Joanna J. Miest

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.

β-Glucan supplemented diets increase poly(I:C)-induced gene expression of Mx, possibly via Tlr3-mediated recognition mechanism in common carp (Cyprinus carpio)

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard®) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection, particularly in the gut. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGard® was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly(I:C)), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response. Subsequently, a set of immune related genes, including mx, were analysed by real-time PCR on liver, spleen, head kidney and mid gut tissues. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of selected cytokines when compared to untreated fish, while mx gene expression remained stable or was slightly up-regulated. Injection with poly(I:C) induced a similar down-regulated gene expression pattern for cytokines in samples from β-glucan fed fish. In contrast, poly(I:C) injection markedly increased mx gene expression in samples from β-glucan fed fish but hardly in samples from fish fed control feed. In an attempt to explain the high induction of mx, we studied Toll-like receptor 3 (TLR3) gene expression in these carp. TLR3 is a prototypical pattern recognition receptor considered important for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through genome data mining, two sequences for carp tlr3 were retrieved (tlr3.1 and tlr3.2) and characterized. Constitutive gene expression of both tlr3.1 and tlr3.2 was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after β-glucan feeding, very high levels of tlr3.1 gene expression were observed in all analysed organs, with the exception of the liver. Our data suggest that β-glucan-mediated protection against viral diseases could be due to an increased Tlr3-mediated recognition of ligands, resulting in an increased antiviral activity of Mx.

Feeding common carp Cyprinus carpio with β-glucan supplemented diet stimulates C-reactive protein and complement immune acute phase responses following PAMPs injection.

The effect of β-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with β-glucan (MacroGard®) with a daily β-glucan intake of 6 mg per kg body weight or with control food for 25 days and then injected with PBS containing either LPS (4 mg/kg) or poly(I:C) (5 mg/kg) or PBS alone. Fish were sampled during the 25 days of the feeding period and up to 7 days post-PAMPs injections for serum and liver, head kidney and mid-gut tissues. Oral administration of β-glucan for 25 days significantly increased serum CRP levels and alternative complement activity (ACP). In addition, the subsequent LPS and poly(I:C) challenges significantly affected CRP and complement related gene expression profiles (crp1, crp2, c1r/s, bf/c2, c3 and masp2), with the greatest effects observed in the β-glucan fed fish. However, in fish fed β-glucan the PAMPs injections had less effects on CRP levels and complement activity in the serum than in control fed fish, suggesting that the 25 days of β-glucan immunostimulation was sufficient enough to reduce the effects of LPS and poly(I:C) injections. Results suggest that MacroGard® stimulated CRP and complement responses to PAMPs immunological challenges in common carp thus highlighting the beneficial β-glucan immunostimulant properties.

C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a highly virulent and lethal disease of common carp Cyprinus carpio and its ornamental koi varieties. However, specific knowledge about immune mechanisms behind the infection process is very limited. We aimed to evaluate the effect of the CyHV-3 infection on the profile of 2 major components of the common carp immune acute phase response: the C-reactive protein (CRP) and the complement system. Common carp were infected with CyHV-3 by bath immersion. Fish were sampled before the infection and at 6, 12, 24, 72, 120 and 336 h post-infection for serum and head kidney, liver, gill and spleen tissues. CRP levels and complement activity were determined from the serum, whereas CRP- and complement-related genes (crp1, crp2, c1rs, bf/c2, c3, masp2) expression profiles were analysed in the tissues by quantitative PCR. Both CRP levels and complement activity increased significantly up to 10- and 3-fold, respectively, in the serum of infected fish during the challenge. Analysis revealed distinct organ- and time-dependent expression profile patterns for all selected genes. These results suggest that CRP and complement behave as acute phase reactants to CyHV-3 infection in common carp with an organ- and time-dependent response.

Temperature effects on gene expression and morphological development of European eel, Anguilla anguilla larvae

Temperature is important for optimization of rearing conditions in aquaculture, especially during the critical early life history stages of fish. Here, we experimentally investigated the impact of temperature (16, 18, 20, 22 and 24°C) on thermally induced phenotypic variability, from larval hatch to first-feeding, and the linked expression of targeted genes [heat shock proteins (hsp), growth hormone (gh) and insulin-like growth factors (igf)] associated to larval performance of European eel, Anguilla anguilla. Temperature effects on larval morphology and gene expression were investigated throughout early larval development (in real time from 0 to 18 days post hatch) and at specific developmental stages (hatch, jaw/teeth formation, and first-feeding). Results showed that hatch success, yolk utilization efficiency, survival, deformities, yolk utilization, and growth rates were all significantly affected by temperature. In real time, increasing temperature from 16 to 22°C accelerated larval development, while larval gene expression patterns (hsp70, hsp90, gh and igf-1) were delayed at cold temperatures (16°C) or accelerated at warm temperatures (20–22°C). All targeted genes (hsp70, hsp90, gh, igf-1, igf-2a, igf-2b) were differentially expressed during larval development. Moreover, expression of gh was highest at 16°C during the jaw/teeth formation, and the first-feeding developmental stages, while expression of hsp90 was highest at 22°C, suggesting thermal stress. Furthermore, 24°C was shown to be deleterious (resulting in 100% mortality), while 16°C and 22°C (~50 and 90% deformities respectively) represent the lower and upper thermal tolerance limits. In conclusion, the high survival, lowest incidence of deformities at hatch, high yolk utilization efficiency, high gh and low hsp expression, suggest 18°C as the optimal temperature for offspring of European eel. Furthermore, our results suggest that the still enigmatic early life history stages of European eel may inhabit the deeper layer of the Sargasso Sea and indicate vulnerability of this critically endangered species to increasing ocean temperature.

Differential effects of Alloherpesvirus CyHV-3 and Rhabdovirus SVCV on apoptosis in fish cells

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.

Cleaner wrasse indirectly affect the cognitive performance of a damselfish through ectoparasite removal

Cleaning organisms play a fundamental ecological role by removing ectoparasites and infected tissue from client surfaces. We used the well-studied cleaning mutualisms involving the cleaner wrasse, Labroides dimidiatus, to test how client cognition is affected by ectoparasites and whether these effects are mitigated by cleaners. Ambon damselfish (Pomacentrus amboinensis) collected from experimental reef patches without cleaner wrasse performed worse in a visual discrimination test than conspecifics from patches with cleaners. Endoparasite abundance also negatively influenced success in this test. Visual discrimination performance was also impaired in damselfish experimentally infected with gnathiid (Crustacea: Isopoda) ectoparasites. Neither cleaner absence nor gnathiid infection affected performance in spatial recognition or reversal learning tests. Injection with immune-stimulating lipopolysaccharide did not affect visual discrimination performance relative to saline-injected controls, suggesting that cognitive impairments are not due to an innate immune response. Our results highlight the complex, indirect role of cleaning organisms in promoting the health of their clients via ectoparasite removal and emphasize the negative impact of parasites on hosts cognitive abilities.

Time and concentration dependency of MacroGard® induced apoptosis

In previous studies an effect of β-glucan on apoptosis in fish was noted and in this investigation we determine the time and concentration dependency of this effect. Primary cell cultures of pronephric carp cells were incubated for 6, 24, 48 h with various concentrations ranging from 0 to 1000 μg/ml of MacroGard(®) β-glucan. Apoptosis was monitored via acridine orange staining. Results indicate a clear effect of time and concentration on the induction of apoptosis in vitro, with only concentration ≥500 μg/ml causing significantly higher percentages of apoptotic cells. Apoptosis was detected after 6 h. This concentration dependent effect has to be considered when studying apoptosis in relation to immunostimulation.

Poly-β-hydroxybutyrate administration during early life: effects on performance, immunity and microbial community of European sea bass yolk-sac larvae

The reliable production of marine fish larvae is one of the major bottlenecks in aquaculture due to high mortalities mainly caused by infectious diseases. To evaluate if the compound poly-β-hydroxybutyrate (PHB) might be a suitable immunoprophylactic measure in fish larviculture, its capacity to improve immunity and performance in European sea bass (Dicentrarchus labrax) yolk-sac larvae was explored. PHB was applied from mouth opening onwards to stimulate the developing larval immune system at the earliest possible point in time. Larval survival, growth, microbiota composition, gene expression profiles and disease resistance were assessed. PHB administration improved larval survival and, furthermore, altered the larva-associated microbiota composition. The bacterial challenge test using pathogenic Vibrio anguillarum revealed that the larval disease resistance was not influenced by PHB. The expression profiles of 26 genes involved e.g. in the immune response showed that PHB affected the expression of the antimicrobial peptides ferritin (fer) and dicentracin (dic), however, the response to PHB was inconsistent and weaker than previously demonstrated for sea bass post-larvae. Hence, the present study highlights the need for more research focusing on the immunostimulation of different early developmental stages for gaining a more comprehensive picture and advancing a sustainable production of high quality fry.