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.     

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.     

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.     

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.     

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.     

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.     

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     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei

We hypothesized that aggregation of bacteria and hemocytes at the gill, which occurs as part of the shrimp's antibacterial immune defenses, would impair normal respiratory function and thereby disrupt aerobic metabolism. Changes in oxygen uptake and lactate accumulation were determined in Litopenaeus vannamei, the Pacific white shrimp, following injection with either saline (control) or a strain of the gram-negative bacterium Vibrio campbellii that is pathogenic in crustaceans. The rate of oxygen uptake was determined during the first 4 h after injection and after 24 h. Injection of bacteria decreased oxygen uptake by 27% (from 11.0 to 8.2 micromol g-1 h-1) after 4 h, while saline-injected shrimp showed no change. Decreased oxygen uptake persisted 24 h after Vibrio injection. In well-aerated water, resting whole-animal lactic acid levels increased in shrimp injected with bacteria (mean=2.59 micromol lactate g-1+/-0.39 SEM, n=8) compared to saline-injected control shrimp, but this difference did not persist at 24 h. Exposure to hypercapnic hypoxia (PCO2=1.8 kPa, PO2=6.7 kPa) also resulted in significant whole-body lactic acid differences (mean=3.99 and 1.8 micromol g-1 tissue in Vibrio and saline-injected shrimp, respectively). Our results support the hypothesis that the crustacean immune response against invading bacteria impairs normal metabolic function, resulting in depression of oxygen uptake and slightly increased anaerobic metabolism.     

'Bright-red' syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi

Since July 2005, recurrent outbreaks of vibriosis have occurred in shrimp farms in northwestern Mexico. Moribund Litopenaeus vannamei associated with mass mortalities were lethargic and displayed red discoloration spots on their abdomen, and hence were called 'bright-reds' by farmers. Shrimp submitted for diagnosis were examined using wet tissue mounts, bacteriological assays and their respective minimum inhibitory concentration (MIC), and histology. A dominant yellow bacterial colony was isolated in thiosulphate citrate bile salts-sucrose (TCBS) agar and identified by molecular methods as Vibrio harveyi strain CAIM 1792. Pathogenicity of the V. harveyi strain was demonstrated in L. vannamei. The lowest MIC against Vibrio isolates from bright-red shrimp was obtained with enrofloxacine (3.01, SD = 5.96 pg ml(-1)). Histology detected severe necrosis in lymphoid organ tubules, muscle fibers, and connective tissue, as well as melanization and hemocytic nodules associate with microcolonies of Gram-negative bacilli. Bacteria from severely affected shrimp were dispersed from the haemocoel to other tissues causing a systemic vibriosis. The data indicate that V. harveyi strain CAIM 1792 is the cause of bright-red syndrome (BRS) and represents a threat to the Mexican shrimp farming industry.     

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and l-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of l-serine ammonia lyase was also examined. Specific activity was highest when l-serine levels in the hemolymph were highest. Thus, it appears that l-serine levels increased under hyposmotic conditions due to the consumption of l-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.     

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.     

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.     

Immunostimulation of white shrimp (Litopenaeus vannamei) following dietary administration of Ergosan

Ergosan an algal product containing 1% alginic acid, developed for use in aquaculture and reported to have immunomodulatory activity, was administered orally to intermoult adult white shrimp (Litopenaeus vannamei) for 15 days. Examination of haemolymph proteins using SDS-PAGE did not reveal any obvious differences between control and Ergosan treated shrimp. Similarly, total haemocyte counts were found to be roughly equivalent for both the control and experimental samples. However, differential analysis of haemocyte populations revealed marked changes in terms of the relative levels of hyaline, semi-granular, and particularly granular haemocytes between the two groups. Moreover, enhancement of the in vitro antimicrobial activity of haemolymph towards two shrimp pathogenic Vibrio isolates was recorded for shrimp fed with Ergosan. Finally, shrimp fed with Ergosan showed a significant increase in relative growth when compared with control groups.     

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5′-terminal untranslated region (UTR) of 60 bp and a 3′-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys⁵³, Cys¹²⁸, Cys¹⁴⁴, Cys¹⁵²) involved in the formation of disulfides bridges and the potential Ca²⁺/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca²⁺, and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.