According to reports by the United Nations' Food and Agriculture Organization (FAO), bacterial disease outbreaks are considered a significant constraint to the development of the aquaculture sector and the terrestrial animal production sector. The ban on the use of antibiotics to control diseases in these production sectors has challenged researchers throughout the world to look for alternative biocontrol strategies1,2. Recently, it has been suggested that short-chain fatty acids (SCFAs) could be useful as biocontrol agents to control bacterial diseases in animal production and more specifically aquaculture3. SCFAs, such as acetate, propionate and butyrate, are the major products of anaerobic bacterial fermentation of non-absorbable carbohydrates in the small intestine4. Several studies have demonstrated that SCFAs inhibit the growth of enterobacteria like Salmonella typhimurium, Escherichia coli and Shigella flexneri5. Recently, luminescent vibrios like Vibrio campbellii were also reported to be as susceptible to SCFAs6. Furthermore, in vivo challenge tests exhibited that SCFAs significantly increased the survival of the aquaculture model organism Artemia franciscana larvae challenged with pathogenic Vibrio campbellii, indicating that these compounds are not only useful in terrestrial animal production, but also in aquaculture6. In another report, the well-known bacterial storage compound poly-ß-hydroxybutyrate (PHB), a polymer of the SCFA ß-hydroxybutyrate, was also shown to protect Artemia larvae from the virulent V. campbellii strain7. Although this compound is insoluble in water, it is likely to be biologically degraded into antibacterial β-hydroxybutyric acid or PHB oligomers in the gastrointestinal tract of organisms7. The degraded product can exert its beneficial effects like other SCFA do7. In several experiments with A. fransiscana, this approach increased the survival of the animals with up to 73% upon challenge with the pathogen V. campbellii7,8.

It has also previously been shown that the SCFA butyrate induces heat shock protein (Hsp) 25 in rat intestinal epithelial cells and protects the latter against oxidant injury9. Hsps are a group of highly conserved proteins of which expression is constitutive or inducible under different conditions. Hsps, particularly Hsp70, have strong cytoprotective effects and behave as molecular chaperones for maintaining proper protein folding, disaggregating and refolding misfolded protein as well as targeting damage proteins for degradation10,11. Besides these, Hsp70 also generate protective immunity against many diseases as demonstrated in a wide variety of animal models12,13,14. For instance, in an axenically hatched A. franciscana, it was well demonstrated that upregulation of endogenous Hsp70 by a Hsp-inducing compound15 or exogenous administration of Artemia Hsp70 or the Escherichia coli Hsp70 equivalent DnaK, protected Artemia against V. campbellii infection by priming the prophenoloxidase system16,17. Similar results were also obtained by Ryckaert et al. (2009) for platyfish Xiphophorus maculates injected with Hsp70 protein and challenged with pathogenic bacteria Yersinia ruckeri.

This study addresses the possibility that the protective effects of PHB could be related to induction of Hsp70. Here, using the Artemia-V. campbellii host-pathogen model, we present novel findings which demonstrate that PHB is indeed a potent in vivo enhancer of Hsp70 and this effect mediates, at least in part, the PHB-induced protection to V. campbellii-challenged Artemia.


PHB confers protection to Artemia against pathogenic V. campbellii

In a previous study, PHB particles (1000 mg/L) with an average diameter of 30 µm were shown to confer protection to Artemia against V. campbellii7. To ascertain the protective effect of PHB and also to re-optimize the effective dose of PHB particles of size 25–30 µm, we carried out an in vivo survival assay using a range of PHB doses from 0 to 1000 mg/L. As shown in Figure 1, PHB significantly enhanced the survival of the challenged Artemia when added at a concentration of 10 (2.6-fold increase), 100 (3.6-fold increase), 250 (2.8-fold increase) and 500 mg/L (2.2-fold increase). A complete protection (no significant differences in survival with negative control) was observed at 100 mg/L concentration. Increasing the PHB concentration to 1000 mg/L did not further increase the survival of the larvae. In contrast, the survival decreased markedly, reaching the level of the negative control.

Figure 1
figure 1

PHB at an optimum concentration confers protection to Artemia larvae against V. campbellii.

PHB particles were added to the Artemia culture water at indicated concentrations. Simultaneously, the larvae were fed with autoclaved LVS3 and challenged with V. campbellii, each at 107 cells/mL, for 48 h. PHB-untreated Artemia challenged with V. campbellii (positive control) and those not challenged with V. campbellii (negative control) served as controls. Values are presented as mean ± standard error and representative of 3 independent experiments. Different letters indicate significant differences (P < 0.05).

PHB induces Hsp70 production in Artemia

To determine whether induction of Hsp70 may be a mechanism for increased resistance of PHB-treated Artemia against V. campbellii challenge, we analyzed the temporal induction profile of Hsp70 by two approaches: qPCR analysis to determine the expression of mRNA for inducible hsp70 and immunoblotting to analyze Hsc70 and Hsp70 proteins. The results of qPCR revealed that at 6 h post treatment, the expression of the hsp70 gene in the PHB and PHB + Vibrio groups did not increase significantly as compared to the negative control (P > 0.05, Figure 2). At 12 and 24 h post treatment, the hsp70 mRNA expression levels in the PHB and PHB + Vibrio groups appeared to decrease compared to the negative control, however, no significant difference was observed among the groups. The expression pattern of hsp70 gene in Artemia challenged with V. campbellii (positive control), relative to the unchallenged (negative) control, tend to increase at 6 h post challenge (P > 0.05), then significantly decreased by 4-fold at 12 h post challenge (P < 0.05). However, at 24 h post challenge, the hsp70 transcripts increased to the same level as that of the control.The Western blot analysis showed that PHB markedly increased Hsp70 production in the Vibrio-challenged and unchallenged Artemia (by 10.5 and 6.2-fold, respectively) when compared with the (negative) control at 6 h post treatment (Figure 3). At 12 and 24 h post treatment, Hsp70 was not detected in the PHB-treated groups (data not shown).

Figure 2
figure 2

PHB at an optimum concentration regulates the expression of hsp70 gene in Artemia larvae challenged with V. campbellii.

PHB particles (100 mg/L) were added to the Artemia culture water at the start of the experiment. Simultaneously, the larvae were fed with autoclaved LVS3 and either challenged with V. campbellii (PHB + Vibrio) at indicated concentrations or not (PHB). Controls were maintained as described in Figure 1. Samples were collected for hsp70 gene expression assay at 6, 12 and 24 h of Vibrio challenge. The expression of hsp70 gene in the negative control was regarded as 1.0. Results, which are the mean of 3 replicates, are presented relative to Artemia actin gene expression, according to the equation of Pfaffl et al. (2002). Bars indicate standard error from the mean. Significant differences between the treatment and (negative) control at corresponding time points are indicated by *(P < 0.05).

Figure 3
figure 3

PHB at an optimum concentration induces Hsp70 production in Artemia larvae challenged with V. campbellii.

For the treatment groups, refer to Fig. 2. for explanation. Artemia samples were collected for analysis of Hsp70 induction at 6 h of Vibrio challenge. (A) Protein extracted from different groups was resolved in SDS-PAGE gel and then transferred to polyvinylidene fluoride membranes and probed with antibody to Artemia Hsp70. Seven microgram of Artemia protein was loaded in each lane. HeLa (heat shocked) cells (6 µg) were loaded on to one well to serve as a positive technical control and for calculating the relative amount of Hsp70 in the sample. Cropped blot image was shown and the full-length blot was presented in Supplementary Figure S1. (B) Quantitative analysis of Hsp70 in the Artemia larvae (expressed relative to the amount in HeLa cells).

PHB regulates the expression of innate immune-related genes in Artemia

Since there exists a correlation between the amount of induced Hsp70 and the degree of improved protective immune responses against diseases in animals19,20, we next determined the temporal expression of the innate immunity-related genes i.e. prophenoloxidase (proPO), transglutaminase (tgase) and ferritin (ftn) in Artemia treated with PHB and simultaneously challenged with V. campbellii. As shown in Figure 4A, there was no significant upregulation of the proPO gene in the PHB-treated groups (PHB and PHB + Vibrio) as compared to the unchallenged (negative) control at any of the time points tested. The expression pattern of the proPO gene in Artemia challenged with V. campbellii (positive control) showed a different trend. The expression levels, relative to that in the negative control, tend to decrease at 6 h post challenge (P > 0.05), significantly down regulated at 12 h post challenge (P < 0.05) and then returned to control level at 24 h post challenge (P > 0.05). We also compared the expression level of the proPO gene between challenged Artemia (positive control) and PHB-treated challenged Artemia (PHB + Vibrio) at all the time points tested. The proPO expression level in the PHB + Vibrio group was significantly higher (2.7-fold, P < 0.05) than the positive control at 6 h post challenge and then remained unaltered at 12 h and 24 h post challenge.

Figure 4
figure 4

PHB regulates the expression of (A) proPO (B) tgase and (C) ftn genes in Artemia larvae challenged with V. campbellii.

For the treatment groups, refer to Fig. 2. for explanation The expression of the proPO, tgase and tgase gene in the negative control was regarded as 1.0. Results, which are the mean of 3 replicates, are presented relative to Artemia actin gene expression, according to the equation of Pfaffl et al. (2002). Bars indicate standard error from the mean. Significant differences between the treatment and untreated control at corresponding time points are indicated by * (P < 0.05).

The mRNA transcript levels of tgase in the PHB + Vibrio group markedly increased by 2.3-fold (P > 0.05) relative to the negative control at 6 h post treatment (Figure 4B). At 12 h post challenge, the transcript levels in this group tend to remain relatively higher (by 1.5 fold, P > 0.05) than that in the negative control. At 24 h post challenge, the transcript level was comparable with the negative control. Relative to the positive control, the tgase expression level in the PHB + Vibrio group remained considerably higher at 6 h (by 1.7-fold) and 12 h (1.4-fold) post challenge, but was not significantly different. At 24 h post challenge, no significant difference was noted between the two groups.

The ftn mRNA transcript levels in the PHB and PHB + Vibrio groups did not increase significantly as compared to the negative control, at any of the time points tested, however the expression level appeared to decrease over time (P > 0.05; Figure 4C). The expression pattern of ftn gene in the Artemia challenged with V. campbellii (positive control), relative to the unchallenged (negative) control, remained at the same level at 6 h post challenge and then significantly decreased by 3.6-fold at 12 h post challenge (P < 0.05). However, the ftn transcripts increased to the same level as that of the negative control at 24 h post challenge. In relative to positive control, the level of ftn transcripts in the PHB + Vibrio group remained unaltered at 6 and 12 h of Vibrio challenge. However, at 24 h, the level appeared to decrease as compared to the positive control (P > 0.05).

PHB increases the activity of phenoloxidase in Artemia challenged with V. campbellii

Besides measuring proPO at the transcriptional level, the assay of proPO at the protein level was also carried out in Artemia that were treated and challenged with PHB and/or V. campbellii for 24 h (Figure 5). The activity of phenoloxidase (PO) in challenged Artemia was significantly enhanced by PHB at both 6 and 12 h post challenge, where an increase of about 2-fold was seen over challenged-Artemia (positive control). The PO activity in PHB-treated Artemia was also significantly higher than that of the untreated Artemia (negative control). However, when compared with Vibrio-challenged Artemia, the PO activity in the PHB-treated Artemia was not significantly different. At 24 h post challenge, no significant differences in the PO activity level were observed among the different groups (P > 0.05).

Figure 5
figure 5

PHB increases the activity of phenoloxidase in Artemia challenged with V. campbellii.

For the treatment groups, refer to Fig. 2 for explanation. Artemia samples were collected for the PO assay after 6, 12 and 24 h of Vibrio challenge. All treatments were carried out in three replicates. Values are presented as mean ± standard error (n = 3). Different letters (small and capital letters for 6 and 12 h, respectively) indicate significant differences among the groups (P < 0.05). Bars (at 12 h) with no letters indicate no significant differences (P > 0.05).


The SCFA ß-hydroxybutyrate and its polymer PHB have in a number of model organisms been shown to induce protective effects against pathogenic stressors resulting in increased survival3,21. Consistent with the previous report7, our results provided clear evidence that addition of PHB particles to the Artemia culture water significantly protected the shrimp against V. campbellii, but significant differences between that study and the current one existed in relation to the effective PHB dose. Maximum and complete protection against V. campbellii was obtained at a PHB concentration of 1000 mg/L in the previous study, whereas in this study, only 100 mg/L was needed to obtain a similar effect. This disparity in the effective doses between the two studies could be attributed to the particle size of the PHB. The particles used in this study are in the range of 25–30 µm (compared to an average diameter size of 30 µM in the earlier study). The brine shrimp Artemia is a continuous, non-selective particle-filtration feeder but particle size has been shown to affect uptake efficiency22. The uptake of the smaller sized particles in this study might have been more efficient resulting in a complete protective effect being obtained at 10 times lesser dose.

The protective mechanisms of PHB and/or its degradation product ß-hydroxybutyrate are not well understood. The main hypotheses relate to the lowering of gut pH resulting in the inhibition of the growth and virulence factor production of pathogenic bacteria3 and the direct delivery of energy to the PHB supplemented animals21. In addition to these, the possibility that the protective effect of PHB might be mediated by the induction of stress proteins, particularly Hsp70, has not been explored previously. In this in vivo study, our results provided conclusive evidence that PHB at a concentration of 100 mg/L induces the production of Hsp70 protein in Vibrio-challenged Artemia and interestingly, at this concentration, it also afforded complete protection to Artemia against V. campbellii. Previous studies using a rat model have demonstrated that butyrate selectively induced an important stress protein Hsp27 (but not Hsp70), both in vitro in cultured rat intestinal epithelial cells (IEC) and in vivo in the colon and that this butyrate-induced expression of Hsp27 resulted in a greater cellular protection against oxidant injury9. In another in vitro study, Parhar et al. (2006) reported that butyrate not only induces Hsp25 but also regulates its phosphorylation in a rat IEC-18 crypt cell line and secondarily increased cellular resistance to apoptosis-inducing agents. From the results of the above-cited and present studies, it can be suggested that some of the protection offered by PHB in our model organism may be derived from the induction of Hsp70. It is worth mentioning that this induction was observed at the protein level but not at the mRNA level. The observed lack of correlation between hsp70 mRNA and protein concentration can be explained by the different lifetimes of the molecules24: hsp70 mRNA half-life is short (about 50 min) in cells after stress, even shorter in cells already containing Hsp70 protein25,26 and few copies per cell are produced, causing their concentrations in cells to fluctuate much more than those of the longer-lived (about 2 h) corresponding protein27. We must also acknowledge that the possible up-regulation of other stress proteins (like Hsp25, Hsp60, Hsp90) by PHB and their implication in conveying resistance to Artemia against Vibrio challenge likely exists as well9. Efforts are currently underway in our laboratory to unravel the effect of the Hsp inducer (PHB) on the expression of other various stress proteins in an attempt to address these possibilities.

Although the Hsp inducing effects of SCFAs are reported9,23,28, yet it is not clear how PHB or SCFAs induce stress proteins expression. At this point, we can argue that the PHB particles are (partially) degraded into different monomeric, dimeric and/or oligomeric forms in the Artemia gut and that this released fatty acid, through nonionic diffusion, might have caused cellular acidification. Lowering of cellular pH might have created a (mild) stress conditions that could have potentially driven the production of inducible Hsp70 in the intestinal epithelial cells9. However, this is pure speculation and needs further investigation.

As an invertebrate that lacks an acquired immune system, the brine shrimp Artemia depends on innate immune factors to build up resistance against pathogens. A growing body of evidence suggests that Hsp70 evokes protective responses in various animals (including in the aquaculture model organism Artemia) against bacterial diseases by inducing these defense factors17,18,29. Having demonstrated the effects of PHB on Hsp70 induction in Artemia host and in view of the association between Hsp70 and Artemia immune system, it is tempting to speculate whether PHB conveyed its protective effect through eliciting the Artemia defense system. To substantiate this hypothesis, we analyzed three important genes proPO, ftn and tgase encoding for immune effector proteins phenoloxidase, TGase and ferritin, respectively. The proPO system is composed, among other proteins, of the prophenoloxidase enzyme, which is the zymogen of phenoloxidase (PO)30,31. The PO induces its protective effect by its role in cuticular melanization, sclerotisation, wound healing, encapsulation and eventual killing of the pathogens32,33. Our results exhibited that the proPO system was markedly induced by PHB at a concentration of 100 mg/L. The induction effect was more prominent when Artemia were treated with PHB in conjunction with Vibrio compared to PHB alone, as indicated by elevation of the proPO mRNA transcript at 6 post challenge by 2.5-fold and PO activity at 6 and 12 h post challenge by 2-fold (see Figure 4A & 5). This result suggests an interaction effect of PHB and Vibrio on the induction of the proPO system in Artemia. Interestingly, this phenomenon correlates well with the Hsp70 protein induction by PHB at 100 mg/L concentration in the Vibrio-challenged group, suggesting that the induced Hsp70 might have accounted for eliciting the proPO immune system. In accordance with these findings, a previous study reported that induced Hsp70 coincides with increased expression of the proPO gene and its product PO enzyme in Artemia thereby promoting resistance to V. campbellii infection in the shrimp17.

In invertebrates with an open circulatory system, including Artemia, there is a need to quickly prevent the loss of blood or equivalent fluids through inflicted injuries (caused by pathogen) as these losses may have a marked impact on the survival of the invertebrate34. Also, there is a need to prevent microbes that have gained access to the body through the wound from disseminating throughout the open circulatory system. Therefore, many invertebrates possess a coagulation system to prevent such injuries from having too serious consequences. The defense molecule TGase is a major component of this system responsible for catalyzing the clotting reaction35,36. Our result revealed that PHB increased the expression of the tgase gene considerably shortly after challenge with Vibrio (markedly at 6 h and slightly at 12 h). This increase in the expression level did not appear to be statistically significant even though the increment was by about 2-fold (possibly due to high standard errors). In the Artemia that were subjected to only Vibrio challenge, no marked increase in the expression level of tgase was recorded at any of the tested time points. Interestingly, the results of tgase expression were supported by survival data especially in the group treated with 100 mg/L of PHB where more than 85% survival (compared to 11% in control) could be obtained. It is possible that the functional protein TGase encoded by the PHB-mediated tgase assists in host defense by preventing tissue damage and simultaneously by blocking (further) progression of Vibrio infection in Artemia. Similar observations have been reported by Babu et al. (2013)37, where shrimp fed a diet containing immunostimulants showed significantly higher expression of tgase and had higher protection against the shrimp pathogen white spot syndrome virus.

Iron has been considered as an essential element required for the survival and growth of most organisms, both hosts and pathogens38. It has almost been certain that tight regulation of iron (regarded as double-edged sword) is a paramount defense mechanism of the host39. For multiplying and inducing pathogenic effects within the host, the pathogen competes for the host's iron40. A variety of iron-withholding defense mechanisms are being used by the host to limit the availability of essential iron from the bacteria without causing deficiency for itself. In this study, we focused on the ferritin (ftn) immune gene encoding for protein ferritin, known to play crucial role as buffer against iron deficiency and iron overload39. Our results revealed that PHB did not up-regulate ftn transcription during Vibrio infection as the expression level was similar to that in the infected/uninfected Artemia. In contrast, in the later period of Vibrio infection (24 h), the ftn expression level was relatively low in the PHB-treated groups. As a major iron-binding protein, it is conceivable that ferritin is a rich nutrient resource and it would be an obvious target for bacteria to pirate iron from39. Unaltered or reduced expression of ftn gene (and presumably in the ferritin protein level) in the PHB-treated group challenged with Vibrio could be a defensive strategy of the host to deprive iron from the bacteria39.

Another interesting observation that was noted in this study was that PHB above a threshold concentration (> 100 mg/L) had an adverse effect upon Vibrio-challenged Artemia as survival of Vibrio-challenged Artemia decreased with PHB concentrations increasing above 100 mg/L. This effect was also associated with a significant decrease in the Hsp70 level in Vibrio-challenged Artemia treated with high PHB dose (1000 mg/L, data not shown). As such toxicity is to be expected, even a benign compound would become toxic after a certain threshold concentration37. However, in this study, it is clear that the effective dose of this compound was 10 times lower than the toxic dose (and through further compound optimization i.e., by the addition of PHB degrading bacteria, this effective dose could possibly be further lowered7). This indicates that PHB has a good safety margin and therefore appeared to be safe for therapeutic use in (aquaculture) animal production sector. A possible explanation for the adverse effects of PHB at high dose could be due to the fact that at higher PHB concentrations, the pH in the Artemia gut might have dropped down remarkably, which could likely contribute to PHB toxicity and thus, more Hsp70 may be utilized to reverse the (deleterious) effect induced by a combination of PHB toxicity and V. campbellii insults than by the latter alone23,41.

In essence, the results presented here provide new insights on the polymer of ß-hydroxybutyrate, PHB, as a novel inducer of Hsp70 biosynthesis under the described experimental conditions. Our findings also imply that this compound-mediated Hsp70 seems to be responsible for generating protective innate immunity through regulating the expression of proPO, tgase and ftn immune genes in the Artemia larvae, offering new clues for the mechanism of the protective effect of PHB against pathogenic V. campbellii. Combining the present study with its outstanding protective effect reported previously, PHB therefore can be a potent bio-control agent in different host–microbe systems, such as for instance Salmonella infections in poultry and swine or bacterial infections in other crustaceans and fishes. To gain more insights into the functional properties of PHB, more detailed studies should be carried out in future by employing RNAi techniques to knock-out Hsp70 gene and determining the effect of PHB treatment on the host innate immune response by performing time-dependent micro-array analysis.


Axenic hatching of Artemia

All challenge tests were performed with high quality hatching cysts of Artemia fransciscana originating from the Great Salt Lake, Utah, USA (INVE Aquaculture, Dendormonde, Belgium) as described previously42. Briefly, Artemia (2.5 g) cysts were hydrated in 89 mL of distilled water for 1 h. Sterile cysts and larvae were obtained via decapsulation using 3.3 mL NaOH (32%) and 50 mL NaOCl (50%). All manipulations were carried out under a laminar flow hood and all tools were sterilized. The decapsulation was stopped after about 2 min by adding 50 mL Na2S2O3 at 10 g/L. The decapsulated cysts were washed with sterile seawater containing 35 g/L of instant ocean synthetic sea salt (Aquarium Systems, Sarrebourg, France). The cysts were suspended in 1-L glass bottles containing sterile seawater and placed in rectangular tank containing water maintained at 28°C using a thermostatic heater for incubation for 18–20 h with constant illumination of approximately 2000 lux. After 20 h of incubation, the axenicity of the Artemia larvae was verified by spread plating 100 mL of the hatching water on Marine Agar (Difco, Detroit, USA) followed by incubating at 28°C for 5 days43. Experiments started with non-sterile larvae were discarded.

Bacterial strains for in vivo challenge tests

Two bacterial strains were in this study: LVS3 (Aeromonas hydrophila) and Vibrio campbellii LMG21363. LVS3 (autoclaved) were used as feed for Artemia and V. campbellii as a pathogen for the challenge assay. Both the strains were stored in 40% glycerol at −80°C. 10 µL of these stored cultures were inoculated into fresh Marine Broth (Difco Laboratories, Detroit, USA) and incubated overnight at 28°C under constant agitation. The grown LVS3 culture was autoclaved, washed in autoclaved artificial seawater and added to the Artemia culture water at approximately 107 cells/mL. Live V. campbellii were also added to the Artemia culture water in a similar fashion at 107 cells/mL concentrations.

Artemia challenge assay

Challenge tests were performed as described previously16, with slight modifications. Briefly, after hatching, groups of 30 larvae were transferred to sterile 40 mL glass tubes that contained 30 mL of sterile artificial seawater. The tubes were inoculated with V. campbellii and the Artemia therein were fed with autoclaved LVS3. PHB particles of 25–30 µm size were added to the Artemia culture water at different concentrations (10, 100, 250, 500 and 1000 mg/L). After feeding and the addition of the appropriate compound and/or bacteria, the glass tubes were put back on the rotor and kept at 28°C. The survival of Artemia was scored 2 days after the addition of the pathogen. All manipulations were done under a laminar flow hood in order to maintain gnotobiotic conditions of the cysts and nauplii. Each treatment was done in quintuplicate.

Sampling and analysis

After 20 h of incubation at 28°C, swimming Artemia larvae were collected, counted volumetrically and transferred to 500 mL sterile glass bottles. The larvae were treated with the dose of PHB that gave the best protection in the Artemia challenge assay and were simultaneously fed and challenged with autoclaved LVS3 and V. campbellii, respectively, as described above. Each treatment was carried out in triplicate. Artemia samples containing live larvae (0.1 g and 60 mg for analysis of protein and genes, respectively) were sampled from all treatments at 6, 12 and 24 h of Vibrio exposure, rinsed in cold distilled water, immediately frozen in liquid nitrogen and then stored at −80°C until further analysis (see the following headings).

Protein extraction and Hsp70 analysis

The Artemia samples were homogenized in cold buffer K (150 mM sorbitol, 70 mM potassium gluconate, 5 mM MgCl2, 5 mM NaH2PO4, 40 mM HEPES, pH 7.4)44 and supplemented with protease inhibitor cocktail (Sigma-Aldrich, USA) as recommended by the manufacturer. Subsequent to centrifugation at 2200 × g for 1 min at 4°C, supernatant protein concentrations were determined by the Bradford method using bovine serum albumin as standard45. Supernatant samples were then combined with loading buffer, vortexed, heated at 95°C for 5 min and electrophoresed in 10% SDS-PAGE gels, with each lane receiving equivalent amounts of protein. HeLa (heat shocked) cells (Enzo Life Sciences, USA) (6 µg) were loaded on to one well to serve as a positive control and for calculating the amount of Hsp70 in the sample. Gels were then transferred to polyvinylidene fluoride membranes (BioRad Immun-BlotTM PVDF) for antibody probing. Membranes were incubated with blocking buffer [50 mL of 1x phosphate buffered saline containing 0.2% (v/v) Tween-20 and 5% (w/v) bovine serum albumin] for 60 min at room temperature and then with mouse monoclonal anti-Hsp70 antibody, clone 3A3 (Affinity BioReagents Inc., Golden, CO), which recognizes both constitutive (heat shock cognate, Hsc70) and inducible Hsp7043, at the recommended dilution of 1:5000. Horseradish peroxidase conjugated donkey anti-mouse IgG was used as secondary antibody at the recommended dilution of 1:2500 (Affinity BioReagents Inc., Golden, CO). The membranes were then treated with enhanced chemiluminescence reagent (GE healthcare, UK) and the signals were detected by a ChemiDoc MP Imaging System (Biorad, Belgium). The relative signal intensity was quantified by densitometry with Biorad Image Lab™ Software version 4.1.

Assay of heat shock protein 70 (hsp70), prophenoloxidase (proPO), transglutaminase (tgase) and ferritin (ftn) gene expression by quantitative real-time PCR (qPCR) analysis

Total RNA was extracted from the Artemia samples using the SV total RNA isolation kit (Promega, Belgium) according to the manufacturer's instructions, after which the RNA was quantified spectrophotometrically (NanoDrop Technologies, Wilmington, DE, USA). First strand cDNA was synthesized from 2 µg total RNA using the RevertAid™ H minus First strand cDNA synthesis kit (Fermentas Gmbh, Germany) according to the manufacturer's instructions. The expression of hsp70, proPO, tgase and ftn genes in Artemia was analyzed by qPCR using a pair of specific primers15. The qPCR amplifications were carried out in a total volume of 25 µL, containing 5.5 µL of nuclease free water, 1 µL of each primer, 12.5 µL of Maxima SYBR Green qPCR Master mix (Fermentas, Cambridgeshire) and 5 µL of cDNA template. The qPCR was performed in a One Step qPCR instrument (Applied Biosystems) using a four-step amplification protocol: initial denaturation (10 min at 95°C); 40 cycles of amplification and quantification (15 s at 95°C, 30 s at 60°C and 30 s at 72°C); melting curve (55–95°C with a heating rate of 0.10°C s−1 and a continuous fluorescence measurement) and cooling (4°C). The β-actin gene was used as a reference gene. Master mixes were prepared in duplicate for each sample and qPCR for target and reference genes was performed. Relative quantification of target gene transcripts with a chosen reference gene transcript was done following the Pfaffl method with the Relative Expression Software tool (REST©) as described previously46.

Assay of phenoloxidase (PO) activity

For PO assay, Artemia samples (0.1 g) were collected after 6, 12 and 24 h of Vibrio challenge from all the treatments. Protein extracts were prepared from sampled larvae16 and their PO activity was determined according to Ashida et al. (1983)47 with some modification. Equal volumes and protein amounts of each extract were added to the wells of 24-well microtitre plates to which 1 mL of the substrate L-DOPA (0.5 mM) dissolved in 100 mM sodium acetate-citric acid buffer (pH 7.1) containing 10 mM CaCl2, was added. The reaction mixture was incubated in the dark at 30°C for 48 h and OD was measured at 490 nm using an ELISA reader (Tecan, Männedorf, Switzerland). Increases in OD490 due to spontaneous non-enzymatic dopachrome production were determined in wells without protein extract. Apparent PO activity was recorded as the change in absorbance over 48 h and it was expressed in units as defined previously16. The protein concentration of the extracts was measured by the Bradford method.

Statistical analysis

Survival data were arcsin transformed to satisfy normality and homocedasticity requirements as necessary. All the data were then subjected to one-way analysis of variances followed by Duncan's multiple range tests using the statistical software Statistical Package for the Social Sciences version 14.0 to determine significant differences among treatments. Results for target gene mRNA quantification are presented as fold expression relative to Artemia actin. The expression level in control was regarded as 1.000 and thereby the expression ratio of the treatments was expressed in relation to the control. Significant differences in expression between control and treatments were analyzed by Relative Expression Software tool–Multiple condition solver (REST–MCS) Version 2 using Pair Wise Fixed Reallocation Randomization Test©43. Significance level was set at P < 0.05.