Seasonal changes of reproductive activity and biochemical composition of pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis> Linnaeus, 1767 in Bohai Sea,China

Seasonal variations in the biochemical composition and the reproductive cycle of pen shell Atrina pectinata in Bohai Sea were investigated from May 2013 to April 2014. Histological analysis indicated that the reproductive cycle of A. pectinata can be divided into two phases, inactive stage and gametogenesis, which were equally and significantly influenced by seawater temperature and food availability. Gametogenesis began in late autumn (October), and completed in June and July. Spawning took place in August, coinciding with the highest water temperature and the richest phytoplankton. The significantly high glycogen content in adductor muscles sustained throughout the late active and ripeness stages, but plummeted during spawning and inactive stages. The protein content in female gonads exhibited a synchronous increase along with oocyte diameter and lipid content, suggesting that the female gonads could accumulate protein and lipid for vitelline in A. pectinata. Furthermore, the RNA/DNA ratio was found to be a useful index to indicate the level of gonad maturation in both males and females. The findings of the present study provided a foundation for the fishery resource administration and the aquaculture development of this species.     

Effects of dietary carbohydrate-to-lipid ratio on the growth performance and feed utilization of juvenile turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

A 9-week feeding trial was conducted to investigate the effects of dietary carbohydrate to lipid ratio(CHO:LIP) on the growth performance and feed utilization of juvenile turbot Scophthalmus maximus(initial body weight 8.75 g ± 0.04 g). Four isonitrogenous and isoenergetic low protein level(39%) diets were formulated with increasing ratios of dietary carbohydrate to lipid(2:18, 6:18, 18:12 and 28:6). A high protein level(50%) diet with the 2:12 ratio of carbohydrate to lipid was used as the control. Results showed that the survival rate, contents of moisture, crude protein and ash in muscle were not significantly affected by dietary treatments. With the dietary CHO:LIP ratio increased from 2:18 to 18:12, weight gain rate significantly increased(P < 0.05). Higher dietary CHO:LIP ratio(28:6) resulted in the significantly decreased weight gain rate(P < 0.05). Meanwhile, this treatment also resulted in the highest daily feed intake and liver glycogen content, as well as the lowest feed efficiency(P < 0.05). Muscle glycogen content in fish fed the diet with 2:12 or 2:18 CHO:LIP ratio was significantly lower than those fed with the other three diets(P < 0.05). The present results confirmed that the juvenile turbot can utilize carbohydrate. Furthermore, the appropriate ratio of dietary carbohydrate to lipid was important to the growth and feed utilization of turbot. The proper CHO:LIP ratio based on the growth performance in the present study was determined to be 18:12 when the dietary protein level was 39%.     

Biodeposition,respiration, and excretion rates of an introduced clam <Emphasis Type="Italic">Mercenaria mercenaria</Emphasis> in ponds with implications for potential competition with the native clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> in Shuangtaizi estuary,China

This study aimed to evaluate the potential impacts of an introduced clam Mercenaria mercenaria on estuarine ecosystem, and implications for the niche competition with a native clam Meretrix meretrix. The biodeposition, respiration, and excretion rates of M. mercenaria were determined seasonally using a sediment trap and a closed respirator in field. The biodeposition rates of M. mercenaria were 0.06–0.37 g/ (ind.·d), and the respiration rates were 0.31–14.66 mg/(ind.·d). The ammonia and phosphate excretion rates were 0.18–36.70 and 1.44–14.87 μg/(ind.·d), respectively. The hard clam M. mercenaria may discharge dry deposits up to 2.1×10⁵ t, contribute 18.3 t ammonia and 9.0 t phosphate to culture ponds, and consume 7.9×10³ t O₂ from ponds annually. It suggested that the hard clam M. mercenaria might play an important role in pelagic-benthic coupling in pond ecosystem through biodeposition and excretion. A comparison of the key physiological parameters of the introduced clam M. mercenaria and the native clam Meretrix meretrix suggested that M. mercenaria had a niche similar to that of Meretrix meretrix in Shuangtaizi estuary and might have a potential competition with Meretrix meretrix for habitat and food if M. mercenaria species escaped from the culture pond or artificially released in estuarine ecosystem.     

Inbreeding depression on growth and survival of full-sib family of Manila clam (<Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>)

In present study, the inbreeding depression (ID) of growth and survival of Manila clam (Ruditapes philippinarum) was investigated at larval and juvenile stages. Nine inbred families (A ₂, B ₂, C ₂, D ₂, E ₂, F ₂, G ₂, H ₂ and I ₂) were established by mating within nine full-sib families with expected inbreeding coefficient of 0.25. Inbred families showed significant differences in shell length and hatching rate of D-larvae (straight-hinged larvae). The larvae of the nine inbred families grew slower than those of control group (CG), and their ID value ranged from 0.81% ± 6.09% to 16.10% ± 1.49%. The ID value of larval survival rate varied between 27.47% ± 9.36% and 70.50% ± 13.66%. The ID was also detected for juvenile growth in A ₂, B ₂, C ₂, and D ₂, which ranged from 4.60 ± 2.21 to 17.71 ± 7.73. The A ₂ family maintained the highest juvenile survival rate, whereas the other inbred families exhibited ID values varying between 62.79% ± 4.54% and 96.14% ± 0.87%. The linear relationship of estimated ID between growth and survival was negatively correlated (R = ?0.434, P < 0.05). The results of this study suggested that the ID of growth was common at the larval stage but was less prevalent at juvenile stage. In contrast, the ID of survival increased from larval to juvenile stage. A better understanding of the effect of inbreeding may aid to selective breeding of Manila clam.     

Use of antioxidant enzymes of clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> as biomarker to polycyclic aromatic hydrocarbon pollution

The typical organic pollutant polycyclic aromatic hydrocarbon (PAH) anthracene was selected as a contaminant to investigate its effects on the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in the clam Ruditapes philippinarum. The results show that SOD, CAT and GSH-Px had diff erent induction and inhibition reactions to anthracene stress, and that three diff erent organs in R. philippinarum (visceral mass, muscle tissue and mantle) had diff erent sensitivities to anthracene stress. This study suggest that SOD activities of the visceral mass, CAT activitities of the mantle and the visceral mass, and GSH-Px activity of the muscle tissue could be used as sensitive indicators of anthracene stress in R. philippinarum.     

Seasonal variation of the glycogen enzyme activity in diploid and triploid Pacific oyster gonad during sexual maturation

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and gly- cogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of trip- loids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.     

Reproductive cycle and seasonal variations in lipid content and fatty acid composition in gonad of the cockle Fulvia mutica in relation to temperature and food

From March 2004 to February 2005,seasonal variations in lipid content and fatty acid composition of gonad of the cockle Fulvia mutica(Reeve) were studied on the eastern coast of China in relation to the reproductive cycle and environment conditions(e.g.,temperature and food availability).Histological analysis as well as lipid and fatty acid analyses were performed on neutral and polar lipids of the gonad.Results showed that gametogenesis occurred in winter and spring at the expense of lipids previously accumulated in summer and autumn,whereas spawning occurred in summer(20.4-24.6℃).The seasonal variation in lipid content was similar to that of the mean oocyte diameter.In both neutral and polar lipids,the 20:5n-3 and 22:6n-3 levels were relatively higher than saturated fatty acids,and polyunsaturated fatty acids were abundant,with series n-3 as the predominant component.Seasonal variations in the 20:5n-3 and 22:6n-3 levels and the principal n-3 fatty acids were clearly related to the reproductive cycle.The ∑(n-3) and ∑(n-6) values were relatively high during January-May,and the associated unsaturation index was significantly higher than that in other months.The results suggest that fatty acids play an important role in the gametogenesis of F.mutica.     

Estimation of age,growth and maturation of purpleback flying squid, <Emphasis Type="Italic">Sthenoteuthis oualaniensis</Emphasis>, 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.     

Trophic Interaction in a <Emphasis Type="Italic">Portunus rituberculatus</Emphasis> Polyculture Ecosystem Based on Carbon and Nitrogen Stable Isotope Analysis

Trophic interaction among various biomass groups in a swimming crab Portunus trituberculatus polyculture pond was investigated using carbon and nitrogen stable isotope analysis. The polycultured animal species also included white shrimp Litopenaeus vannamei, short-necked clam Ruditapes philippinarum, and redlip mullet Liza haematochila. The mean δ¹³C value for all the biomass groups in polyculture ecosystem ranged from ?25.61‰ to ?16.60‰, and the mean δ¹⁵N value ranged from 6.80‰ to 13.09‰. Significant difference in the δ¹³C value was found between particulate organic matter (POM) and sediment organic matter (SOM) (P < 0.05), indicating that these two organic matter pools have different material sources. Assuming that a ¹³C-enrichment factor of 1.00‰ and a ¹⁵N-enrichment factor of 2.70‰ existed between consumer and prey, diets of the four cultured animals were estimated using a stable isotope mixing model. The estimated model results indicated that P. trituberculatus mainly feed on Aloidis laevis; L. vannamei mainly feed on shrimp feed; while A. laevis, R. philippinarum and L. haematochelia mainly feed on POM. Shrimp feed was also an important food source of R. philippinarum and L. haematochelia. The diets of P. trituberculatus, L. vannamei, R. philippinarum, and L. haematochila showed complementary effects in this polyculture ecosystem. Our finding indicated that the polyculture of these four organisms with suitable farming density could make an effective use of most of the food sources, which can make a highly efficient polyculture ecosystem.     

The relationship between fat content and biological parameters of bigeye tuna (<Emphasis Type="Italic">Thunnus obseus</Emphasis>) in the Western Central Indian Ocean

Comparing fat content with physiological status can throw light on the reproduction and feeding behavior of the fish. The biological data of 586 bigeye tuna were collected from the longline fishery in the waters of Western Central Indian Ocean from November, 2012 to March, 2013. The spatial or temporal distribution of the fat content, and the relationships of fat content with gender, round weight, gonadal maturity and fork length were analyzed. A generalized additive model (GAM) was used to analyze the relationships between fat content and fork length (FL), gonadosomatic index (GSI), condition factor (K), and somatic index (SI). Results showed that: 1) the fat content of bigeye tuna was in the range of 3.1%–27.1% with the average 12.8%; 2) there were no significant geographical differences of average fat content (P > 0.05) among 1° squares in general; 3) there were no significant differences (P > 0.05) of the fat content for different genders, months, or gonad maturity stages; 4) there was an extremely significant correlation (P = 0.000) between fat content and FL and GSI. There was no significant correlation (P = 0.051) between fat content and K. There was a significant correlation (P = 0.003) between fat content and SI. The results of this study suggest that the fat content of the matured fish was relatively stable. The survey area was in a spawning region, and the survey period was the spawning season for bigeye tuna.     

Biochemical Composition and Nutritional Value of Different Shell Color Strains of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The shell color of Pacific oyster (Crassostrea gigas) is a desirable trait; but the nutritional studies on C. gigas with different shell colors have not been conducted. Through successive selective breeding, five shell color strains of black (B), purple (P), orange (O), golden (G) and white (W) C. gigas have been developed. The aim of this study was to evaluate the chemical composition and nutritional value of five shell color strains and one commercial population with a common color. The biochemical composition including moisture, total protein, glycogen, ash, total fat, fatty acids (FA), amino acids and minerals was detected. The results indicated that the protein (50.76%–56.57%) was the major component. The content of glycogen showed a significant difference between orange shell and golden shell strains, as well as between commercial population and golden shell strain. In addition, all shell color strains contained a large amount of essential amino acids (12.20–14.15 g (100 g)^?1), of them leucine (2.81–3.29 g (100 g)^?1) and lysine (2.79–3.28 g (100 g)^?1) were predominant. The oysters were rich in polyunsaturated fatty acids (42.26%–45.24% of total fatty acid) with high levels of DHA (18.53%–21.16% of total fatty acid) and EPA (17.23%–18.68% of total fatty acid). Significant differences of mineral contents (Mg, Zn, Fe and Cu) were identified among the six populations. These results indicated that C. gigas with different shell colors presented rich nutritional value with high protein, glycogen, essential amino acids and polyunsaturated fatty acids. The biochemical composition obtained in this study is useful for selective breeding of C. gigas with different shell colors.     

Effects of ocean acidification driven by elevated CO<Subscript>2</Subscript> on larval shell growth and abnormal rates of the venerid clam, <Emphasis Type="Italic">Mactra veneriformis</Emphasis>

The venerid clam (Mactra veneriformis Reeve 1854) is one of the main cultured bivalve species in intertidal and shallow subtidal ecosystems along the west coast of Korea. To understand the effects of ocean acidification on the early life stages of Korean clams, we investigated shell growth and abnormality rates and types in the D-shaped, umbonate veliger, and pediveliger stages of the venerid clam M. veneriformis during exposure to elevated seawater pCO₂. In particular, we examined abnormal types of larval shell morphology categorized as shell deformations, shell distortions, and shell fissures. Specimens were incubated in seawater equilibrated with bubbled CO₂-enriched air at (400±25)×10^-6 (ambient control), (800±25)×10^-6 (high pCO₂), or (1 200±28)×10^-6 (extremely high pCO₂), the atmospheric CO₂ concentrations predicted for the years 2014, 2084, and 2154 (70-year intervals; two human generations), respectively, in the Representative Concentration Pathway (RCP) 8.5 scenario. The mean shell lengths of larvae were significantly decreased in the high and extremely high pCO₂ groups compared with the ambient control groups. Furthermore, under high and extremely high pCO₂ conditions, the cultures exhibited significantly increased abundances of abnormal larvae and increased severity of abnormalities compared with the ambient control. In the umbonate veliger stage of the experimental larvae, the most common abnormalities were shell deformations, distortions, and fissures; on the other hand, convex hinges and mantle protuberances were absent. These results suggest that elevated CO₂ exerts an additional burden on the health of M. veneriformis larvae by impairing early development.     

Effects of dietary genistein on GH/IGF-I axis of Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

There is considerable concern that isoflavones, such as genistein in fish feed composed of soybean protein, aff ects somatic growth in fish. Our previous works demonstrated that 30 and 300 μg/g dietary genistein had no significant eff ect on growth performance in Nile tilapia (Oreochromis niloticus), but the higher level of genistein (3 000 μg/g) significantly depressed growth. This study was conducted to further examine the eff ects of dietary genistein on the endocrine disruption on growth hormone/insulin-like growth factor-I (GH/IGF-I) axis in Nile tilapia (O. niloticus). Juvenile fish were fed by hand twice daily to satiation with one of four isonitrogenous and isoenergetic diets, each containing either 0, 30, 300 or 3 000 μg/g genistein. Following an 8-week feeding period, plasma GH and IGF-I levels were investigated by radioimmunoassay and gene expression levels of gh, ghrelin, gnrhs, ghr, npy, npyrs, pacap, ghrs, igf-I, igf-Ir, and igfbp3 were examined by real-time PCR. The results show that no significant change in plasma GH and IGF-I levels in fish fed with diets containing 30 μg/g and 300 μg/g genistein. mRNA expression of genes along the GH/IGF-I axis remained unaff ected, except for igf-Ir, which was stimulated by the 300 μg/g genistein diet. While in fish fed the 3 000 μg/g genistein diet, the plasma GH and IGF-I levels decreased, and mRNA expression of gh, ghr2, npyr1, igf-I, and igf-Ir were also significantly depressed. In contrast, npy and igfbp3 mRNA expression were enhanced. This study provides convincing evidence for growth impediment by genistein by disturbing the GH/IGF-I axis in Nile tilapia O. niloticus.     

Loss of preferred habitat and pollen limitation threatens reproduction in a rare mountain <Emphasis Type="Italic">Paeonia delavayi</Emphasis>

Paeonia delavayi is a wild tree peony species endemic to high-altitude regions in southwestern China. Recent agricultural land expansion, however, not only causes its dwindling population size, but also poses a severe threat to its long-term persistence. Since our knowledge of the reproductive biology of P. delavayi is very limited, and some management misconceptions have further exacerbated the already worrisome situation, the aim of the present study is to provide some scientific evidence regarding habitat preference and breeding system of P. delavayi, and to help correct some management misconceptions. Specifically, morphological traits of P. delavayi populations growing in two different habitats (i.e., the open area versus forest understory), including basal stem diameter, plant height and flower numbers per plant, were measured and compared with each other. A range of floral manipulation experiments was further conducted to study the extent of self-compatibility and pollen limitation. The results showed that P. delavayi preferred the open habitat over forest understory as it generally grew bigger with thicker stems, and produced a higher number of flowers in the former habitat. Therefore, the open habitat should receive immediate priority for conservation action instead of being converted to agricultural land; also, P. delavayi is pollen limited, as evidenced by the fact that experimental flowers receiving supplemental non-self pollen generally displayed elevated seed production than flowers subjected to other treatments (e.g., non-supplemental self pollen, non-supplemental non-self pollen and supplemental self pollen), which is consistent with reported observations that alpine plants are more likely to be pollen-limited than lowland plants. We suggest that human intervention might be necessary to guarantee the long-term persistence of P. delavayi as harsh alpine environment, intense competition for pollinators and different anthropogenic perturbations co-limit its reproductive success.     

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Periphytic biofilms in aquaculture waters are thought to improve water quality, provide an additional food source, and improve the survival and growth of some reared animals. In the Asia- Pacific region, particularly in China, artificial reefs are commonly used in the commercial farming of sea cucumbers. However, few studies have examined the epilithic biofilms on the artificial reefs. To gain a better understanding of the succession of epilithic biofilms and their ecological processes in sea cucumber culture waters, two experiments were conducted in culture waters of the sea cucumber Apostichopus japonicus in Rongcheng, China, using artificial test panels. On the test panels of succession experiment, more than 67 species were identified in the biofilms. On the test panels of seasonal variation experiment, more than 46 species were recorded in the biofilms. In both experiments, communities of epilithic biofilms were dominated by diatoms, green algae and the annelid Spirorbis sp. In the initial colonization, the dominant diatoms were Cocconeis sp., Amphora spp. and Nitzschia closterium in June, which were succeeded by species of Navicula, Cocconeis and Nitzschia (July to September), and then by Licmophora abbreviata, Nitzschia closterium and Synedra spp. in the following months. A diatom bloom in the autumn and filamentous green algae burst in the summer were also observed. Ecological indices well annotated the succession and seasonal changes in epilithic communities. Multidimensional scaling (MDS) analysis found significant differences in diatom community composition among months and seasons. Fast growth of biofilms was observed in the summer and autumn, whereas the biomass of summer biofilms was largely made up of filamentous green algae. Present results show that the components of epilithic biofilms are mostly optimal foods of A. japonicus, suggesting that biofilms on artificial reefs may contribute important nutritional sources for sea cucumbers during their growth seasons. Future works should include quantitative determination of the contribution of epilithic biofilms to the diet of A. japonicus, potential roles of epilithic biofilms in regulating the water quality of sea cucumber ponds, and the regulation of epilithic biofilms in sea cucumber culture ponds.     

Seasonal Changes of Reproductive Activity and Biochemical Composition of Pen Shell Atrina pectinata Linnaeus,1767 in Bohai Sea,China

Seasonal variations in the biochemical composition and the reproductive cycle of pen shell Atrina pectinata in Bohai Sea were investigated from May 2013 to April 2014. Histological analysis indicated that the reproductive cycle of A. pectinata can be divided into two phases, inactive stage and gametogenesis, which were equally and significantly influenced by seawater temperature and food availability. Gametogenesis began in late autumn(October), and completed in June and July. Spawning took place in August, coinciding with the highest water temperature and the richest phytoplankton. The significantly high glycogen content in adductor muscles sustained throughout the late active and ripeness stages, but plummeted during spawning and inactive stages. The protein content in female gonads exhibited a synchronous increase along with oocyte diameter and lipid content, suggesting that the female gonads could accumulate protein and lipid for vitelline in A. pectinata. Furthermore, the RNA/DNA ratio was found to be a useful index to indicate the level of gonad maturation in both males and females. The findings of the present study provided a foundation for the fishery resource administration and the aquaculture development of this species.     

<Emphasis Type="Italic">Grateloupia ramosa</Emphasis> Wang &amp; Luan sp. nov. (Halymeniaceae,Rhodophyta), a new species from China based on morphological evidence and comparative <Emphasis Type="Italic">rbc</Emphasis>L sequences

Grateloupia ramosa Wang & Luan sp. nov. (Halymeniaceae, Rhodophyta) is newly described from Hainan Province, southern China. The organism has the following morphological features: (1) purplish red, cartilaginous and lubricous thalli 5–10 cm in height; (2) compressed percurrent axes bearing abundant branches with opposite arrangement; (3) claw-like apices on top, constricted to 2–4 cm at the base; (4) cortex consisting of 3–6 layers of elliptical or anomalous cells and a medulla covered by compact medullary filaments; (5) reproductive structures distributed throughout the thallus, especially centralized at the bottom of the end portion of the branches; and (6) 4-celled Carpogonial branches and 3-celled auxiliary-cell branches, both of the Grateloupia-type. The morphological diff erences were supported by molecular phylogenetics based on ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL) gene sequence analysis. There was only a 1 bp divergence between specimens collected from Wenchang and Lingshui of Hainan province. The new species was embedded in the large Grateloupia clade of the Halymeniaceae. The pairwise distances between G. ramosa and other species within Grateloupia ranged from 26 to 105 bp, within pairwise distances of 13–111 bp between species of the large genus Grateloupia in Halymeniaceae. Thus, we propose this new species as G. ramosa Wang & Luan sp. nov.     

Biodegradation of <Emphasis Type="Italic">Enteromorpha</Emphasis> polysaccharides by intestinal micro-community from <Emphasis Type="Italic">Siganus oramin</Emphasis>

Micro-communities are supposed to have more potential functions of biodegradation of polysaccharides than single strain; however, the intestinal micro-communities involved in the biodegradation of Enteromorpha polysaccharides (EP) were seldom reported. In order to obtain the EP-degrading micro-community, the intestines of Siganus oramin was obtained to isolate the micro-communities, which were enriched by 0.3% of EP as the sole carbon source. A stable micro-community with EP degradative capability was achieved after seven generations of subculture, named H1. Results showed that H1 was able to degrade 75% of EP within 24 hours, and the activity of EP lyases reached 500 U mL^?1 in 32 hours. With denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analysis, ten bacteria closely related to Marinomonas pontica, Microbacterium sp., Leucobacter chironomi, Cyclobacterium sp., Algoriphagus winogradskyi, Pseudoalteromonas sp. and Vibrio sp. were determined. Furthermore, compared with the DGGE bands sequence and the clone library analysis, the dominant bacteria of the EP-biodegrading micro- community were Pseudoalteromonas sp. and Vibrio sp., with the respective proportion of 38% and 46%, and they should play an important role in EP degradation together with other degrading bacteria in the micro-community H1.     

Impact of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) is an important viral pathogen that infects farmed penaeid shrimp, and the threat of Vibrio parahaemolyticus infection to shrimp farming has become increasingly severe. Viral and bacterial cross or superimposed infections may induce higher shrimp mortality. We used a feeding method to infect Litopenaeus vannamei with WSSV and then injected a low dose of V. parahaemolyticus (WSSV+Vp), or we first infected L. vannamei with a low-dose injection of V. parahaemolyticus and then fed the shrimp WSSV to achieve viral infection (Vp+WSSV). The eff ect of V. parahaemolyticus and WSSV co-infection on survival of L. vannamei was evaluated by comparing cumulative mortality rates between experimental and control groups. We also spread L. vannamei hemolymph on thiosulfate citrate bile salt sucrose agar plates to determine the number of Vibrio, and the WSSV copy number in L. vannamei gills was determined using an absolute quantitative polymerase chain reaction (PCR) method. LvMyD88 and Lvakt gene expression levels were detected in gills of L. vannamei by real-time PCR to determine the cause of the diff erent mortality rates. Our results show that (1) the cumulative mortality rate of L. vannamei in the WSSV+Vp group reached 100% on day 10 after WSSV infection, whereas the cumulative mortality rate of L. vannamei in the Vp+WSSV group and the WSSV-alone control group approached 100% on days 11 and 13 of infection; (2) the number of Vibrio in the L. vannamei group infected with V. parahaemolyticus alone declined gradually, whereas the other groups showed significant increases in the numbers of Vibrio (P<0.05); (3) the WSSV copy numbers in the gills of the WSSV+Vp, Vp+WSSV, and the WSSV-alone groups increased from 10⁵ to 10⁷ /mg tissue 72, 96, and 144 h after infection, respectively. These results suggest that V. parahaemolyticus infection accelerated proliferation of WSSV in L. vannamei and vice versa. The combined accelerated proliferation of both V. parahaemolyticus and WSSV led to massive death of L. vannamei.