Article

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

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Abstract

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.

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... Junior et al. (2017) reported that L. vannamei fed an ethanolic extract of C. dactylon showed considerably elevated levels of THC and AA, as well as significant variations in PO over time. In addition to terrestrial plants, seaweeds are a possible source of immunostimulants, as studies have demonstrated that crude extracts of seaweeds or chemicals derived from them can boost shrimp innate immunity and make shrimp farming more environmentally friendly (Sirirustananun et al., 2011;Wongprasert et al., 2014;Afsharnasab et al., 2016;Jasmanindar et al., 2018). Gracilaria spp. ...
... are essential red algae with a high mineral and bioactive component content, including alkaloids, glycosides, lectins, flavonoids, terpenoids, saponins, phytic acid, and tannins (Sangeetha et al., 2014). For instance, the extract of G. tenuistipitata or G. corticata (hot water) with food could increase L. vannamei's resistance to WSSV by boosting immunological markers, such as THC, SOD, RB, PO, LA, and the mitotic index of hematopoietic tissue (Sirirustananun et al., 2011;Houshmand et al., 2022) (Table 2). In a separate investigation, immersing shrimp L. vannamei in saltwater with an aqueous extract of Gracilaria tenuistipitata (600 mg/L) enhanced their resistance and immunological response (THC, PO, RB, SOD, and LA) to WSSV . ...
... For instance, a diet containing extracts of A. Mexicana, C. tagal, and C. dactylon could boost the survival rate of shrimp exposed to WSSV ( Table 2). The extract and powder of G. tenuistipitata and G. corticata reduced mortality or increased survival of L. vannamei when they were fed or immersed in the extract (Sirirustananun et al., 2011;Lin et al., 2011;Afsharnasab et al., 2016) (Table 2). It has been shown that Sargassum spp. ...
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Disease outbreaks in shrimp have increased as the global shrimp aquaculture industry has become more intensive. The white spot syndrome virus (WSSV) is a highly virulent and lethal infection that causes the most harm to global penaeid shrimp production, with a massive mortality rate of 80 to 100% within 3 to 10 days of the onset of clinical symptoms. As there is no effective treatment for the disease, numerous control or management measures have been implemented to eradicate the virus. Plants can serve as an eco-friendly alternative to antibiotics or as immunostimulants for shrimp against WSSV with significant enhancement of immune parameters. This review focuses on terrestrial and marine plants/algae that can stimulate shrimp immunity against WSSV, screening of plant extracts against WSSV, and various plant forms (powder, crude extract, fraction, and their compounds) that can inhibit WSSV infection in shrimp by increasing their immune response. The Sargassaceae family has been reported as an immunostimulant against WSSV the most frequently, followed by the Gracilariaceae, Poaceae, and Leguminosae families. Further studies are necessary to identify the active compounds and to apply them on a large scale in shrimp aquaculture.
... Shrimp depend only on non-specific (innate) immune processes for resistance against infections; they lack specialized white blood cells (WBCs) involved in antibody production and immunological memory (lymphocytes) [46][47][48]. Medicinal plants can exert immunostimulant effects thanks to compounds that activate immune mechanisms and increase resistance against diseases [49][50][51][52][53]. These plants are administered in their entirety or as parts (leaf, seed or root), as extracts or compounds; with feed additives, as a single extract or as the mixture of extracted compounds, and could even be applied together with prebiotics or other immunostimulants [47,54]. ...
... Besides terrestrial plants, seaweed extracts have been applied as therapeutics and prophylactics to manage fish or shrimp health (Vatsos & Rebours 2014). For instance, the extract of Gracilaria -and Aspargopsis has been used as an alternative to synthetic antibiotics against pathogen attacks, and to develop the immunity of shrimp [50,109,110]. Gracilaria verrucosa and Gracilaria tenuistipitata are presently being used in Taiwan as [47] (continued on next page) the primary red seaweeds for those purposes [111]. ...
... For centuries, medicinal plants have been applied as immunostimulants due to the presene of bioactive molecules that activate white blood cells (WBC) and boost an animal's resistance [30]. Interest in using plant extracts as shrimp immunostimulants has increased over the previous decade [50,51]. Numerous studies have demonstrated that administration in shrimp species of plants or their extracts could improve the immunological parameters, including phagocytic activity (PA), phenoloxidase activity (PO), respiratory burst (RB) activity, clearance efficiency, total haemocyte counts (THC), superoxide dismutase (SOD) activity, complement activity, lysozyme activity (LA), total protein (globulin and albumin) (TP) and antiprotease activity [49,51,55,109,155,157]. ...
Article
Disease epidemics in shrimp aquaculture increase apace with the development of aquaculture systems throughout the world. The disease caused by Vibrio spp. (vibriosis) is considered the most devastating, which has made it the most feared bacterial disease in the shrimp sector. In aquaculture, several strategies have already been applied to control Vibrio strains, including chemicals, probiotics, antibiotics, natural products from plants, including plant oils; hence, there has been considerable attention for using plants in shrimp aquaculture to provide sustainable, eco-friendly and safe compounds, such as alkaloids, saponins, terpenoids and flavonoids for replacing chemical compounds and antibiotics in current aquaculture. Medicinal plants may also have immunostimulating activity, increase growth and resistance in shrimps. The present paper aims to review the inhibition of Vibrio spp. in shrimp by medicinal plants, using both in vitro or/and in vivo techniques. Several medicinal plants appear capable of inhibiting growth of Vibrio pathogens outside living shrimp or in the body of shrimp, through enhancing growth and immune capacity when shrimps are fed or injected with them. In the current review Gracilaria spp. (Gracilariaceae family) and Sargassum spp. (family Sargassaceae) have been used most for in vitro and in vivo experiments. Among the terrestrial plants, Eucalyptus camaldulensis, Psidium guajava, Rhodomyrtus tomentosa, and Syzygium cumini (Myrtaceae family) had significant activity against Vibrio.
... Previous studies have demonstrated that sodium alginate from S. duplicatum and G. tenuistipitata and four types of alginates from S. siliquosum could increase the phagocytic activity and ROS production of hemocytes [20,32,36,66]. Similarly, we found oral administration of SHE can enhance both immune parameters in hemocytes. ...
... For example, shrimp that received carrageenan via immersion or oral ingestion gradually increased THC, PO activity, superoxide anion production, SOD activity, and lysozyme activity from hour 1 to 3 or week 1-3 during the treatment period, which then decreased after 5 h or 5 weeks, respectively [49]. Likewise, immune parameters increased from day 1-28 in white shrimp that received G. tenuistipitata extract in their diet, after which they showed a decline by day 35 [66]. Phagocytic index and ROS production increased progressively in P. monodon fed a diet containing β-glucan from day 1-24 and returned to control levels on day 40 [69]. ...
... Macroalgae derived extracts, including the water-soluble polysaccharides extract of Ulva rigida [70], fucoidan [34,71], alginate from S. siliquosum [35,36], polysaccharide extract of S. fusiforme [31], and hot-water extracts of Gelidium amansii [72] and G. tenuistipitata [66], have been reviewed as potential feed additives for enhancing shrimp immunity. However, little is known about the effects of these immunostimulants on shrimp growth performance. ...
Article
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.
... Maftuch et al. (2012) and Vienna et al. (2015) reported that seaweed Gracilaria verrucosa have antibacterial activity and can enhance innate immune of shrimp. Sirirustananun et al. (2011) demonstrated that the supplementation of G. tenuistipitata in shrimp feed at a dose of 0.5-2.0 g/kg for 14 days treatment can increase the immune system of shrimp that infected by WSSV. ...
... Phagocytosis activity was associated with an increased respiratory burst. Respiratory burst activity is a mechanism of particle removal by phagocytic hemocyte cells involving the release of degradative enzymes into the phagosome (an oxygen-dependent killing mechanism) and generating reactive oxygen intermediates (Sirirustananun et al., 2011). Respiratory burst of the dose extract has increased compared to the initial measurement at first and second day after challenge. ...
... The best survival is the treatment of the dose extract A (0.5 g/kg of feed). Previous research has shown that G. verrucosa extract is to maintain the survival of white shrimp against diseases caused by Vibrio bacterial infection (Kanjana et al., 2011;Sirirustananun et al., 2011;Jasmanindar et al., 2018). Seaweed Gracilaria genus is a potential source for alternative natural medicines for the fish cause this species had been reported had potential as anti-oxidant, anti-bacterial, anti-tumor, anti-viral, antibacterial activity and enhances innate immune of shrimp (Maftuch et al., 2016;Saraswaty et al., 2015). ...
Article
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The objectives of this study were to investigate the antibacterial activity of G. verrucosa extract in test inhibitory zone with different concentrations (500, 1000, 1500, and 2000 mg/L) and to examine G. verrucosa extract with different dosage (0.5, 1.0, 1.5, 2.0 g/kg) in feed on immune responses (total hemocytes count, phagocytic activity, phenoloxidase activity, respiratory burst) and survival rate in the Litopenaeus vannamei against the pathogenic Vibrio harveyi. Pacific white shrimp with an initial body weight of 5.25±0.55 g was reared in the aquarium (60×30×30 cm3) with a density of 10 shrimp/aquarium. Pacific white shrimp had been fed three times a day as much as 3% in at satiation for 14 days after challenged with V. harveyi. The first results of the inhibitory test showed that all the concentration of G. verrucosa extract was able to inhibit the growth of V. harveyi and the second result showed that the extract of G. verrucosa can increase the immune responses of shrimp. In the result of survival showed that shrimp fed with 0.5, 1.0, 1.5, and 2.0 g/kg has 80, 73, 70, and 70%, respectively. In conclusion, the seaweed extract of G. verrucosa has antibacterial activity and can induce the immune responses and resistance of Pacific white shrimp against V. harveyi infection.Keywords: Gracilaria verrucosa, seaweed, Vibrio harveyi, vibriosis, Litopenaeus vannamei ABSTRAKTujuan dari penelitian ini adalah untuk menguji aktivitas antibakteri ekstrak G. verrucosa dalam uji zona hambat dengan konsentrasi yang berbeda (yaitu 500, 1000, 1500, dan 2000 mg/L) dan studi perlakuan pengobatan untuk menguji ekstrak G. verrucosa pada pakan dengan dosis yang berbeda (yaitu 0,5; 1,0; 1,5; dan 2,0 g/kg) pada respons imun (yaitu jumlah total hemosit, aktivitas fagositik, aktivitas fenoloksidase, respiratory burst) dan tingkat kelangsungan hidup pada udang vaname terhadap bakteri patogen Vibrio harveyi. Udang vaname dengan berat badan awal 5,25 ± 0,55 g dipelihara di akuarium (60 × 30 × 30 cm3) dengan kepadatan 10 udang/akuarium. Udang vaname pasifik diberi makan tiga kali sehari 3% at satiation selama 14 hari setelah di uji tantang V. harveyi. Hasil pertama dari uji zona hambat menunjukkan bahwa semua konsentrasi ekstrak G. verrucosa mampu menghambat pertumbuhan V. harveyi dan hasil kedua menunjukkan bahwa pemberian ektrak G. verrucosa dapat meningkatkan respon imun udang. Hasil tingkat kelangsungan hidup menunjukkan bahwa perlakuan pakan udang dengan dosis 0,5; 1,0; 1,5; dan 2,0 g/kg memiliki tingkat kelangsungan hidup masing-masing 80, 73, 70, dan 70%. Kesimpulannya, ekstrak rumput laut G. verrucosa memiliki aktivitas antibakteri dan dapat menginduksi respons imun & ketahanan udang terhadap infeksi V. harveyi.Kata kunci: Gracilaria verrucosa, rumput laut, Vibrio harveyi, vibriosis, udang vaname
... Gracilaria tenuistipitata and Gracilaria verrucosa are the primary species of red seaweed currently being cultured in Taiwan (Chen, 1990). The administration of G. tenuistipitata extract increases the immune response and resistance to V. alginolyticus and WSSV in white shrimp L. vannamei Sirirustananun et al., 2011). The administration of G. verrucosa extract increases total hemocytes count (THC) and resistance against V. harveyi and WSSV in white shrimp L. vannamei and tiger shrimp Penaeus monodon (Maftuch et al., 2012;Zahra et al., 2017). ...
... The survival rates of white shrimp L. vannamei immersed in seawater containing GTE at 400 and 600 mg L −1 for 3 h and challenged with WSSV were significantly higher than in controls over 24-120 h . The survival rates of white shrimp fed GTE-containing diets at 0.5, 1.0, and 2.0 g kg −1 for 14 days and challenged with V. alginolyticus or WSSV were significantly higher than in controls over 72-144 h (Sirirustananun et al., 2011). The dietary administration of G. verrucosa extract shows resistance to WSSV infection in white shrimp L. vannamei (Zahra et al., 2017). ...
... The hemocyte count, PO activity, RB, SOD activity, and lysozyme activity in white shrimp immersed in seawater containing 400 mg L −1 GTE for 3 h and receiving WSSV injections were significantly higher than in controls at 6-120 h post challenge . The HCs, GCs, THC, PO activity, RB, and lysozyme activity in white shrimp L. vannamei fed a diet containing GTE at 2.0 g kg −1 after 14 days were significantly higher than in controls (Sirirustananun et al., 2011). The dietary administration of G. verrucosa extract increases THC, PO activity, RB, and phagocytic activity in white shrimp (Zahra et al., 2017). ...
... There are many more candidates like this, such as Pongamia pinnata [146], Ulva intestinalis [147], and Gracilaria tenuistipitata [148]. WSSV is one of the most thoroughly studied aquatic viruses to date, as the major disaster it has brought to shrimp and crabs is one of the problems that fisheries and biologists still have to face. ...
... It should be noted that the antiviral mechanisms of some of these medicinal plants and active pharmaceutical ingredients have not been confirmed, but are merely inferences made by researchers on the basis of past results. Rhus verniciflua Stokes Flavonoids Inducing apoptosis of cells [47] Celosia cristata and Raphanus sativus Extract Inducing gene expression involved in the innate immune response [57] Licorice (Glycyrrhiza uralensis, GUF) Extract; glycyrrhizin (GL); glycyrrhetinic acid (GLA) Inhibiting the early fusion steps [59] / Curcumin Inhibiting virus entry in cells [61] Olive tree leaf (Olea europaea) (LExt) Extract; oleuropein (Ole) Inactivating virus particles; inhibiting cell-to-cell spread [63] / α-Lipoic acid (LA) Inducing antiviral gene expression; reducing VHSV-induced oxidative stress [65] SVCV / β-Glucose Regulating the innate immune response [76] Herbaspirillum camelliae Selenium Activating IFN-related gene expression [77] Astragalus membranaceus Astragalus polysaccharide (APS) Activating IFN-related gene expression [80] Astragalus membranaceus APS Stimulating the immune response of host; reducing SVCV-induced apoptosis [150] / Palmitic acid (PA) Inhibiting autophagy [81] Psoralea corylifolia Bavachin (BVN) Blocking SVCV-induced apoptosis [84] Bupleurum yinchowense Saikosaponin D (SSD) Reducing SVCV-induced apoptosis [85] Viola philippica Extract Disturbing virus binding, entry, and replication in host cells [129] Glycyrrhiza uralensis (GUF) Extract Impacting the binding of virus particles to cell receptors and the replication of viruses in host cells [130] WSSV Green tea EGCG Inducing gene expression involved in the innate immune response [138,139] Typha angustifolia Naringenin (NAR) Restraining early viral gene replication [140] Pericarpium Citri Reticulatae Hesperidin Improving nonspecific immunity [142] Gardenia jasminoides Extract Blocking viral immediate-early stage gene transcript [143] Gardenia jasminoides Genipin (GN) Attenuating oxidative stress and inflammatory; decreasing signal transducer and activator of transcription gene expression [151] Eucommia ulmoides Geniposidic acid (GPA) Restraining early viral gene replication; promoting apoptosis [144] Pongamia pinnata Bis(2-methylheptyl)phthalate Improving nonspecific immunity [146] Gracilaria tenuistipitata Extract Enhancing the innate immunity [148] Kappaphycus alvarezii Carrageenan Improving nonspecific immunity [152] ...
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Aquaculture offers a promising source of economic and healthy protein for human consumption, which can improve wellbeing. Viral diseases are the most serious type of diseases affecting aquatic animals and a major obstacle to the development of the aquaculture industry. In the background of antibiotic-free farming, the development and application of antibiotic alternatives has become one of the most important issues in aquaculture. In recent years, many medicinal plants and their active pharmaceutical ingredients have been found to be effective in the treatment and prevention of viral diseases in aquatic animals. Compared with chemical drugs and antibiotics, medicinal plants have fewer side-effects, produce little drug resistance, and exhibit low toxicity to the water environment. Most medicinal plants can effectively improve the growth performance of aquatic animals; thus, they are becoming increasingly valued and widely used in aquaculture. The present review summarizes the promising antiviral activities of medicinal plants and their active pharmaceutical ingredients against aquatic viruses. Furthermore, it also explains their possible mechanisms of action and possible implications in the prevention or treatment of viral diseases in aquaculture. This article could lay the foundation for the future development of harmless drugs for the prevention and control of viral disease outbreaks in aquaculture.
... In the year 1992, the WSSV virus that was first isolated in Taiwan had been reported to cause deaths extensively in crustaceans like crabs and shrimps and also other aquatic animal species (Lo et al. 1996;Hossain et al. 2001). Recently, several studies have reported the activity of various seaweed species against WSSV virus Sirirustananun et al. 2011). The extracts of red seaweed Gracilaria tenuistipitata have been found to increase significantly the immunological activities of the whiteleg shrimp Litopenaeus vannamei against WSSV, and hence, the shrimp mortality was reduced (Sirirustananun et al. 2011). ...
... Recently, several studies have reported the activity of various seaweed species against WSSV virus Sirirustananun et al. 2011). The extracts of red seaweed Gracilaria tenuistipitata have been found to increase significantly the immunological activities of the whiteleg shrimp Litopenaeus vannamei against WSSV, and hence, the shrimp mortality was reduced (Sirirustananun et al. 2011). Similarly, significant antiviral activity has also been reported in freshwater crab Paratelphusa hydrodomous against WSSV infection when treated with the methanolic extract of red macroalga Hypnea spinella (Dinesh et al. 2014). ...
Article
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Aquaculture is growing post-haste in recent years particularly in the fish and shrimp production. The rapid growth of aquaculture and increasing demand for fish have led to a rapid development of the fish and shrimp industry, resulting in increased production of both fish and shrimps. As a result, there is a greater risk of disease outbreaks. Mass mortalities in aquaculture are primarily due to infectious diseases caused by bacteria, viruses, and fungi. Among them, viral diseases are the most devastating, causing huge loss in the production of both cultured fish and shellfishes. There are several effective methods of treatment for these disease outbreaks. This review focuses on various methods of controlling the viral pathogens using various treatment methods like use of medicinal plants and seaweed extracts, bioactive compounds from actinomycetes, vaccines, probiotic microbes, chemicals, nanoparticles, and green synthesis of nanoparticles.
... (Hoa et al. 2018) or L. vannamei integrated with G. vermiculophylla (Anaya-Rosas et al. 2019) induced immune responses and enhanced protection against V. parahaemolyticus infection. Moreover, L. vannamei receiving G. tenuistipitata extract (GTE) via injection (Hou and Chen 2005) or immersion (Yeh et al. 2010) coupled with a diet containing GTE (Sirirustananun et al. 2011) resulted in improved resistance to V. alginolyticus and white spot syndrome virus (WSSV) challenges. Similarly, L. vannamei fed a diet containing G. verrucosa extract (Jasmanindar et al. 2018), sulphated polysaccharides from G. birdiae (Cantelli et al. 2019), G. lemaneiformis meal (Niu et al. 2019) or Ulva prolifera (Ge et al. 2019b) induced better disease resistance to WSSV. ...
... L. vannamei that received GTE via immersion were shown to stimulate the earlier recovery of immune parameters following a V. alginolyticus challenge and the combined stresses of a V. alginolyticus challenge and temperature change (Yeh et al. 2010). Similarly, L. vannamei shrimp were fed a diet containing GTE, which was observed to elevate protection against V. alginolyticus and WSSV challenges (Sirirustananun et al. 2011). Additionally, P. monodon shrimp fed a diet containing sulphated galactans (SG) isolated from G. fisheri triggered immunostimulatory and antiviral activities that could protect shrimp from WSSV infection since immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase were enhanced in shrimp . ...
Article
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The integration of seaweeds (extractive, low-footprint species) into aquaculture systems has been considered an alternative approach for the sustainable development of the shrimp industry, which aims to expand in an environmentally friendly way. Two consecutive experiments were conducted to evaluate the effects of integrating various densities of black tiger shrimp Penaeus monodon post-larvae (PL) and red seaweed Gracilaria tenuistipitata on water quality and shrimp performance in the nursery phase. The first experiment involved a 3 × 4 factorial design with three levels of shrimp density (1000; 2000 and 3000 PL m−3) and four levels of red seaweed density (0, 1.0, 1.5 and 2.0 kg m−3) randomly allocated in triplicate tanks for 30 days. Shrimp PL (with a mean weight of 0.012 ± 0.002 g and length of 1.12 ± 0.09 cm) and red seaweed were reared in 150 L tanks at a salinity of 15 g L−1. Significant synergistic effects between shrimp and seaweed densities were observed for the NO3− total nitrogen (TN) and PO43− contents, as well as for the survival (p < 0.05) and production of shrimp (p < 0.01). The integration of shrimp and red seaweed significantly decreased the concentrations of nitrogen and phosphorus in the rearing tanks and greatly enhanced the survival and growth rate of shrimp. In particular, increased stocking density resulted in poorer growth performance but enhanced production. Applying stocking densities of 1000 and 2000 PL m−3 obtained larger shrimp size, while a density of 3000 PL m−3 achieved the highest production output in the integrated system. In the second experiment, which followed the 30-day growth trial, shrimp quality was assessed via an immersion challenge test using pathogenic Vibrio parahaemolyticus (a concentration of 2 × 108 CFU mL−1) on the shrimp groups previously stocked at a density of 3000 PL m−3 and integrated with different quantities of seaweed. After 14 days of challenge, the cumulative mortality in the monoculture system averaged 75.6%, which was significantly higher (p < 0.05) than those in the integrated groups (17.8–31.1%). It is concluded that the integration of P. monodon shrimp and G. tenuistipitata improved tank water quality as well as shrimp survival and growth while also enhancing the antibacterial activity of shrimp against V. parahaemolyticus infection in the nursery phase.
... In high-density farming environments, aquatic animals leave organic waste such as feces and unconsumed feed in the water system and ammonia is produced when proteins in the waste are mineralized (Ren et al., 2015;Sirirustananun et al., 2011;Koul, 2001). Ammonia exists mainly in the form of ionic (NH 4 + ) and non-ionic ammonia (NH 3 ), the latter is the main hazard to aquatic animals (Eddy, 2010;Ip et al., 2001;Liew et al., 2013). ...
... Ammonia exists mainly in the form of ionic (NH 4 + ) and non-ionic ammonia (NH 3 ), the latter is the main hazard to aquatic animals (Eddy, 2010;Ip et al., 2001;Liew et al., 2013). Because of its lipid solubility and lack of charge, NH 3 is more likely to spread to the blood or hemolymph through the cell membrane and affected the survival of organisms (Sirirustananun et al., 2011). Studies have found that high concentration of ammonia nitrogen can downregulate the activity of non-specific immune enzymes in aquatic animals (Farrelly et al., 2015), lead to different degrees damage of organs (Benli et al., 2008;Miron et al., 2008;Li et al., 2014) and decrease feeding rate whereas increase mortality (Rodríguez-Ramos et al., 2008). ...
Article
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As one of the main toxic substances in aquaculture water, ammonia causes seriously physiological harm to aquatic animals. In order to investigate the effects of ammonia exposure on the antioxidant defense, immune response, and NF-κB signaling pathway in the Chinese Strip-necked Turtle (Mauremys sinensis), we designed two experimental groups (control and 6.45 mM ammonia), and sampled at 6 h, 24 h, 48 h, re 24 h (recover 24 h), and re 48 h. The results showed that the blood ammonia (BA) content was significantly increased when the turtles were subjected to ammonia, and the activities of cholinesterase (CHE) and aspartate aminotransferase (AST) in the blood also showed a significant upward trend. The malondialdehyde (MDA) content continuously increased during ammonia exposure, and more than doubled at 48 h compared with the control group. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and their corresponding mRNA expression levels in liver during ammonia exposure were obviously increased when compared to control group, but most decreased to the normal levels at re 48 h. In addition, the mRNA and protein expression levels of NF-E2 related factor 2 (Nrf2) showed similar up-regulation patterns to antioxidase during ammonia exposed periods; whereas Nrf2 negative regulator kelch-like ECH-binding protein 1 (Keap1) showed opposite patterns. Moreover, the mRNA expression levels of heat shock proteins (HSP70, HSP90) significantly elevated upon the exposure of ammonia. Furthermore, ammonia increased the mRNA levels of p50 and p65 at different exposed times. Similarly, the protein expression levels of p50 and p65 also significantly increased at exposed times. The transcription levels of immune cytokines (BAFF and IL-6) were upregulated. In the recovery periods, there was a decline but did not return to normal levels. Taken together, these results indicated that antioxidation, immunity, and NF-κB signaling played a certain protective role for Mauremys sinensis under ammonia exposure. Our results will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in turtles.
... The mechanism of particle removal by phagocytic cells called a respiratory burst involves the release of degradative enzymes into the phagosome (an oxygen-dependent killing mechanism) and the production of ROI (reactive oxygen intermediates) ( Martín et al ., 2012). An increase in a respiratory burst is correlated with an increase in phagocytosis activity and vice versa (Jasmanindar, 2009;Sirirustananun et al ., 2011). During phagocytosis, viral particles are recognized by receptors on the cell surface and are engulfed by cells that re-prepare the cytoskeleton for phagosome formation. ...
Article
The aquaculture field continues to grow from year to year. This is because aquaculture fulfills the world's food needs. Aquaculture intensification continues. As a result of this intensification, the aquaculture environment experienced a decrease in water quality. This led to the rapid growth of bacteria, fungi, parasites, and viruses, resulting in a high incidence of disease outbreaks. An alternative solution that has been extensively researched recently is using immunostimulants to increase the non-specific immunity of fish and shrimp, which will help fish and insects prevent disease outbreaks. One of the natural ingredients that have the potential to become an immunostimulant is from the brown algae group. Immunostimulants from brown algae such as Hot-water Extract of Tropical Brown Seaweed, Sargassum cristaefolium, Sargassum glaucescens, Sargassum duplicatum, Sargassum wightii, Sargassum sp. Polysaccharides from seaweed, such as fucoidan extracts from brown algae, can enhance the non-specific immune system in shrimp Litopenaneus vannamei, Penaeus monodon, Fenneropenaeus indicus, and tilapia Oreochromis niloticus. These immunostimulants increase non-specific immunity, such as total hemocyte count, phagocytosis activity, phenoloxidase, phagocytic, respiratory burst, superoxide dismutase, and total plasma protein. Immunostimulants from brown algae can also fight bacterial disease attacks Vibrio parahaemolyticus, Streptococciasis, Streptococcus iniae, and White spot syndrome virus (WSSV) by increasing survival rate and can increase the growth and feed efficiency of cultivated commodities that are not given immunostimulants.
... The mechanism of particle removal by phagocytic cells, called the respiratory burst, involves the release of degradative enzymes into the phagosome (an oxygen-dependent killing mechanism) and the production of ROI (reactive oxygen intermediates) (Rodriguez & Moullac, 2000). An increase in the respiratory burst is correlated with an increase in phagocytosis activity and vice versa (Jasmanindar, 2009;Sirirustananun et al., 2011). During phagocytosis, viral particles are recognized by receptors on the cell surface and are engulfed by cells that reprepare the cytoskeleton for phagosome formation. ...
Article
Vanamei shrimp Litopeneus vannamei is a commodity with high economic value. Shrimp is Indonesia's main export commodity. The high value of sales and production of vanamei shrimp is constrained by the many disease attacks in the vanamei shrimp farming industry. An alternative solution that has been continuously researched for several years to combat shrimp disease is using immunostimulants from natural ingredients. Using natural ingredients for immunostimulants can increase the non-specific immunity of vanamei shrimp to ward off disease and be environmentally friendly. Immunostimulants can be administered orally, by immersion, or by injection. Immunostimulants from natural ingredients such as spirulina platensis, polysaccharides from seaweed such as carrageenan, sodium alginate, and fucoidan, extracts from red seaweed, as well as the use of polysaccharides from the bacterium Nodulisporium sp. KT29, Trichoderma sp. able to increase the non-specific immune system in L. vannamei shrimp. These immunostimulants increase non-specific immunity, such as total hemocyte count, phagocytosis activity, phenoloxidase, phagocytic, respiratory burst, superoxide dismutase, and total plasma protein. Immunostimulants from these natural ingredients can also fight infectious disease attacks.
... The marine fishing and aquaculture of L. vannamei are affected by weather changes and diseases [2]. The deteriorating water environment has seriously affected the shrimp industry due to stress and diseases in recent years [3,4]. Therefore, in the process of shrimp culture, it is very important to develop feed additives to improve the resistance and survival rate of shrimp for better growth and development. ...
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Litopenaeus vannamei was divided into seven groups (defined as diets A0–A6) and fed with diets respectively containing 0, 0.5, 1, 2, 4 and 8 g/kg Yarrowia lipolytica (astaxanthin content: 1.5%) and 3 g/kg Haematococcus pluvialis (astaxanthin content: 2%). After an eight-week feeding trial, the results reflected that different levels of Y. lipolytica and H. pluvialis could significantly increase the weight gain rate of L. vannamei (p < 0.05). The condition factor and weight gain rate of group A4 were significantly higher than those of the other groups (p < 0.05); the HSI significantly decreased with the increase of Y. lipolytica (p < 0.05). The addition of Y. lipolytica to the diet had significant effects on total protein (TP), albumin (ALB), glutathione peroxidase (GSH-Px), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) (p < 0.05). The total protein and albumin of the A5 and A6 groups were significantly higher than those of the other groups (p < 0.05). The GSH-Px activity of the A5 group was the highest and the T-AOC of the A0 group was the lowest. Inducible nitric oxide synthase (I-NOS) increased with the addition of Y. lipolytica (p < 0.05). Y. lipolytica inclusion had no negative effect on physiological and biochemical parameters and some serum immune and antioxidant indexes (p > 0.05). Astaxanthin in Y. lipolytica had an obvious effect on body color. After cooking, the body color of the shrimp deepened with increasing Y. lipolytica content. The red body color of L. vannamei was significantly improved by adding yeasts hydrolysate 2~8 g/kg to the diet. According to the regression analysis between the level of Y. lipolytica added to the diets and the weight gain rates, the optimal level of Y. lipolytica is 4.64 g/kg.
... Owing to the tiny size of larvae and their susceptibility to stress, oral vaccination was considered a better method of immunization in comparison to injection or immersion. And the oral method adopted was bio-encapsulation of bacteria utilizing live Artemia where the recombinant E. coli that possesses and simultaneously expresses the NNV capsid gene was incorporated in a natural feeder diet for fish larvae, which showed complete antigen absorption and subsequent immune response (Sirirustananun et al. 2011). The protective immune response was due to the effective uptake through feed and double layer protection (the cell wall of artemia and artemia cuticle) of the antigen which protects it from the digestive enzymes and effective delivery into the hindgut. ...
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The prevalence of infectious diseases in the aquaculture industry and a limited number of safe and effective oral vaccines has imposed a challenge not only for fish immunity but also a threat to human health. The availability of fish oral vaccines has expanded recently, but little is known about how well they work and how they affect the immune system. The unsatisfactory efficacy of existing oral vaccinations is partly attributable to the antigen degradation in the adverse gastrointestinal environment of fishes, the highly tolerogenic gut environment, and inferior vaccine formulation. To overcome such challenges in designing: an easier, cost-efficient, and effective vaccination method, several encapsulation methods are being adopted to safeguard antigens from the intestinal atmosphere for their immunogenic functions. Oral vaccination is easily degraded by gastric acids and enzymes before reaching the immunological site; however, this issue can be solved by encapsulating antigens in poly-biodegradable nanoparticles, transgenic designed bacteria, plant systems, and live feeds. To enhance the immunological impact, each antigen delivery method operates at a different level. Utilizing nanotechnology, it has been possible to regulate vaccination parameters, target particular cells, and lower the antigen dosage with potent nanomaterials such as chitosan, poly D,L-lactic-co-glycolic acid (PLGA) as vaccine carriers. Live feeds such as Artemia salina can be utilized as bio-carrier, owing to their appropriate size and non-filter feed system, through a process called bio-encapsulation. It ensures the protection of antigens over the fish intestine and ensures complete uptake by immune cells in the hindgut for increased immune response. This review comprises recent advances in oral vaccination in aquaculture in terms of an encapsulation approach that can aid in future research.
... In our previous study showed that when M. rosenbergii were fed banana peel extract at 6.0 g kg − 1 , lead to an increase in levels of THCs, DHC, PO activity, and RBs [27]. A similar result was found in L. vannamei fed with Gracilaria tenuistpitata extract-containing diets showed increased HCs, GCs, and THCs, together with increases in mitotic cells and the mitotic index of haematopoietic tissue (HPTs) [59]. This fact suggests that dietary supplemented BBH might also be capable of enhancing THC of prawns with proliferation of haemocytes in HPTs and may promote mobilization of mature GCs, which can result in an increase in THC in prawn at 28 days of feeding trial and then returned to the basal level after 56 days of feeding trial. ...
Article
Banana (Musa acuminata) blossom contains high nutritional value and bioactive compounds. In this study, Macrobrachium rosenbergii were fed with diets containing banana blossom powder (BBP) at 10 and 20 g kg⁻¹, hot-banana blossom (BBH) extract at 10 and 20g kg⁻¹, and the basal diet for 56 days. The growth performance, physiological response and immune parameters were evaluated. The results showed that a significantly higher percentage weight gain (PWG) and percentage length gain (PLG) in prawns fed with BBH diet. The feed efficiency (FE) significantly increased in prawns fed BBP. The prawn fed both BBH and BBP diet showed higher survival rate than control group. The prawn fed with BBH showed a significant increase in total haemocyte count (THC) and different haemocyte count (DHC), whereas phenoloxidase (PO) activity and respiratory bursts (RBs) significant increase in prawns fed both BBP and BBH diet. Furthermore, M. rosenbergii fed with both BBP and BBH diets showed significantly higher phagocytic activity and clearance efficiency against Lactococcus garvieae infection. At the end of the 56 days of feeding trial, the susceptibility of prawns to L. garvieae infection and hypothermal (18 °C) stress were evaluated. The results showed that prawns fed BBH diets had a significantly higher survival rate against L. garvieae than those of fed with the basal diet. Anti-hypothermal stress was observed in prawns fed both BBP and BBH diets showing no significant difference in haemolymph glucose in prawns subjected to 18 °C and 28 °C, whereas the norepinephrine level in haemolymph of prawns fed with BBH diets subjected to 18 °C was significantly lower than in prawns subjected to 28 °C. In summary, we recommend addition of hot-banana blossom extract to the diet of M. rosenbergii at 20 g kg⁻¹ to promote growth performance, improve physiological function, enhance immunity, increase anti-hypothermal stress, and to increase resistance against L. gavieae.
... Previous studies have shown that dietary manipulation can regulate metabolic processes in aquatic animals and enhance their ability to resist stress (Mai et al. 2004;Martins et al. 2012;Zhang et al. 2012;Dawood et al. 2017). Natural feed organisms are rich in a variety of nutrients and active substances, which can be used as nutritional fortifiers in shrimp culture to enhance resistance to stress (Sirirustananun et al. 2011;Xiao et al. 2017). Ampithoe sp. is a type of nutritious natural feed organism that is rich in crude protein (51.2%), crude lipids (5.8%), and essential amino acids (23.4%) (Peng et al. 2015). ...
Article
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Ammonia can easily accumulate in water during the culture period, and the accumulated concentrations can reach high levels, which is one of the main stress factors with severely negative effects on the growth and health of cultured shrimp. Dietary manipulation, such as feeding Ampithoe sp. meal, has been proven to be an effective method for reducing the mortality of Litopenaeus vannamei under sublethal ammonia stress and thus improving the ammonia tolerance of the shrimp, but the underlying mechanism needs to be further studied. In this study, L. vannamei with increased ammonia tolerance (PV_T) were obtained by feeding Ampithoe sp. meal, shrimp not fed Ampithoe sp. meal were used as a control (PV_C), and a comprehensive transcriptome analysis of the response of L. vannamei exposed to ammonia-N at the 50% lethal concentration for 96 h was performed. A total of 318,815,732 clean reads, including 170,156,604 and 148,659,128 clean reads from the shrimp in the PV_T and PV_C groups, were obtained. The expression of genes involved in carbohydrate and lipid metabolism pathways was upregulated in the PV_T group compared with the PV_C group, which indicated that the shrimp in the PV_T group had a higher energy supply level than those in the PV_C group, and this difference was the main cause of the observed improvement in ammonia tolerance. Additionally, the upregulated expression of genes involved in the AMP-activated protein kinase (AMPK) pathway in the PV_T group was responsible for the observed upregulation of the expression of genes involved in the carbohydrate and lipid metabolism pathways. However, among the GO terms that were significantly enriched in the DEGs identified from the comparison of the PV_T and PV_C groups, only one GO term was related to the immune system process. In conclusion, feeding Ampithoe sp. meal to L. vannamei exposed to sublethal ammonia can enhance the carbohydrate and lipid metabolism pathways to increase the energy supply and thus reduce the mortality of the shrimp.
... The mechanisms by which feed additives have such beneficial effects on shrimp health include stimulating the innate immune system, providing micro/essential nutrients, and maintaining a healthy microbiome. For example, sulphated polysaccharides derived from seaweed (e.g., Gracilaria sp.) and phloroglucinol added to shrimp feeds stimulated several innate immune parameters and led to greater resistance to bacterial and viral agents [196][197][198][199]. Lipopolysaccharides coated on feed pellets were shown to increase shrimp survival following exposure to Vibrio harveyi [200]. ...
Article
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Intensification of the shrimp sector, also referred to as vertical expansion, has been predominately driven by consecutive incidences of global disease outbreaks, which have caused enormous economic loss for the main producer countries. A growing segment of the shrimp farming industry has opted to use production systems with higher density, biosecurity, and operating control to mitigate the risks posed by disease. However, successful super-intensive shrimp production is reliant on an advanced understanding of many important biological and economic parameters in the farming system, coupled with effective monitoring, to maintain optimal production. Compared to traditional extensive or semi-intensive systems, super-intensive systems require higher inputs of feed, energy, labor, and supplements. These systems are highly sensitive to the interactions between these different inputs and require that the biological and economical parameters of farming are carefully balanced to ensure success. Advancing nutritional knowledge and tools to support consistent and efficient production of shrimp in these high-cost super-intensive systems is also necessary. Breeding programs developing breeding-lines selected for these challenging super-intensive environments are critical. Understanding synergies between the key areas of production systems, nutrition, and breeding are crucial for super-intensive farming as all three areas coalesce to influence the health of shrimp and commercial farming success. This article reviews current strategies and innovations being used for Litopenaeus vannamei in production systems, nutrition, and breeding, and discusses the synergies across these areas that can support the production of healthy and high-quality shrimp in super-intensive systems. Finally, we briefly discuss some key issues of social license pertinent to the super-intensive shrimp farming industry.
... As shown in Figure 2, shrimp have three types of blood cells, semi-granular, granular, and hyaline cells (Chen et al., 2014). These circulating hemocytes perform a significant function in shrimp innate immune responses, including pattern-recognition, release of antimicrobial peptides, prophenoloxidase (proPO) activation, phagocytosis, encapsulation, and nodule formation (Sirirustananun et al., 2011). ...
Article
Probiotics are increasingly used in aquaculture to protect cultivated organisms from pathogens and improve water quality and feed efficiency. Shrimp are the most commonly cultivated aquaculture species worldwide. In this current review, we will explore the current challenges that impact shrimp aquaculture and summarize how probiotics are utilized to improve production. Probiotics are live microorganisms that are described as friendly bacteria or healthy bacteria supplemented to the shrimp gut through feeds. Probiotics have been demonstrated to increase gastrointestinal stability, secrete antibacterial compounds, compete with pathogens to prevent intestinal adhesion, compete for the nutrients required for pathogen survival and produce antitoxin effects. Probiotics can also modulate the immune system and control the allergic response of the body. Therefore, this review sheds light on the challenges faced in shrimp production as well as the different types of probiotics, their methods of application, their modes of action, their role in improving shrimp production, and their influence on water quality.
... Litopenaeus vannamei, one of the most profitable shrimp species in south China, is very popular with customers because of its delicious flesh and high nutritional value (Sirirustananun et al., 2011). Previous studies demonstrated that both lutein and astaxanthin play essential roles in growth performance, anti-resistance as well as pigmentation of aquatic animals (Kalinowski et al., 2005;Meilisza et al., 2017). ...
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An 8-week feeding experiment was conducted to investigate whether diet supplementation of lutein could result in similar growth performance, antioxidant capacity, and immunity of Litopenaeus vannamei when compared to dietary astaxanthin. Juvenile L. vannamei (initial body weight: 0.64 ± 0.04 g) were fed with one of five isonitrogen and isolipids diets with/without lutein or astaxanthin [control group (C); the lutein (L) groups contained 0, 62.5, 75, 87.5 ppm lutein, respectively, the astaxanthin (A) group contained 50 ppm astaxanthin]. Results showed that dietary supplementation of lutein ranging from 62.5 to 75 ppm resulted in similar growth performance (WGR, SGR, FCR, and SR) of L. vannamei compared with the A group (P > 0.05). Apart from that, no statistical difference was observed in antioxidant parameters (hemolymph T-AOC, hemolymph MDA, and RNA expression level of GSH-PX, CAT), anti-inflammatory ability (Relish, Rho, and HSP70) and apoptosis-related gene expression (Caspase3) among lutein treatments ranging from 62.5 to 87.5 ppm and the A group (P > 0.05). These results indicate that a dose of 62.5–75 ppm of lutein was suitable in the diet of L. vannamei for substituting dietary astaxanthin.
... Previous studies concerning the role of algal extracts as immunostimulants reported that fish and shrimp fed the diets supplemented with algal extracts at a concentration as low as 0.1% showed an improvement in innate immune responses, including increased complement and lysozyme activities, and plasma protein (del Rocío Quezada-Rodríguez & Fajer-Ávila, 2017;Sirirustananun et al., 2011). ...
Article
The search for natural immunotherapeutic agents has become increasingly important for fish disease treatment and prophylaxis. The present research explored the effects of macro- and microalgal extracts on zebrafish juvenile growth and immune status. A plant-based diet was used as a control (diet C), and seven other diets were prepared similar to diet C, supplemented with 10 g Kg−1 of each algal extract (Fucus vesiculosus, F; Nannochloropsis gaditana, N; and Ulva rigida, U), or a combination of extracts (diets FN, FU, NU and FNU). A fishmeal-based diet was included as a positive control (diet C+). Diet C+ outperformed the remaining dietary treatments, growth-wise. None of the extracts improved diet C negative effects. However, the inclusion of U. rigida and N. gaditana extracts promoted an immunomodulatory action after 1 week of trial, by upregulating cytokine expression. This effect subsided after 30 days, suggesting tolerance may be developed over time. In contrast, effects on growth-related genes were still observed after 5 weeks of feeding F, N, U and FN diets. Algal extract dietary inclusion did not compromise intestinal integrity. These findings support the potential of algal extracts as functional feed additives during short periods (<30 days) to enhance immunomodulation.
... Additionally, plant derived compounds such as sulphated polysaccharides derived from seaweed (e.g. Gracilaria sp.) and phloroglucinol stimulate innate immune parameters and can lead to greater resistance to bacterial and viral agents Kumar et al., 2018;Lim et al., 2020;Sirirustananun et al., 2011;Wongprasert et al., 2014). Lipopolysaccharide from the cell wall of Gram-negative bacteria also stimulates the innate immune system of shrimp and when coated on feed pellets was shown to increase shrimp survival following exposure to V. harveyi (see Rungrassamee et al., 2013). ...
Article
Acute hepatopancreatic necrosis disease (AHPND) caused by pathogenic isolates of Vibrio parahaemolyticus (VPAHPND) carrying a plasmid encoding two pir-like toxins, is one of the most serious diseases affecting shrimp aquaculture. This study investigated if the microbial feed ingredient “Novacq™” improves resilience of Penaeus vannamei to VPAHPND infection and mortality. Two independent challenge trials were conducted where shrimp were fed a diet containing Novacq™ or a control diet for either 10 or 15-days (for trial 1 and trial 2 respectively) prior to exposure to VPAHPND. Mortalities were monitored every 3 h over the challenge periods (trial 1–99 h and trial 2–120 h) and foregut samples were collected from individual shrimp at different stages of the disease challenge to compare pirA and pirB toxin expression and V. parahaemolyticus abundance via qPCR. Shrimp fed Novacq™ had higher survival compared to control fed shrimp in both trial 1 and trial 2 with the relative percent survival (RPS) compared to the control 34% and 51% respectively. However, differences in Kaplan Meier survival estimates between the two diet treatments were only significant in trial 2. There was no significant difference in expression of pirA or pirB toxin genes or V. parahaemolyticus abundance between the two diet treatments in either trial, however, both toxin expression and V. parahaemolyticus abundance significantly changed throughout the disease time course. Results of this study along with previous studies of Novacq™ effects on a viral pathogen and other performance traits (i.e. growth), suggests Novacq™ may promote a general resilience phenotype that offers greater protection against both bacterial and viral agents of which the mode of action is still unknown.
... Several studies have reported that seaweed and its compounds have caused positive effects on the health of marine shrimp. Improved growth performance, immunological parameters, resistance to bacteria, viruses, and thermal shock were some of the positive effects observed (Chen et al., 2014;Elizondo-González, 2018;Schleder et al., 2017;Sirirustananun et al., 2011;Suantika et al., 2017;Yudati et al., 2016;). Kotya et al. (2011) observed significant improvements in daily growth rate and survival when K. alvarezii was used as a feed additive for Penaeus monodon. ...
Article
This study evaluated the effect of carrageenan from Kappaphycus alvarezii on the growth and health parameters of the Pacific white shrimp Litopenaeus vannamei. Shrimp (initial weight 4.5 ± 0.5 g) were farmed in 800 L tanks for five weeks, at a density of 30 shrimp tank⁻¹, under controlled temperature (28.5 °C), constant aeration, 100% water exchange four times week⁻¹, and fed four times day⁻¹. Five experimental diets containing 0%, 0.5%, 1.0%, 1.5%, and 2.0% of carrageenan were used and followed a complete randomized design (all in triplicate). After the 5-week feeding trial period, we assessed shrimp growth parameters (survival, feed conversion, total, weekly, and final weight gain) as well as immunological and gut microbiota profiles. A total of 30 shrimp from each feeding treatment were infected with a inoculum of the white spot syndrome virus (WSSV), and the resulting cumulative mortality was monitored for 96 h. WSSV infection results showed that shrimp that received carrageenan supplementation had higher survival rates (80%, 75%, 85%, and 70%, respectively, increasing carrageenan, 0.5 to 2%) compared to the control group (45%). No significant differences in weight gain and food conversion were observed among the feeding treatments relative to the control diet. Final weight varied from 8.50 g to 10.30 g, and the food conversion rate from 1.42 to 1.67. Also, no significant differences were found in intestinal microbiota and immunological parameters. Metagenomic analysis showed that adding 0.5% of carrageenan in the diet caused an increase in the relative abundance of an unassigned bacterium (from Rhodobacteraceae family) and bacteria from two other families, Rubritaleaceae and Caldilineaceae, with 1.0% and 1.5% addition levels, respectively. Adding low levels of carrageenan (up to 1.5%) to the feed of the Pacific white shrimp might benefit their microbiota composition as well as their capacity to respond to WSSV infection, without causing any loss in growth performance and health status.
... Thus, a growing interest in the potential of macro-and microalgae and their extracts as functional ingredients in aquafeeds has emerged (Van Hai 2015). For instance, it was shown that juvenile fish and shrimp fed diets supplemented with algal extracts at concentrations as low as 0.1% showed increased lysozyme, phagocytic, complement, and respiratory burst activities, and increased plasma protein (Sirirustananun et al. 2011;del Rocío Quezada-Rodríguez and Fajer-Ávila 2017). Although there are no reports on algal extract impact in intestinal morphology, several authors have reported that the inclusion of whole algae does not negatively affect intestinal morphology (Guerreiro et al. 2019;Passos et al. 2021). ...
Article
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Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.
... One effort in the prevention of disease is through enhancing the body's defense system against shrimp attack pathogens [6] by using immunostimulants [7] [8]. Shrimp body defense against disease is not only done through feed with a balanced composition but can be accompanied by giving immunostimulants in the feed. ...
... One of the most frequently disease which attacked white shrimp is vibriosis caused by Vibrio harveyi (Widanarni et al., 2012). The most common ways to control the disease are using antibiotics (Defoirdt et al., 2011), SPF juvenile selection (Lightner, 2005), using normal ingredients, and sinbiotics which acts as immunostimulant (Sirirustananun et al., 2011;Zubaidah et al., 2015). The antibiotics application was prohibited nowadays because of the food security issue. ...
Article
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The purpose of this study is to evaluate the effectiveness of Nodulisporium sp. KT29 supplementation with variousVibrio harveyi induction in feed against vibriosis in vannamei white shrimp. The study design included KP (positivecontrol without supplementation Nodulisporium sp. KT29 and infected with V. harveyi), KN (negative controlswithout Nodulisporium sp. KT29 and infected with physiological solution), NT (treatment of supplementationNodulisporium sp KT29 20 mL/kg and infected with V. harveyi), NM (treatment of supplementation testNodulisporium sp. KT29 induction of V. harveyi dead cell 20 mL/kg and infected V. harveyi), NH (treatment ofsupplementation Nodulisporium sp. KT29 induction of V. harveyi live cell 20 mL/kg and infected with V. harveyi).The study parameters included inhibition zone, resistance, immune responses, and hemolim glucose. The resultsshowed Nodulisporium sp. KT29 with induction treatment raised antibacterial activity with best treatment of NMand NH (P<0.05). The results of V. harveyi infection resistance presented NM treatment of 20 mL/kg increasesurvival in vannamei shrimp reached 72.2% (P<0.05). In addition, the same treatment increase the immuneresponse activity and decrease the activity of hemolim glucose. It could be concluded that providing NM 20 mLtreatment boosted the resistance and the immune system in vaname shrimp to control vibriosis reared at the sea.Keywords: antibacterial, β-glucan, induced, Nodulisporium sp. KT29, Vibrio harveyi ABSTRAK Tujuan penelitian yaitu mengevaluasi efektivitas suplementasi Nodulisporium sp. KT29 dengan berbagai perlakuaninduksi Vibrio harveyi dalam pakan terhadap pengendalian vibriosis pada udang vaname yang dibudidayakan dilaut. Rancangan penelitian meliputi KP (kontrol positif tanpa suplementasi Nodulisporium sp. KT29 dan diinfeksiV. harveyi), KN (kontrol negatif tanpa Nodulisporium sp. KT29 dan diinfeksi larutan fisiologis), NT (perlakuanuji suplementasi Nodulisporium sp. KT29 20 mL/kg dan diinfeksi V. harveyi), NM (perlakuan uji suplementasiNodulisporium sp. KT29 diinduksi sel mati V. harveyi 20 mL/kg dan diinfeksi V. harveyi), NH (perlakuan ujisuplementasi Nodulisporium sp. KT29 induksi sel hidup V. harveyi 20 mL/kg dan diinfeksi V. harveyi). Parameterpenelitian meliputi zona hambat, resistensi, respons imun, dan glukosa hemolim. Hasil penelitian menunjukkanNodulisporium sp. KT29 dengan perlakuan induksi dapat meningkatkan aktivitas antibakteri dengan perlakuanterbaik NM dan NH (P<0.05). Hasil pengamatan resistensi infeksi V. harveyi menunjukkan perlakuan NM 20 mL/kg dapat meningkatkan kelangsungan hidup pada udang vaname mencapai 72.2% (P<0.05). Perlakuan yang samajuga meningkatkan respons imun dan menurunkan aktivitas glukosa hemolim. Disimpulkan bahwa pemberianNM 20 mL dapat meningkatkan resistensi dan sistem imun udang vaname terhadap pengendalian vibriosis di laut.Kata kunci : antibakteri, β-glucan, induksi, Nodulisporium sp. KT29, Vibrio harveyi
... This can be attributed to the accelerated maturation of haemocyte precursors in the haematopoietic tissue, followed by release of new cells into the circulation system to maintain the haemocyte population and functionality in the shrimp [56]. Similarly, L. vannamei provisioned for 14 days on a diet containing Gracilaria tenuistipitata extract and a minor bupleurum decoction showed higher THCs than control groups [57,58]. ...
Article
This study investigates the effects of Phyllanthus amarus extract (PAE) on immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp (Litopenaeus vannamei). In vitro PAE treatment did not alter the cell viability of haemocytes and significantly enhanced immune parameters such as phenoloxidase (PO) activity, phagocytic activity, and superoxide anion (O2-) production. We conducted two feeding trials to examine the effects of PAE on the growth, disease resistance, and innate immune parameters of white shrimp. In the first in vivo trial, shrimps (4.01 ± 0.03 g) were fed a diet containing 0 g (control), 10 g (PAE10), 20 g (PAE20), or 40 g (PAE40) of PAE per kilogram of feed for 56 days. After the feeding period, the PAE20 group showed a significantly higher weight gain and specific growth rate than shrimp fed the control diet. Furthermore, after challenge with V. alginolyticus, shrimp fed a diet containing PAE showed significantly higher survival than those fed the control diet. The second in vivo trial (28 days) was performed to identify the mechanisms of enhanced immunity in PAE-fed shrimp. Shrimp fed the PAE20 diet generally had the highest total haemocyte count, PO activity, phagocytic activity, and O2- production, followed by the PAE40 and PAE10 groups. Thus, our results suggest that administration of 20 g of PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white shrimp.
... Therefore, the development of alternative methods to replace the current employed methods has attracted increasing attention (Huynh et al., 2018). Recently, some remedial addition agents were applied to stimulate the capacities of disease-resistance and immune responses in L. vannamei, such as prebiotics, probiotics, seaweed and plant extracts (Harikrishnan et al., 2011;Kapka-Skrzypczak et al., 2012;Sirirustananun et al., 2011). Furthermore, synbiotics, the combination of single or more probiotics and prebiotics, were also highly recommended (Li et al., 2009 T effects on the health of the host through cultured water or feed (Kumar et al., 2018). ...
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To explore the impacts of dietary hydrolyzed yeast (Rhodotorula mucilaginosa) and Bacillus licheniformis (B. licheniformis) on the growth, intestinal histology, innate immune response and ammonia resistance of Litopenaeus vannamei, four experimental diets (basal diet (Control); 0.5 % hydrolyzed yeast (HY); 0.1 % B. lincheniformis (BL) and 0.5 % hydrolyzed yeast + 0.1 % B. licheniformis (SYN)) were fed shrimps for 8 weeks. Results showed no significant differences on growth and body composition among all treatments, while a significantly higher feed efficiency (FE) in shrimp fed BL diet and a significantly higher protein efficiency ratio (PER) in shrimp fed BL and SYN diets were found compared to those in shrimp fed the Control diet (P
... So far, many herbal extracts have been assayed for these purposes. Administration of Sargassum polycystum (see Bright Singh et al., 2005), S. weighti (see Balasubramanian et al., 2006), Gracilaria tenuistipitata (see Nuttarin et al., 2011), Cynodon dactylon (see Citarasu et al., 2006), Ceriops tagal (see Sudheer et al., 2012), Rhizophora mucronata (see Chakraborty et al., 2014), and Uncaria tomentosa (see Júnior et al., 2018) to crustaceans infected with WSSV improved the survival rate, hematological, immunological, and oxidative stress parameters. Three routes explained their observed antiviral effect including reacting between extract ingredients and the proteins of the viral envelope, inhibition of virus proliferation, and the stimulation of the host's innate immunity, such as prophenoloxidase (proPO), superoxide dismutase (SOD), etc. (Balasubramanian et al., 2007). ...
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The main goal of this study was to investigate the biochemical, hematological, and immunological impacts of olive (Olea europaea) leaf extract (OLE) in Penaeus vannamei experimentally infected by white spot syndrome virus (WSSV). A total of 160 juvenile whiteleg shrimp (11.1 ± 0.2 g), obtained from a private sector in Hormozgan Province (Iran), were kept for two weeks to acclimatize to conditions in the facility aquarium. The health status of the shrimp was assured by random sampling before experiment action; PCR testing confirmed the absence of common shrimp diseases. Three groups, each consisting of 20 shrimp fed with three different doses of OLE (50, 100, 200 mg/kg of the shrimp biomass), and a positive control group (not feeding with OLE), were all orally subjected to a virus challenge two weeks later. For comparison, three negative control groups with 20 shrimp were fed three different doses of OLE, and another group was fed with a diet lacking OLE. The PCR test confirmed the presence of the WSS virus in the treatment and positive control groups, while the negative control groups did not show any infections. The hemolymph of shrimp was taken on days 1, 8, and 22 from all groups after challenge for evaluation of hemolymph total protein, glucose, oxyhemocyanin (OxH), superoxide dismutase (SOD), phenoloxidase (PO) activity, coagulation time, and total hemocyte count (THC). Results showed that shrimp survival significantly increased up to 65% when fed with high doses of OLE for seven days. The maximum hemolymph total protein and glucose levels significantly belonged to the treatment that was challenged by WSSV without receiving OLE. The present findings indicate WSS infection caused a significant decrease in OxH level compared to that of the control groups. Therapeutic feeding with OLE could significantly decrease the SOD and PO activities, and the hemolymph clotting time. We did not see no remarckable effects of OLE on the total hemocyte count. In conclusion, the various indices studied in this research showed that the olive leaf extract would be useful in the control and prevention of white spot syndrome.
... The Pacific white shrimp (Litopenaeus vannamei) is the most commonly cultured crustacean in South China. In recent years, the deteriorating environments have seriously affected shrimp farming, especially in the subtropical regions of Guangdong province in China where the climate is humid and the temperature is high throughout the year [25,26]. In commercial situations, feedstuffs or diets are often stored in paper bags, making the lipids readily susceptible to oxidation. ...
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The present study investigated the effect of dietary astaxanthin (AX) on the growth performance, antioxidant parameters, and repair of hepatopancreas damage in Pacific white shrimp (Litopenaeus vannamei). To evaluate the hepatopancreas protective function of AX in shrimps, we compared the effect of five isonitrogenous and isoenergetic diets under oxidized fish oil conditions with varying AX levels during the 50-day experimental period. The formulated diets were as follows: (i) OFO (oxidized fish oil); (ii) OFO/AX150 (oxidized fish oil + AX150 mg/kg); (iii) OFO/AX250 (oxidized fish oil + AX250 mg/kg); (iv) OFO/AX450 (oxidized fish oil + AX450 mg/kg); and, (v) control group (fresh fish oil). Results showed that the oxidized fish oil with 275.2 meq/kg peroxide value (POV) resulted in a substantial decrease in the final body weight of L. vannamei (P > 0.05) and induced some visible histopathological alterations in the hepatopancreas. Growth performance was significantly higher in shrimps fed with the OFO/AX450 diet than those fed with the OFO diet (p < 0.05). However, no significant difference was observed when the OFO/AX450 diet was compared to the control diet containing fresh fish oil (p > 0.05). Moreover, shrimps under the OFO/AX450 diet displayed a significant improvement in hepatopancreatic health and showed a reduction of malondialdehyde (MDA) compared to those under the OFO diet (p < 0.05). Dietary AX improved the antioxidant capacity of L. vannamei by increasing the catalase (CAT) activity in the hemolymph. Acute salinity change test showed a higher shrimp survival rate under OFO/AX450 diet than the OFO diet (p < 0.05), suggesting that AX can contribute to enhanced stress tolerance. In conclusion, our data suggest that AX confers dose-dependent protection against OFO-induced oxidative insults and hepatopancreatic damage in shrimp.
... It has been indicated that the stress tolerance of aquatic animals can be improved by nutritional enhancement (Mai et al., 2004;Tahmasebi-Kohyani et al., 2012;Zhang et al., 2012;Dawood et al., 2017). Natural feed organisms with high nutrient levels and palatability have been used as nutritional fortifiers to enhance the stress tolerance of shrimp (Sirirustananun et al., 2011). Ampithoe sp., a small crustacean rich in crude protein (51.2 % of dry weight), polyunsaturated fatty acids (41.9 % of total fatty acids) and essential amino acids (22.2 % of total amino acids) (Peng et al., 2015), has been demonstrated to be an excellent natural feed organism to reduce the accumulation of ammonia-N and urea-N in hemolymph and enhance ammonia-N tolerance in L. vannamei (Shan et al., 2018;Shan et al., 2019a, Shan et al., 2019b. ...
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The aim of this experiment was to investigate the mechanism of the improved ammonia-N tolerance in Litopenaeus vannamei induced by dietary supplementation with freeze-dried powder of Ampithoe sp. (FDPA). In the present study, shrimp were divided into three groups and fed an FDPA-supplemented diet for 0 days (S0 group), 21 days (S21 group) or 42 days (S42 group). Then, the three groups of shrimp were exposed to ammonia-N (1.61 mg/L nonionic ammonia) for 96 h, and changes in oxidative stress, endoplasmic reticulum (ER) stress and lipid metabolism in the hepatopancreas were investigated. The cumulative mortality rates of the S0, S21 and S42 groups at the end of the ammonia-N stress test were 46.7 %, 30.0 % and 33.3 %, respectively. The activity levels of superoxide dismutase (SOD) and catalase (CAT) in the hepatopancreas of shrimp were increased in the FDPA diet groups compared with the S0 group. The level of malondialdehyde (MDA) and the mRNA expression of binding protein (Bip), eukaryotic initiation factor 2α (eIF2α), inositol requiring enzyme-1 (IRE1), spliced form of X box binding protein 1 (XBP1s) and activating transcription factor 4 (ATF4) were significantly decreased in the hepatopancreases of the S21 and S42 groups compared with those of the S0 group. Moreover, the activity of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and malonyl-CoA (MCoA) was decreased; the activity of carnitine palmitoyltransferase (CPT) was increased; and the levels of free fatty acid (FFA) and triglyceride (TG) were decreased in the hepatopancreases of the shrimp fed the FDPA diet compared with those of the shrimp fed the control diet. The results indicated that the FDPA diet could alleviate the oxidative stress and ER stress induced by ammonia-N exposure, ensure the normal metabolism and energy supply of lipids in the hepato-pancreas, and thus enhance the ammonia-N tolerance of L. vannamei.
... Bacillus cereus ATCC 14579 (FB332900.1) was used as an outgroup to root the tree from Gracilaria likely trigger the innate immune response of shrimps against pathogens such as V. alginolyticus, V. harveyi and WSSV [72][73][74]. For an example, Jasmanindar et al. [75] showed that the dietary administration of Gracilaria verrucosa increased the immunity parameters (total haemocyte count, phenoloxidase activity, respiratory burst, superoxide dismutase activity and lysozyme activity) of shrimps infected with V. harveyi. ...
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The outbreak of acute hepatopancreatic necrosis disease (AHPND) has caused great economic losses to the shrimp culture sector. However, the use of antibiotics to fight this disease has resulted in negative impacts on human health and the environment. Thus, the use of natural alternatives to antibiotics may be a better solution. In this study, four Bacillus species obtained from the guts of shrimps (Fenneropenaeus penicillatus and Penaeus monodon) showed antimicrobial activity against the AHPND-causing Vibrio parahaemolyticus strain 3HP using the cross-streaking and agar spot methods. Two of the Bacillus isolates, B2 and BT, also showed good probiotic properties, exhibiting tolerance to bile, good adhesion to shrimp mucus, non-hemolytic, susceptibility to antibiotics and being safe towards hosts. Moreover, a seaweed-probiotic blend (a combination of Bacillus B2 and 20 mg/ml of the red seaweed Gracilaria sp.) exhibited synergistic in vitro inhibition against V. parahaemolyticus strain 3HP, with an observed inhibition zone of 5.0 mm. The broth co-culture experiment results further indicated that the seaweed-probiotic blend inhibited V. parahaemolyticus through competitive exclusion. The in vivo challenge trials also confirmed that this seaweed-probiotic blend significantly reduced the mortality of shrimps post-challenge with the AHPND-causing V. parahaemolyticus strain 3HP (p < 0.05) compared to the negative control (mortality rate = 13.88% vs 72.19%). Thus, this seaweed-probiotic blend may serve as an alternative to antibiotics in controlling the outbreak of AHPND.
... Furthermore, L. vannamei that received GTE via immersion has been shown to promote earlier the recovery of immune parameters following a V. alginolyticus challenge and the combined stresses of a V. alginolyticus challenge and temperature change (Yeh et al. 2010). In another study, white shrimp were fed a diet containing GTE, which was found to increase resistance to V. alginolyticus and white spot syndrome virus (WSSV) challenges (Sirirustananun et al. 2011). Additionally, white shrimp immersed in seawater containing GTE displayed a capability for maintaining homeostasis by regulating cellular and humoral immunity against ammonia stress, as evidenced by upregulated gene expression and the earlier recovery of immune parameters . ...
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The effects of integrating different densities of white leg shrimp Litopenaeus vannamei postlarvae (PL) and red seaweed Gracilaria tenuistipitata on water quality and shrimp performance were assessed in the nursery phase. A 3 × 4 factorial experiment with three levels of shrimp density (1000, 2000, and 3000 PL m⁻³) and four levels of red seaweed density (0, 1.0, 1.5, and 2.0 kg m⁻³) was randomly designed in triplicate. Shrimp PL12 (mean weight of 4.27 ± 0.28 mg) and red seaweed were stocked in 150-L tanks at a salinity of 15 g L⁻¹ for 30 days. The results indicated that the integration of shrimp PL and red seaweed significantly reduced the nitrogen and phosphorus contents in the culture tanks and improved the survival and growth rate of shrimp, although not significantly. A significant interaction effect (p < 0.01) between shrimp and seaweed densities was only observed for shrimp production. Notably, higher stocking density resulted in lower growth performance but enhanced production. Applying stocking densities of 1000 and 2000 PL m⁻³ reared shrimp that were bigger in size, while a density of 3000 PL m⁻³ obtained the highest production output in the integrated system. Following the 30-day growth trial, shrimp quality was examined through a bacterial challenge test using pathogenic Vibrio parahaemolyticus on the shrimp groups previously reared at a density of 3000 PL m⁻³ and integrated with different amounts of seaweed. After 14 days of immersion challenge, the cumulative mortality in the control group was significantly higher than that of other groups. This result suggests that the presence of G. tenuistipitata improved the antibacterial activity of L. vannamei against V. parahaemolyticus during the nursery phase.
... Steroid sebagai antivirus mampu mengganggu replikasi virus (Al Jaber et al. 2011) Wongprasert et al. (2013) menyatakan sulfated galactan yang diisolasi dari rumput laut Gracilaria fisheri dengan dosis 100-200 µg/mL selama 7 hari dapat meningkatkatkan sistem imun dan aktifitas antiviral pada udang vaname yang diinfeksi dengan WSSV. Sirirustananun et al. (2011) menyatakan bahwa penambahan Gracilaria tenuistipitata pada pakan dengan dosis 0.5 hingga 2.0 g/kg selama 14 hari dapat meningkatkan sistem imun pada udang vaname yang diinfeksi dengan WSSV. Menurut Jasmanindar (2009) penggunaan ekstrak G. verrucosa dengan dosis 50 µg/g bobot udang dalam meningkatkan sistem ketahanan udang vaname memberikan hasil dapat meningkatkan resistensi udang vaname terhadap infeksi bakteri Vibrio harveyi hingga 73.3% dan 2 kali pemberian (interval 14 hari) ekstrak G. verrucosa selama 30 hari pemeliharaan mampu memberikan kelangsungan hidup hingga 86.7%. ...
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One of diseases in the white shrimp is white spot disease that caused by White Spot Syndrom Virus (WSSV). Thus, effective effort to prevent WSSV outbreak in shrimp farming is required, and one of which is the administration of immunostimulant. This study aims to examine the effect of feed containing G. verrucosa extract at different doses on the survival of white shrimp infected with WSSV. This study consisted of six treatments of the dose of G. verrucosa and each of three replications, namely KN (without extract), KP (without extract + WSSV infection), A (extract of 2,000 mg / kg + WSSV infection), B (dose of 3,000 mg / kg + WSSV infection), C (4,000 mg / kg + WSSV infection), and D (5,000 mg / kg + WSSV infection). White shrimp with initial body weight of 6-10 g/shrimp were reared in the (60×30×30) cm with density of 10 shrimps/aquarium. Shrimp were given feed (32% protein) containing G. verrucosa extract with a 3% feeding rate of biomass weight three times a day for 14 days. On the 15th day tested challenged with WSSV at a dose of 0.1 mL / intramuscularly. The result showed that feed containing G. verrucosa extract was able to significantly increase the survival of white shrimp compared to the positive control treatment. The best survival after challenge test on treatment C (4,000 mg / kg), which is 56.67 ± 5.74%. It was concluded that the dosage of 4,000 mg / kg of feed gave the best results to improve the survival of white shrimp infected with WSSV.
... Hyaline cells are involved in phagocytosis, while semi-granular and granular cells are active in encapsulation, the storage and release of the prophenoloxidase, and cytotoxicity . White shrimp L. vannamei that received the extracts of S. duplicatum and Gracilaria tenuistipitata and sodium alginate of S. siliquosum all showed increased hemocyte counts in different routes Sirirustananun et al. 2011;Yudiati et al. 2016), while some researchers found it in contrast (Cheng et al. 2005;Liu et al. 2004). The present study indicated that shrimps fed with three different doses of S. siliquosum sodium alginate for 14 days showed an increase in hemocyte counts which indicates the higher proliferation of hemocytes when compared to the control treatment (Fig. 3). ...
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Although alginate is known as an immunostimulant in shrimp, the comprehensive and simultaneous study on its activity to resolve the relationship of the hematological parameters, upregulation of immune-related gene expression, and resistance to pathogen has not been found in shrimp. We performed experiments to evaluate the effect and mechanism of alginate from S. siliquosum on Pacific white shrimp immune system. Hematological parameters were examined after oral administration of Na alginate in the shrimp. White spot syndrome virus (WSSV) was injected to the shrimp at 14 days, and its copy number was examined quantitatively (qRT-PCR). Immune-related gene expression was evaluated by qRT-PCR. Alginate increased some hematological immune parameters of shrimp. Before WSSV infection, expression levels of Toll and lectin genes were upregulated. The lectin gene were upregulated post infection, and the Toll gene in all the treatments were downregulated, except the shrimps fed with alginate at 6.0 g kg⁻¹ at 48 h post infection (hpi). The shrimps fed with alginate at 6.0 g kg⁻¹ were the most resistant and gave the least WSSV copy number at 48 hpi. Resistance of shrimps fed the alginate-supplemented diets against WSSV was significantly higher compared to that of the control treatment with 56% and 10% of survival rates, respectively. Oral administration of alginate did not affect the growth and total protein plasma. At 120 h post challenge, alginate treatment at 6.0 g kg⁻¹ exhibited the highest survival rate. It is concluded that oral administration of alginate enhanced the innate immunity by upregulating immune-related gene expression. Consequently, the enhancement of the shrimp innate immunity improves the resistance against WSSV infection.
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The extract from Theobroma cacao L, pod husk served as the immunostimulant to enhance the immunity and resistance against Lactococcus garvieae of Macrobrachium rosenbergii. In this study, we employed the injection method and dietary administration method to determine the effect of cocoa pod husk (CPH) on M. rosenbergii. The non-specific immune parameters and disease resistance were evaluated after the prawn injected with 1 μg prawn-1 CPH extract (C1), 3 μg prawn-1 CPH extract (C3), and 5 μg prawn-1 CPH extract (C5) for 1, 3, and 7 days. The results showed a significant increase of total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to L. garvieae. The non-specific immune parameters, physiological parameters, and disease resistance and growth performance were evaluated after the prawn fed with 1 g kg-1 CPH extract diet (CD1), 3 g kg-1 CPH extract diet (CD3) and 5 g kg-1 CPH extract diet (CD5). The results showed a significant increase in all immune parameters and showed a significant decrease in physiological parameters. No significant difference was observed in growth performance of prawn fed with the CPH containing diet. Both injection and dietary method showed a significant increase in disease resistance against to L. garvieae. We therefore recommend that CPH extract can be used as a immunostimulant for prawn by dietary administration to regulate immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen.
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This study aimed to evaluate the dietary supplementation of various levels of Bacti-nil®Aqua on growth performance, nutrient utilization, immune response, antioxidant activities, mortality rate, and chemical composition of the Pacific white shrimp Litopenaeus vannamei. Four hundred and eighty L. vannamei fries (3.0 ± 0.002 g) were randomly divided into 4 treatments (T1-T4). T1 represented the negative control fed on a basal diet, the fries in treatments T2, T3 and T4 were fed on Bacti-nil®Aqua supplemented diet at three concentrations of 2, 3, and 4g/kg diet, respectively, for 60 days. The results showed that the morphometrics measurements (body and antenna length), the growth parameters (BW, BWG, ADG, and RGR) and the condition factor values were significantly (P < 0.05) higher in the Bacti-nil®Aqua supplemented treatments than those of the control group. Moreover, the different Bacti-nil®Aqua supplemented diets had no significant effect on the survival rate (SR) of L. vannamei. The chemical composition of the L. vannamei and feed utilization parameters exhibited no significant differences in DM and ash. The protein content of L. vannamei increased significantly when fed on Bacti-nil®Aqua, compared to T1. In contrast, the highest values of lipids were recorded in the control treatment (T1). Different concentrations of Bacti-nil®Aqua (T2-T4) resulted in a significant improvement in PER and PPV%, the best FCR, and the highest nonspecific immune responses (THC, phagocytosis, lysozyme activity, and phenoloxidase activity), in addition, superoxide dismutase activity compared to the control diet (T1). Overall, the current study concluded that the highest levels of Bacti-nil®Aqua (3 and 4g/kg) resulted in improved growth parameters, survival rate, feed utilization, chemical composition, and nonspecific immune responses of L. vannamei.
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This research aimed to optimize the extracting condition of banana peel (BP) and to investigate the effect of organic banana peel extract (BPE) on the immunity of giant freshwater prawn (Macrobrachium rosenbergii). The result demonstrated that the optimum extracting condition of organic BPE was 50% v/v of methanol at 100°C for 10 min. Under this condition, the maximum total phenolic content and extraction yield of 10.44 mg of gallic acid equivalents (GAE) per g of dry matter (mg GAE/g DM) and 33% w/v were obtained respectively. The organic BPE could inhibit an aquatic pathogen Aeromonas hydrophila at the minimum inhibitory concentration of 625 µg/disc. The main phytochemicals in organic BPE showed two major biological functions, which are antioxidant and antipathogenic activities. Subsequently, the effects of organic BPE on the immunity of M. rosenbergii were investigated. The results demonstrated that the organic BPE could increase immunity and phagocytic activity and decrease the susceptibility of M. rosenbergii. Moreover, the organic BPE could also increase total as well as differential haematocyte count, decrease the coagulation time and increase the total protein in serum of M. rosenbergii. The phagocytic activity of prawn was enhanced to 82.00% by using an organic BPE at 6 µg/g. At the same time, the cumulative mortality was declined to lower 20% after 6 days of organic BPE injection. In conclusion, organic BPE can be a potential immunostimulant in giant freshwater prawn culture. The utilization of BP in aquaculture can further add the value of BP and reduce organic pollutants.
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The present study examined the effects of the hydro-alcoholic extraction of the red seaweed Gracilaria corticata (GCE), as food additive on growth, antioxidant defence and immunity in the goldfish, Carassius auratus . Four experimental treatments in three replications were established and fed the experimental diets for 60 days. The groups were: a control (fish fed only a basal diet), GCE1: fish supplemented with 0.5 % GCE/kg diet, GCE2: fish supplemented with 1 % GCE/kg diet, GCE3: fish supplemented with 1.5 % GCE/kg diet. After feeding period, the antioxidant [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] and immune responses were evaluated. Dietary GCE had no effect on growth performance ( P >0.01). The plasma immune responses including alternative complement (ACH 50 ) and lysozyme activities elevated in fish supplemented with 1 % and 1.5 % GCE compared to those fed 0.5 % GCE and basal diet ( P <0.01). The plasma and mucosal immunoglobulin (Ig) significantly elevated in all GCE supplemented fish ( P <0.01). The plasma peroxidase activity significantly increased only in the fish receiving GCE at dietary level of 1.5 % GCE compared to control and those supplemented with 0.5 % and 1 % GCE ( P <0.01). The mucosal lysozyme, protease activity and alkaline phosphatase significantly increased in fish supplemented with 1 % GCE compared to other experimental diets ( P <0.01). The activity of antioxidant enzymes (SOD, CAT, GPx) showed significant increases in fish supplemented with 1 % and 1.5 % GCE ( P <0.01). Furthermore, the expression of the immune-related genes, complement and lysozyme significantly elevated in the treatments of 1 % and 1.5 % GCE compared to other experimental diets ( P <0.01). The results of this study revealed that the use of GCE as a food additive in the diet of goldfish diet can improve the fish immunity without negative impacts on growth.
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The sustainable uses of cacao, Theobroma cacao L, pod husk (CPH) produced from cacao bean industry bring environment great benefits. Botanical polysaccharides possessing a variety of physiological functions are therefore referred to as biological response modifiers, and those are also found in CPH. In this study, the weight gain rate (WGR), length gain rate (LGR), feeding efficiency (FE), and survival rate (SR) of shrimp, Litopenaeus vannamei, fed diets containing CPH pectin at 0 (CON), 1.0 (CPH1), 3.0 (CPH3) and 6.0 (CPH6) g/kg were calculated every 7 days within 56 days of feeding. The non-specific immune parameters, disease resistance, tolerance to hypothermal stress and stress indicators were also evaluated after the shrimp fed with CPH pectin containing diets. Shrimp that were fed with CPH3 after 56 days had higher WGR, LGR, and FE than those in CPH0. The SR significantly increased for shrimp challenged with Vibrio alginolyticus, and subjected to hypothermal (14 °C) stress when they were fed diets containing CPH3 and CPH6, and CPH6 after 14 or 28 days of feeding, respectively. Dietary CPH3 and/or CPH6 showed a significant increase of total hemocyte count (THC), differential hemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), and phagocytic activity and clearance efficiency to V. alginolyticus at 14 and/or 28 days. Furthermore, shrimp fed with CPH pectin-contained diets after 14 and 28 days retarded secretion of dopamine (DA) and norepinephrine (NE), and decreased the level of glucose and lactate in plasma under cold stress. We therefore recommend that CPH pectin can be used as a physiomodulator for shrimp by dietary administration at 3.0 g/kg for 56 days to promote growth; and at 3.0–6.0 g/kg within an appropriate time frame (28 days) to regulate neuroendocrine and immune responses, and carbohydrate metabolism lead to enhance resistance against pathogen and hypothermal stress.
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This study was conducted to evaluate the effect of Haematococcus pluvialis powder on astaxanthin content and isomer composition, antioxidant ability and immunity of Litopenaeus vannamei after short‐term feedings. Experimental diets include a commercial diet (control) and diets containing H. pluvialis powder (HP‐35 and HP‐70, 35 and 70 mg of carotenoids kg⁻¹). After feeding for 15 days, the content of astaxanthin in the muscle, cephalothorax and shell of shrimp increased significantly with increasing addition of H. pluvialis powder (p < 0.05). Dietary addition of H. pluvialis powder significantly increased the activity of total antioxygenic capacity (T‐AOC), superoxide dismutase (SOD) and glutathione S‐transferase (GST) as well as the relative mRNA levels of immune‐related genes, Toll, MyD88, IMD, Crustin, PO and Lysozyme in hepatopancreas (p < 0.05). The proportion of astaxanthin isomers in different body parts was different, whereas it has no significant difference between same body parts of different groups. This indicated that the accumulation of astaxanthin among body parts may be accompanied with selection and isomerization. In conclusion, feeding with H. pluvialis powder diets containing 35 and 70 mg of carotenoids kg⁻¹ can enhance the carotenoid and astaxanthin content, antioxidant and immune capacity of L. vannamei.
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The impacts of dietary mannan oligosaccharide (MOS) and Bacillus lincheniformis (B. lincheniformis) on growth performance, immune responses, intestinal health and ammonia resistance of Litopenaeus vannamei were explored. Four resultant diets (control diet (CON), 0.2 % MOS (MOS), 0.1 % B. lincheniformis (BL), 0.2 % MOS plus 0.1 % BL (SYN)) were produced to feed shrimps for 8 weeks. Results showed that significantly higher weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) and protein productive value (PPV) were found in shrimp fed the SYN diet (P < 0.05). The villus number (VN) and villus height (VH) of shrimps fed the SYN diet were significantly higher than those of shrimps fed the CON diet (P < 0.05). Moreover, significantly thicker submucosa (SM) of shrimp fed the MOS diet than that of shrimps fed BL and CON diets were found (P < 0.05). Intestinal acetic acid content was significantly higher in shrimps fed supplemented diets (P < 0.05). Meanwhile, the propionic acid content in shrimps fed BL and SYN diets were significantly higher than those in shrimps fed the other diets (P < 0.05). The expression levels of catalase (CAT), glutothion peroxidase (GPX), superoxide dismutase (SOD), penaeidin -3a (Pen-3a) and heat shock protein (Hsp-70) were significantly upregulated by supplemented diets at some time points (P < 0.05). The survival rates of shrimps fed supplemented diets after ammonia challenge were significantly higher (P < 0.05). The expression levels of CAT, SOD, Pen-3a and Hsp-70 in shrimps fed supplemented diets were upregulated after ammonia challenge. In conclusion, dietary synbiotic could enhance growth performance, feed utilization and intestinal morphology, while dietary MOS and/or B. lincheniformis supplementation could positively influence the intestinal SCFAs content, increase immune responses and ammonia resistance of Litopenaeus vannamei.
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The aim of this study was to determine the influence of dietary glucan and vitamin C, alone and in combination, on the cellular and humoral immune responses of rainbow trout. After 4 weeks of adaptation on a control diet containing glucan at 0ppm and vitamin C at 150ppm, four groups of rainbow trout were fed the experimental diets containing vitamin C at 150 and 1000ppm plus a fixed amount of glucan, or no glucan for 2 weeks. The fish were then switched back to the control diet. Non-specific immunity was investigated at the end of the two-week experimental feeding period, and then again 4 weeks later. Macrophage activities such as oxidative burst and pinocytosis were determined along with complement activation and lysozyme levels. The specific immune response was evaluated as: (1) proliferation of lymphocytes induced by mitogen and (2) the antibody response after vaccination against enteric redmouth disease. Fish were vaccinated at the end of the 2-week experimental feeding period and antibody titres were determined every second week for the 16 following weeks. The results of this trial showed a significant effect of dietary vitamin C but not glucan on the non-specific immune response of rainbow trout such as increased oxidative burst, pino-cytosis and lysozyme activity. Dietary treatments had no effect on alternative pathway of complement activation. A significant enhancing effect of dietary beta-1,3/1,6 yeast glucan was observed on concanavalin A-induced proliferation of lymphocytes and the antibody response after vaccination against enteric redmouth disease. When considering the antibody titres at the peak time, glucan or vitamin C significantly enhanced the response. This was also observed on serum complement levels measured at the same time. Results from this trial helped to establish the beneficial effect of a dietary combination of glucan and vitamin C on the immune response of rainbow trout, as well as to evaluate the specific effect of each substance.
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In the marine eco system, seaweeds are directly exposed and are susceptible to ambient micro organisms such as bacteria, fungi and viruses. Seaweed species of kappaphycus (red algae) and padina (brown algae) from the coast of Tamilnadu, India were tested in vitro for their antibacterial activities against different types of bacteria using disc diffusion method. Methanol was used for inhibition of different bacterias such as pseudomonas flouresences, staphylococcus aureus, vibriochloera and proteus mirabilis in the case of red algae. In the study, it is observed that kappaphycus maximum activity against pseudomonas flouresences, staphylococcus aureus and less inhibition on vibriochloera and proteus mirabilis. Benzene, n-hexane, ethylacetate, methanol, chloroform : methanol solvents were used for inhibition of staphylococcus aureus and E-coli. It is noted that chloroform : methonal is the best solution for extracting the effective antibacterial materials from the brown algae species. The chloroform: methanol solvent further used for antibacterial activity against eleven pathogenic bacterias. It is observed from the experiments that the extract residues of algae recorded maximum activity against staphylococcus aureus with an inhibition zone compared to other bacterias. The extract residues of brown algae did not show any effect on the growth of proteus vulgaris and psedudomonoaeruginosa.(Journal of American Science 2009:5(3) 20-25) ( ISSN: 1545-1003)
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A lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to β-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and β-1,3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the β-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.
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A massive ephtic of vibriosis, named “sindroma gaviota” (or sea gull syndrome = SGS) by local shrimp fanners, began in November 1989 and continued through May 1990 in nursery and grow-out ponds in a number of shrimp farms located in the Gulf of Guaynquil near Cunyaquil, Ecuador. The level of cumulative mortalities ranged from slight in some ponds to more than 90% in others. Examination of affected shrimp, Penaeus vannamei Boone, led to the isolation of several closely related Vibrio species. The predominant species were identified as Vibrio parahaemolyticus, V. vulnificus, and V. alginolyticus using the API-NFT system. Several of the strains exhibited a transient luminescence and some were positive for urease, an unusual trait for these species. Comparisons of the antimicrobial sensitivity patterns between farm and hatchery isolates from 1989–1990 and hatchery isolates from 1987–1989 indicate that the 1989–1990 pond isolates were not of hatchery origin. The epizootic coincided with the second year of a severe drought in Ecuador. Thus, the discharge of fresh water by the Guayas River was reduced and salinities and relative nutrient concentrations were elevated in the Gulf of Guaynquil. These environmental conditions were ideal for the increased growth of the causative vibrios in the estuarine waters and are believed to be factors that contributed to the 1989–1990 SGS epizootic.
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This paper reports on the feasibility of utilizing the abundant marine alga Rhizoclonium as a substitute for wood fiber, based on studies on its morphology and chemical composition. The alga appears as wood fiber-like filaments consisting of tubular end-to-end connections of individual cells. In the population studied, each cell averaged 82 μm long, 76 μm wide and had cell wall 7.4 μm thick. The composition was 15.9% ash, 9.72% extractable by 90% acetone, 9.43% extractable by alcohol-benzene, 3.8% acid insoluble fraction, 17.8% pentosan, 36.3% 1% NaOH soluble fraction and 57.4% carbohydrate. The composition of its carbohydrates is similar to that of wood fiber. After hydrolysis, reduction, and acetylation of the sugars, and GC-MS analysis the components showed glucose (65.8%), xylose (19.8%), galactose (12.5%) and mannose (1.3%). There were high contents of cold- and hot-water extractables, 31.1% and 34.6%, respectively. These consisted of xylose, galactose and glucose. The crystallinity index (CI%) of its holocellulose was as high as 86.5%, close to the 90.5% value of wood fiber. The 1091 cm-1 peak intensity increased with reaction cycles, suggesting decreasing absorptivity and increasing crystallinity. This corresponds to terrestrial plant fibers. Taken together, these features suggest that Rhizoclonium has good potential as a raw material for pulp.
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There are five different viruses which are currently being studied for their impact on commercial farming of the black tiger prawn (Penaeus monodon) in Thailand. Some of these viruses cause disease in other penaeid shrimp species and even other crustacean species. Some occur not only in cultivated shrimp in other Asian countries, but also in those from Australia and the western hemisphere. In descending order from greatest to least economic impact on the Thai shrimp industry, the five viruses are: white-spot baculovirus, yellow-head virus, hepatopancreatic parvo-like virus, infectious hypodermal and hematopoeitic necrosis virus and monodon baculovirus. The purpose of this review is to summarize recent work on these viruses and to suggest future directions of research that may be useful in the effort to develop a sustainable shrimp industry.
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The question as to how the immune defence of an invertebrate animal is initiated and coordinated has largely been unanswered. This short review focuses on recent discoveries about crayfish hemolymph proteins, which may play roles in cell adhesion events leading to initiation of phagocytosis and encapsulation. Focus will also be made on anti-oxidative enzymes that may participate in the production of reactive oxygen compounds used in the destruction of engulfed or encapsulated parasites. Peroxinectin is stored in semi-granular and granular hemocytes and released concomitant with activation of prophenoloxidase (proPO). It is a cell adhesion protein, enhancing phagocytosis and encapsulation and triggers degranulation. It is also a peroxidase, belonging to the same protein family as mammalian myeloperoxidase. Peroxinectin binds a 90-kDa peripheral cell surface superoxide dismutase (SOD) of crayfish blood cells. Integrins are transmembrane proteins present on crayfish hemocytes and commonly known to be acting as cell adhesion receptors in many events. After its release and activation, peroxinectin may opsonize foreign surfaces where it is recognized by integrins on the hemocyte. This can be a starting point for phagocytosis or encapsulation. Peroxinectin and extracellular SOD (EC-SOD) may then cooperate during a respiratory burst to destroy an ingested or encapsulated parasite.
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The total haemocyte count (THC), phenoloxidase activity, respiratory burst and phagocytic activity and clearance efficiency to Vibrio alginolyticus were examined when the white shrimp Litopenaeus vannamei were injected with different types (type I to type V) of carrageenan at 6 µg g− 1 shrimp. Results showed that the THC, phenoloxidase activity, respiratory burst, and phagocytic activity against V. alginolyticus were significantly higher in shrimp that received any type of carrageenan after 24 h. Results also showed that the THC and PO activity maintained significantly higher in shrimp that received type IV carrageenan as compared to those that received other types of carrageenan after 96 h. In a separate experiment, L. vannamei which had been injected with different types of carrageenan after 3 h were challenged with V. alginolyticus at 7.34 × 106 cfu shrimp− 1, and placed in seawater. The survival of shrimp that received type IV carrageenan was significantly higher than that of shrimp that received type I, II, III and V carrageenan, and shrimp that received saline as well as the control shrimp after 120 h. It was concluded that L. vannamei that received type IV carrageenan (λ-carrageenan) exhibited higher immune ability of L. vannamei as well as resistance against V. alginolyticus infection.
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Shrimp farming constitutes an important source of revenue and employment in many developing countries. However, infectious diseases have affected the profitability of the shrimp industry. For this reason, disease prevention is a priority and shrimp immunology has become a prime area of research. In such a perspective, studies into the value of cellular and humoral parameters as indicators of shrimp condition are being carried out, with the intention of developing criteria for sanitary surveys, immunomodulation studies and selection programs for shrimp with high resistance to pathogens. Several quantitative, fast and easy procedures are being adapted to evaluate the expression of the immune response of shrimp. In regard to cellular parameters, the hemogram and two cellular mechanisms, the radical oxygen intermediates (ROIs) generated during postphagocytic events and phenoloxidase (PO) activity have been considered as potential markers. Concerning humoral parameters, the antibacterial activity of plasma and the concentration of plasma proteins can be considered as criteria of health status. Information is presented about the immunological tools used to evaluate these health markers and the results concerning the clinical significance of response modifications.
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Significant amounts of marine macroalgal (seaweed) polysaccharides are used in food, pharmaceuticals and other products for human consumption. Thus, the global seaweed polysaccharide industry operates in a highly regulated environment. Genetic manipulation of macroalgae to alter composition or growth characteristics may lead to products that do not fall within the current regulations: research that is readily translatable to industrial application is generally restricted to seaweed cultivation and processing and new applications of the approved polysaccharides. There is a great need, however, for research into the genome structure and metabolic pathways of commercially important marine macroalgae. This precompetitive research may not be immediately applicable to the seaweed polysaccharide industry but is critical for sustaining future commercial growth.
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A spectrophotometric nitroblue tetrazolium (NBT) reduction assay was used to demonstrate the production of superoxide anions (O2−) by haemocytes of the white shrimp Penaeus vannamei. It was found that haemocytes, without receiving an experimental stimulant, showed a rather high background activity. Therefore, optimal parameters (number of haemocytes, type of incubation medium, type and concentration of stimulants) were first established, in order to obtain a reliable and reproducible quantitative assay. With this optimized assay, and using specific inhibitors, it was shown that it is indeed the production of O2− that was measured. Activities varied strongly among individual shrimp specimens. Live bacteria, among these Vibrio strains, induced O2− production in the haemocytes, in a dose-dependent manner. Whereas Vibrio anguillarum and a probiotic strain of V. alginolyticus evoked clear reactions, a pathogenic strain of V. harveyi failed to elicit O2− production in the haemocytes. It is discussed that this may explain this strain's capability of evading the host's oxidative microbicidal activity, which would be a virulence factor in these bacteria. Heat-killed bacteria hardly induced NBT reduction in the cells. The fungicide propiconazole or Tilt®, found as a pollutant in the aquatic environment where the shrimp are reared, was tested for its effect on NBT reduction by the haemocytes. In haemocytes that did not receive an experimental stimulant, Tilt® induced the reduction of NBT in a dose-dependent manner. In experimentally stimulated haemocytes, however, Tilt® strongly reduced the reaction upon the stimulant PMA. Probable explanations for these seemingly controversial effects of Tilt® are discussed, as are possible consequences of this sort of pollutants for shrimp aquaculture. This easy to perform and relatively cheap and simple quantitative assay for measuring the activity of an oxidative microbicidal mechanism in shrimp haemocytes, appears quite reliable and may therefore prove to be a valuable tool for monitoring shrimp health and immunologic status.