Histological characterization of gonadal sex differentiation in Nibea albiflora

The aim of this study was to investigate the period of sex differentiation and the pattern of gonadal differentiation in Nibea albiflora using histological techniques. The fish were raised in temperature between 19.5 and 21.5°C. The promordial gonads formed at the posterior region of the abdomen and the fish remained undifferentiated till the 36 day post hatching (dph) with a total length (TL) of 21.20 ± 3.90 mm. Ovarian differentiation precedes testicular differentiation, as usual in teleost fish. Oocytes undergoing meiosis at zygotene (bouquet) and pachytene stages were firstly observed when the total length reached 33.05 ± 4.61 mm at 40 dph, and the formation of the ovarian cavity began simultaneously at 46 dph with total length of 49.40 ± 4.83 mm. When the mean total length reached 78.05 ± 5.24 mm (61 dph), ovarian cavity grew bigger, and oocytes were firstly detected. By contrast, signs of morphological differentiation in the presumptive testis are not observed until their total length reached 69.18 ± 5.26 mm at 55 dph. The unrestricted lobular organization of the testis is evident at the stage of TL = 78.05 ± 5.24 mm (61 dph). These findings indicate that N. albiflora is a differentiated gonochorist and the critical period of sex differentiation in N. albiflora occurs at the stage of TL = 21.20 ± 3.90 mm (36 dph).     

The effects of dietary vitamin C on growth performance,serum enzymes activities and resistance to Vibrio alginolyticus challenge of yellow drum Nibea albiflora

A 9‐week feeding trial was conducted to determine the optimal dietary vitamin C requirement and its effects on serum enzymes activities and bacterial resistance in the juvenile yellow drum Nibea albiflora (initial weight 33.2 ± 0.10 g). Six practical diets were formulated containing vitamin C 2.1, 45.3, 89.6, 132.4, 178.6 and 547.1 mg kg^?1 diet supplied as l ‐ascorbyl‐2‐monophosphate. The fish fed 547.1 mg kg^?1 diet showed a significantly higher survival than that fed 2.1 mg kg^?1 diet. The weight gains and specific growth rate of the fish fed 2.1 mg kg^?1 diet were significantly lower than those of the fish fed 89.6–547.1 mg kg^?1 diets. The liver vitamin C concentration firstly increased with increasing dietary vitamin C supply from 2.1 to 178.6 mg kg^?1 diet and then stabilized. The serum superoxide dismutase activities of the fish fed 547.1 mg kg^?1 diet were significantly lower than those of the fish fed 2.1–89.6 mg kg^?1 diet. The fish fed 2.1 mg kg^?1 diet had a significantly higher alkaline phosphatase activity than those in the other groups except the 45.3 mg kg^?1 group. Fish that received diets containing vitamin C at 547.1 mg kg^?1 had significantly higher nitro blue tetrazolium and lysozyme activity, and fish that received diets containing vitamin C at 45.3–547.1 mg kg^?1 exhibited resistance against Vibrio alginolyticus infection. The dietary vitamin C requirement of the juvenile yellow drum was established based on broken‐line model of weight gain to be 142.2 mg l ‐ascorbyl‐2‐monophosphate kg^?1 diet.     

Partial replacement of fish oil with terrestrial lipid blend and effects on growth performance,body composition,immune parameter and growth‐related genes in yellow drum (Nibea albiflora)

The imbalance of fish oil (FO) supply and demand has motivated efforts to identify an alternative for aqua feed. An 8‐week feeding trial was conducted to evaluate the effects of dietary fish oil partial replacement with mixed oil from linseed and lard (1:1) on growth, body composition and immunity when the N3 long‐chain polyunsaturated fatty acid (N3 LC‐PUFAs) requirement is met for Nibea albiflora. Two types of experimental diets were formulated with 100% fish oil (FO) or 69% mixed oil (Mix). The results indicated that the Mix diet significantly improved the specific growth rate (SGR), weight gain (WG), feed efficiency ratio (FER), muscle tissue growth hormone receptor (GHR), insulin‐like growth factor 1 (IGF1) and insulin‐like growth factor 1 receptor (IGF1R) gene expression of the yellow drum (p < .05). No significant difference in the survival rate (SR), feed intake (FI), hepatosomatic index (HSI), viscerosomatic index (VSI), condition factor (CF), N3 PUFA percentage, liver antioxidant enzyme activities or pro‐inflammatory gene expression was observed between the two treatments. Therefore, terrestrial blend lipid from linseed and lard could be used as an alternative for dietary fish oil without compromising growth performance and immunity for the juvenile yellow drum.     

Formulated diet alters gut microbiota compositions in marine fish Nibea coibor and Nibea diacanthus

Nibea, an economical marine fish, is generally fed on trash fish (the low‐value fish), which can cause high feed costs and waste pollutions in high‐density aquaculture. To assess the effect of formulated diet on the gut microbiota in Nibea coibor and Nibea diacanthus, denaturing gradient gel electrophoresis, clone libraries analysis and Illumina sequencing of the 16S ribosomal RNA gene were used in this study. Two Nibea fishes were both dominated by Gammaproteobacteria (especially Photobacterium) and shared a set of gut microbiota, including Bacilli, Mollicutes, Alphaproteobacteria and Fusobacteriia. Statistical analyses revealed that formulated diet led to lower feed conversion ratio (p < 0.001), lower abundance of Vibrio (p = 0.040) and infectious diseases pathways (p = 0.001), higher abundance of polysaccharide‐degrading bacterium Cellvibrio (p = 0.006) in two Nibea species, with higher weight gain rate (p = 0.023) and microbial diversity (Shannon, p = 0.049 and Simpson, p = 0.044) and more carbohydrate metabolism (p = 0.020) observed in N. coibor. The distribution and correlation network of 17 potential short‐chain fatty acid producing bacteria were obtained and visualized in all treatment groups. The results reveal that formulated diet has beneficial effects on the gut microbial ecology in two Nibea fishes, which suggests the possibility of replacing trash fish diet with formulated diet in Nibea aquaculture.     

Efficacy of 17 α‐methyl testosterone and letrozole on sex reversal of protogynous grouper,Epinephelus tauvina (Forskal, 1775) and spawning performance of sex‐reversed males

This study aimed to test the efficacy of 17 α‐methyl testosterone (17 α‐MT) alone and in combination with letrozole, an aromatase inhibitor, for the induction of sex reversal in protogynous greasy grouper, Epinephelus tauvina. Further, the long‐lasting effects of these treatments and spawning performance of sex‐reversed males were also investigated. Greasy grouper with oocytes in the perinucleolus stage were implanted with 5 mg 17 α‐MT kg^?1 body weight (T₁), 5 mg 17 α‐MT and 0.2 mg letrozole kg^?1 body weight (T₂) and 5 mg 17 α‐MT with 0.4 mg letrozole kg^?1 body weight (T₃) and no androgens/enzyme inhibitor implanted (C). The 17 α‐MT alone and in combination of letrozole‐induced sex reversal in greasy grouper, whereas untreated control fish (C) showed normal ovarian development. However, T₂ and T₃ group showed 100% sex reversal and completion of spermatogenesis up to functional male phase in 2 and 3 months, respectively, whereas T₁ group resulted in only 66.67% functional male with motile spermatozoa after 4 months. Sex‐reversed males successfully fertilized the eggs during induced spawning. There were significant differences on fertilization and hatching rates between T₂ group (79.00 ± 4.36%; 77.67 ± 2.87%, respectively) and T₁ group (57.67 ± 3.17%; 63.87 ± 2.91%, respectively). The result suggested that 17 α‐MT (5.0 mg kg^?1 BW) in combination with letrozole (0.2 mg kg^?1 BW) has the potential to produce 100% sex‐reversed male in short period in greasy grouper, which might greatly help in seed production of greasy grouper.     

Role of sex steroids in gonadal differentiation of the mussel Choromytilus chorus (Bivalvia Mytilidae) (Molina 1782)

In Chile, the species Choromytilus chorus stands out for its ecological and economic importance. This mussel has a very particular condition in the colour of the gonad, being cream‐yellow colour the male gonad and dark brown the female which is hardly desired by the consumers. In this context, the aim of this research is to determine the role that sex steroids have in gonadal differentiation of the mussel C. chorus. For this, juveniles with sizes 15–22 mm were selected, which were subjected to acute exposure in the form of bath for 60 days to four treatments: T1: dihydrotestosterone (DHT); T2: 17β‐estradiol (E2), T3: fadrozole (F) and T4: DHT‐F (DF), plus a control without chemicals. Each treatment as well as control included three replicates with 90 individuals each. The mussels undergoing treatment with E2 had a sex ratio (male:female) of 0.47 compared to 1:1 in the control group. In contrast, in the groups treated with DHT, F and DF the sex ratio changed to 2.0, 1.60 and 1.70 respectively. In the fertilization trials, all the mussels produced functional gametes, as they were able to generate gametes that were developed to morula, and veliger larva. Nevertheless, it should be noted that the size of oocytes from the group exposed to estradiol (61.12 ± 2.40 μm) was significantly higher than in other groups. These results support the hypothesis that sex steroids would be involved in sexual differentiation of marine bivalves.     

舟山近海黄姑鱼胚胎发育及前期仔鱼形态的观察

通过对黄姑鱼胚胎发育全过程的连续观察,描述了黄姑鱼胚胎发育的特征、发育速度及前期仔鱼的形态特征等。观察结果表明,黄姑鱼的胚胎发育与其它硬骨鱼浮性卵鱼类基本相似,属端黄卵,盘状分裂。在水温22.0~22.5℃、盐度26.5的条件下,黄姑鱼受精卵经24h15m in孵出仔鱼。初孵仔鱼全长1.75±0.09mm,孵出后约50h,卵黄囊完全被吸收。     

Effects of dietary proline on swim bladder collagen synthesis and its possible regulation by the TGF‐β/Smad pathway in spotted drum,Nibea diacanthus

Swim bladder is an ideal source of collagen production in fish for improved human health. Proline (Pro) is the main proteinogenic amino acid needed for collagen production. However, the effects of Pro supplementation on the swim bladder collagen synthesis have rarely been evaluated in fish. We determined the effects of dietary Pro supplementation on swim bladder collagen synthesis and its possible signalling pathway in spotted drum, Nibea diacanthus. A total of 450 N. diacanthus (100 ± 3.05 g) were randomly assigned into six treatments and fed with diets supplemented with different levels of Pro (0, 2.5, 5, 7.5, 10 and 12.5 g/kg of dry diet, hereafter P0, P1, P2, P3, P4 and P5, respectively) for 8 weeks. At the end, we evaluated collagen synthesis in swim bladder and the expression of genes related to TGF‐β/Smad pathway in the fish. Dietary Pro levels increased significantly the contents of crude protein, total collagen (TC) and the levels of some amino acids in swim bladder than the control diet (p < .05). The optimum amount of dietary Pro inclusion in diets for swim bladder collagen synthesis in N. diacanthus was 7.6 and 7.5 g/kg based on crude protein and TC in swim bladder, respectively. Dietary Pro levels increased significantly the proline 4‐hydroxylase (P4H) content in fish serum, swim bladder, muscle and liver tissues than control (p < .05). The relative expression of collagen type I alpha 1 (COL1A1), alpha 2 (COL1A2) and mothers against decapentaplegic homolog 2 (Smad2) genes in liver and swim bladder initially increased significantly as the concentration of Pro and later decreased (p < .05). Similarly, the relative expression of transforming growth factor beta (TGF‐β), P4Ha2 and P4Ha3 genes in the swim bladder increased significantly as dietary Pro levels increased (p < .05). Using K‐means clustering analysis, dietary proline partly promoted collagen accumulation in swim bladder through upregulation of Smad2 and TGF‐βRT genes. Taken together, Pro affected the collagen metabolism in swim bladder, probably by regulating the TGF‐β/Smad pathway, most likely via transient overexpression of Smad2 gene.     

Increased gonad growth of the purple sea urchin (Strongylocentrotus purpuratus) fed the giant kelp (Macrocystis pyrifera) and the sea lettuce (Ulva lactuca) enriched with nutrients

Adult (Strongylocentrotus purpuratus) purple sea urchins were fed giant kelp (Macrocystis pyrifera) and sea lettuce (Ulva lactuca) macroalgae that were unenriched and enriched with nutrients. Urchins were fed over 10 weeks (5% body weight per day) under laboratory conditions to determine the effect of macroalgae species and treatment (enriched and unenriched) on growth, gonad index and gonad quality. Significant differences were observed when urchins were fed enriched Macrocystis pyrifera (MPE). Gonad weight increased from 0.7 ± 0.1 g (mean ± SD) to 5.9 ± 1 g, and gonad index increase from 3.5 ± 1.5% to 14.9 ± 2.3% over 10 weeks. Significant differences were also noted in gonad quality, and gonad colour and firmness were better in urchins that were fed unenriched M. pyrifera (MPUE) and MPE – whereas texture did not differ between M. pyrifera treatments and unenriched U. lactuca (ULUE). With regard to taste, gonads were sweeter in MPE‐fed individuals. Enriched U. lactuca (ULE) failed to grow gonads significantly; as a result, their quality was unacceptable for the market. These results suggest that S. purpuratus experience increased gonad production and quality in short periods when fed nutrient‐enriched M. pyrifera.     

Effect of hydroxyproline supplementation on growth performance,body composition,amino acid profiles,blood‐biochemistry and collagen synthesis of juvenile chu's croaker (Nibea coibor)

Hydroxyproline (Hyp) may play an important role in collagen metabolism, as it is almost exclusively found in collagen. To examine this possibility, a 56‐day feeding trial was conducted to evaluate the effect of graded supplementaries of Hyp (0, 2.5, 5, 7.5, 10 and 12.5 g/kg dry diet weight) on growth performance, feed utilization, biochemical composition and collagen synthesis of chu's croaker, Nibea coibor (13.6 ± 0.28 g). The growth performance and feed utilization were improved with increased levels of dietary Hyp, reaching a maximum at 5 g/kg (diet H2) followed by a slight decrease. Polynomial regression analysis showed that the maximum specific growth rate (SGR) was attained at 7.177 g/kg dietary Hyp. Increasing levels of Hyp did not affect body composition of juvenile Nibea coibor. However, dietary Hyp supplementation significantly affected the levels of Leu, Ile, Met, Lys, Ser, Glu and Pro in muscle and the levels of Met, Lys, Glu, Pro and Gly in swim bladder. The serum calcium (SC) level decreased significantly with increasing levels of dietary Hyp. However, the triglycerides (TG) and total cholesterol (T‐CH) levels in serum and liver were relatively stable. Importantly, the acid‐soluble collagen (ASC) and total collagen (TC) levels in swim bladder increased significantly initially and then decreased after reaching a maximum. Polynomial regression analysis revealed that the maximum TC level in swim bladder was attained at 9.736 g/kg of dietary Hyp. Collectively, these results suggest that the appropriate provision of hydroxyproline is essential for maximal collagen synthesis and growth.     

The development of a formulated feed containing Ulva (Chlorophyta) to promote rapid growth and enhanced production of high quality roe in the sea urchin Tripneustes gratilla (Linnaeus)

This study investigated growth and gonad production of Tripneustes gratilla fed four protein‐rich artificial diets supplemented with varying amounts (0%, 5%, 15% and 20% weight/weight (w/w); designated 0, 5, 15 and 20U, respectively) of the macroalga Ulva over a 12‐week period. Gonad size, texture, colour and a number of production performance parameters were quantified and compared with urchins fed fresh Ulva (FU) and a combination of FU and artificial feed (FB). All artificial diets significantly (P ≤ 0.05) increased gonad somatic indices (GSI) compared with the FU treatment. The 20U treatment increased GSI by 205% by week 9, compared with a 57% increase in the GSI of urchins fed FU. Gonad colour was calculated using three colour parameters, namely L* (lightness), a* (redness) and b* (yellowness). Whereas L* and a* values did not differ significantly between treatments, b* values for all treatments, with the exception of the 20U and FB treatments, were significantly (P ≤ 0.05) lower or less yellow than the FU treatment by week 12. These results show that we have successfully formulated a feed (20U) which can produce commercially acceptable gonads in terms of both size and colour, indicating the potential for this artificial feed to support commercial echinoculture.     

Effect of dietary vitamin C on growth performance,body composition and biochemical parameters of juvenile Chu's croaker (Nibea coibor)

Vitamin C is an essential micronutrient for normal physiological and immune functions of fish. However, its requirements and effects in Chu's croaker (Nibea coibor) are currently unknown. A 56‐day feeding trial was conducted to evaluate the optimal dietary vitamin C requirements based on its effects on growth performance, body composition and biochemical parameters in juvenile Chu's croaker (14.17 ± 0.1 g). Six isoproteic (450 g/kg crude protein) and isolipidic (100 g/kg crude lipid) diets were formulated to contain 2.24 (basal diet), 39.03, 85.01, 171.16, 356.49 and 715.46 mg/kg of vitamin C. The results showed that fish fed on 171.16 mg/kg vitamin C diet had the highest growth performance and feed utilization. Fish fed on the basal diet had higher malondialdehyde (MDA) content and lower activities of antioxidant enzymes in the serum and liver as compared with those fed on vitamin C diets. Polynomial analysis indicated that the optimal dietary vitamin C requirements of juvenile Chu's croaker were 102.28, 98.21, 150.26, 165.38, 71.46, 176.19, 84.84 and 103.78 mg/kg based on weight gain, specific growth rate, liver storage, muscle storage, liver MDA content, liver alanine aminotransferase activity, liver alkaline phosphatase activity and liver superoxide dismutase activity, respectively. We recommend an inclusion level in the range of 71.46–150.26 mg/kg vitamin C in the diets of juvenile Chu's croaker for optimum growth performance, liver function, antioxidant capacity and innate immunity functions.     

Effect of ration on gonad development of the Pacific geoduck clam,Panopea generosa (Gould, 1850)

The effect of ration on Panopea generosa gonad development was tested over 52 days. Clams were fed Isochrysis sp. and Chaetoceros muelleri (50 : 50 cell count) at rations of 0.8 × 10⁹, 2.4 × 10⁹, 4.0 × 10⁹, 5.6 × 10⁹, 7.2 × 10⁹ and 10.0 × 10⁹ cells clam^?1 day^?1 (R₁, R₂, R₃, R₄, R₅ and R₆, respectively). The highest ration (R₆) caused a 25% die‐off within 3 days and was discontinued. Ration did not significantly affect condition index, gonadosomatic index, connective tissue occupation index or oocyte diameter. Clams fed the R₅ ration (85% of which spawned from day 26 to 52) were more spent than clams in any other treatment with significantly fewer oocytes mm^?2 than those fed the R₁, R₂ and R₃ rations and significantly lower levels of sperm occupation than clams fed any other ration. Spawn percentages were low from day 26 to 52 in R₁, R₂ and R₄ (15, 0 and 0%, respectively). Clams in the R₃ treatment had a similar spawn percentage (100% from day 26 to 52) to those in the R₅ treatment yet maintained gonads in a more ripened condition with higher levels of gamete occupation, making R₃ the most likely ration to maximize gamete output over time.     

Effect of temperature and salinity on sex inversion in Asian Seabass (Lates calcarifer): relationship with plasma sex steroids concentration and aromatase activity of gonad and brain

Asian Seabass which shows precocious sex inversion in cultured environment is a major impediment to maintain a proper broodstocks. Seabass grown commercially in freshwater were transported to the research facility and held in freshwater at 29°C for 4 weeks and acclimatized to the experimental conditions. Fish were daily fed with a commercial pellet (50% protein, 18 MJ kg^?1) to satiety. Blood, brain and gonad collected before and at the end of the experiment were analysed for sex steroids level and aromatase activity. There was an increase in Plasma E₂ levels with temperature in fish at 34°C whereas no significant difference was observed at 24 and 29°C although the highest plasma T level was detected in fish at 34°C which had significantly lower level than at the beginning, except those held at 24°C in freshwater. Plasma 11‐KT was significantly greater in fish at 24°C compared with 29 or 34°C which had clear opposite to the E₂. Aromatase activity in brain was higher at 29°C than at either 24 or 34°C, whereas gonadal aromatase recorded highest at 34°C. These findings concluded that high temperature in culture facility induces sex inversion of Asian Seabass.     

Effects of androgenic gland ablation on growth,sexual characters and spermatogenesis of the white shrimp,Litopenaeus vannamei (Decapoda: Penaeidae) males

This research was implemented to study the effects of androgenic gland ablation in the white shrimp Litopenaeus vannamei to explore sex reversal potential as an alternative technology for monosex female mariculture based on the sex dimorphic growth of this species. The surgical procedure was applied to male postlarvae (PL) at different ages, after external sex differentiation, as well as in sex undifferentiated PL. Andrectomized males regenerated normal appendices including pereiopods and pleopods; however, body growth and relative size of regenerated petasmas and appendices masculinae were statistically inferior (P ? 0.05) to control males. Spermatogenesis in andrectomized males was active, but a phenomenon of degradation of spermatids and reproductive tissues was detected. No sex reversal was accomplished regardless of PL age from sex undifferentiated stages (PL38) to sex differentiated stages (?PL55). The complete regeneration of sexual characters in andrectomized L. vannamei (Dendrobranchiata) is different from previous reports from Decapoda.     

Effects of dietary sodium butyrate on growth,diet conversion,body chemical compositions and distal intestinal health in yellow drum (Nibea albiflora,Richardson)

The present study was conducted to investigate the effects of sodium butyrate (SB) on the physical barrier function, pro‐inflammatory response and possible underlying mechanisms in the distal intestine (DI) of yellow drum when fed a high‐SBM diet. Three iso‐proteic and iso‐lipidic diets were formulated with fish meal (FM, the control), 45% fish meal protein replaced by SBM (SBM) and 45% fish meal protein replaced by SBM but supplemented with 0.15% SB (SBM + SB). Fish were fed twice daily for 10 weeks. Results indicated that SB supplementation significantly increased the specific growth rate (SGR) and feed efficiency ratio (FER) and methionine content of muscle when compared with those of fish fed the SBM diet (p < .05). The morphologic histology results showed that SB dramatically improved the physical barrier structure, characterized as increases of fold height (FH) and muscular thickness (MT) (p < .05). RT‐qPCR data were accordant with morphologic histology results, in which claudin 3, claudin 4 and occludin were increased while claudin 7 and myosin light chain kinase (MLCK) mRNA expression levels were decreased (p < .05). Sodium butyrate also exerted anti‐inflammatory function, which may be attributed to the suppression of nucleus p65 protein expression. Results suggest SB can be incorporated in high‐SBM diets to ameliorate the negative consequences of alternative dietary ingredients on yellow drum physiology.     

大黄鱼雌雄性腺长链非编码RNA的挖掘与差异分析

为探究长链非编码RNA在大黄鱼性腺发育与分化中的作用,从雌雄各3尾大黄鱼(Larimichthys crocea)的性腺中提取总RNA,进行去除rRNA的链特异性转录组建库和二代测序。将测序数据比对到大黄鱼参考基因组,经比对、组装共得到来自31675个基因的66088个转录本,严格筛选得到来自3984个基因位点的5162条lncRNA。进一步分析获得了在大黄鱼雌雄性腺中差异表达的mRNA9341个,lncRNA2782个,高度相关的lncRNA-mRNA对1227个;有多个lncRNA靶向已知的性别分化和发育相关基因,其中lncRNA MSTRG.24346与大黄鱼的性别决定候选基因dmrt1距离相近,且相关性极显著。该研究表明lncRNA可能在大黄鱼性别分化中起到重要作用,值得深入研究阐明机制。     

The annual reproductive cycle and sex inversion of the Picnic seabream,Acanthopagrus berda (Forsskål 1775) from Indian waters: Histological and morphometric description

The annual reproductive cycle of picnic seabream, Acanthopagrus berda (Forsskål, 1775), one of the potential aquaculture candidate from estuarine waters of Calicut, Kerala (India) was studied. Based on the morphological and histological studies, the ovotestes of A. berda were classified as active male, active female, inactive male, inactive female, transitional and undifferentiated. Histological observation of transitional gonads showed signs of degeneration in the testicular lobe, proliferation of connective tissue and empty sperm ducts indicating protandrous hermaphroditism in A. berda. Ovary was classified into seven maturity stages (virgin, developing virgin, developing, maturing, mature, running, spent) and testis into five maturity stages (resting, maturing, mature, running, spent). Gonadal development in A. berda indicated resting phase (February–July), pre‐spawning phase (March–August) and spawning phase (August–December). Inactive (24.6%) and active males (21.6%) were observed as dominant in smaller length classes (140–250 mm TL), whereas inactive (18%) and active females (51%) were observed as dominant in larger length classes (251–450 mm TL). Few primary females (28.1%) were observed in smaller (below 250 mm TL) and few primary males (28.5%) were observed in larger length classes (above 250 mm TL). From the present study, it can be concluded that in A. berda, most of the individuals function first as males and then change sex to female, but few continue to function as either male or female throughout their lifespan indicating digynous protandrous hermaphroditism.