Comparison of five commercial kits for isolation of total RNA in samples of WSSV-infected shrimp.

Viral diseases are the most serious threat to the expansion and development of shrimp aquaculture. Rapid diagnosis of the white spot syndrome virus (WSSV), a lethal shrimp pathogen, is essential to restrict its spread and reduce the mortality of infected shrimp. This virus has globally affected the shrimp farming industry, with a devastating economic impact. Several studies have focused on the expression of WSSV transcripts to understand the molecular mechanisms governing the pathological development of the disease. Since gene expression studies and molecular diagnostics at the early stages of infection depend on the efficient isolation of high-quality RNA, the extraction methods should be carefully selected. However, previous comparisons of the performance of RNA isolation kits have yet to be systematically investigated. In this study, 5 commercial RNA extraction methods were compared in WSSV-infected shrimp. The highest total RNA yield (ng mg-1 tissue) was obtained using TRIzol. Even though the 260/280 nm absorption ratios showed significant differences, the methods showed good purity values (>2.0). RNA integrity was evaluated in a denaturing agarose gel electrophoresis, and degradation was observed after the total RNA samples were treated with DNase I. Finally, the method that allowed the earlier detection of WSSV transcripts by qRT-PCR was the Zymo Direct-zol RNA MiniPrep kit. This study shows that the amount of observed (or estimated) WSSV transcripts might be affected because of the RNA isolation method. In addition, these results may contribute to improve the accuracy of the results obtained in gene expression studies, for more sensitive and robust detection of WSSV.

Multiple reproduction forms in the polyps of the cannonball jellyfish Stomolophus sp. 2: Probable life-cycle reversal.

The jellyfish genera Stomolophus spp. is one of the most abundant in the Pacific Ocean, yet it has not been thoroughly studied. Until recently, research has been developed and directed to its knowledge because of the economic interest in its exploitation. The genus Stomolophus in the Pacific Ocean is composed of five species (S. agaricus, S. chunii, S. collaris, S. fritillaria, and S. meleagris), and Stomolophus sp. 2 has been recently reported in the central part of the Gulf of California. Therefore, this study aimed to describe in vivo the different developmental stages of Stomolophus sp. 2 life cycle. As a result, multiple polyp reproduction forms were described, such as polyp-stolon formation, polydisc strobilation with more than 20 ephyrae formed by each strobila, and polyp formation directly from juvenile ephyra. In the degenerating phase, the polyps turned into cysts induced by stress conditions, such as changes in temperature, oxygen, and food availability. The life cycle of Stomolophus sp. 2 can be distinguished from that of S. meleagris by showing various asexual reproduction mechanisms and polydisc-like strobilation. The formation of polyps directly from the ectoderm of degenerating juvenile medusae suggests the possibility of a reversion cycle. Because of the different life cycles between S. meleagris and S. sp. 2, in addition to their morphological and genetic differences, this study proposes that Stomolophus sp. 2 should be considered a new species and suggests the name Stomolophus yaquilli, in reference to the indigenous community that lives in the species distribution area.

Understanding the Digestive Peptidases from Crustaceans: from Their Biochemical Basis and Classical Perspective to the Biotechnological Approach.

Scientific studies about decapod crustaceans' digestive physiology have increased, being an important topic with novel results in the last years. This revision aims to show how the study of crustacean peptidases has evolved, from the classical biochemical characterization studies to the assessment of their usefulness in biotechnological and industrial processes, with emphasis on commercial species of interest to world aquaculture and fisheries. First studies determined the proteolytic activity of the midgut gland crude extracts and evaluated the optimum biochemical properties of specific enzymes. Peptidase's identity was determined using inhibitors and specific protein substrates on tube tests and electrophoresis gels. Later, various studies focused on the characterization of purified peptidases and their gene expression. Recently, the integrated mechanisms of enzyme participation during the digestive process of food protein have been established using novel techniques. Scientific research has revealed some of the potential biotechnological applications of crustacean peptidases in the food industry and other processes. However, the knowledge field is enormous, and there is much to explore and study in the coming years.

Growth performance, carcass traits, muscle fiber characteristics and skeletal muscle mRNA abundance in hair lambs supplemented with ferulic acid.

Ferulic acid (FA) is a phytochemical with various bioactive properties. It has recently been proposed that due to its phytogenic action it can be used as an alternative growth promoter additive to synthetic compounds. The objective of the present study was to evaluate the growth performance, carcass traits, fiber characterization and skeletal muscle gene expression on hair-lambs supplemented with two doses of FA. Thirty-two male lambs (n = 8 per treatment) were individually housed during a 32 d feeding trial to evaluate the effect of FA (300 and 600 mg d-1) or zilpaterol hydrochloride (ZH; 6 mg d-1) on growth performance, and then slaughtered to evaluate the effects on carcass traits, and muscle fibers morphometry from Longissimus thoracis (LT) and mRNA abundance of ß2-adrenergic receptor (ß2-AR), MHC-I, MHC-IIX and IGF-I genes. FA increased final weight and average daily gain with respect to non-supplemented animals (p < 0.05). The ZH supplementation increased LT muscle area, with respect to FA doses and control (p < 0.05). Cross-sectional area (CSA) of oxidative fibers was larger with FA doses and ZH (p < 0.05). Feeding ZH increased mRNA abundance for ß2-AR compared to FA and control (p < 0.05), and expression of MHC-I was affected by FA doses and ZH (p < 0.05). Overall, FA supplementation of male hair lambs enhanced productive variables due to skeletal muscle hypertrophy caused by MHC-I up-regulation. Results suggest that FA has the potential like a growth promoter in lambs.

Effects of Ferulic Acid Supplementation on Growth Performance, Carcass Traits and Histochemical Characteristics of Muscle Fibers in Finishing Pigs.

FA dietary supplementation on the growth performance, carcass traits and histochemical characteristics of the Longissimus thoracis muscle from finishing pigs was investigated. Four hundred and twenty pigs were used in this study, and 105 animals (with five replicate pens and 21 pigs per pen) were assigned to one of four treatments: basal diet (BD) without additives (C-); BD + 10 ppm ractopamine hydrochloride + 0.97% lysine (C+); BD + 25 ppm of FA (FA); and BD + 25 ppm of FA + 0.97% lysine (FA-Lys). Dietary supplementation with FA or ractopamine increased both the average daily gain (14%) and loin muscle area (19%), while fat deposition decreased by 53%, in comparison with C- (p < 0.05). The growth performance of pigs treated with FA was similar to those of ractopamine (p > 0.05). The histochemical analysis showed that FA and C+ treatments induced a shift in muscle fiber types: from fast fibers to intermediate (alkaline ATPase) and from oxidative to glycolytic fibers. Muscle tissues from animals treated with FA or ractopamine had a lower cross-sectional area and a greater number of muscle fibers per area (p < 0.05). Findings regarding growth performance and carcass traits indicate that FA supplementation at 25 ppm without extra-lysine can replace the use of ractopamine as a growth promoter in finishing pigs.

Adaptive mitochondrial response of the whiteleg shrimp Litopenaeus vannamei to environmental challenges and pathogens.

In most eukaryotic organisms, mitochondrial uncoupling mechanisms control ATP synthesis and reactive oxygen species production. One such mechanism is the permeability transition of the mitochondrial inner membrane. In mammals, ischemia-reperfusion events or viral diseases may induce ionic disturbances, such as calcium overload; this cation enters the mitochondria, thereby triggering the permeability transition. This phenomenon increases inner membrane permeability, affects transmembrane potential, promotes mitochondrial swelling, and induces apoptosis. Previous studies have found that the mitochondria of some crustaceans do not exhibit a calcium-regulated permeability transition. However, in the whiteleg shrimp Litopenaeus vannamei, contradictory evidence has prevented this phenomenon from being confirmed or rejected. Both the ability of L. vannamei mitochondria to take up large quantities of calcium through a putative mitochondrial calcium uniporter with conserved characteristics and permeability transition were investigated in this study by determining mitochondrial responses to cations overload. By measuring mitochondrial swelling and transmembrane potential, we investigated whether shrimp exposure to hypoxia-reoxygenation events or viral diseases may induce mitochondrial permeability transition. The results of this study demonstrate that shrimp mitochondria take up large quantities of calcium through a canonical mitochondrial calcium uniporter. Neither calcium nor other ions were observed to promote permeability transition. This phenomenon does not depend on the life cycle stage of shrimp, and it is not induced during hypoxia/reoxygenation events or in the presence of viral diseases. The absence of the permeability transition phenomenon and its adaptive meaning are discussed as a loss with biological advantages, possibly enabling organisms to survive under harsh environmental conditions.

Muscle fiber morphometry and physicochemical characteristics of the Longissimus thoracis muscle of hair male lambs fed zilpaterol hydrochloride and implanted with steroids.

Muscle fiber morphometry and physicochemical characteristics were evaluated in LT muscles obtained from entire male lambs treated with zilpaterol hydrochloride (ZH, 0 and 0.15 mg/kg body weight) and/or steroidal implant (SI, with and without trenbolone acetate/estradiol). ZH and SI acted synergistically to increase LT area, type-IIb fiber cross-sectional area and soluble collagen content, likewise to decrease metmyoglobin concentration and insoluble collagen content. Ash content and ultimate pH showed a decrease due to an antagonistic effect between ZH and SI. Content of total collagen, protein, fat, moisture, oxidized lipids and water-holding capacity were unaffected by ZH and SI. Supplemental ZH, but not SI, decreased all color parameters and tended to increase shear force. Overall, the SI implantation of male lambs followed by a ZH supplementation promoted greater LT hypertrophy, without affecting protein and fat content, and physicochemical characteristics in their meat.

Protective Effect of Lacticaseibacillus casei CRL 431 Postbiotics on Mitochondrial Function and Oxidative Status in Rats with Aflatoxin B1-Induced Oxidative Stress.

Studies have shown that the intracellular content of probiotic (postbiotics) has antioxidant properties, which can improve the antioxidant status in vivo. However, its absorption and mechanisms underlying the protective effects are still unknown. The antioxidant capacity of Lacticaseibacillus casei CRL431 (IC-431) postbiotics was determined after an in vitro simulated digestive process. Permeability of antioxidant constituents of IC-431 was determined by an ex vivo everted duodenum assay. Aflatoxin B1-induced oxidative stress rat models were established and treated with IC-431; biomarkers of hepatic mitochondrial function and H2O2 levels, oxidative stress, and oxidative stress index (OSi) were examined. The antioxidant capacity of IC-431 (477 ± 45.25 µmol Trolox Equivalent/L) was reduced by exposure to the simulated digestive process. No difference (p > 0.05) was found among digested and the permeate fraction of IC-431. A protective effect was observed by significantly lower OSi and higher liver glutathione peroxidase and catalase activities. Lower H2O2 production, a higher degree of mitochondrial uncoupling, and lower mitochondrial respiration coefficient were also observed (p < 0.05). These results suggest that IC-431 antioxidant components permeate intestinal barriers to enter the bloodstream and regulate antioxidant status during AFB1-induced oxidative stress by reducing hepatic mitochondrial dysfunction, thus enhancing antioxidant enzyme response.

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.

Metabolism, ATP production and biofilm generation by Staphylococcus epidermidis in either respiratory or fermentative conditions.

Staphylococcus epidermidis is a Gram-positive saprophytic bacterium found in the microaerobic/anaerobic layers of the skin that becomes a health hazard when it is carried across the skin through punctures or wounds. Pathogenicity is enhanced by the ability of S. epidermidis to associate into biofilms, where it avoids attacks by the host and antibiotics. To test the effect of oxygen on metabolism and biofilm generation, cells were cultured at different oxygen concentrations ([O2]). As [O2] decreased, S. epidermidis metabolism went from respiratory to fermentative. Remarkably, the rate of growth decreased at low [O2] while a high concentration of ATP ([ATP]) was kept. Under hypoxic conditions bacteria associated into biofilms. Aerobic activity sensitized the cell to hydrogen peroxide-mediated damage. In the presence of metabolic inhibitors, biofilm formation decreased. It is suggested that at low [O2] S. epidermidis limits its growth and develops the ability to form biofilms.