The effect of ethanol on some hematological parameters of the australian red claw crayfish (Cherax quadricarinatus)

Purpose: to study the effect of ethanol on the parameters of THC, the percentage of granulocytes and total protein in the hemolymph of the Red claw crayfish (Cherax quadricarinatus).Materials and methods. The object of this experiment was 26 males of the Australian red-clawed crayfish (Cherax quadricarinatus) weighing from 23 to 83 g. The individuals were evenly divided into two experimental groups - with an injection of ethanol and a control group without an injection of 13 crayfish for each group. The injection dose was 2515 mg per 100 g of body weight. A day after the introduction of ethanol, hemolymph was taken with a syringe from the ventral sinus, the syringe was pre-washed with a 4% EDTA-Na2 solution. Three parameters were determined: the total hemocyte count (THC), percent granulocytes and percent total protein content. Counting of hemocytes and determination of granulocytes were performed in a Goryaev chamber under a light microscope. The total protein was determined by the refractometric method.Results. Differences in THC and total protein between the groups were statistically unreliable (p>0,05). THC in the experimental group is 36% more than in the control group. The total protein after the introduction of ethanol actually increased by 0,7%, and relatively by 14%. There were statistically different indicators of the proportion of granulocytes (p<0,05) - the average value of 33,1% in the experimental group versus 24,5% in the control group. A reliable (p<0,05) strong feedback was revealed between the total protein and the mass of individuals in both experimental groups, while in the experimental group there is a visible shift in the values of dependent hemolymph indicators towards an increase in smaller individuals.Conclusion. A single injection of ethyl alcohol with a dosage of 2515 mg per 100 g of body weight into the hemolymph of C. quadricarinatus does not cause significant changes in the THC and total protein after 24 hours. At the same time, the proportion of granulocytes actually increases by 9%, relative to 37%. This may indicate that granulocytes are involved in the formation of cancer defense mechanisms when exposed to toxic substances. The effect of different dosages of ethanol injections and the duration of its effect on hematological parameters requires additional consideration. It is necessary to investigate its effect on other indicators, such as the pH and buffer capacity of the hemolymph, the concentration of hemocyanin, glucose, lactates and calcium.

Impacts of pH on the Fitness and Immune System of Pacific White Shrimp

The atmospheric partial pressure of CO2 (pCO2) has been increasing dramatically since the beginning of the industrial revolution and about 30% of the CO2 produced by anthropogenic activities was absorbed by the ocean. This led to a perturbation of the seawater carbonate chemistry resulting in a decrease of the average surface ocean pH by 0.1 and termed ocean acidification (OA). Projections suggest that pCO2 may reach 900 μatm by the end of the twenty-first century lowering the average pH of the surface ocean by 0.4 units. The negative impacts of OA on many species of marine invertebrates such as mollusks, echinoderms, and crustaceans are well documented. However, less attention has been paid to the impacts of low pH on fitness and immune system in crustaceans. Here, we exposed Pacific white shrimps to 3 different pHs (nominal pH 8.0, 7.9, and 7.6) over a 100-days experiment. We found that, even though there were no significant effects on fitness parameters (survival, growth and allometries between length and weight), some immune markers were modified under low pH. A significant decrease in total hemocyte count and phenoloxidase activity was observed in shrimps exposed to pH 7.6 as compared to pH 8.0; and phagocytosis rate significantly decreased with decreasing pH. A significant increase in superoxide production was also observed at pH 7.6 as compared to pH 8.0. All these results suggest that a 100-days exposure to pH 7.6 did not have a direct effect on fitness but lead to a modulation of the immune response.

Effects of spray-dried animal plasma on growth performance, survival, feed utilization, immune responses, and resistance to Vibrio parahaemolyticus infection of Pacific white shrimp (Litopenaeus vannamei)

Spray-dried animal plasma (SDP) in feed for several animal species provides health benefits, but research about use of SDP in shrimp feed is very limited. The objectives of the present study were to investigate the effects of dietary SDP on growth performance, feed utilization, immune responses, and prevention of Vibrio parahaemolyticus infection in Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the post-larvae were divided into five groups (four tank/group and 80 shrimp/tank) and fed four times daily diets with porcine SDP at 0, 1.5, 3, 4.5, and 6% of the diet for 45 days. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups: four SDP groups as in Experiment 1 plus the positive and negative controls (four tank/group and 30 shrimp/tank). They were then challenged with V. parahaemolyticus by immersion at 105 colony-forming units (CFU)/mL and were fed with the same diets for another 4 days. In Experiment 1, shrimp fed 4.5% or 6% SDP diets had significantly higher body weight, survival rate, and improved feed conversion ratio. The immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) of the shrimp fed 3–6% SDP diets also showed significant enhancement compared to the control. In Experiment 2, the survival rates of the 3–6% SDP groups were significantly higher than the positive control at day 4 after the immersion challenge. Likewise, the histopathological study revealed milder signs of bacterial infection in the hepatopancreas of the 3–6% SDP groups compared to the challenged positive control and 1.5% SDP groups. In conclusion, shrimp fed diets with SDP, especially at 4.5–6% of the diet, showed significant improvement in overall health conditions and better resistance to V. parahaemolyticus infection.

miR-151 Affects Low-Temperature Tolerance of Penaeus vannamei by Modulating Autophagy Under Low-Temperature Stress

MicroRNAs (miRNAs) play key roles in many physiologic and pathologic processes, including autophagy. Autophagy is cellular in an emergency response mechanism of environment stress, but their complex molecular regulatory mechanism under low-temperature stress is largely unknown in shrimp, especially miRNA-mediated regulation of autophagy in low-temperature tolerance. In this article, a shrimp PvTOR and miRNA pva-miR-151 cooperation in response to low-temperature stress has been reported. Pva-miR-151 showed expression patterns opposite to target PvTOR under low-temperature stress. The pva-miR-151 targets the 3′-UTR region of PvTOR, regulate the formation of autophagosome, which contribute to the degradation and recycling of damaged organelles. In addition, the low-temperature tolerance was correlated positively with autophagy in shrimp. Silenced pva-miR-151 increased sensitivity to low-temperature stress, whereas overexpression pva-miR-151 decreased the expression of PvTOR and p-TOR and increased tolerance to low-temperature stress by improving the formation of autophagosome and total hemocyte count. In addition, the TOR activator 3BDO can partially rescue autophagy induced by overexpression of pva-miR-151; these results indicate that miR-151 was necessary for the low-temperature tolerance in shrimp. Taken together, we provide a novel strategy and mechanism for shrimp breeding to improve shrimp low-temperature tolerance.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Effect of gallic acid on the larvae of Spodoptera litura and its parasitoid Bracon hebetor

The antibiosis effect of gallic acid on Spodoptera litura F. (Lepidoptera: Noctuidae) and its parasitoid evaluated by feeding six days old larvae on artificial diet incorporated with different concentrations (5 ppm, 25 ppm, 125 ppm, 625 ppm, 3125 ppm) of the phenolic compound revealed higher concentration (LC50) of gallic acid had a negative impact on the survival and physiology of S. litura and its parasitoid Bracon hebetor (Say) (Hymenoptera:Braconidae). The mortality of S. litura larvae was increased whereas adult emergence declined with increasing concentration of gallic acid. The developmental period was delayed significantly and all the nutritional indices were reduced significantly with increase in concentration. Higher concentration (LC50) of gallic acid adversely affected egg hatching, larval mortality, adult emergence and total development period of B. hebetor. At lower concentration (LC30) the effect on B. hebetor adults and larvae was non-significant with respect to control. Gene expression for the enzymes viz., Superoxide dismutase, Glutathione peroxidase, Peroxidase, Esterases and Glutathione S transferases increased while the total hemocyte count of S. litura larvae decreased with treatment. Our findings suggest that gallic acid even at lower concentration (LC30) can impair the growth of S. litura larvae without causing any significant harm to its parasitoid B. hebetor and has immense potential to be used as biopesticides.

Effects of Larval Density on Plutella xylostella Resistance to Granulosis Virus

It has been reported that some phase-polyphenic insects from high-density conditions are more resistant to pathogens than those from low-density conditions. This phenomenon is termed “density-dependent prophylaxis” (DDP). However, whether non phase-polyphenic insects exhibit DDP has rarely been elucidated. The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. In this study, the resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus when reared at densities of 1, 2, 5, 10, 15, and 20 larvae per Petri dish were investigated under laboratory conditions. Compared with larvae reared at lower densities, larvae reared at moderate density showed a significantly higher survival rate, but the survival rate significantly decreased with further increases in rearing density. Furthermore, the phenoloxidase, lysozyme and antibacterial activity and total hemocyte count in the hemolymph of the larvae, regardless of whether they were challenged with the virus, from different larval densities corresponded to the observed differences in resistance to Plxy GV. These results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.

Activation of cellular immune response in insect model host Galleria mellonella by fungal α-1,3-glucan

Alpha-1,3-glucan, in addition to β-1,3-glucan, is an important polysaccharide component of fungal cell walls. It is reported for many fungal species, including human pathogenic genera: Aspergillus, Blastomyces, Coccidioides, Cryptococcus, Histoplasma and Pneumocystis, plant pathogens, e.g. Magnaporthe oryzae and entomopathogens, e.g. Metarhizium acridum. In human and plant pathogenic fungi, α-1,3-glucan is considered as a shield for the β-1,3-glucan layer preventing recognition of the pathogen by the host. However, its role in induction of immune response is not clear. In the present study, the cellular immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan was investigated for the first time. The changes detected in the total hemocyte count (THC) and differential hemocyte count (DHC), formation of hemocyte aggregates and changes in apolipophorin III localization indicated activation of G. mellonella cellular mechanisms in response to immunization with A. niger α-1,3-glucan. Our results, which have clearly demonstrated the response of the insect immune system to this fungal cell wall component, will help in understanding the α-1,3-glucan role in immune response against fungal pathogens not only in insects but also in mammals, including humans.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.