Genetic diversity status of Pacific white shrimp (<Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>) using SSR markers in Iran

Genetic diversity is vital for the maintenance of genetic pool in cultured shrimps. In order to estimate the current status of genetic diversity in Litopenaeus vannamei shrimp in Iran, as an exotic species, a total of 45 individuals from Amiri and Gorgeaj farms in Jask port of Hormozgan province and one hatchery in Gomishan city of Golestan province, were detected using four microsatellite loci. The number of alleles per locus was 5–10, and the mean effective number of alleles (N _e) across populations and loci ranged from 4.834 to 5.148. The overall mean observed heterozygosity (H _o) ranged from 0.450 to 0.479, which was lower than the expected one (0.789–0.794). There was nothing remarkable about any of the allele frequencies across populations or loci. The mean inbreeding coefficient (F _IS) and pairwise genetic differentiation (F _ST) among populations were 41.6 % and 0.133, respectively. The three studied populations departed from Hardy–Weinberg equilibrium (HWE). Analysis of molecular variance revealed 14 % variability among and 86 % within populations. However, considering departing from HWE and the high F _IS and F, the moderate pairwise F _ST values, importance of introducing genetically diverse broodstock and monitoring to control inbreeding is discussed.     

Comparison of flavor components in shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> cultured in sea water and low salinity water

To compare the flavor components of shrimp Litopenaeus vannamei cultured in sea water and low salinity water, the chemical composition of muscle was analyzed, muscle extracts prepared, and their sensory components and sensory properties were assessed. Shrimp cultured in sea water had a higher content of crude protein, lower moisture, and a higher flesh pH compared to the low salinity samples (P<0.05), whereas no significant differences were found in crude lipid and ash. The free amino acid composition of muscle extracts and the amino acid composition of peptides from shrimp cultured in sea water and low salinity water were similar, that is, the former had a very high content of glycine, arginine, proline and alanine, and the latter a very high content of glutamate, glycine, arginine, proline and alanine. The major components of nucleotides were adenosine monophosphate and inosine monophosphate, and the major components of organic acids were acetic acid and malic acid. Extracts from shrimp cultured in sea water had enhanced umami, sweetness and overall flavor, and less of an earthy-musty taste compared to samples cultured in low salinity water (P<0.05). Aftertaste did not differ between the two shrimp extracts (P>0.05).     

The effectiveness of a commercial microbial product in poorly prepared tiger shrimp, Penaeus monodon (Fabricius), ponds

The efficacy of a commercial microbial product was tested in commercial tiger shrimp, Penaeus monodon (Fabricius), ponds for one culture period in Kuala Selangor, Malaysia. Four ponds with replicates for treatment and control were used. The pond bottom was dried but the organic sludge was not removed as normally practised in pond preparation. The ponds were stocked with 15 post‐larvae at the rate of 31.m^?2. Physical, chemical and biological parameters of the pond were analysed every 2 weeks during the culture period. Water quality parameters remained within the optimum range for shrimp culture except for ammonia‐nitrogen being significantly higher in control ponds and silica in treated ponds. Benthic organisms were not found in any of the ponds. The average counts of different bacteria were not significantly higher in treated ponds than control. Because of poor health, the shrimp were harvested earlier (72 days) than the usual 120 days. An average of 875.60 ± 67.00 kg shrimp ha^?1 was obtained in treated ponds with a feed conversion ratio (FCR) of 1.57 ± 0.10 and survival rate of 42.35 ± 5.37% compared with 719.50 ± 130.94 kg shrimp ha^?1, 2.99 ± 0.70 and 21.25 ± 3.26%, respectively, in control ponds. Neither the microbial product nor the frequent water exchange was effective in overcoming the problems caused by the poor pond bottom.     

Effect of microencapsulated thyme essential oil on white spot virus-infected <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

The present work was performed to investigate if feeding Litopenaeus vannamei with microencapsulated thyme essential oil (TEM, 1.05 g thyme essential oil per 100-g powder) adsorbed on commercial pellet feed was able to protect shrimps against white spot syndrome virus (WSSV) disease. Five treatments were tested: uninfected shrimp fed with commercial pelleted feed (T_C, negative control), WSSV-infected shrimp fed with commercial pelleted feed (T₁, positive control), WSSV-infected shrimp fed with commercial pelleted feed with 0.1% TEM (T₂), WSSV-infected shrimp fed with commercial pelleted feed with 0.5% TEM (T₃), and WSSV-infected shrimp fed with commercial pelleted feed with 1% TEM (T₄). At 72 h post infection, phenoloxidase activity of shrimps treated with 1% TEM did not show significant differences with T_C values but it was significantly higher than that of the other treatments (T₁, T₂, and T₃). Moreover, shrimps treated with T₄ presented absence of clinical signs of WSSV infection and their survival rate was significantly higher than that of T₁, T₂, and T₃ treatments. Therefore, 1% TEM seemed to protect shrimps against WSSV symptoms. Using microencapsulated thyme essential oil may help to fight against WSSV in shrimp farms.     

Prevalence of white spot syndrome virus infection detected by one-step and nested PCR in selected tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) hatcheries

White spot syndrome (WSS) is considered as a great threat to commercial farming of the tiger shrimp (Penaeus monodon). The causal agent of WSS is a DNA virus called white spot syndrome virus (WSSV). The prevalence of this dreadful virus infection has been studied in five randomly selected hatcheries located in the Cox’s Bazar district of Bangladesh. Both one-step and nested polymerase chain reaction (PCR) involving two pairs of primers, namely, 146F1/146R1 and 146F2/146R2, amplifying the 1447 bp and 941 bp fragments, respectively, were conducted to detect the WSSV. Out of 60 randomly collected shrimps, 12 (20%) were found to be positive by one-step PCR, while 18 (30%) were found to be positive by nested PCR. The nested PCR was found to be much more sensitive than the one-step PCR. The shrimp specimens showing clinical signs of WSS were positive for WSSV by both one-step and nested PCR. Some of the apparently healthy samples were also found to be positive for WSSV by nested PCR. Among the two primer-pairs, the inner pair amplifying the 941 bp fragment was more sensitive than the outer primer pair amplifying the 1447 bp fragment when used in one-step PCR.     

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in Pacific white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> after multiple-dose oral administration

The present study was designed to explore pharmacokinetics (PK) of enrofloxacin and its metabolite ciprofloxacin in Pacific white shrimp Litopenaeus vannamei after multiple-dose oral administration of enrofloxacin (30 mg/kg dose per 12 h and continuous feeding for 3 days). Enrofloxacin and ciprofloxacin concentrations in hemolymph, hepatopancreas, and muscle of the shrimp were simultaneously determined by high-performance liquid chromatography. PK parameters were analyzed based on statistical moment theory. Meanwhile, the relationship of pharmacokinetics/pharmacodynamics (PK/PD) was established based on drug concentration of hemolymph and in vitro antibacterial activity (MIC value). Results showed faster absorption of enrofloxacin in hemolymph (T_max?=?1 h) and muscles (T_max?=?1 h) than that in hepatopancreas (T_max?=?3 h) after the first oral administration. In multiple-dose oral administration, slight accumulation of enrofloxacin occurred in the hemolymph and hepatopancreas, while in the muscle, enrofloxacin concentration showed a significant decline following multiple administration. Tissue distribution of enrofloxacin and ciprofloxacin both followed the order hepatopancreas?>?hemolymph?>?muscle, with significantly higher ciprofloxacin concentration in hepatopancreas than in hemolymph (approximately 10-fold) and muscles (approximately 50-fold), indicating that the hepatopancreas is the main organ involved in metabolism of enrofloxacin in Pacific white shrimp. After multiple-dose administration, C_max/MIC and AUC_0–24/MIC values showed that the therapeutic regimen in this study could be remarkably effective in prevention and treatment of Vibrio infection in Pacific white shrimp.     

Enhancement of <Emphasis Type="Italic">Penaeus monodon</Emphasis> shrimp postlarvae growth and survival without water exchange using marine <Emphasis Type="Italic">Bacillus pumilus</Emphasis> and periphytic microalgae

We have investigated the possibility of using a consortium of marine bacterium and periphytic microalgae to improve the water quality and increase the growth and survival of the shrimp Penaeus monodon in a hatchery system. Three treatments were evaluated for their effect on P. monodon postlarvae (PL) when the culture water was not changed: Bacillus pumilus alone (B); periphytic microalgae alone (M); B. pumilus + periphytic microalgae (BM). P. monodon PL raised in a tank of unchanged water without bacterium and periphytic microalgae served as the control. The water in tanks of the M and BM treatments had significantly low levels of total ammonia-nitrogen (TAN) (0.03 and 0.01 mg l⁻¹, respectively) and nitrite-nitrogen (NO₂-N) (0.03, 0.01 mg l⁻¹, respectively) than that in the B (TAN 0.80, NO₂-N 0.68 mg l⁻¹) and control (TAN 1.11, NO₂-N 1.12 mg l⁻¹) tanks. Moreover, PL cultured in tanks M and BM had significantly higher survival and specific growth rates and a significantly higher resistance to the reverse salinity stress test than those in the B and control tanks. Compared to the control PL, the PL cultured in the BM tanks had significantly higher levels of protein, lipid, polyunsaturated fatty acids, ecosapentaenoic acid, and docosahexaenoic acid. The culture water in tanks BM also contained significantly less Vibrio than the control water. Our results illustrate the beneficial effects of a B. pumilus and periphytic microalgae consortium on improving the water quality and the growth and survival of shrimp PL grown in a hatchery system.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Use of a <Emphasis Type="Italic">Noctiluca</Emphasis>-killing bacterium <Emphasis Type="Italic">Marinobacter salsuginis</Emphasis> strain BS2 to reduce shrimp mortality caused by <Emphasis Type="Italic">Noctiluca scintillans</Emphasis>

The ability of an algicidal bacterium Marinobacter salsuginis strain BS2, isolated from shrimp pond water, to reduce shrimp mortality was investigated under laboratory conditions. When two species of shrimp (Penaeus monodon and Litopenaeus vannamei) (body length 1.5–1.8 cm) were cultured together with the dinoflagellate Noctiluca scintillans, nearly 80 % of the shrimps died within 7 days. However, when bacterial strain BS2 was also added to the culture, N. scintillans was killed within 48 h, and shrimp survival rates on the 7th day improved from 23 to 87 % for both P. monodon and L. vannamei. The bacterium BS2 alone had no effect on shrimp condition. Under conditions of increased dissolved oxygen, the effect of using BS2 was greater, and shrimp survival rates improved even more dramatically, from 26 to 98 %. These studies provide the first evidence that the use of killing bacteria, isolated from shrimp culture water, can suppress harmful algal blooms (HABs) and thus restore the efficiency of shrimp production. The control of HABs in this way in shrimp culture farms would be a major benefit for shrimp production.     

Carbonic anhydrase and Na/K-ATPase activities during the molt cycle of low salinity-reared white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Changes in hemolymph osmolality, ion concentrations, and enzymatic activities of carbonic anhydrase (CA) in the gills and epidermal tissue, and Na/K-ATPase in the gills during the molt cycle were investigated in the white shrimp Litopenaeus vannamei. Hemolymph osmolality was high in the intermolt and early premolt stages, but started to decrease prior to ecdysis through to postmolt stages A and B. Changes in Na⁺ and Cl⁻ ion concentrations paralleled those in hemolymph osmolality. CA activity levels in the anterior and posterior gills were low at intermolt stage C₀ and premolt stage D₀, and maximum at premolt stage D₃. In the epidermal tissue, activity was relatively high at intermolt stage C₀ and premolt stage D₀, but fluctuated towards premolt stage D₃ and postmolt stage A. On the other hand, Na/K-ATPase activity in the gills decreased between intermolt stage C₀ and premolt stage D₂, but increased at premolt stage D₃ and postmolt stage A. The changes in patterns of CA activity during the molt cycle suggest that CA may be involved in supplying counter-ions for Na⁺ and Cl⁻ uptake during molting. Branchial Na/K-ATPase appears to be involved in producing local osmotic gradients in order to support water influx across the epithelium.     

Effect of VP28 DNA vaccine on white spot syndrome virus in <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. Considering the global environmental, the economic and sociological importance of shrimp farming, and the constraints of high intensity cultivation, development of novel control measures against the outbreak of WSSV become inevitable. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the recombinant plasmid in immunized Litopenaeus vannamei. The VP28 gene was cloned in the eukaryotic expression vector pVAX1, and the construct vector was named as lpv28. The protective effect of lpv28 against WSSV was evaluated in L. vannamei by injecting lpv28 construct and later challenging with WSSV. Expression of these proteins from the recombinant plasmids was confirmed in vitro by RT-PCR and Western blot analysis. The result of vaccination trials showed that a survival rate in shrimp vaccinated with lpv28 was 52.5% at most compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups showed that the vaccinated groups owned a high level of lysozyme, alkaline phosphatase, and total superoxide dismutase when compared to the control group. Furthermore, protein expression analysis indicated that VP28 can be detected in gill, muscle and head soft tissue of the shrimps in the immunized group after 14th day injection. Thus, the result indicated that DNA vaccination strategy has a potential utility against WSSV.     

Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

<Emphasis Type="Italic">Virgibacillus proomii</Emphasis> and <Emphasis Type="Italic">Bacillus mojavensis</Emphasis> as probiotics in sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) larvae: effects on growth performance and digestive enzyme activities

This study examined the effects of two probiotics (Virgibacillus proomii and Bacillus mojavensis) on the digestive enzyme activity, survival and growth of Dicentrarchus labrax at various ontogenetic stages in three separate experiments. These probiotics were incorporated as single or mixed into fish feed for a period of 60 days. The growth parameters, proximate composition of whole body, digestive enzymes and gut microbiology were monitored at regular. The increments in length and weight and the survival were significantly higher (P < 0.05), and the values of food conversions were significantly lower (P < 0.05) in fishes fed the probiotic. The administration of V. proomii and B. mojavensis in diet resulted in an increase (P > 0.05) in body ash and protein content and in the specific activity of phosphatase alkaline and amylase in the digestive tract of all the fishes. V. proomii and B. mojavensis persisted in the fish intestine and in the feed in high numbers during the feeding period (group 1: 5.8 × 10⁴ CFU/ml, group 2: 9.6 × 10⁴ CFU/ml, and group 3: 9.8 × 10⁴ CFU/ml day 60). The two probiotics V. proomii and B. mojavensis were adequate for improved growth performance and survival and for healthy gut microenvironment of the host.     

Enhancement of survival and metamorphosis rates of <Emphasis Type="Italic">Penaeus monodon</Emphasis> larvae by feeding with the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

The purpose of this study was to compare the performance of two species of diatoms, Thalassiosira weissflogii and Chaetoceros gracilis, in the larviculture of the black tiger shrimp Penaeus monodon. Shrimp larvae were fed with either C. gracilis, T. weissflogii, or a combination of the two species of diatoms. The larvae fed solely with T. weissflogii or a combination of the two types of diatom had significantly higher survival rates and faster metamorphosis than those fed solely with C. gracilis. The numbers of diatom cells consumed by larvae during 3-h periods were determined, revealing that larvae consumed significantly higher numbers of C. gracilis than T. weissflogii. However, when the protein, total fatty acid, and polyunsaturated fatty acid content of the two species of diatom are compared, significantly higher amounts of each are found in T. weissflogii. Converting the number of diatom cells consumed into equivalent protein, total fatty acid, eicosopentaenoic acid, and decosahexaenoic acid reveals that larvae fed with T. weissflogii received significantly higher amounts of all the nutrients, compared to those consuming C. gracilis. The results showed an advantage of feeding T. weissflogii to C. gracilis in enhancing survival and metamorphosis in P. monodon larvae.