Establishment of a cobia (Rachycentron canadum) gill cell line: A valuable tool for immune response studies.

Cobia (Rachycentron canadum), a commercially important marine fish, has been used to develop a novel gill cell line, designated CG, for the first time. The CG cell line was cultured in Leibovitz's-15 medium with 5% fetal bovine serum (FBS) and successfully sub-cultured more than 110 passages. It underwent verification through sequencing of the mitochondrial cytochrome C oxidase subunit I (COI) gene. Optimal growth rate was achieved when the CG cell line was cultured in a medium supplemented with 5% FBS, 1% Penicillin-Streptomycin (P/S), and 5 parts per thousand (ppt) of coral sea salt water, maintained at a temperature of 27 °C. The addition of 5 ppt of salt in the growth medium suggests that this cell line could be a viable in vitro tool for marine ecosystem toxicological studies or for culturing marine parasitic microorganisms. The CG cell line was also successfully transfected using the pTurbo-GFP plasmids, showing an 18% efficiency, with observable GFP expression. Furthermore, the cell line has been effectively cryopreserved. Gene expression analysis indicated that the CG cell line exhibits responsive regulation of immune gene expression when exposured to various stimulants, highlighting its potential as an in vitro platform for immune response studies. This makes it suitable for exploring dynamic immune signaling pathways and host-pathogen interactions, thereby offering valuable insights for therapeutic development.

Assessment of Bacillus subtilis fermented Caulerpa microphysa byproduct as feed additive on the growth performance, immune regulation and disease resistance of white shrimp (Litopenaeus vannamei).

In this study, the immunomodulatory and antioxidant activity of fermented Caulerpa microphysa byproduct (FCMB) by Bacillus subtilis was evaluated, and its potential as a feed additive for white shrimp (Litopenaeus vannamei) was explored. In vitro experiments showed that the FCMB supernatant contained polysaccharides, polyphenols and flavonoids, and exhibited antioxidant properties as assessed by various antioxidant assays. Additionally, the FCMB supernatant was found to increase the production rate of reactive oxygen species and the activity of phenoloxidase in hemocytes in vitro. Furthermore, the results of the in vivo feeding trial showed that dietary 5 g kg-1 FCMB significantly improved the weight gain and specific growth rate of white shrimp after 56 days of feeding. Although there were no significant differences in total hemocyte count, phagocytosis, superoxide anion production rate, and phenoloxidase activity among the experimental groups, upregulation of immune-related genes was observed, particularly in the hepatopancreas and hemocytes of shrimps fed with 5 g or 50 g FCMB per kg feed, respectively. In the pathogen challenge assay, white shrimp fed with 5 % FCMB exhibited a higher survival rate compared to the control group following Vibrio parahaemolyticus challenge. Therefore, it is concluded that the fermented byproduct of C. microphysa, FCMB, holds potential as a feed additive for enhancing the growth performance and disease resistance against V. parahaemolyticus in white shrimp.

Optimizing inorganic carbon and salinity for enhanced biomass and pigment production in Colaconema formosanum: Implications for sustainable carbon sequestration and stress responses.

This study investigates a cultivation strategy for the macroalga Colaconema formosanum by determining optimal inorganic carbon concentration and salinity for maximizing biomass and photosynthetic pigment production while also facilitating carbon sequestration. The response surface method was used with a central composite design (CCD-RSM) to determine the optimal conditions. Results showed that adding 1.2 g/L of carbon increased the specific growth rate to 18%-19% per day. The maximum amount of pigment, including phycobiliprotein and chlorophyll, was achieved by adjusting both carbon content and salinity. This strategy enables mass pigment production and offers an eco-friendly approach to carbon sequestration while reducing culture period. This study also sheds light on algal mechanisms against enriched inorganic carbon and salinity content, contributing to an enhanced understanding of these vital processes.

Modulation of immune genes in the mucosal-associated lymphoid tissues of cobia by Sarcodia suae extract.

Rachycentron canadum (cobia) is a marine fish species of high economic value in aquaculture due to its fast growth rate and good feed conversion efficacy. Regrettably, the industry has been affected by significant setbacks from high mortality due to diseases. Consequently, an improved perception of innate immunity correlated to each mucosal-associated lymphoid tissue (MALT) in teleost fish is necessary to understand hosts' response towards infections better. The utilization of polysaccharides in seaweed to stimulate the immune system has gathered unprecedented attention. The present study examined the immunostimulatory effects of Sarcodia suae water extracts (SSWE) on in vivo gill-, gut- and skin-associated lymphoid tissues (GIALT, GALT, and SALT) via immersion and oral ingestions. The GIALT genes (TNF-α, Cox2, IL-1ß, IL-6, IL-8, IL-17 A/F1-3, IL-11, IL-12, IL-15, IL-18, MHCIa, IgM, and IgT) except IL-10 recorded positive upregulations in a dose-dependent manner post 24 h immersion in SSWE, indicating the algae extract contained bioactive compounds that could stimulate the immune genes. The upregulation of IL-12, IL-15, and IL-18 in the gills and hindgut post-SSWE immersion indicated that the extract could promote Th1-related responses in the MALTs. The modulation of immune gene expressions in the feeding trial was less potent than in the SSWE immersion. These findings indicated that the SSWE stimulated robust immune responses in both the GIALT and GALT of cobia. This suggests that the SSWE could be further explored as an effective immersive stimulant for fish, enhancing their immune system against pathogens.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

Determination of the growth performance and reproductive season of wild Channa lucius (Cuvier, 1831) populations of genus Channa (Channidae, Perciformes) in the Mekong Delta, Vietnam.

This study aimed to investigate the growth and spawning characteristics of wild Channa lucius (Cuvier, 1831) populations in the Mekong Delta, Vietnam. The study was carried out from March 2011 to February 2012. The length-weight relationship of C. lucius was determined according to the equations W = 0.0044L3.2494 , R2  = 0.9157 (female fish) and W = 0.0047L3.2178 , R2  = 0.9434 (male fish). The length and weight of the body for female fish ranged from 16.3 to 36.0 cm and 49.0 to 550.0 g, and for male fish, from 14.6 to 38.2 cm and 46.0 to 639.0 g, respectively. Female and male fish had almost similar lengths at first maturity (21.3958 cm for females and 21.3952 cm for males). The condition factor of female fish ranged from 0.842 to 0.864 and for male fish ranged from 0.848 to 0.874. The gonadosomatic index of female fish was highest in June (1.68%) and lowest in November (0.69%); that of male fish was highest in June (0.53%) and lowest in September (0.15%). The sexual maturity rate (SMR) of female fish peaked in May, with SMRs of 33.3% (stage III) and 58.3% (stage IV). In conclusion, C. lucius has a positive allometric range (b > 3), reaching sexual maturity when its length reaches 21.39 cm for both sexes, and the spawning season begins in December but peaks in May and June.

First Report of Screening of Novel Angiotensin-I Converting Enzyme Inhibitory Peptides Derived from the Red Alga Acrochaetium sp.

ACE inhibitors generated from food proteins have recently become the most well-known subclass of bioactive peptides, and their bio-functionality can be a potential alternative to natural bioactive food components and synthetic drugs. The bioactivities of Acrochaetium sp., the red alga used in this investigation, have never been reported before. Screening of bioactive peptides from Acrochaetium sp. as ACE inhibitors were hydrolyzed with various proteolytic enzymes. Protein hydrolysates were fractionated separately using reversed phased (RP) and strong cation exchange (SCX) chromatography and identified as VGGSDLQAL (VL-9) using α-chymotrypsin. It comes from Phycoerythrin (PE), an abundant protein in a primarily red alga. The peptide VL-9 shows the ACE inhibitory activity with IC50 value 433.1 ± 1.08 µM. The inhibition pattern showed VL-9 as a non-competitive inhibitor. Molecular docking simulation proved that VL-9 was non-competitive inhibition due to the interaction peptide and ACE was not in the catalytic site. Moreover, VL-9 derived from Acrochaetium sp. is a natural bioactive peptide that is safer and available for food protein; also, the ACE inhibitory peptide derived from Acrochaetium sp. could be the one alternative resource to develop functional food for combating hypertension.

TLR23, a fish-specific TLR, recruits MyD88 and TRIF to activate expression of a range of effectors in melanomacrophages in Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is an important food fish species that is mainly cultivated in tropical and subtropical countries. However, microbial diseases have created various difficulties for this industry. The fundamental prerequisite for tackling disease outbreak prevention and disease resistance is to know how hosts' immune responses against invading microbes are initiated. Toll-like receptors (TLRs) are vital pattern recognition receptors and play pivotal roles in the cellular innate immunity defense that is able to recognize pathogen-associated molecular patterns (PAMPs). In this study, Oreochromis niloticus TLR23 (OnTLR23) was cloned and bioinformatic analyses revealed that OnTLR23 is not an ortholog of mammalian TLR13 as previously suggested. The basal transcript level of OnTLR23 was found to be higher in the immune-related organs and was upregulated in the spleen and/or head kidney following Aeromonas hydrophila, Streptococcus agalactiae or poly I:C injections, and increased in the melanomacrophage-like tilapia head kidney (THK) cell line after LPS and zymosan stimulation. Furthermore, we demonstrated for the first time that OnTLR23 locates mainly in the intracellular region in fish cells and the constitutively active form of OnTLR23 promotes the expression of molecules related to antigen presentation, proinflammatory cytokines, antimicrobial peptides and type I interferon in THK cells. A co-immunoprecipitation assay revealed that OnTLR23 can interact with both OnMyD88 and OnTRIF, but not with OnTIRAP. A luciferase assay showed that the NF-κB activity was not elevated in the OnTLR23 overexpressed THK cells after treatment with ligand for TLR13 as well as other known purified bacterial-derived ligands of TLRs. Taken together, OnTLR23 is likely to recruit OnMyD88 and OnTRIF as adaptors to induce the expression of various effectors in melanomacrophages, but its corresponding ligand is an issue awaiting further investigation.

Increasing arsenic accumulation as an implication of climate change: a case study using red algae.

Climate change due to an increasing concentration of carbon dioxide in the atmosphere is a global issue. It can impact aquatic environments by affecting water flow, pollutant transformation and migration, and other toxicant-related effects. We assessed the interactive effects of temperature warming and pH changes on variations in accumulation of total arsenic (AsT) in the red alga Sarcodia suae at different levels of arsenite (AsIII). Result showed that AsT variations in the alga were moderated by significant joint effects of warming temperature and/or increasing pH levels and their interactions with increasing AsIII concentrations. Our study suggests possible deleterious impacts on macroalgal populations due to toxicological effects associated with prevailing environmental conditions. Therefore, improved pollution management, climate change adaptation, and mitigation strategies are needed to deal with current environmental issues and As aggravation.

Enhanced Colaconema formosanum biomass and phycoerythrin yield after manipulating inorganic carbon, irradiance, and photoperiod.

Due to an increasing CO2 concentration leading to global warming, the techniques as carbon capture utilization and storage are currently critical issues. This study aimed to investigate a cultivation strategy using optimal inorganic carbon level, irradiance, and photoperiod for producing the highest biomass and photosynthesis pigment contents (chlorophyll and phycobiliprotein) in the macroalga Colaconema formosanum. The results revealed that adding 1 g L-1 carbon increases phycoerythrin ratio by 12.52-13.74% and decreases allophycocyanin by 10.4-9.57%. Optimal conditions can increase algal growth by 60%, providing 5-6 mg g-1 total phycobiliprotein and 650-680 µg g-1 total chlorophyll. The results in this study illustrate the sensitivity of photosynthesis pigment after treatment with carbon, and suggest a hypothesis explaining the mechanism. The results also provide a feasible use of carbon for high-value large-scale production of pigment in the macroalgae industry.

Effect of Methylmercury Exposure on Bioaccumulation and Nonspecific Immune Respsonses in Hybrid Grouper Epinephelus fuscoguttatus × Epinephelus lanceolatus.

Mercury (Hg) is a dangerous heavy metal that can accumulate in fish and is harmful when consumed by humans. This study investigated the bioaccumulation of mercury in the form of methylmercury (MeHg) and evaluated nonspecific immune responses such as phagocytic activity and superoxide anion (O2-) production in hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus). The hybrid grouper leukocytes were incubated with methylmercury chloride (CH3HgCl) at concentrations of 10-10,000 µg/L to determine cell viability, phagocytic activity, and O2- production in vitro. Subsequently, the grouper were exposed daily to CH3HgCl mixed in the experimental diets at concentrations of 0, 1, 5, and 10 mg/kg for 28 days. The bioaccumulation of MeHg in the liver, head kidney, and muscle tissue was measured, and the phagocytic activity and O2- production were evaluated. In vitro results indicated that cell viability was significantly lower than that of the control group at concentrations > 500 µg/L. The phagocytic rate and O2- production at concentrations ˃ 500 and ˃ 200 µg/L, respectively, were significantly lower than those of the control group. The dietary exposure demonstrated that MeHg accumulated more substantially in the liver and head kidney compared with the muscle tissue in the treatment groups. Moreover, the cumulative concentration significantly increased with higher concentrations and more days of exposure. The phagocytic rate and O2- production in the treatment groups were significantly lower than those in the control group from days 2 and 1, respectively. In conclusion, hybrid grouper accumulated significant MeHg in the liver and head kidney compared with the muscle tissue, and higher concentrations and more exposure days resulted in decreased cell viability, phagocytic activity, and O2- production.

Fish-specific TLR18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages.

Toll-like receptors (TLRs) are evolutionarily conserved proteins of pattern recognition receptors (PRRs) and play a crucial role in innate immune systems recognition of conserved pathogen-related molecular samples (PAMPs). We identified and characterized TLR18 from Nile tilapia (Oreochromis niloticus), OnTLR18, to elucidate its role in tissue expression patterns, modulation of gene expression after microbial challenge and TLR ligands, subcellular localization in fish and human cells, and the possible effectors TLR18 induces in a melanomacrophage-like cell line (tilapia head kidney (THK) cells). OnTLR18 expression was detected in all tissues examined, with the highest levels in the intestine and the lowest in the liver. OnTLR18 transcript was up-regulated in immune-related organs after bacterial and polyinosinic-polycytidylic acid (poly I:C) challenges and in the THK cells after lipopolysaccharide (LPS) stimulation. In transfected THK and human embryonic kidney (HEK) 293 cells, OnTLR18 localizes in the intracellular compartment. OnMyD88 and OnTRIF, but not OnTIRAP, were co-immunoprecipitated with OnTLR18, suggesting that the former two molecules are recruited by OnTLR18 as adaptors. The constitutively active form of OnTLR18 induced the production of pro-inflammatory cytokines, type I interferon (IFN), and antimicrobial peptides such as tumor necrosis factor α, interferon (IFN) d2.13, tilapia piscidin (TP)2, TP3, TP4, and hepcidin in THK cells. Our results suggest that OnTLR18 plays an important role in innate immunity through initiating nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and IFN signaling pathways via OnMyD88 and OnTRIF and induces the production of various effectors in melanomacrophages.

Extraction Procedure, Characteristics, and Feasibility of Caulerpa microphysa (Chlorophyta) Polysaccharide Extract as a Cosmetic Ingredient.

The green alga Caulerpa microphysa, which is native to Taiwan, has a relatively high economic value and a well-developed culture technique, and is used mainly as a foodstuff. Its extract has been shown to exhibit antitumor properties, but the polysaccharide content of the extract and its anti-inflammatory and wound-healing effects and moisture-absorption and -retention capacity remain unknown. Hence, the objective of this study was to evaluate the potential of the polysaccharides in C. microphysa extract (CME) for use in cosmetics. The overall polysaccharide yield from the CME was 73.93% w/w, with four molecular weight fractions. The polysaccharides comprised 59.36 mol% mannose, 27.16 mol% glucose, and 13.48 mol% galactose. In addition, the CME exhibited strong antiallergic, wound-healing, transdermal-delivery, and moisture-absorption and -retention effects. In conclusion, the results suggested that CME potentially has anti-inflammatory and wound-healing effects and a good moisture capacity, which can be used in cosmetic applications.

Phycoerythrin from Colaconema sp. Has Immunostimulatory Effects on the Whiteleg Shrimp Litopenaeus vannamei and Increases Resistance to Vibrio parahaemolyticus and White Spot Syndrome Virus.

We investigated whether phycoerythrin (PE), a pigment sourced from marine algae, could act as an immunomodulatory agent in whiteleg shrimp (Litopenaeus vannamei). To this end, PE was extracted and purified from a PE-rich macroalgae, Colaconema sp. Our in vitro analysis demonstrated that PE enhanced prophenoloxidase and phagocytosis activity but inhibited the production of reactive oxygen species in hemocytes. Additionally, the PE signal could be detected using an in vivo imaging system after its injection into the ventral sinus of the cephalothorax of whiteleg shrimp. The expression profiles of fourteen immune-related genes were monitored in hemocytes from whiteleg shrimp injected with 0.30 µg of PE per gram of body weight, and crustin, lysozyme, penaiedin 4, and anti-lipopolysaccharide factor showed up-regulated post-stimulation. The induction of immune genes and enhancement of innate immune parameters by PE may explain the higher survival rates for shrimp that received different doses of PE prior to being challenged with Vibrio parahaemolyticus or white spot syndrome virus compared to controls. Combined, these results show that PE from Colaconema sp. can differentially stimulate the immune response of whiteleg shrimp in vitro and in vivo and could potentially be used as an immunomodulator in shrimp culture.

Hippocampus kuda protein hydrolysate improves male reproductive dysfunction in diabetic rats.

The global prevalence of diabetes mellitus is rapidly increasing. This disease is associated with many complications including male reproductive dysfunctions and infertility. Seahorse ( Hippocampus kuda) is a marine teleost fish well known for its beneficial effects on the reproductive system in traditional Chinese medicine books. Recently, several studies have been shown that the enzymatic hydrolysate of seahorse has multiple pharmacological activities. This study aimed to investigate the seahorse peptide hydrolysate (SH) ameliorative effects on the diabetic-induced male reproductive dysfunction in rat models. The in vivo studies were carried out with three different doses of SH (4, 8, and 20 mg/kg) and the diabetes condition was induced by administrating with streptozotocin (35 mg/kg) and fed a 40% high-fat diet. Seahorse hydrolysate (20 mg/kg) inhibited lipid peroxidation, increased antioxidant enzyme activity, and restored seminiferous tubules morphology in testis. Moreover, it improved reproductive dysfunction by increasing the level of testosterone, follicle-stimulating hormone, luteinizing hormone, sperm count, and motility. According to these results, we suggested that SH exhibited amelioration effects on the reproductive dysfunction.

Enhancing growth, phycoerythrin production, and pigment composition in the red alga Colaconema sp. Through optimal environmental conditions in an indoor system.

Phycoerythrin (PE) is a compound with strong potential for both basic research and industrial applications, but short supply and high prices have so far hindered its development. One common problem is a shortage of biomass for extraction. The aim of the present study was to determine a cultivation strategy (optimizing temperature, irradiance, photoperiod, and light quality) to produce greater biomass and higher PE concentrations in the alga Colaconema sp. We found that an optimized culture process could increase algae growth 7-9 fold while allowing extraction of 9-10 mg g-1 total phycobiliproteins, containing 60%-65% PE. Low energy costs make this approach economically feasible and competitive when compared with existing methods. Our results suggest an improved strategy for the large-scale production of PE and offer valuable applications in the algae industry.

Expression, signal transduction, and function analysis of TIRAP and TRIF in Nile tilapia (Oreochromis niloticus).

Toll/interleukin 1 receptor domain-containing adaptor protein (TIRAP) and toll/interleukin 1 receptor-domain-containing adapter-inducing interferon-ß (TRIF) are crucial adaptors of signal transduction for the signaling pathways of toll-like receptors (TLRs). TIRAP and TRIF perform an essential function in an antimicrobial immune response; however, their function in Nile tilapia remains unknown. Herein, TIRAP and TRIF from Nile tilapia were identified and functionally characterized. Phylogenetic analysis showed that OnTIRAP and OnTRIF clustered with corresponding homologs from other fish species, with comparable gene structures to those of select vertebrate TIRAP and TRIF genes, respectively. The expression profiles of OnTIRAP and OnTRIF were broadly distributed in the ten tissues investigated, with high transcript levels noticed in immune organs. The transcription levels of OnTIRAP and OnTRIF were upregulated in response to bacterial and poly (I:C) challenges. GFP signals were only detected in the cytoplasmic region of fish cells transfected with OnTIRAP-GFP and OnTRIF-GFP expression plasmids. Moreover, overexpression of OnTIRAP and OnTRIF activated interferon-ß (IFN-ß) and activator protein 1 (AP1) reporters in HEK 293 cells. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) reporter was only observed in OnTRIF-overexpressing HEK 293 cells. Furthermore, the results of the co-immunoprecipitation analysis showed that OnTRIF, but not OnTIRAP, was recruited as an adaptor protein by OnTLR25. This study provides the first evidence on the functions of OnTIRAP and OnTRIF in the immune system of Nile tilapia against pathogens and may serve as the basis for further investigations on TLR signaling in fish.

Sarcodia suiae Water Extract Promotes the Expression of Proinflammatory and Th1-Type Cytokines and Delay the Onset of Mortality in Cobia (Rachycentron canadum) During Photobacterium damselae subsp. damselae Infection.

Cobia (Rachycentron canadum) is a marine fish of high economic value that grows at a fast rate. However, intensive fish farming has led to disease outbreaks in cobia cultures, which is highly costly to the industry. The impact of infectious diseases on cobia production has led to the inappropriate and increased use of chemicals and antibiotics, which negatively affects the environment and human health and promotes the spread of drug-resistant pathogens. Hence, prophylactic measurements, such as the use of immunomodulators, are required to improve the health of cultured animals against pathogens. In this study, we examined the effects of Sarcodia suiae water extract (SSWE) in cobia in vitro and in vivo. We found that treatment with SSWE could significantly increase the expression of cytokines (e.g., IL-1ß, IL-6, IL-10, IL-12, and TNF-α) and chemokines (e.g., IL-8) in primary cultured head kidney leukocytes. Intraperitoneal injection of SSWE (20 µg/g body weight) promoted higher expression of IL-6, IL-8, IL-10, IL-12, chemokines (e.g., CC1), and antibodies (e.g., IgT) in head kidney and spleen tissues of the fish compared with other dose levels. Additionally, we describe for the second time (only after India) of the isolation of Photobacterium damselae subsp. damselae (Phdd) from a deadly epizootic in cage-farmed cobia. An intraperitoneal inoculation of SSWE before Phdd challenge showed that SSWE treatment could delay the onset of mortality of cobia. Finally, fish that received SSWE intraperitoneally before infection with Phdd exhibited elevated expression of Th1-type cytokines, namely, IL-8, IL-12, TNF-α, and IFN-γ. At the same time, the expression of Th2-related factors (such as IL-10 in the head kidney, and IgM and IgT in the spleen) were lower for the fish that received SSWE instead of PBS before the Phdd challenge. The results indicate that SSWE treatment facilitates the induction of Th1-type cytokines in cobia to fight against Phdd infection and has the potential to be used as an immunostimulant and vaccine adjuvant for fish.

Effects of three medicinal herbs Bidens pilosa, Lonicera japonica, and Cyathula officinalis on growth and non-specific immune responses of cobia (Rachycentron canadum).

This study investigates the effects of three medicinal herbal extracts, namely Bidens pilosa (BPE), Lonicera japonica (LJE), and Cyathula officinalis (COE), on nonspecific immune parameters of cobia (Rachycentron canadum) in vitro and in vivo. During in vitro tests, BPE treatment increased reactive oxygen species (ROS) production in a dose-dependent manner in primary head kidney leukocytes. Similarly, ROS production rates were enhanced by LJE (50 and 100 mg/ml) and COE (100 mg/ml). This suggests that these three herbal extracts possess immunostimulating properties. We then conducted two feeding trials to examine the effects of these three herbal extracts on growth and innate immune parameters of cobia, and sought an optimal dietary supplementation proportion required for activating the non-specific immune responses. In the first trial, we supplemented the diet with 1, 5, or 10% of the individual extracts. After a ten-week feeding trial, no negative impacts on weight gain, feed conversion rate, and survival rate were observed in fish offered experimental diets. Further, ROS production, phagocytic capacity of the head kidney leukocytes, and serum lysozyme activity were enhanced by differing degrees in fish fed the herbal extracts compared to fish in the control group. A similar albumin/globulin ratio was seen between each experimental group and the control group regardless of the type and dose of herbal extract used, indicating these medicinal herbal extracts are safe for cobia. We then performed a 30-day feeding trial with lower extract concentrations (1, 3, and 5% of the diet) to identify dose responses in cobia at various time points so that we could establish a cost-effective manner of administering the three extracts for cobia. All BPE fed fish had higher ROS production compared to the control group, while phagocytosis rate and index were simultaneously raised in only the BPE30 group (3% BPE). Immune parameters such as ROS production, phagocytic rate, and serum lysozyme activity were triggered when fish received 30 g LJE per kg of feed. However, ROS production only increased in the LJE10 group (1% LJE) on day 30 and was not enhanced in the LJE50 group (5% LJE). Additionally, although the phagocytic rate and phagocytic index were induced in the LJE50 group, serum lysozyme activity was not elevated in this group (LJE50) at any time point examined. ROS production was greatly improved in all COE fed groups, but only the COE30 group (3% COE) showed prolonged enhanced phagocytic rate over the 30-day feeding trial.

Sargassumhorneri extracts stimulate innate immunity, enhance growth performance, and upregulate immune genes in the white shrimp Litopenaeus vannamei.

The white shrimp Litopenaeus vannamei is one of the most commercially important aquatic species. However, intensive farming to meet economic demands has been linked to animal stress and subsequent disease outbreaks. In this study, we explore the immunomodulatory effects of Sargassum horneri hot-water extract (SHE) on the immune parameters of L. vannamei. Hemocytes incubated in vitro with 10 mg/ml of SHE showed the highest response in phenoloxidase (PO) activity and reactive oxygen species production. In the in vivo trials, shrimp were fed diets containing 0 (control), 2.5 (SHE 2.5), 5.0 g (SHE 5.0), and 10 g (SHE10.0) of lyophilized SHE per kg feed for four weeks, after which immune parameters were measured. Group SHE10.0 had the highest total hemocyte count, reactive oxygen species production, and phagocytic rate during the 4-week feeding period, followed by groups SHE5.0 and SHE2.5. However, group SHE5.0 showed a better growth performance among all examined groups after four weeks of rearing. Furthermore, gene expression analysis revealed that L. vannamei fed diets containing 0.5% SHE over 28 days showed significant modulation of 11 immune-related genes, including prophenoloxidase I, prophenoloxidase II, peroxinectin, α2macroglobulin, clotting protein, lysozyme, superoxide dismutase, glutathione peroxidase, penaiedin2-4, and crustin. Combined, these results show that SHE is a functional feed additive that can be applied orally to enhance innate immunity and growth performance of white shrimps.