Nutritional evaluation of two marine microalgae as feedstock for aquafeed

Using microalgae for animal nutrition provides an economically viable route for microalgae‐based technological innovation, especially in combination with CO₂ fixation given current global warming. However, this technology still lacks sufficient evaluation for screening microalgae for specific animals; meanwhile, current studies show some prejudice regarding ‘essential’ or ‘non‐essential’ ingredients. The results show that Dunaliella salina and Nannochloropsis salina were able to accumulate high protein (30%–57%) and lipid (20%–46%) content without affecting the performance of CO₂ fixation, which reached 0.28 and 0.23 g L^?1 day^?1 respectively. Both species exhibited high quality of lipids and proteins for Penaeus monodon based on the profiling. The essential fatty acid indexes (EFAI) for N. salina and D. salina were 3.81 and 9.02 respectively. Butyric acid was found to be present in both D. salina (12.03%) and N. salina (4.87%) based on the total fatty acids (FAs). The essential amino acid indexes (EAAI) for D. salina and N. salina were 2.23 and 1.29 respectively. Arginine was the most abundant essential amino acid (EAA) in both D. salina (10.83%) and N. salina (13.35%) on the basis of total amino acids (AAs). This study comprehensively compares the nutritional quality of the two commercial marine microalgae of D. salina and N. salina with the potential to be used as sustainable sources of lipids and proteins to reduce or even replace the traditional fish oil and fish meal in aquafeeds.     

Flocculated microalgae concentrates as diets for larvae of the tiger prawn Penaeus monodon Fabricius

Fresh algal culture is a major bottleneck in the aquaculture industry. Substitutes that are cost-effective and simplify hatchery procedures, such as algae concentrates need to be evaluated. Four species of alga – Chaetoceros muelleri , C. calcitrans , a tropical Skeletonema sp. and Thalassiosira pseudonana – were concentrated by flocculation, stored for 6 weeks at 4 °C and then compared with their fresh counterparts as feeds for Penaeus monodon larvae. The algae were fed at either high or low cell densities with no supplements.
Fresh C. muelleri promoted the highest survival, greatest weight gain and fastest development to mysis 1. Larvae fed this diet were twice as heavy as those fed most of the other diets. Concentrated C. muelleri or T. pseudonana promoted similar survival rates to that of larvae fed fresh C. muelleri although development rates were slower. Larvae fed fresh C. calcitrans had high survival and intermediate development, but those fed the concentrate had very poor survival and development. Skeletonema sp., whether in the fresh or concentrated form, was a poor diet for prawn larvae: it resulted in high mortality and slow development. Cell density did not affect survival or dry weight of larvae but did affect development in some cases. Flocculated algal concentrates show promise as replacement feeds for fresh algae.     

A reference diet for nutritional studies of the giant tiger prawn Penaeus monodon

Critical evaluation of the nutrient requirements of an animal requires the use of a purified reference diet. In this study, the effects of either a purified reference experimental diet, a practical experimental diet and an imported commercial diet on the growth and nutritional condition of the prawn Penaeusmonodon were compared. The reference diet, based on widely available ingredients of consistent and known nutrient content, supported excellent prawn growth when fed either ad libitum or subsatiation (≈ 75% of the mean initial satiation intake). In a 6-week experiment, growth (as percentage increase in weight, ± SEM) of the prawns fed ad libitum differed significantly (P < 0.05) between each diet in the order: practical (142 ± 10%), reference (121 ± 9%) and commercial (91 ± 16%). Growth for the reference diet fed subsatiation was 116 ± 4%, and was not significantly (P > 0.05) different from that obtained with the same diet fed ad libitum. Dry matter food conversion for the reference diet was significantly (P < 0.05) better when fed subsatiation (1.58 ± 0.08) rather than ad libitum (2.08 ± 0.06) and better than that obtained with either the practical (3.40 ± 0.15) or commercial (3.02 ± 0.28) diets, which were not significantly (P > 0.05) different from each other. The lipid content (mg g^–1 of prawn) of the digestive gland of prawns fed ad libitum was similar for reference, practical and commercial diets (6.03 ± 0.38, 4.92 ± 0.90 and 4.92 ± 0.95 mg g^–1 of prawn, respectively), but significantly (P < 0.05) higher than that for the reference diet fed subsatiation (4.73 ± 0.38 mg g^–1 of prawn).     

斑节对虾增殖细胞核抗原基因克隆及表达分析

为研究增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)在斑节对虾(Penaeus monodon)卵巢发育中的功能,利用RACE技术克隆得到了斑节对虾PCNA基因(PmPCNA)的cDNA全长序列。该序列全长978 bp,包括135 bp的5′非编码区(5′UTR)、60 bp的3′UTR和编码260个氨基酸的783 bp的开放阅读框(ORF)。同源性分析结果表明,PmPCNA与其他物种具有很高的蛋白同源性。组织表达模式分析表明,PmPCNA在所监测的各组织中为组成性表达,其中在卵巢和脑中表达量较高;卵巢发育不同阶段基因的表达分析则表明PmPCNA在Ⅲ期表达量最高,是其他各期表达量的2倍;注射五羟色胺(5-hydroxy tryptamine,5-HT)后,PmPCNA在卵巢中表达量明显上升,其中在48 h表达量升高幅度最大,是对照组的5倍左右。利用原核表达技术获得了PCNA的体外重组蛋白,Western Blot分析证实该重组蛋白为PCNA蛋白。以上研究结果表明,PmPCNA蛋白是斑节对虾卵巢发育过程中的重要调节因子,本研究为进一步认识斑节对虾卵巢发育机制提供了基础资料。     

Dietary biofloc supplementation in black tiger shrimp,Penaeus monodon: effects on immunity,antioxidant and metabolic enzyme activities

A 60‐day indoor experiment was conducted to study the effect of dietary supplementation of biofloc on metabolic enzyme activities and immune responses in Penaeus monodon juveniles. Biofloc developed in indoor fibreglass‐reinforced plastic (FRP) tanks (1000 L) was used as dietary supplement in P. monodon (2.90 ± 0.10 g) reared in 1000‐L FRP tanks. Graded level of dried biofloc was included in shrimp basal diets, 0% (control, B0), 4% (B4), 8% (B8) and 12% (B12). The level of metabolic enzymes like malate dehydrogenase (MDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was not significantly different with control up to 8% dietary supplementation. A higher level of total haemocyte count (THC) was noticed in B8 (22.16 ± 2.17 × 10⁶ cells mL^?1) and B4 (21.11 ± 0.56 × 10⁶ cells mL^?1) compared with control, C (14.61 ± 2.74 × 10⁶ cells mL^?1). Biofloc‐supplemented groups recorded significantly higher (P < 0.05) serum SOD and catalase activity (P < 0.01) in comparison with control. The groups fed with 4% dietary biofloc supplement recorded highest relative percentage survival (RPS), 45% after challenge with Vibrio harveyi followed by 36% and 27% RPS in B8 and B12 groups. Based on these results, it can be concluded that supplementation of biofloc even at 4% level in the feed improves immune responses and metabolic activities in black tiger shrimp juveniles.     

斑节对虾细胞周期蛋白E基因的克隆与表达分析

为了解细胞周期蛋白E(cyclin E)基因在斑节对虾(Penaeus monodon)发育过程中的作用, 构建和测序了斑节对虾全组织cDNA文库, 获得了斑节对虾细胞周期蛋白(Pmcyclin) E的部分表达序列。通过RACE技术克隆得到1条全长1 706 bp的cDNA序列。Pmcyclin E开放阅读框(ORF)为1 263 bp, 编码420个氨基酸。生物信息学分析表明, 该氨基酸序列含有周期蛋白家族特有的CYCLIN保守区并含有N-糖基化位点及磷酸化位点。经BLAST同源性分析表明, 该基因编码蛋白与红火蚁(Solenopsis invicta)、切叶蜂(Megachile rotundata)等多种节肢动物细胞周期蛋白E有很高的同源性。组织分布分析表明, 该基因在斑节对虾精巢、卵巢、肠、脑、肝胰腺、胃和心脏组织中均有表达, 其中卵巢中表达量较高, 而心脏、胃及肝胰腺中表达量相对较低。分别对蜕皮抑制激素(MIH)注射和眼柄切除的对虾进行表达分析, 发现眼柄切除后, Pmcyclin E大量表达, 而注射MIH后细胞Pmcyclin E的表达量相对降低。结果表明, 细胞cyclin E基因在斑节对虾卵巢发育过程中起重要作用, 且眼柄切除对细胞cyclin E的表达具有影响, 该结果为进一步探究斑节对虾卵巢的发育机理提供了理论依据。

     

Integration of substrate in biofloc based system: Effects on growth performance,water quality and immune responses in black tiger shrimp,Penaeus monodon culture

To evaluate effect of substrate integration in biofloc based system, a 52‐day growth experiment was conducted using black tiger shrimp, Penaeus monodon juveniles (3.32 ± 0.07 g). The factorial design consisted of floc, F (with or without) as first factor and substrate (bamboo mat, B; nylon mesh, N; and without substrate) as second factor. This resulted six treatments; F + B, F + N, F, B, N and a control without biofloc and substrate. Shrimps were stocked at 110 nos. m^–3 in Fibre Reinforced Plastic (FRP) tanks and, rice flour was used as carbon source in biofloc based treatments. Incorporation of nylon mesh and bamboo mat in biofloc system trapped 31.3%–38.6% and 8.5%–13.5% total suspended solids respectively and reduced bottom solid deposition. Among the substrate based groups, significantly better development of biofilm with higher microbial population noticed in F + B compared with nylon mesh. Similarly, significantly higher final growth (p < 0.01) was recorded in F + B system followed by F + N while no significant difference in body weight recorded among floc, F or substrate based groups (B, N). Biofloc and substrate integration (F + B and F + N) resulted significantly (p < 0.01) lower feed conversion ratio compared to control and floc. Incoporation of bamboo substrate in biofloc, (F + B) improved shrimp immune responses through higher hemocyte counts and prophenoloxidase activity compared to other treatments. The study revealed that integration of substrate in the biofloc system improved growth performance, FCR and immune parameters in shrimp by trapping the suspended biofloc particles, better water quality parameters, enhanced biofilm growth and provision of quality natural food.     

“水陆互补”理念下的水产品营养健康功效

水产养殖在全球粮食安全中发挥着至关重要的作用,为人类提供优质蛋白源持续稳定供应提供了重要保障。益生菌是一类可用于水产养殖的有益微生物,不仅能够提高水产动物的生长性能、免疫能力,而且具有抑制致病菌、改善养殖水质等功效。益生菌在水产养殖中的推广应用可促进水产养殖业的健康可持续发展。因此,本文首先介绍了水产养殖中益生菌的定义与来源,详细阐述了益生菌对水产可持续养殖的意义以及益生菌在水产养殖中的实际应用,并对其未来研究方向进行了展望,以期为益生菌在水产养殖可持续发展中的推广和应用提供可靠的理论依据。

     

Estimates of heritability and genetic correlations for growth‐related traits in the tiger prawn Penaeus monodon

Heritabilities and genetic and phenotypic correlations were estimated for body weight (BW), body length (BL), carapace length (CL), carapace width (CW) and carapace height (CH) of the Penaeus monodon from measurements on progeny at 6 months of age resulting from a nested mating design. Estimation was made on data from 41 full‐sib and 10 half‐sib families. The estimated heritabilities for these traits were 0.13–0.24, and the significantly phenotypic and genetic correlations were 0.81–0.94 and 0.88–1.00, respectively. The present results indicate that genetic improvement through selective breeding conducted on P. monodon might be feasible and would be an efficient method to obtain a positive response to selection.     

Acrosome reaction in the sperm of the black tiger shrimp Penaeus monodon (Decapoda, Penaeidae)

Owing to the problem of male infertility in the domesticated shrimp Penaeus monodon, this study was conducted to reveal the morphological events of an acrosome reaction (AR) of sperm of this highly valuable species. The AR observed in an in vitro incubation of sperm with egg water (EW) and that during actual spawning was compared. Under transmission and scanning electron microscopy, sperm taken from the female thelycum was composed of a posterior main body, a central cap and an anterior single spike. Upon contact with EW, the sperm underwent two phases of AR: acrosomal exocytosis and spherical mass formation. The former was composed of a degeneration of the spike, swelling of the cap region and rupture of the acrosomal pouch. The latter began with polymerization of materials within the subacrosomal region and ended with re‐configuration of the subacrosomal region into an electron‐dense spherical mass. The AR of the sperm observed during spawning revealed similar morphological events, with degeneration of the spike upon contact with the vitelline envelope and formation of the spherical mass while penetrating into jelly material produced by protruding cortical rods. The results suggest the presence of AR inducers derived from the vitelline envelope and cortical rods of the egg. This study forms the basis for an evaluation of infertility regarding to AR in the domesticated P. monodon male.     

Effects of dietary mannan oligosaccharide on growth,survival, physiological,immunological and gut morphological conditions of black tiger prawn (Penaeus monodon Fabricius 1798)

The aim of this experiment was to determine the effects of dietary inclusion with mannan oligosaccharide (Bio‐Mos, Alltech, Nicholasville, KY, USA) on growth, survival, physiological and immunological conditions and gut morphology of the black tiger prawn (Penaeus monodon). Five diets supplemented with MOS at 0 g kg^?1 (control diet), 1, 2, 4 and 8 g kg^?1 were fed to the prawn juveniles (0.4 ± 0.06 g, total weight) for the duration of 63 days. Growth was the highest (P < 0.05) when the prawns were fed the 1 g kg^?1 MOS included diet. Wet tail muscle index (Tw/B), dry tail muscle index (Td/B) and tail muscle protein (Tmp) were higher (P < 0.05) in the prawns fed MOS included diets MOS compared with the prawns fed the control diet. Total haemocyte counts (THCs) of the prawns fed MOS included diets were higher (P < 0.05) than THCs of the prawns fed the control diet. Epithelium layer and epidermal cell density of the gut of the prawns fed 1 g kg^?1 and 2 g kg^?1 MOS diets were better than the prawns fed the control and other MOS diets. The results imply a positive effect of dietary supplementation of 1–2 g kg^?1 MOS in the culture of black tiger prawns.     

Hatching envelope formation in the egg of the black tiger shrimp,Penaeus monodon (Decapoda,Penaeidae)

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)‐labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti‐FITC‐conjugated gold‐lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar‐associated components of the HE. This revealed that both Con A and WGA‐binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold‐labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N‐acetyl‐β‐d ‐glucosamine (sugar affinity to WGA).     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

Identification and characterization of Alix/AIP1 interacting proteins from the black tiger shrimp,Penaeus monodon

Apoptosis is proposed to be a major cause of death in shrimp viral infections. From our previous study, an apoptosis-related gene, Pm-Alix, was identified from the black tiger shrimp. Its expression was high in defence-related tissues including haemocytes and the lymphoid organ. To clarify its possible role in shrimp, we used Pm-Alix as bait in a yeast two-hybrid analysis to search for Alix interacting proteins in shrimp. Two cDNA sequences discovered had homology to a predicted ubiquitin C of the purple sea urchin, Strongylocentrotus purpuratus, and to a guanylyl cyclase of the red swamp crayfish, Procambarus clarkii. In vitro pull-down assays confirmed positive interaction between Pm-Alix and both proteins. Tissue distribution analysis revealed that Pm-Alix and the two binding partners were widely expressed in various tissues but more highly expressed in haemocytes. However, no significant positive or negative correlation was found in the expression of these genes as shrimp approached morbidity and death after challenge with white spot syndrome virus. Thus, the results suggested that Alix and its interacting partners did not play a direct role related to shrimp death.     

Effects of biofloc under different carbon sources and protein levels on water quality,growth performance and immune responses in black tiger shrimp Penaeus monodon (Fabricius, 1978)

A 75‐day 2 × 3 factorial experiment was conducted to evaluate the effect of two levels of dietary protein (32 and 40%) and two different carbon sources (rice flour–R and molasses‐M), and without carbohydrate (control–C) in black tiger shrimp Penaeus monodon juveniles (3.37 ± 0.04 g) at 100 nos. m^?3 in 100 L fibre reinforced plastic tanks. Biofloc volume and total suspended solid were higher in molasses added groups (32 + M and 40 + M) followed by rice flour (32 + R and 40 + R) and controls (32C and 40C). Molasses and rice flour addition significantly reduced (P < 0.01) the total ammonia–N compared to controls. The highest Vibrio, Bacillus and Lactobacillus counts were recorded in 40 + M, 32 + M and 32 + R respectively. Among the treatments, significantly higher (P < 0.01) final body weight was recorded in 40 + R (8.5 ± 0.3 g), 40 + M (7.8 ± 0.3 g) and 32 + R (7.5 ± 0.4 g) compared to control groups, 32C (6.1 ± 0.3 g), 40C (6.4 ± 0.3 g) and molasses added group, 32 + M (5.7 ± 0.4 g). Rice flour supplementation significantly increased (P < 0.01) the total haemocyte count (×10⁶ cells mL^?1) in 32 + R (45.7 ± 3.7) and 40 + R (44.3 ± 3.1) compared to controls, 32C (27.3 ± 3.4) and 40C (25.8 ± 0.9). Similarly, higher superoxide dismutase, catalase, serum protein and glucose were recorded in the rice flour added groups, 40 + R followed by 32 + R. Among the treatments, the highest level of prophenoloxidase (OD 490 nm, 0.3 ± 0.0) and survival after challenge with Vibrio harveyi (55.6%) was observed in 32 + R. The study elucidates that rice flour addition produces optimum level of biofloc with better growth and immune responses compared to molasses and control. Furthermore, rice flour addition at 32% protein level could replace 40% protein feed.     

Pharmacokinetics of oxolinic acid in black tiger shrimp, Penaeus monodon Fabricius, and the effect of cooking on residues

This study examined the pharmacokinetics and bioavailability of oxolinic acid (OA) in black tiger shrimp Penaeus monodon Fabricius, in brackish water (salinity 10 g L^?1) at 28–29°C, after intra‐sinus (10 mg kg^?1) and oral (50 mg kg^?1) administration and also investigated the net changes of OA residues in the shrimp after cooking (boiling, baking and frying). The haemolymph concentrations of OA after intra‐sinus dosing were best described by a two‐compartment open model. The distribution and elimination half‐lives were 0.84 and 17.7 h respectively. The apparent volume of distribution at a steady state and the total body clearance were estimated to be 2061 mL kg^?1 and 90.1 mL kg^?1 h^?1 respectively. The bioavailability of OA after an oral administration was 7.9%. The peak haemolymph concentration, the time to peak haemolymph concentration and the elimination half‐life after oral administration were 4.20 μg mL^?1, 4 h and 19.8 h respectively. Oxolinic acid muscle and shell levels increased to a maximum (muscle 1.76 μg g^?1 and shell 8.17 μg g^?1) at 4 h post administration and then decreased with the elimination half‐life value of 20.2 and 21.9 h respectively. Residual OA in muscle and shell was reduced by 20–30% by each cooking procedure examined.     

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane,acrosome and DNA of black tiger prawn (Penaeus monodon) spermatozoa

To explore the impact of moulting and short‐term chilled storage of spermatophores on the sperm quality for a commercially important penaeid prawn, spermatozoa of Penaeus monodon from early (B‐C), middle (D0‐1) and late (D2‐3) moult stages were compared for sperm quality parameters relating to the structural integrity of plasma membrane (Viab%), acrosome (AR%) and DNA (SDF%) after being stored at 4°C for 0–26 days. The three different sperm extenders used for chilled storage included artificial lobster haemolymph (AH), calcium free saline (CS) and filtered seawater (FS); two storage conditions were applied either as a free sperm suspension or retained within the intact spermatophore. Results showed that (a) the lowest natural AR% was shown for B‐C spermatozoa in CS whereas the highest levels were for B‐C, D0‐1 and D2‐3 spermatozoa in FS and D2‐3 spermatozoa in AH; (b) the calcium ionophore A23187 agent used in this study was able to increase the mean AR% by 6.22%; (c) the Viab% was significantly lower in CS than that in FS; (d) the SDF% significantly increased over the period of chilled storage for B‐C and D0‐1 spermatozoa, while the SDF% of D2‐3 spermatozoa was initially elevated and did not change significantly over time; and (e) there was no difference in sperm quality between two storage conditions. This study has successfully demonstrated the moult‐related variance in the percentages of acrosome‐reacted and DNA‐damaged spermatozoa, providing evidence of moult‐related spermatophore renewal cycle in this species.     

The use of green fluorescent protein as a marker for monitoring a probiotic Bacillus S11 in the black tiger shrimp Penaeus monodon

To monitor the probiotic Bacillus S11 (BS11) in vivo , wild-type cells were transformed with the green fluorescent protein (GFP)-expressing plasmid, pAD44-12, carrying the gfp mut3a gene under the constitutive Bacillus cereus UW85 promoter, and its non-GFP control plasmid pAD. Transformants with pAD44-12 (BS11-GFP), not pAD (BS11-pAD), expressed detectable but not too high levels of green fluorescence. Chloramphenicol resistance (BS11-GFP, BS11-pAD) and GFP fluorescence (BS11-GFP) as markers suggested that plasmid retention was 78–79% for both BS11-GFP and BS11-pAD cells after approximately 50 generations of growth without antibiotic selection. When mixed into shrimp feed at a final concentration ∼10⁵ CFU g⁻¹, the inclusion of viable transformed bacteria in fed shrimps was observed. After feeding shrimp three times daily in 400-L cement tanks for 9 weeks, no significant differences in the average shrimp weight, the number of BS11 in either the culture water or in the shrimp's gut were seen between shrimp fed BS11-GFP and BS11-pAD or BS11, suggesting that expression of the gfp mut3a gene has no detectable effect on BS11 properties and shrimp growth. Histological examination of sections of shrimp's intestines following feeding with BS11-GFP demonstrated that BS11-GFP in shrimp feed survived and adhered onto the shrimp intestines' surface. BS11-GFP thus has good potential as a non-invasive marker tag for short-term experiments.     

基于InDel标记的斑节对虾早期性别鉴定方法的建立

为分辨斑节对虾(Penaeus monodon)发育早期的性别,利用改良的插入/缺失(InDel)标记追踪了从受精卵、无节幼体、溞状幼体、糠虾幼体、仔虾后1 d(PL1)、仔虾后30 d(PL30)及亚成虾个体的性别,建立了测序和荧光定量PCR熔解曲线的检测方法。结果表明,InDel标记可扩增获得雌雄特异性序列,用测序法判定亚成虾性别的结果与外部观察结果一致,准确率达100%,对受精卵、无节幼体、溞状幼体及糠虾幼体均可得到雌雄特异性序列;进而建立了基于SYBR Green实时荧光定量PCR(Quantitative real-time polymerase chain reaction,q RT-PCR)熔解曲线的快速鉴定斑节对虾性别的方法,其中雄性特异序列的熔解温度(Melting temperature,Tm)为(79.10±0.10)℃,雌性为(78.45±0.20)℃,通过特异的熔解曲线可准确区分PL30、亚成虾和无节幼体Ⅵ期个体,准确率可达96.3%以上。