.This study provided insights into the reproductive patterns and biology of O.mirabilis and is an essential basis for the quantity control of this species in aquaculture areas.     

Effect of hydraulic loading rate on the efficiency of effluent treatment in a recirculating puffer aquaculture system coupled with constructed wetlands

Constructed wetlands(CWs) were integrated into an indoor recirculating aquaculture system of obscure puffer(Takifugu obscurus) for effluent treatment. The effect of hydraulic loading rate(HLR) on the efficiency of effluent treatment by CWs was examined for over a month. The CWs were operated under brackish conditions(salinity 7.4–7.6) at 3 different HLRs(0.762, 0.633, and 0.458 m d–1) 3 times, 10 days each. Overall, the CWs exhibited high efficiency in removal of total ammonium nitrogen(by 81.03–92.81%) and nitrite nitrogen(by 99.40%–99.68%). The efficiency of CWs in removal of total ammonium nitrogen, nitrate nitrogen, total Kjeldahl nitrogen, total phosphorous, and total suspended solids(TSS) increased with the decrease of HLR. The CWs operated at the 3 HLRs in a decreasing trend proves to be effective, providing a useful method for effluent treatment in commercial puffer aquaculture systems.     

Reproductive ecology of the seabob shrimp Xiphopenaeus kroyeri(Heller,1862) in a coastal area of Southern Brazil

The predictability of certain environmental factors that affect the life cycle of the seabob shrimp Xiphopenaeus kroyeri(Heller,1862) was evaluated in a study of its reproductive biology in an area adjacent to Babitonga Bay,State of Santa Catarina,Brazil. Monthly sampling was conducted from July 2010 through June 2011 at depths of 5,8,11,14,and 17 m.76 004 individuals were obtained,with a pronounced peak in absolute abundance in austral autumn(34 208),coinciding with the annual closed season from March to May. Grain size composition of the sediment showed the closest relationship to the distribution of individuals(multiple linear regression,P 0.05),related to their burying habit. The observed correlations between the abundance of reproductive males(bearing spermatophores) and females with spent gonads(cross-correlation,P 0.05),and between reproductive males and reproductive females(with a 1-month lag) suggest that the peak of reproductive males preceded the peak offemale ones. This result agrees with the pattern expected for females,which copulate in post-ecdysis(spent gonads). Spawning seemed to take place at greater depths,as evidenced by the concentration of reproductive females in these areas. The reproductive activities observed here confirm that this species follows a tropical/subtropical reproductive pattern,spawning continuously throughout the year,with the highest peaks in spring and autumn. The data indicate that the juvenile recruitment period observed in August-September resulted from the reproductive output noted in April–May. Additionally,the reproductive period recorded in November led to the juvenile peak observed in March–May.     

Estimated reproductive success of brooders and heritability of growth traits for large yellow croaker (Larimichthys crocea) using microsatellites

We estimated the reproductive success of adult brood fish and the heritability of growth-related traits for large yellow croaker (Larimichthys crocea). We created two independent full-factorial cross groups (1 and 2) by crossing 4 males×4 females and 4 males×3 females, respectively. We measured the body weight (BW), body length (BL), and body height (BH) of 281 individuals from group 1 and 318 individuals from group 2 at 20 months post hatch (harvest age). We also collected a tissue sample from each individual. The parents and offspring were genotyped using six polymorphic microsatellites. Of the 599 offspring, 99.2% were assigned to a single pair of parents. In both groups, some parent pairs produced a large number of offsprings while other pairs did not produce any offspring. The genetic diversity and putative Ne were lower in the offsprings than in the parents in both groups. The heritability estimates at 20-month age were 0.13±0.10 for BW, 0.19±0.13 for BL, and 0.09±0.06 for BH. The genetic and phenotype correlation between BW, BL, and BH was close to 1. The results provide basic information for selective breeding and further genetic characterization of large yellow croaker.     

Genetic variation of natural and cultured stocks of Paralichthys olivaceus by allozyme and RAPD

Population genetics of the left-eyed flounder, Paralichthys olivaceus, including natural and cultured stocks distributed in the coastal waters near Qingdao of eastern maritime China, was analyzed in allozyme and RAPD. The results showed that among total 29 gene loci of 15 isozymes, 9 and 7 were po- lymorphic in natural and cultured stocks, respectively. The status of genetic diversity in P. olivaceus is low in terms of polymorphic loci in chi-square test and genetic departure index of Hardy-Weinberg equi- librium. More alleles in IDHP, CAT, GDH and Ldh-C allozymes were found in the fish, which could be used as markers in assortive breeding and distinguishing stock, population or species evolution. Total 88 and 86 RAPD bands ranging from 200 to 2 500 bp were recognized individually in average of 7.8–8.0 bands per primer. The genetic diversity in cultured stock is lower than that in natural ones showing an ob- viously decreasing genetic divergence. Therefore, effective countermeasures must be taken to protect ge- netic resources of marine cultured fishes. The 2 markers have their own pros and cons. Combining the 2 markers to investigate the genetic variation of populations is suggested. The results provide basic data of this flounder and they are useful for studying genetic improvement and genetic resources of the fish.     

Transcriptome Analysis of Crassostrea sikamea (♀) × Crassostrea gigas (♂) Hybrids Under and After Thermal Stress

Crossbreeding is an effective approach to manage the genetic decline in aquaculture.One-way hybrids of Crassostrea sikamea(♀)and Crassostrea gigas(♂)have advantages in growth traits and adaptation to high temperature.Here,we used high-throughput sequencing to analyze the molecular processes in the hybrids under and after thermal stress.The hybrids were cultured in the seawater with an increasing temperature from 25℃to 40℃during 10 hours,which is regarded as the thermal stress stage.Then the temperature decreased from 40℃to 25℃within 2 h,which is regarded as the recovery stage.In this study,1293 significant diffe-rentially expressed genes(DEGs)were obtained under thermal stress,of which 576 were upregulated and 717 were downregulated,and 740 significant differentially expressed genes(DEGs)were obtained in the recovery stage,of which the number of upregulated and downregulated genes was 409 and 331,respectively.The antigen processing and presentation,NOD-like,and NF-kappa B path-ways were significantly enriched during the thermal stress stage.The MAPK and PPAR signaling pathways were significantly enrich-ed during the recovery stage.The HSP70,HSP90,and CANX genes were strongly and rapidly upregulated in the control/thermal stress groups but were slightly less upregulated in the thermal stress/recovery group.These results indicate that the innate immune system or nonspecific immunity was deployed to protect interior tissues from thermal stress.In addition,85%of the mutual DEGs were involved in bidirectional regulation(up/down or down/up)when the oysters were removed from the thermal stress to recover.This study provides preliminary insight into the molecular response of C.sikamea(♀)and C.gigas(♂)hybrids to thermal stress and provides a basis for future studies on temperature-adaptation and the possible expansion of hybrid breeding.     

Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus(Coccolithoviruses) serine palmitoyltransferase(SPT) gene by electroporation

Emiliania huxleyi is the most prominent modern coccolithophore,a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry.Coccolithoviruses are large double stranded DNA viruses,which is responsible for the demise of large oceanic blooms formed by E.huxleyi.E.huxleyi virus(EhVs) acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transferbetweenvirus-host Curre ntly,there is limited experimental validation identifying the functions of these genes in EhV.Genetic transformation of eukary otic cells is a powerful tool to get an insight into gene functions of the studied organisms.Serine palmitoyltransferase(SPT) catalyzes the first committed step in de novo sphingolipid biosynthetic pathway.Here,a novel vector system for the transformation of E.huxleyi was designed.It contained fragments of promoter and terminator sequences of E.huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene "fcp" and harbored EhV-99 B1 spt gene.The resultant recombinant transformation vectors pEhux-Ⅰ-spr and pEhux-Ⅱ were co-transferred into E.huxleyi BOF92 by electroporation.Transformants were obtained upon glufosinate-ammonium selection,and confirmed by Southern hybridization,genome PCR,qRT-PCR and Western blot screening of spt gene,which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels.The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography(TLC).The results present the genetic transformation system for E.huxleyi,providing additional genetic resource with potential for exploring basic biological questions such as the virus-ho st interactions.     

Estimation of Age,Growth and Maturation of Purpleback Flying Squid,Sthenoteuthis oualaniensis,in Bashi Channel,Central Pacific Ocean

The age, growth and maturation of Sthenoteuthis oualaniensis were determined with statolith data collected with a light purse seine from the Bashi Channel of central Pacific Ocean. The estimated longevity of the squid was no more than 6 months for females, and no more than 5 months for males. Growth in mantle length(ML) was best described by logistic models for both females and males, while growth in body weight(BW) was best fitted by power curves. The maximum absolute growth rate(AGR) and instantaneous growth rate(IGR) in ML or BW both occurred at 91–105 days for females and 76–90 days for males. Back calculated hatching dates were from October to January, with a peak in December, although the short duration of sampling date might have had an influence on the result. The lower percentage of mature females(37.2%) suggested that the study area during the sampling date was not a spawning ground for the species. Size and age at first maturity were 183 mm ML and 136 days for females, whereas they were 156 mm ML and 85 days for males.     

Steroid sex hormone dynamics during estradiol-17β induced gonadal differentiation in Paralichthys olivaceus(Teleostei)

Steroid sex hormones,such as estradiol-17β(E2)and testosterone(T),are important regulators of sex change in fish.In this study,we examined the effects of E2 treatment on the dynamics of E2 and T during gonadal differentiation in the olive flounder Paralichthys olivaceus using histology and radioimmunoassay(RIA).Flounder larvae were divided into five groups(G0–G4),and fed with 0 (control),0.2,2,20 and 100 mg E2/kg feed from 35 to 110 day post hatching(dph).Fish growth in the G1 and G2 groups was not significantly different from that of the control group(P0.05),while fish in the G3 and G4 groups were less active and showed growth depression and high mortality.The gonads of fish in the G3 and G4 groups were smaller and surrounded by hyperplastic connective tissue.The frequency of females in the G0–G4 groups was 54.5%,75.0%,100%,100%and 93.3%,respectively.The RIA analyses of E2 and T showed that T levels decreased during gonadal differentiation,and increased slightly at the onset of ovarian differentiation,while E2 levels increased gradually and peaked at the onset of ovarian differentiation in the control group.In the E2-treated groups,T levels decreased before the onset of ovarian differentiation.E2 levels were high on the 48 dph,but declined to a lower level on the 54 dph, and then increased gradually during gonadal differentiation.And a sharp increase of E2 levels were observed in all E2-treated groups at the onset of ovarian differentiation.The data suggest that T and E2 play important roles during gonadal differentiation,and an E2 dose of 2 mg/kg feed could induce sex reversal in P.olivaceus.     

Land use and land cover changes in small Carpathian catchments between the mid-19~(th) and early 21~(st) centuries and their record on the land surface

Land use and land cover(LULC) changes and their impact on the mountain environment were studied in six catchments(～10 km~2 each) in the Polish Western Carpathians from the mid-19~(th) century to the early 21~(st) century. The analysis of cadastral and orthophoto maps indicates that during the investigated period, the forest area increased, quantified by an annual change index(Annch), between +0.12% to +0.27%, with a decrease of arable land index to –0.45% and –2.28% in the analysed catchments. LULC changes were accompanied by a continuous increase in settlement developments(residential and farming houses) by 50%-140% as well as significant changes related to their spatial distribution. Abandonment of arable land and forest succession have resulted in the geomorphological transformation of hillslopes, which predominantly includes a decrease in used road density, their transformation to road cuts and gorges. Overpopulation and the domination agriculture in the past caused the expansion of unpaved roads density and then the fragmentation of hillslopes, as well as the development of agricultural terraces.     

Effect of Hydraulic Loading Rate on the Efficiency of EffluentTreatment in a Recirculating Puffer Aquaculture SystemCoupled with Constructed Wetlands

Constructed wetlands （CWs） were integrated into an indoor recirculating aquaculture system of obscure puffer （Takifuguobscurus） for effluent treatment. The effect of hydraulic loading rate （HLR） on the efficiency of effluent treatment by CWs was ex-amined for over a month. The CWs were operated under brackish conditions （salinity 7.4-7.6） at 3 different HLRs （0.762, 0.633, and0.458 m d-1） 3 times, 10 days each. Overall, the CWs exhibited high efficiency in removal of total ammonium nitrogen （by81.03-92.81%） and nitrite nitrogen （by 99.40%-99.68%）. The efficiency of CWs in removal of total ammonium nitrogen, nitratenitrogen, total Kjeldahl nitrogen, total phosphorous, and total suspended solids （TSS） increased with the decrease of HLR. The CWsoperated at the 3 HLRs in a decreasing trend proves to be effective, providing a useful method for effluent treatment in commercialpuffer aquaculture systems.     

Genetic study of Kelp “901” strain

Based on DNA extraction and optimization of random amplified reaction (RAPD) to the gametophytes and sporophytes of Kelp “901” strain, genetic study on variation was conducted to its parents and offsprings of F6, F7,Fs, and F9 generation. RAPD results have shown that among 30 selected primers for gametophytes, 297 loci ranging from 200 to 3 000 bp were obtained in the average of 9,9 loci for each primer, This indicated a high polymorphic rate with RAPD detection. UPGMA (unweighted pair-group method arithmetic average) analysis showed that each male and female gametophyte of a generation could be clustered into one pair separately. The genetic distances of the Kelp 901 generation were 0.321 2-0.476 7, and the maximum was between F7 and Fs (0.476 7). Identity analysis showed that F6 generation was more close to the female parent (0.659 3), and F7 generation was more close to the male parent (0.5788). To the sporophytes study in 24 selected primers for RAPD amplification, 191 loci ranging from 230-2 800 bp were obtained, in the average to each primer of 8.0 loci. The heterozygosity to six populations were male parent (0.223 9),female parent (0.107 2), F6 (0.216 4), F7 (0.228 6), F8 (0.229 6) and F9 (0.317 2). The nearest genetic distance was 0.083 5 (Fs, F9). Total heterozygosity (HT) ofF6, F7, F8 and F9 generations was 0.318 6, the average heterozygosity (Hs) for F6,F7, F8 and F9 generations was 0.248 0, and deduced coefficient of population differentiation (Gst) was 22.2%. Six sequence characterized amplified regions (SCAR) were preliminary screened through RAPD analysis. It needed to be verified in detail as they are significant for molecular marker assistance in breeding and selecting Laminaria.     

Seasonal Variation of Biochemical Components in Clam(Saxidomus purpuratus Sowerby 1852) in Relation to Its Reproductive Cycle and the Environmental Condition of Sanggou Bay,China

Seasonal variation of biochemical components in clam(Saxidomus purpuratus Sowerby 1852) was investigated from March 2012 to February 2013 in relation to environmental condition of Sanggou Bay and the reproductive cycle of clam.According to the histological analysis,the reproductive cycle of S.purpuratus includes two distinctive phases:a total spent and inactive stage from November to January,and a gametogenesis stage,including ripeness and spawning,during the rest of the year.Gametes were generated at a low temperature(2.1℃) in February.Spawning took place once a year from June to October.The massive spawning occurred in August when the highest water temperature and chlorophyll a level could be observed.The key biochemical components(glycogen,protein and lipid) in five tissues(gonad,foot,mantle,siphon and adductor muscle) were analyzed.The glycogen content was high before gametogenesis,and decreased significantly during the gonad development in the gonad,mantle and foot of both females and males,suggesting that glycogen was an important energy source for gonad development.The protein and lipid contents increased in the ovary during the gonad development,demonstrating that they are the major organic components of oocytes.The lipid and protein contents decreased in the testis,implying that they can provide energy and material for spermatogenesis.The results also showed that protein stored in the mantle and foot could support the reproduction after the glycogen was depleted.     

Genetic differentiation of Japanese sardinella (Sardinella zunasi) populations in the Northwest Pacific revealed by ISSR analysis

Knowledge of population genetic structure plays an important role in fisheries management.In this research,In-ter-Simple-Sequence-Repeat(ISSR)markers were employed to evaluate the genetic structure of Japanese sardinella(Sardinella zun-asi)populations in the Northwest Pacific.Eighty seven individuals from 5 locations were screened using 4 highly polymorphic prim-ers.A total of 173 polymorphic loci were detected out of 191 loci amplified.Small but significant genetic differentiation was de-tected between the Chinese and Japanese populations by both AMOVA and pairwise FST analyses,which was further supported by cluster analysis.We consider that climate change during glaciations should be responsible for the genetic differentiation.Isolation by geographic distance among populations was observed,indicating that the distance might also lead to the genetic differentiation.However,no genetic structure was found within the populations off both the Chinese and Japanese coasts,indicating a high-level along-coast gene flow,which might result from ocean current transport and common ground for over-wintering.