Transcriptome analysis and immune gene expression of channel catfish (Ictalurus punctatus) fed diets with inclusion of frass from black soldier fly larvae.

The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. In this study, we subjected channel catfish (Ictalurus punctatus) to a 10-week feeding trial to assess how different dietary amounts of frass inclusion would affect both systemic and mucosal tissue gene expression, especially in regard to growth and immune-related genes. Fish were divided in quadruplicate aquaria, and five experimental diets comprising 0, 50, 100, 200, and 300 g of frass per kilogram of feed were fed twice daily. At the end of the trial, liver, head kidney, gill, and intestine samples were collected for gene expression analyses. First, liver and intestine samples from fish fed with a no frass inclusion diet (control), low-frass (50 g/kg) inclusion diet, or a high-frass (300 g/kg) inclusion diet were subjected to Illumina RNA sequencing to determine global differential gene expression among diet groups. Differentially expressed genes (DEGs) included the upregulation of growth-related genes such as glucose-6-phosphatase and myostatin, as well as innate immune receptors and effector molecules such as toll-like receptor 5, apolipoprotein A1, C-type lectin, and lysozyme. Based on the initial screenings of low/high frass using RNA sequencing, a more thorough evaluation of immune gene expression of all tissues sampled, and all levels of frass inclusion, was further conducted. Using targeted quantitative PCR panels for both innate and adaptive immune genes from channel catfish, differential expression of genes was identified, which included innate receptors (TLR1, TLR5, TLR9, and TLR20A), proinflammatory cytokines (IL-1ß type a, IL-1ß type b, IL-17, IFN-γ, and TNFα), chemokines (CFC3 and CFD), and hepcidin in both systemic (liver and head kidney) and mucosal (gill and intestine) tissues. Overall, frass from black soldier fly larvae inclusion in formulated diets was found to alter global gene expression and activate innate and adaptive immunity in channel catfish, which has the potential to support disease resistance in this species in addition to demonstrated growth benefits.

Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrioparahaemolyticus.

Vibrio parahaemolyticus (Vpara) is the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND), or Early Mortality Syndrome (EMS) in shrimp. Shrimp, like other invertebrates, lack an adaptive immune system and depend solely on innate immunity against invading pathogens. To better understand the defense mechanisms of shrimp to this problematic pathogen, we evaluated the changes in hematology, immunology and biochemical values of the hemolymph from shrimp challenged with V. parahaemolyticus up to 8 days post-challenge. Thirty-six shrimp (12 g) were distributed in 9 tanks (75 L), divided into three groups (non-challenged, challenged with 5 × 102 cfu/shrimp and challenged with 1 × 103 cfu/shrimp) in triplicate. Pacific white shrimp, Litopenaeus vannamei, were administered an inoculum of V. parahaemolyticus under the shell between the 5th and 6th abdominal segment to assess cellular and humoral immune responses. Total hemocyte count (THC) significantly decreased in shrimp challenged with Vpara at 6 h, 12 h and 24 h-post infection. Hemocyte lysate phenoloxidase (PO) activity in Vpara-challenged shrimp at 48 h post challenge was significantly increased compared to that of control shrimp. No significant differences were observed in total plasma protein between plasma from control and Vpara-challenged shrimp. However, shrimp challenged with 5 × 102, and 1 × 103 cfu/shrimp had significantly lower hemocyanin at 6 h and 48 h sampling point, respectively. At 24 h post-challenge, the ≥140 kDa and 70 kDa bands from SDS-PAGE of hemocyanin-concentrated hemolymph lysate samples showed a higher and lower intensity, respectively, in Vpara-challenged group than those of the control group. Plasma from Vpara-challenged shrimp at 6 h and 12 h-post infection significantly suppressed V. parahaemolyticus growth. However, significantly less bacterial growth suppression was observed in plasma of shrimp challenged with higher dose compared to control shrimp at the 192 h post-challenge point. Plasma chemistry parameters did not significantly differ among treatments. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp.

A novel paper-based and pH-sensitive intelligent detector in meat and seafood packaging.

The annual amount of food waste or loss is about one-third of the total edible food globally produced for human consumption. Continuous and real-time monitoring by spoilage detectors can significantly reduce food waste. A novel paper-based pH-sensitive meat spoilage detector was developed. A mixture of soybean hulls (SBHs) (hydrothermally-treated in an acidic environment), bentonite, and bromocresol purple (BCP) was coated on paper to produce the detector. The resultant meat spoilage detector was evaluated as a real-time freshness and spoilage indicator of catfish fillets (Ictalurus punctatus). Freshness and spoilage of fish meat with varying weights and headspace were determined by tailoring the detector's pH. Elemental, structural, and functional analysis verified the formation of a packed SBH-bentonite matrix with enhanced gas adsorption capacity and effective BCP-immobilization. Binder nanofibrillation increased the overall visual color vibrancy and decreased the binder demand in the coating formulation. Headspace volume in the studied range (40 and 160 cm3) did not affect the activation time of the detectors. However, increasing fish weight decreased the detectors' optimum activation time and pH. The findings of this study show that the developed detectors can be tailored for a wide range of sample and packaging sizes by simply adjusting the pH of the detector.

Antimicrobial activity of the biopolymer chitosan against Streptococcus iniae.

The antimicrobial activity and mode of action of chitosan were evaluated against Streptococcus iniae, a pathogenic Gram-positive bacterium of fish worldwide. Cell proliferation kinetics were examined following exposure to varying concentrations of chitosan. The action of chitosan on S. iniae was also investigated by measuring agglutination activity, conductivity, and extracellular and intracellular bacterial adenosine triphosphate (ATP) levels. Chitosan exhibited antibacterial activity against S. iniae at concentrations of 0.1% and above and was lethal at a concentration of 0.4% and higher. The mechanism of antibacterial activity of chitosan at the inhibitory level of bacterial growth appears to hinge upon the interaction between chitosan and the oppositely charged bacterial surface. This interplay causes agglutination, which was readily observed grossly and microscopically. After interacting with the cell surface via adsorption, an efflux of intracellular ATP was documented, which suggests that chitosan disrupts the bacterial cell causing leakage of cytosolic contents and ultimately cell death. Results suggest chitosan may be worth evaluating as a natural alternative to antibiotic against S. iniae infection of fish.

Examining the interplay between Streptococcus agalactiae, the biopolymer chitin and its derivative.

Streptococcus agalactiae is a highly pathogenic bacterium of aquatic species and terrestrial animals worldwide, whereas chitin and its derivative chitosan are among the most abundant biopolymers found in nature, including the aquatic milieu. The present investigation focused on the capability of S. agalactiae to degrade and utilize these polymers. Growth of S. agalactiae in the presence of colloid chitin, chitosan, or N-acetyl-glucosamine (GlcNAc) was evaluated. Chitosanase production was measured daily over 7 days of growth period and degraded products were evaluated with thin later chorography. Chitin had no effect on the growth of S. agalactiae. Degraded chitin, however, stimulated the growth of S. agalactiae. S. agalactiae cells did not produce chitinase to degrade chitin; however, they readily utilize GlcNAc (product of degraded chitin) as sole source of carbon and nitrogen for growth. Chitosan at high concentrations had antibacterial activities against S. agalactiae, while in the presence of lower than the inhibitory level of chitosan in the medium, S. agalactiae secrets chitosanase to degrade chitosan, and utilizes it to a limited extent to benefit growth. The interaction of S. agalactiae with chitin hydrolytes and chitosan could play a role in the diverse habitat distribution and pathogenicity of S. agalactiae worldwide.

Attempt to develop live attenuated bacterial vaccines by selecting resistance to gossypol, proflavine hemisulfate, novobiocin, or ciprofloxacin.

In an attempt to develop attenuated bacteria as potential live vaccines, four chemicals (gossypol, proflavine hemisulfate, novobiocin, and ciprofloxacin) were used to modify the following four genera of bacteria through chemical-resistance strategy: (1) Aeromonas hydrophila (9 isolates); (2) Edwardsiella tarda (9 isolates); (3) Streptococcus iniae (9 isolates); and (4) S. agalactiae (11 isolates). All bacteria used in this study were able to develop high resistance to gossypol. However, only some bacteria were able to develop resistance to proflavine hemisulfate, novobiocin, or ciprofloxacin. When the virulence of resistant bacteria was tested in tilapia or catfish, none of the gossypol-resistant isolate was attenuated, whereas majority of the proflavine hemisulfate-resistant isolates were attenuated. However, all proflavine hemisulfate-attenuated bacteria failed to provide significant protection to fish. Eight novobiocin- or ciprofloxacin-resistant Gram-positive bacteria (S. agalactiae and S. inaie) were found to be attenuated. However, none of them offered protection higher than 70%. Of seven attenuated novobiocin- or ciprofloxacin-resistant Gram-negative isolates (A. hydrophila and E. tarda), only one (novobiocin-resistant E. tarda 30305) was found to safe and highly efficacious. When E. tarda 30305-novo vaccinated Nile tilapia were challenged by its virulent E. tarda 30305, relative percent of survival of vaccinated fish at 14- and 28-days post vaccination (dpv) was 100% and 92%, respectively. Similarly, E. tarda 30305-novo offered 100% protection to channel catfish against challenges with virulent parent isolate E. tarda 30305 at both 14- and 28-dpv. Our results suggest that the development of live attenuated bacterial vaccines that are safe and efficacious is challenging, although it is feasible.

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections.

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.

Chemotactic factors of Flavobacterium columnare to skin mucus of healthy channel catfish (Ictalurus punctatus).

To gain an insight into the chemotactic factors involved in chemotaxis, we exposed a virulent strain of Flavobacterium columnare to various treatments, followed by analysis of its chemotactic activity. The chemotactic activity of F. columnare was significantly (P<0.05) inhibited when cells were pretreated by sodium metaperiodate, and a major portion of the capsular layer surrounding the cells was removed. Pretreatment of F. columnare with D-mannose, D-glucose and N-acteyl-D-glucosamine significantly (P<0.05) inhibited its chemotaxis activity, whereas pretreatment of cells with d-fructose, L-fucose, D-glucosamine, d-galactosamine, D-sucrose and N-acetyl-D-galactosamine failed to inhibit its chemotactic activity. These results indicate that at least three carbohydrate-binding receptors (D-mannose, D-glucose and N-acteyl-D-glucosamine) associated with the capsule of F. columnare might be involved in the chemotactic responses. The relative transcriptional levels of three gliding motility genes (gldB, gldC, gldH) of F. columnare compared with 16S rRNA gene following the exposure of F. columnare to catfish skin mucus were evaluated by quantitative PCR (qPCR). qPCR results revealed that the transcriptional level of gldH was significantly (P<0.001) upregulated in normal F. columnare at 5 min postexposure to the catfish mucus. However, when F. columnare were pretreated with D-mannose, there was no upregulation of gliding motility genes. Taken together, our results suggest that carbohydrate-binding receptors play important roles in the chemotactic response to catfish mucus.

Growth performance, vitamin E status, and proximate and fatty acid composition of channel catfish, Ictalurus punctatus, fed diets containing various levels of fish oil and vitamin E.

A study was conducted to determine the effect of increasing dietary levels of fish oil on vitamin E requirement and their effect on growth performance, liver vitamin E status, and tissue proximate and fatty acid compositions of channel catfish. Basal purified diets (42% protein and 3,800 kcal DE/kg) supplemented with 6, 10, and 14% menhaden fish oil were each supplemented with 50, 100, and 200 mg vitamin E/kg (3×3 factorial experiment). Each diet was fed to juvenile channel catfish in three random aquaria to apparent satiation twice daily for 12 weeks. Weight gain, feed intake, and feed efficiency ratio were not affected by dietary levels of fish oil, vitamin E, or their interaction. Survival rate at the end of week 12 was significantly lower for fish fed diets containing 14% fish oil, regardless of vitamin E content. Whole-body moisture significantly decreased and lipid increased when dietary lipid levels were increased to 10 or 14%. Dietary vitamin E levels had no effect on body proximate composition. Lipid content of liver was not influenced by dietary levels of fish oil and vitamin E or their interaction. Hepatosomatic index significantly decreased with increasing lipid levels but was not affected by dietary levels of vitamin E. Liver vitamin E increased with increasing dietary vitamin E but decreased with increasing fish oil levels. Fatty acid composition of whole body and liver reflected that of dietary lipid but was not influenced by dietary levels of vitamin E. Whole-body saturates increased, whereas MUFA decreased with increasing dietary levels of fish oil. Liver saturates were not affected by fish oil levels, but MUFA and n-6 decreased and increased, respectively, with increasing fish oil levels. Total n-3 and n-3 HUFA in both tissues increased with increasing fish oil levels in diets, but liver stored much higher levels of these fatty acids.