Genetic differentiation in hatchery strains and wild white shrimp Penaeus (Litopenaeus) vannamei (Boone, 1931) from northwest Mexico

Ten enzymatic systems were analyzed to determine allozyme genetic differentiation among three hatchery strains (A, B and C) of white shrimp, Penaeus (Litopenaeus) vannamei, commonly used in shrimp farming in northwest Mexico. A wild population from northern Sinaloa was used as a reference. Fifteen loci were detected, nine of which were polymorphic (ACP-1*, ACP-2*, AKP-2*, EST-2*, EST-3*, EST-4*, EST-5*, LAP*, and LDH*). Polymorphism of A, B and C were 53, 53, and 40%. The mean observed heterozygosity per locus was 0.071, 0.093, and 0.050 without any significant difference among them or with respect to the wild population (0.056). In all samples observed heteroxygosity was smaller than expected. The mean number of alleles per locus was 1.8 in A and 1.87 in both B and C. Strain A was the only sample without rare alleles. Only EST-3* and LAP* of strain A were in Hardy-Weinberg equilibrium; the other loci were in disequilibrium in all samples. High inbreeding values and heterozygote deficiencies were detected in all samples. Distribution of allelic frequencies was heterogeneous among the samples (G-test), involving all polymorphic loci and suggesting a genetic differentiation. According to Fst, a moderate genetic differentiation (7.4%) was detected among the samples. Greater differences were between strains A and C. Based on genetic distance, the samples were grouped into two pairs, B–C and A-wild. Strain A is a young strain related to the wild sample, whereas strains B and C have a different geographic origin than the wild sample.     

Immunomodulatory effects of dietary Bacillus coagulans in grouper (Epinephelus coioides) and zebrafish (Danio rerio) infected with Vibrio vulnificus

Groupers bred for mariculture are of high economic value in Taiwan. Unfortunately, these fish are highly susceptible to bacterial infection, and the use of antibiotics to control disease has had the unintended consequence of generating antibiotic-resistant bacteria. Herein, we examined whether it is possible to enhance grouper immunity through application of Bacillus coagulans, a probiotic bacterial species. In order to study its effects on immunity, B. coagulans was isolated from lactobacillus mixtures and administered live to grouper (Epinephelus coioides) and zebrafish (Danio rerio). Fish were fed for 30 days on a diet consisting of B. coagulans (10⁴, 10⁶, 10⁸, or 10¹⁰ colony-forming units (cfu) of B. coagulans in 50 ml of media) mixed with 50 g of eel powder. After 30 days, disease resistance against Vibrio vulnificus (204) infection and expression of immune-related genes were determined. We found that oral administration of B. coagulans significantly enhanced expression of several immune-related genes, including myeloid differentiation factor (MyD)88, interleukin (IL)-1β, tumor necrosis factor (TNF)-2, and IL-1β in grouper, and Toll-like receptor (TLR) 4, TNF-α, TRAM 1, and nuclear factor (NF)-κB in zebrafish. Treatment with B. coagulans significantly reduced mortality at 24, 48, 72, and 96 h after challenge with V. vulnificus (204). In vitro studies revealed that B. coagulans possesses bactericidal activity against Enterococcus faecalis (10066), Staphylococcus aureus subsp. (10780), Streptococcus agalactiae (S47, S48, 819), Escherichia coli (10675), Klebsiella oxytoca (13985), Pseudomonas aeruginosa (19660), and V. vulnificus (204, 12905, YJ016). On average, 10⁷ colony-forming units of B. coagulans were required for bactericidal activity. In conclusion, our results indicate that dietary intake of B. coagulans enhances expression of immune-related genes and protects grouper and zebrafish against V. vulnificus (204) infection.     

Susceptibility by amberjack (Seriola dumerili), yellowtail (S. quinqueradiata) and Japanese flounder (Paralichthys olivaceus) to Neobenedenia girellae (Monogenea) infection and their acquired protection

This study investigated: 1) susceptibility differences to infection by Neobenedenia girellae (Capsalidae) between amberjack Seriola dumerili (Carangidae), yellowtail S. quinqueradiata and Japanese flounder Paralichthys olivaceus (Paralichthyidae); 2) growth and egg production of N. girellae on each fish species; 3) acquired protection of each fish species against this parasite. The number of N. girellae on S. dumerili was significantly higher than on S. quinqueradiata and P. olivaceus when these fishes were exposed to oncomiracidia in the same aquarium. Neobenedenia girellae growth on S. dumerili was fastest and, thus the number of eggs laid by parasites on S. dumerili was greater than on the other two species. Seriola dumerili and P. olivaceus, which were previously infected with N. girellae and treated by freshwater bath, acquired partial protection against re-infection by N. girellae. The relative re-infection of three S. dumerili individuals out of eleven individuals was markedly low compared with the initial infection, and the relative initial infection and re-infection on two P. olivaceus out of eleven individuals was markedly low. The results of this study could be useful to control N. girellae infections when cultivating S. dumerili, S. quinqueradiata and P. olivaceus.     

Diet trials on tehuelche scallop Aequipecten tehuelchus (d'Orb) larvae

Laboratory spawned one week old veliger larvaeof the tehuelche scallop, Aequipectentehuelchus (D'Orb., 1846), were reared topedivelger stage under three different dietaryregimes in order to determine the best algalcombination under standard hatchery conditions.Veligers were cultured at mean densities of 2.7larvae/ml. Larval growth and survival wereassessed on larvae fed the following dietarytreatments: (1) Isochrysis galbana(T-Iso)+ Chaetoceros calcitrans, (2)Pavlova lutheri + C calcitrans, and (3)P. lutheri + I. galbana T-Iso +Ch. calcitrans. The highest growth rateswere detected on larvae fed diets includingP. lutheri.     

Extinction of Daphnia lumholtzi (Sars) in Lake Kinneret (Israel)

Daphnia lumholtzi (Sars) was found in Lake Kinneret until the late nineteen-fifties. Lake Kinneret was the northern-most limit of the distribution of this species. It is suggested that fingerlings of grey mullets and Sarotherodon aureus that were introduced into Lake Kinneret caused the extinction of D. lumholtzi.     

Biological evaluation of phytoplankton (Chlorella sp., Tetraselmis sp. and Isochrysis galbana) as food for milkfish (Chanos chanos) fry

Phytoplankton cultures of Chlorella sp., Tetraselmis sp. and Isochrysis galbana were used alone as feed to rear separate batches of newly caught milkfish (Chanos chanos) fry (about 21 days old). Ultrastructural studies of fry hepatocytes and pancreatic acinar cells indicated that they cannot directly utilize Chlorella, which has a rigid cell wall. The fry can directly utilize Tetraselmis and Isochrysis, but neither is nutritionally adequate for growth and survival if used as the only feed. Tetraselmis is nutritionally inferior to Isochrysis.     

Inhibitory effects of Bacillus licheniformis (DAB1) and Pseudomonas aeruginosa (DAP1) against Vibrio parahaemolyticus isolated from Fenneropenaeus indicus

In aquaculture industries, there is an urgent need to develop microbial control strategies, to control disease outbreaks. In recent years, probiotics are considered as a valid alternative for the use of antibiotics in aquaculture to prevent high mortality and promote growth. In the present study, seven strains of bacteria such as Bacillus licheniformis (DAB1), Bacillus pumilus (DAB2), Bacillus sp. (DAB3), Pseudomonas aeruginosa (DAP1), Pseudomonas sp. (DAP2), Pseudomonas aeruginosa (DAP3), Pseudomonas aeruginosa (DAP4), and three pathogenic Vibrio parahaemolyticus (DAV1, DAV2, DAV3) were isolated from healthy and diseased Fenneropenaeus indicus collected from the east coast of Tamilnadu, India. The strains were identified by biochemical analysis and 16S rRNA sequence methods. Among the seven probiotic strains tested, the cell-free extract from DAB1 and DAP1 exhibited higher inhibitory activity of V. parahaemolyticus than other isolates under in vitro conditions. The LC₅₀ of DAV1, DAV2, and DAV3 was found to be ～10³ CFU mL^?1. Pathogenicity of three V. parahaemolyticus DAV1, DAV2, and DAV3 showed significant mortalities (40 %) in Artemia nauplii at inoculation densities of 10³ CFU mL^?1 when compared to the controls (unchallenged nauplii). A significant reduction in mortality (P < 0.001) was found by addition of 10⁶ CFU mL^?1 of DAB1 and DAP1 strains in nauplii against the pathogens. In conclusion, the present study result reveals that DAB1 and DAP1 have potential applications for controlling pathogenic V. parahaemolyticus in Artemia culture systems and aquaculture practices.     

Effects of Pro-Tex on zebrafish (Danio rerio) larvae,adult common carp (Cyprinus carpio) and adult yellowtail kingfish (Seriola lalandi)

Aquaculture practices bring several stressful events to fish. Stressors not only activate the hypothalamus–pituitary–interrenal-axis, but also evoke cellular stress responses. Up-regulation of heat shock proteins (HSPs) is among the best studied mechanisms of the cellular stress response. An extract of the prickly pear cactus (Opuntia ficus indica), Pro-Tex, a soluble variant of TEX-OE^®, may induce expression of HSPs and reduce negative effects of cellular stress. Pro-Tex therefore is used to ameliorate conditions during stressful aquaculture-related practices. We tested Pro-Tex in zebrafish (Danio rerio), common carp (Cyprinus carpio L.) and yellowtail kingfish (Seriola lalandi) exposed to aquaculture-relevant stressors (thermal stress, net confinement, transport) and assessed its effects on stress physiology. Heat shock produced a mild increase in hsp70 mRNA expression in 5-day-old zebrafish larvae. Pro-Tex increased basal hsp70 mRNA expression, but decreased heat-shock-induced expression of hsp70 mRNA. In carp, Pro-Tex increased plasma cortisol and glucose levels, while it did not affect the mild stress response (increased plasma cortisol and glucose) to net confinement. In gills, and proximal and distal intestine, stress increased hsp70 mRNA expression; in the distal intestine, an additive enhancement of hsp70 mRNA expression by Pro-Tex was seen under stress. In yellowtail kingfish, Pro-Tex reduced the negative physiological effects of transport more efficiently than when fish were sedated with AQUI-S^®. Overall, our data indicate that Pro-Tex has protective effects under high levels of stress only. As Pro-Tex has potential for use in aquaculture, its functioning and impact on health and welfare of fish should be further studied.     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

Recruitment control and production of Tilapia aurea (Steindachner) with the predator,Cichlasoma managuense (Günther)

The effects of different stocking rates and ratios of Tilapia aurea (Steindachner) in combination with Cichlasoma managuense (Günther) on tilapia recruitment and production were evaluated in 440 m² ponds. The research was conducted at the National Fisheries Station in El Salvador, Central America.The tilapia A_T value, the total weight of marketable tilapia (14 cm or larger) expressed as a percentage of the total weight of the whole tilapia population, averaged 96 or greater at a stocking ratio of 4:1 (fingerling tilapia: fingerling C. managuense) and at a stocking ratio of 8:1 (fingerling tilapia: adult C. managuense). Tilapia A_T values at a stocking ratio of 8:1 (fingerling tilapia: fingerling C. managuense) ranged from 71–89.Highest estimated annual production of marketable tilapia (3611 kg ha^?1) occurred at the highest stocking rate of 12000 tilapia ha^?1 in populations with an average tilapia A_T value of 98. Greatest weight gain of marketable tilapia (167 g of 1.17 g day^?1) occurred at the lowest stocking rate of 6000 tilapia ha^?1 in populations with an average tilapia A_T value of 99.Estimated annual production of marketable C. managuense ranged from 212–414 kg ha^?1. The average weight gain of marketable fish varied from 48–60 g (0.32–0.40 g day^?1). The average C. managuense, A_T value was 54, ranging from 29–84.The estimated production of marketable fish of both species at stocking rates of 6000, 9000 and 12000 tilapia ha^?1 in populations with tilapia A_T values of 96 or greater averaged 3295, 3290 and 4025 kg ha^?1 per year, respectively.     

Molecular cytogenetic study in Octopus (Amphioctopus) areolatus from Japan

Octopus (Amphioctopus) areolatus is an important marine cephalopod in Japan. We examined its diploid chromosome number, karyotype, and genome size (C value), and performed fluorescence in situ hybridization (FISH) using a vertebrate telomeric probe. The diploid chromosome number was 60 in embryonic cells, with 24 pairs of metacentric chromosomes, four pairs of metacentric/submetacentric chromosomes, and two pairs of submetacentric chromosomes. The mean C value was estimated to be 5.47 pg. From these findings, and those reported previously for other octopus species, we suggest that the factor causing the quantum change of C value in O. (A.) areolatus was genome duplication, and not polyploidy. In telomeric FISH analysis, hybridization signals were clearly observed in the telomeric regions of the chromosomes. This is the first report of FISH analysis of cephalopod chromosomes, and our findings suggest that the telomere sequence of O. (A.) areolatus is (TTAGGG) _n , which may allow gene mapping in the future.     

Population dynamics of Melicertus kerathurus (Decapoda: Penaeidae) in Thermaikos Gulf (N. Aegean Sea)

The east Atlantic-Mediterranean penaeid Melicertus kerathurus constitutes a valuable resource for Mediterranean fisheries. In recent years, its world production is almost totally based on the yields from Greece and Tunisia. A two-year study of prawn stock in Thermaikos Gulf (NW Aegean Sea) provided information on age, growth and mortality of the species. M. kerathurus exhibits a marked sexual growth dimorphism, with females showing an overall wider size range and greater size than males. Mean monthly size, CL_max were also in favor of females. Monthly age classes varied from one to four for females and from one to three for males, and corresponded to three generations (0⁺, 1⁺, 2⁺). New generation (0⁺) cohorts recruited into fishery in waves, from late summer to February. According to the von Bertalanffy growth curve, females grow larger than males, but there is a prominent seasonal growth oscillation in both sexes (females: CL_∞ = 62.48, K = 1.15, C = 0.87, WP = 0.16, Rn = 0.170; males: CL_∞ = 47.78, K = 1.28, C = 0.97, WP = 0.12, Rn = 0.205). Growth performance indices (φ′, φ) and longevity were in favor of females. K values for both sexes in Thermaikos Gulf are the highest reported for the species, suggesting a higher growth rate and a lower longevity. Estimated values for total, natural and fishery mortality were greater in males. In both sexes, an exploitation rate E > 0.5 was found, implying an intense fishing impact on M. kerathurus stock in Thermaikos Gulf. Intense exploitation in other parts of the Mediterranean and eastern Atlantic, along with the replacement of the species by Eritrean penaeid prawns in the eastern Mediterranean, has resulted in a severe reduction in catches, in most countries. Thus, existing stocks of M. kerathurus should be considered as potentially endangered, and species-specific initiatives towards sustainable management of this resource should be implemented.     

The isolation of intestinal microflora of farmed red seabream (Pagrus major) and black seabream (Acanthopagrus schlegeli) at larval and juvenile stages

The aerobic bacterial flora in the intestine of farmed red seabream and black seabream was investigated at their larval and juvenile stages in relation to the microflora of ambient water and diets. The total bacterial number in the intestine was 7.4×10⁴ and 3.4×10⁴ colony-forming units (CFU)/fish for red seabream and black seabream, respectively. Among the intestinal flora, Vibrio (ca. 45%) and Pseudomonas (30%) were the most dominant bacteria.The total bacterial counts were 3.1×10⁴ CFU/ml for rearing water, 2.1×10⁷ CFU/g for live diets (rotifer and brine shrimp) and 1.2×10⁴ CFU/g for artificial diets. Among the water bacteria, Pseudomonas (22%) and Moraxella (29%) constituted dominant groups, and in the live diets Pseudomonas (48%) was predominant. The incidence of Vibrio in waters and live diets was 7% and 11%, respectively.     

Food preferences in cultured rabbitfishes (Siganidae)

Four species of rabbitfishes, Siganus concatenata, S. oramin, S. striolata and S. virgata were held in rearing tanks and fed with benthic algae. Out of 101 algae species and 4 vascular plants offered, the fishes ate only 67 species. The electivity indices E, for 23 algae were determined. The species Enteromorpha intestinalis, Gracilaria confervoides and G. cylindrica proved to have the highest indices with E = 0.594, E = 0.269 and E = 0.182, respectively.     

Comparaison de la croissance et de la composition chimique d'Oreochromis (Tilapia) niloticus L. et de Cichlasoma (Theraps) melanurum Gth. Nourris avec Azolla

The growth of two species of Cichlidae, the African Oreochromis niloticus and the Central American Cichlasoma melanurum, fed with three different diets (100% pellets, 50% pellets and 50% Azolla, 100% Azolla) was compared. The growth performances of O. niloticus (G=2.3) fed only with pellets were higher than those of C. melanurum (G=0.9). With the mixed diet (50% pellets and 50% Azolla) growth was reduced for both species but this reduction was proportionally lower with C. melanurum. The fish fed exclusively with Azolla showed negative growth but fish weight loss was least for C. melanurum.Comparison of the chemical composition of the two fish species reveals significant differences. Generally moisture is lower for C. melanurum and lipid concentration is higher for this species with all three diets. Fed with Azolla, both species have a higher moisture and lower lipid concentration. Crude protein concentration (±15%) is very similar for both species regardless of diet.In conclusion, for intensive fish culture based on commercial pellets, the African species O. niloticus performs better than the American C. melanurum. On the other hand, the use of Azolla as feed for these fish does not seem very satisfactory but experiments should be conducted with typical herbivorous Cichlidae which may be able to utilize this fern in agro-piscicultural ecosystems.     

Characterization and expression analysis of two distinct neuropeptide Ya paralogues in Jian carp (Cyprinus carpio var. Jian)

Two distinct neuropeptide Ya paralogues (jlNPYa1 and jlNPYa2) were cloned and characterized in Jian carp (Cyprinus carpio var. Jian), with a highly conserved organization encoded by four exons and three introns. The cDNAs for jlNPYa1 and jlNPYa2 were 693 and 730 bp in size, respectively. jlNPYa1 and jlNPYa2 both encoded a 96-amino acid protein, which shared 97.9 % identity. Phylogenetic tree showed that it has two NPYa genes, called jlNPYa1 and jlNPYa2, that presumably resulted from the tetraploidization event in the carp lineage. Analysis of expression profiles of jlNPYa1 and jlNPYa2 showed that the two NPY genes had a broad tissue distribution but expressed primarily in the forebrain, hypothalamus, testis and liver. The expression pattern was different in juvenile and adult (female and male) Jian carp. In juvenile, the highest expression level of jlNPYa1 and jlNPYa2 was detected in the testis. In adult, it was detected in the forebrain. In female hypothalamus, the expression level of jlNPYa1 was significantly higher than that of jlNPYa2. However, the opposite was true in male hypothalamus. The differing distribution patterns of the two NPY genes suggested that jlNPYa1 and jlNPYa2 might play different roles in Jian carp.     

Movement patterns and growth estimates of big skate (Raja binoculata) based on tag-recapture data

From 2003 through 2006, 18,180 big skate (Raja binoculata) were tagged in three regions in British Columbia, Canada: northern Hecate Strait, Queen Charlotte Sound and the west coast of Vancouver Island. To date, this is the largest tagging program conducted for skates or rays worldwide. As of December 31, 2008, 7% of the tagged fish (n = 1238) have been recaptured through the commercial fisheries. Generally, 75% of the recaptured fish were recaptured within 21 km of the tagging location. Long-range movements (up to 2340 km) were undertaken by a small percentage (1.5%) of the recaptured fish. Tagged big skate were recaptured in waters off of Oregon, Washington, throughout the Gulf of Alaska and the Bering Sea. The majority of big skate recaptured outside of Canadian waters were female (83%, n = 15), of which 80% (n = 12) were likely immature at release. Three methods of estimating growth parameters were employed and produced varying results. The Gulland and Holt method was not able to produce growth parameter estimates. The GROTAG method for combined sexes produced von Bertalanffy growth curve parameters L_∞ (294.7 cm), K (0.05) and t₀ (?1.44) that were similar to published estimates for British Columbia big skates. The von Bertalanffy growth curve parameters produced by Fabens method for combined sexes were L_∞ = 168.6 cm, K = 0.16 and t₀ = ?0.81.     

Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases XbaI, KpnI and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism.     

太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用

为研究太平洋鳕发育早期特异免疫系统形成的机制,通过RAG1和IgM基因的转录水平衡量特异免疫系统的发育特点.根据GenBank中RAG1和IgM的序列信息,分别设计1对特异引物,从太平洋鳕头肾中扩增得到RAG1和IgM的基因片段.将所获基因片段分别插入到克隆载体pMD18-T中,从而构建太平洋鳕RAG1和IgM基因的质粒标准品.建立并优化太平洋鳕RAG1和IgM基因绝对荧光定量PCR方法.为进一步验证该方法的可靠性,分别利用绝对定量和相对定量检验目的基因在太平洋鳕早期发育过程不同组织内的表达差异.以优化后的绝对荧光定量PCR方法检测不同发育时期太平洋鳕RAG1和IgM的表达情况.结果显示,RAG1的回归方程为y=-3.266x+33.77,回归系数R2=0.996;IgM的回归方程为y=-3.119x +27.61,回归系数R2 =0.998.绝对定量和相对定量结果在基因转录趋势上显现出一致性,即RAG1基因在胸腺和头肾中表达,且在胸腺中的表达量显著高于头肾中的表达量,在肝脏和脾脏中无表达;IgM基因在胸腺、头肾、肝脏和脾脏中均有表达,其中脾脏中表达量最高,其次是头肾.RAG1基因在太平洋鳕发育早期的表达水平很低,到61日龄(days posthatching,dph)至95 dph表达量显著提高;IgM基因在早期表达水平同样很低,到33 dph至61 dph才有明显表达,在95 dph时表达量显著提高.研究表明,本实验方法可靠,特异性较强,可成功对目标基因转录水平进行检测.