Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

Immune parameters, haemocyte lifespan, and gene expressions of lipopolysaccharide and β-glucan-binding protein (LGBP), peroxinectin (PX), integrin β, and α2-macroglobulin (α2-M) were examined in white shrimp Litopenaeus vannamei juveniles (0.48 ± 0.05 g) which had been reared at different salinity levels of 2.5‰, 5‰, 15‰, 25‰, and 35‰ for 24 weeks. All shrimp survived during the first 6 weeks. The survival rate of shrimp reared at 2.5‰ and 5‰ was much lower (30%) than that of shrimp reared at 15‰, 25‰, and 35‰ (76%~86%) after 24 weeks. Shrimp reared at 25% grew faster. Shrimp reared at 2.5‰ and 5‰ showed lower hyaline cells (HCs), granular cells (GCs), phenoloxidase activity (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity, but showed a longer haemocyte lifespan, and higher expressions of LGBP, PX, integrin β, and α2-M. In another experiment, shrimp which had been reared at different salinity levels for 24 weeks were challenged with Vibrio alginolyticus (6 × 10(6) cfu shrimp(-1)), and WSSV (10(3) copies shrimp(-1)) and then released to their respective seawater. At 96-144 h, cumulative mortalities of shrimp reared at 2.5‰ and 5‰ were significantly higher than those of shrimp reared at 15‰, 25‰, and 35‰. It was concluded that following long-term exposure to 2.5‰ and 5‰ seawater, white shrimp juveniles exhibited decreased resistance against a pathogen due to reductions in immune parameters. Increases in the haemocyte lifespan and gene expressions of LGBP, integrin β, PX, and α2-M indicated that shrimp had the ability to expend extra energy to modulate the innate immune system to prevent further perturbations at low salinity levels.     

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei

In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.     

Effects of dopamine on the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (20.0+/-1.5 g) were injected individually with dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1), respectively. For the shrimp that received dopamine at 10(-7) and 10(-6)mol shrimp(-1), the THC decreased by 25% and 39%, phenoloxidase activity decreased by 15% and 32%, respiratory burst decreased by 21% and 36%, and SOD activity decreased by 50% and 63%, respectively, after 4 h. The phagocytic activity and clearance efficiency of shrimp that received dopamine at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 16, 8, 8, 24, 16 and 4 h, respectively, for the shrimp that received dopamine at either dose. In another experiment, L. vannamei which had received dopamine at 10(-8), 10(-7) and 10(-6)mol shrimp(-1) were challenged after 1 h by injection with V. alginolyticus at 1.0x10(5) colony-forming units (cfu) shrimp-1 and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received dopamine at either dose was significantly higher than that of shrimp that received saline after 8 h, and of shrimp that received saline at the termination of the experiment (48 h after the challenge). It is therefore concluded that dopamine administration at 10(-6)mol shrimp-1 or less causes immune modulation of L. vannamei.     

Effects of phosphatidyl serine on immune response in the shrimp Litopenaeus vannamei

Phosphatidyl serine plays an important role in animal innate immunity. Given its important functions, numerous investigations have been carried out on its immunological function in many animals. However, studies of phosphatidyl serine in the white shrimp Litopenaeus vannamei, an economically important animal, are rare. In this paper, we demonstrated influences of injecting phosphatidyl serine (PS) on immune response including some parameters from pro-phenol oxidase activating system (pro-PO system) and hemocyanin-derived phenol oxidase activity (Hd-PO) along with antibacterial and bacteriolytic activities in the white shrimp Litopenaeus vannamei with different PS concentrations (5, 10 and 20 μg mL^?1). The results showed that PS could affect immune response of L. vannamei significantly (P<0.05), including total hemocyte counts (THC), PO activity from hemocyte, phenol oxidase (PO) activity from plasma, hemocyanin concentration, Hd-PO activity as well as antibacterial and bacteriolytic activities in the plasma. Among the lines, 20 μg mL^?1 PS had the strongest effect on the above parameters, whereas 5 μg mL^?1 had the least effect. The experimental results indicated that PS was able to activate exocytosis of pro-PO and formation of Hd-PO in white shrimp after injection, further regulating the immune process reflected by variation of antibacterial and bacteriolytic activities in a certain way.     

Effect of methyl parathion on the susceptibility of shrimp Litopenaeus vannamei to experimental vibriosis

Following increasing calls for environmental safety over the past 2 decades, persistent pesticides are being replaced by more rapidly degradable products. However, even these pesticides can affect non-target species, and may be associated with slow growth and increased susceptibility to viral and bacterial infections. In this study, juvenile white shrimp Litopenaeus vannamei (also named Penaeus vannamei) were challenged by intramuscular injection with Vibrio parahaemolyticus after 4 d prior exposure to methyl parathion in feed pellets at 0.080 microg g(-1). The bacterial injection control group consisted of shrimp fed pellets containing the methyl parathion-carrier solvent acetonitrile. Three additional control groups comprised 2 sterile saline-injection groups fed pellets containing methyl parathion or acetonitrile prior to injection, and 1 uninjected group fed normal pellets. Cumulative mortalities were recorded on the 4th and 8th days, and the presence of histological lesions was recorded on the 8th day. Cumulative mortalities were significantly higher in the group exposed to methyl parathion and bacteria on Day 8. Histological lesions, typical of vibriosis, were significantly associated with the injection of V. parahaemolyticus. The study provides strong experimental evidence that prior exposure to methyl parathion can increase the severity of Vibrio infections.     

Reconsideration of phenoloxidase activity determination in white shrimp Litopenaeus vannamei

Though phenoloxidase (PO) activity has been used as an important index in immunological research of crustaceans, methods for the determination of PO activity are not consistent even for the same species. Plasma, the major location of PO activity, should be the most reasonable sample, instead of hemocytes or serum, for the determination of PO activity of shrimp. The current study provided a thorough characterization and reconsideration for PO activity assay in the plasma of Litopenaeus vannamei. Results show that the final concentration of l-dihydroxyphenylalanine (l-DOPA) for PO activity assay should be no less than 1.5 mg ml^?1, and pH 6.6 should be used to maintain the stability of l-DOPA solution. This study provides direct evidence that PO activity is significantly inhibited by EDTA, and it is suggested to use EDTA-free anticoagulant in separating plasma for PO activity assay in future studies. Repeated measurements indicated that the assayed PO activities are significantly affected by preservation conditions, and plasma is quite unstable with spontaneous activation when put in ice or stored at ?20 °C. Thus samples need to be measured immediately or preserved at ?80 °C with assay as soon as possible after it is thawed, and should not be preserved for a second time for measuring PO activity.     

Isolation and characterization of new microsatellite loci in the Pacific white shrimp Litopenaeus vannamei and cross‐species amplification in other penaeid species

The goal of the present study was to obtain valuable microsatellite markers useful in genetic approaches of the shrimp Litopenaeus vannamei, an important aquaculture species of the world. Eight loci produced suitable polymorphic patterns with allele number ranging from two to 12 and expected heterozygosity from 0.18 to 0.89. Cross‐species amplification was successfully performed in five other penaeid species (Litopenaeus schmitti, Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Xiphopenaeus kroyeri and Rimapenaeus constrictus), indicating that some these loci can be useful in studies of other related species.     

Recombinant shrimp (Litopenaeus vannamei) trypsinogen production in Pichia pastoris

Shrimp (Litopenaeus vannamei) trypsinogen has never been isolated from its natural source. To assess the production of L. vannamei trypsinogen, we engineered Pichia pastoris strains and evaluated two culture approaches with three induction culture media, to produce recombinant shrimp trypsinogen for the first time. The trypsinogen II cDNA was fused to the signal sequence of the Saccharomyces cerevisiae alpha mating factor, placed under the control of the P. pastoris AOX1 promoter, and integrated into the genome of P. pastoris host strain GS115. Using standard culture conditions for heterologous gene induction of a GS115 strain in shake flasks, recombinant shrimp trypsinogen was not detected by SDS‐PAGE and Western blot analysis. Growth kinetics revealed a toxicity of recombinant shrimp trypsinogen or its activated form over the cell host. Thus, a different culture approach was tested for the induction step, involving the use of high cell density cultures, a higher frequency of methanol feeding (every 12 h), and a buffered minimal methanol medium supplemented with sorbitol or alanine; alanine supplemented medium was found to be more efficient. After 96 h of induction with alanine supplemented medium, a 29‐kDa band from the cell‐free culture medium was clearly observed by SDS‐PAGE, and confirmed by Western blot to be shrimp trypsinogen, at a concentration of 14 μg/mL. Our results demonstrate that high density cell cultures with alanine in the induction medium allow the production of recombinant shrimp trypsinogen using the P. pastoris expression system, because of improved cell viability and greater stability of the recombinant trypsinogen. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

凡纳滨对虾繁殖中不同亲本对子代遗传贡献率的差异

利用5个含有稀有等位基因的高度多态性微卫星位点比较了凡纳滨对虾繁殖中不同亲本对子代遗传贡献率的差异。通过稀有等位基因的5个微卫星位点能够对亲代和子代的谱系进行明确的鉴别。10个亲代个体中有8个个体对子代群体的基因库有贡献,不同个体之间的贡献率存在差别,最高为54.28%,最低为8.57%。在亲代和子代群体遗传结构的分析中,子代等位基因的数目与亲代相比降低了11.11%。子代的平均期望杂合度(He)、平均观测杂合度(Ho)和平均多态性信息含量(PIC)等指标均低于亲代。实验结果表明:亲本对子代基因库的贡献率的差异也是造成子代群体遗传变异水平降低的原因之一;微卫星标记可作为一种有效的工具用于对虾系谱的确认、人工繁育群体遗传多样性水平的监测等方面     

Hemocytic immune responses triggered by CpG ODNs in shrimp Litopenaeus vannamei

CpG oligodeoxynucleotides (CpG ODNs), also called bacterial DNA or synthetic oligodeoxynucleotides, can induce apparent immunity protection against various pathogens, and they are widely used as functional immunostimulant or vaccine adjuvant in mammals. In the present study, CpG-rich plasmid pUC57-CpG was constructed and employed to stimulate the shrimp Litopenaeus vannamei, and the total hemocyte count, percentage of apoptotic hemocytes, regeneration of circulating hemocytes, the ability of phagocytosis and generation of reactive oxygen species (ROS) were measured to reveal the possible protection mechanism of CpG ODNs. After the injection of pUC57-CpG, the total hemocyte count significantly decreased (p < 0.01) to 2.56 × 10⁷ cell/mL at the first day post stimulation, while the apoptosis increased (p < 0.01), which was 1.72-fold of that in control group. At the same time, the regeneration of circulating hemocytes fluctuated in a similar trend, and a significant increase was observed at the first day post stimulation. The phagocytotic activity including the percentage of phagocytosis and phagocytotic index, experienced an upward tend during the whole experimental period and the ROS level increased by 22% (p < 0.05) compared to that in the control group at first day post stimulation. These results together suggested that pUC57-CpG could promote the apoptosis and regeneration of circulating hemocytes, and enhance the phagocytosis and ROS production, which might contribute to the boosted immunity against the infection of pathogens.     

Enhanced cellular immunity in shrimp (Litopenaeus vannamei) after 'vaccination'

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.     

Swimming ability and physiological response to swimming fatigue in whiteleg shrimp, Litopenaeus vannamei

Some penaeids are active swimmers, undertaking migrations of hundreds of nautical miles. At present, however, very little is known of swimming ability in penaeid shrimps. The aim of the present study is to investigate swimming endurance of whiteleg shrimp, Litopenaeus vannamei, against one of five flow velocities (5.41, 6.78, 8.21, 10.11, and 11.47 cm s(-1)) for up to 9000 s at 20 degrees C in a swimming channel. Body mass, hemolymph total protein concentration, and hemolymph glucose level were measured before swimming and immediately following swimming to evaluate physiological effect of swimming in L. vannamei. No shrimp swam the full 9000 s at any of the velocities tested. The swimming endurance decreased as swimming speed was increased. The relationship between swimming endurance (t, in s) and swimming speed (v, in cm s(-1)) can be described by the Curve Estimation: v.t0.38 = 159.64 (R2 = 0.94). The swimming ability index (SAI), defined as SAI = integral 0-9000 vdt x 10(-4) (cm) was found to be 7.28 cm for the shrimp tested. Swimming to fatigue leads to severe loss of body mass, hemolymph total protein concentration, and hemolymph glucose level in L. vannamei (P < 0.05). Furthermore, these decreases and swimming speed showed significantly polynomial relationships (P < 0.05). The results suggest that the power model fits well to the observed endurance estimates and the SAI is a good index to quantitatively describe the overall swimming ability of L. vannamei. Furthermore, hemolymph total protein concentration may be used as a rapid and reliable indicator to assess the penaeid shrimps' swimming speed and hence swimming ability.     

Functional characterization of the mitochondrial uncoupling proteins from the white shrimp Litopenaeus vannamei

Mitochondrial uncoupling proteins (UCPs) play an essential role in dissipating the proton gradient and controlling the mitochondrial inner membrane potential. When active, UCPs promote proton leak across the inner membrane, oxidative phosphorylation uncoupling, oxygen uptake increase and decrease the ATP synthesis. Invertebrates possess only isoforms UCP4 and UCP5, however, the role of these proteins is not clear in most species since it may depend on the physiological needs of each animal. This study presents the first functional characterization of crustacean uncoupling proteins from the white shrimp Litopenaeus vannamei LvUCP4 and LvUCP5. Free radicals production in various shrimp organs/tissues was first evaluated, and mitochondria were isolated from shrimp pleopods. The oxygen consumption rate, membrane potential and proton transport of the isolated non-phosphorylating mitochondria were used to determine LvUCPs activation/inhibition. Results indicate that UCPs activity is stimulated in the presence of 4-hydroxyl-2-nonenal (HNE) and myristic acid, and inhibited by the purine nucleotide GDP. A hypoxia/re-oxygenation assay was conducted to determine whether UCPs participate in shrimp mitochondria response to oxidative stress. Isolated mitochondria from shrimp at re-oxygenation produced large quantities of hydrogen peroxide and higher levels of both LvUCPs were immunodetected. Results suggest that, besides the active response of the shrimp antioxidant system, UCP-like activity is activated after hypoxia exposure and during re-oxygenation. LvUCPs may represent a mild uncoupling mechanism, which may be activated before the antioxidant system of cells, to early control reactive oxygen species production and oxidative damage in shrimp.     

Chronic effect of nitrite on the rearing of the white shrimp Litopenaeus vannamei in two salinities

The aim of this study was to determine the effects of nitrite on the growth and survival of the white shrimp L. vannamei in two different salinities. Nitrite concentrations tested in salinity 8 g/L were 0 (control), 2.5, 5.0, 10.0, and 20.0 mg NO₂^?-N/L, and in salinity 24 g/L were 0 (control), 5.0, 10.0, 20.0, and 40.0 mg NO₂^?-N/L. For these experiments, 30 experimental units with 30?L of useful volume were stocked with 20 juvenile L. vannamei (8.0 ± 0.50 g), corresponding to a stocking density of 100 shrimp/m², and cultivated for an experimental period of 30 days. A significant difference was found between the control and treatment groups with respect to growth and survival. The 2.5 mg NO₂^?-N/L treatment showed the best performance indexes in salinity 8 g/L, while the best growth performance indexes were found in the control and 5.0 mg NO₂^?-N/L treatments in salinity 24 g/L. Total mortality was observed in the 10 and 20 mg NO₂^?-N/L treatment groups from salinity 8 g/L and in the 40 mg NO₂^?-N/L treatment group in salinity 24 g/L. This study determined that concentrations of nitrite of up to 2.5 and 10 mg/L are acceptable for the rearing of L. vannamei in salinities of 8 and 24 g/L, respectively.     

Seaweed meal as a protein source for the white shrimp Litopenaeus vannamei

The rhodophytes Hypnea cervicornis and Cryptonemia crenulata are abundant along the Brazilian coastline and are rich in nutrients. They may therefore be used as a source of protein in shrimp diets. The aim of the present study was to test this hypothesis. The experiment was conducted in a laboratory, where 10-day-old post-larvae aged underwent 7 days of acclimation in a 1,000 L tank. They were then kept in plastic aquariums, each containing 10 L, and 20 larvae were fed daily (10% of biomass) in four equal portions with one of four diets (five repetitions of each) for a period of 45 days. All diets contained 30% crude protein (isoprotein) and 300 kcal 100 g⁻¹ (isocaloric), with different percentages of seaweed powder: Diet “A” 39%; Diet “B” 26%, Diet “C” 13%, and Diet “D” without seaweed (control diet). Algae were collected, rinsed, dried and ground up for the feed formulations. Weight of the animals was measured at the beginning of the experiment and at 15-day intervals to assess their growth. The physico-chemical variables of the water were measured every 2 days. Final biomass, biomass gain and specific growth rate (SGR) exhibited no significant differences between treatments (P > 0.05). Survival rate was equal under the four experimental conditions, being consistent within four decimal places 95.2% to 97.00% (P > 0.05). Diets “A” and “B”, with a greater content of algae, exhibited better feed conversion (1.79:1 and 1.82:1) than Diets “C” and “D” (2.04:1 and 2.08:1) (P < 0.05). The physical-chemical variables of the water showed no significant variation and remained within the standards necessary for the wellbeing of the animals. If sufficient biomass of beached algae can be practically and economically collected, it may be used as a component in the making of shrimp feed.