Aquaculture Nutrition
2016
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doi: 10.1111/anu.12410

1

1,2 2

1

1,† 1,†

1 Laboratory of Aquaculture & Artemia Reference Centre, Faculty of Bioscience Engineering - Department of Animal Production, Ghent University, Gent, Belgium; 2 Southeast Asian Fisheries Development Centre/Aquaculture Department (SEAF-
DEC/AQD), Tigbauan, Iloilo, The Philippines

The beneficial effects of PHB as supplement for giant tiger prawn Penaeus monodon postlarvae using a shortterm enrichment strategy via Artemia were examined. The effects of co-supplementing with a lipid emulsion were also evaluated to determine whether it yielded an additional benefit. Results on the average weight and larval development were not significantly different among postlarvae fed the different dietary treatments, indicating that PHB supplementation could not be used to stimulate growth in P. monodon postlarvae while such positive results have been reported in other aquaculture species. Nonetheless, significantly higher survival was obtained in postlarvae fed PHB-enriched Artemia irrespective of lipid enrichment. In addition, PHB increased the survival of the postlarvae after exposure to a lethal dose of ammonia. Lipid supplementation nullified this effect. The cumulative mortality of postlarvae subjected to a sublethal concentration of ammonia for 24 h and subsequent exposure to pathogenic Vibrio campbelli showed that PHB but not lipids could effectively enhance the resistance of the postlarvae. Co-supplementing lipids even significantly decreased this outcome. Our study indicates that PHB supplementation increases the quality of larval P. monodon and their chance of surviving under adverse environmental conditions. The short-term co-supplementation with lipid emulsion did not add to these effects.
KEY WORDS: ammonia stress test, lipid emulsion, Penaeus monodon postlarvae, poly-beta-hydroxybutyrate, short-term enrichment, Vibrio challenge test
Received 28 April 2015; accepted 7 December 2015

Correspondence: P. De Schryver, Laboratory of Aquaculture & Artemia Reference Centre, Faculty of Bioscience Engineering - Department of Animal Production, Ghent University, Rozier 44, B-9000 Gent, Belgium. E-mail: peter.deschryver@ugent.be †Senior author.
Diseases are a major constraint to aquaculture production of invertebrates (Bachere 2003). Crustacean larvae are often exposed to stressful conditions making them susceptible to infections. In the shrimp aquaculture industry, bacterial diseases are often considered as major problems resulting in production and economic loss (Balakrishnan et al. 2011). Mainly Vibrio species have been associated with low survival in hatchery or grow-out conditions. The natural abundance of Vibrio spp., their multiplication rates and their ability to adapt to environmental changes in shrimp culture ecosystems contribute to the problem (Saulnier et al. 2000).
Environmental parameters (e.g. temperature, pH, levels of dissolved oxygen) play an important role in disease susceptibility because they may influence the capacity of the immune system (Le Moullac & Haffner 2000). In addition, farming of aquatic animals commonly involves displacement from their natural habitat to an environment that is new and sometimes stressful. It also necessitates the use of feeds that are often unnatural or artificial and it requires culturing at stocking densities that are much higher than what is naturally occurring. These are conditions that can increase the chance for exposure to pathogens, can compromise defensive responses and can facilitate pathogen replication and disease transmission (Walker & Mohan 2009). Cheng & Chen (1998) found

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ª 2016 John Wiley & Sons Ltd

suboptimal temperature and pH to increase mortality in freshwater prawn resulting from infection with Enterococcus-like bacteria.
A number of measures have been developed to mitigate the impact of diseases in the shrimp aquaculture industry. Common practices range from disinfection of the rearing water to the application of chemotherapy (e.g. antibiotics) (Smith et al. 2003). The latter has become undesirable as they promote the selection for antibiotic resistance in both the target pathogenic bacteria and all other microorganisms present in the environment. The application of preventive approaches such as vaccines, immunostimulants, prebiotics and probiotics to enhance the shrimp’s disease resistance is becoming increasingly important and will be essential for the further development of more sustainable aquaculture practices. Recently, an alternative approach was suggested implying the use of the bacterial storage compound poly-beta-hydroxybutyrate (PHB). Beta-hydroxybutyric acid (b-HB), the monomer of PHB, is known to exhibit some antimicrobial, insecticidal and antiviral activities (Tokiwa & Ugwu 2007), comparable to other short-chain fatty acids (SCFA) (Defoirdt et al. 2006). Disruption of the bacterial cell wall resulting in leakage, interference with nutrient transport and altered energy or molecule synthesis are examples of less direct growth interfering effects associated with SCFA (Ricke 2003).
PHB and/or its degradation product b-HB can probably also be used as an energy source by crustaceans (Sui et al. 2014). Organic acids, mainly butyric acid, have also been mentioned as typical energy sources for the growth of intestinal epithelial cells (Biagi et al. 2007). Defoirdt et al. (2007) found a prolonged survival in case starved nauplii were supplied with PHB and an increased survival and growth in the case PHB was used as a feed additive. It remains, however, to be determined whether the energy delivery effect of PHB may increase the robustness of crustaceans to counteract adverse rearing conditions. It is therefore the goal of this study to demonstrate the potential application of PHB for increasing the robustness and resistance of the giant tiger prawn P. monodon postlarvae during culture. The effects of co-supplementing lipid emulsion rich in highly unsaturated fatty acids (HUFAs) were also examined to determine whether the addition of dietary nutrients can increase the potential beneficial effects of PHB. Survival, growth and larval quality of the postlarvae were also examined.

P. monodon larvae at late protozoea stages (PZ2-3) were obtained from the Shrimp hatchery of the Southeast Asian Fisheries Development Centre/Aquaculture Department (SEAFDEC/AQD) (Tigbauan, Iloilo Philippines). Larvae were transported to the laboratory using a double-layered plastic bag half filled with natural seawater and oxygenated before sealing. This bag was placed in one 25-L styrofoam box (36 cm long 9 26 cm wide 9 27 cm deep and 2.0 cm thickness). The lid of the styrofoam box was sealed using packing tape. Upon arrival, larvae were acclimatized to laboratory conditions in a 250-L cylindroconical fibreglass tank filled with 32 g LÀ1 UV-treated seawater at 30 Æ 2 °C for 1 week. During the acclimatization period, larvae were fed on algal mix mostly composed of Chaetoceros spp. obtained at the Larval Food Laboratory of SEAFDEC/AQD (Parado-Estepa et al. 1991). The overall status of the test animals was also examined. Randomly collected samples obtained from the same original batch were submitted to the Fish Health Section of SEAFDEC/AQD for diagnosis of white spot syndrome virus (WSSV) following the methods of Kimura et al. (1996) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) using the IQ2000TM IHHNV Detection System (Farming IntelliGene, Taiwan). The presence of the monodon-type baculovirus (MBV) using malachite green staining was also examined. Other morphological characteristics such as gut diameter (6th abdominal segment), muscle width and rostral spine count (dorsal and ventral) were also evaluated to ensure a good overall health status of the larvae (Solis 1988).
At the end of the acclimatization period, larvae now at early mysis stage were randomly distributed and stocked at a density of 100 individuals LÀ1 in to the experimental setup consisting of 10-L plastic tanks (31 cm long 9 27 cm wide 9 27 cm deep) filled with 8-L UV-treated seawater. The seawater used in the set-up was passed through a filtration system consisting of a series of filter cartridges (5, 10 and 15 lm) before passing through an ultraviolet filter at a capacity of approximately 30 L minÀ1 (Mega Fresh, Taiwan). Water was changed every 2 days at 25% of the water volume with fresh UV-sterilized seawater to remove waste. When changing the water, a fabricated siphon (Diplex polyvinylchloride hose) was used to carefully drain the water into a separate bucket to trap mysis that could be siphoned up as well. The rearing water was analysed on

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Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

a regular basis to determine the total ammonia-nitrogen, nitrite-nitrogen and nitrate-nitrogen using JBL Test Kits (Neuhofen, Germany), and the concentrations of total ammonia-nitrogen, nitrite-nitrogen and nitrate-nitrogen were maintained below 0.2, 0.1 and 10.0 mg LÀ1, respectively. Each experimental tank was supplied with air through an air diffuser to maintain dissolved oxygen above 5 mg LÀ1. Water temperature and salinity measured using a YSI 556 MPS Multiprobe System (Japan) averaged to 30 Æ 2 °C and 32 g LÀ1, respectively. A linear fluorescent lamp (40 W; Philips, Andover, MA, USA) was used to give a daily light regime of 10-h light and 14-h dark.
HIGH 5 Artemia cysts (INVE Aquaculture, Thailand) were incubated at 5 g LÀ1 in UV-sterilized sea water and allowed to hatch in a 30-L hatching tank for 24 h. Vigorous aeration was provided with illumination set at approximately 27 lE mÀ2 sÀ1. At approximately 36-h incubation, Artemia nauplii (now Artemia instar II) were washed with UV-sterilized seawater, transferred and stocked at a density of 100 nauplii mLÀ1 in new seawater. Artemia instar II nauplii were enriched with either crystalline PHB particles (98% poly-beta-hydroxybutyrate-2% poly-beta-hydroxyvalerate, Goodfellow, Huntingdon, England) at 1 g LÀ1, standard lipid emulsion (ICES 30/0.6/C, Han et al. 2000) at 0.3 g LÀ1 or the combination of crystalline PHB + ICES 30/0.6/C (at 1 and 0.3 g LÀ1, respectively). Nonenriched Artemia nauplii were also maintained. PHB particles prior to utilization were sieved over a sterile 50-lm sieve. PHB particles bigger than 50 lm were not used during enrichment. The enrichment of the Artemia was carried out following a drip method whereby the enrichment products were initially suspended in separate bottles containing 200 mL of UV-sterilized seawater. Drips containing the enrichment suspensions were delivered to the enrichment tanks over a period of approximately 30 min through a narrow tube attached to the bottle and controlled via a valve. Bottles were provided with aeration to ensure constant mixing of the enrichment products. As opposed to the standard practice of enriching Artemia nauplii for 24 h, the differentially enriched Artemia Instar II were harvested after 6 h and thoroughly rinsed with

UV-sterilized seawater to remove excess enrichment product.
Enriched and non-enriched Artemia nauplii administered through the rearing water were used as live food for the giant tiger prawn from mysis to early postlarval stages. The experiment included the following dietary treatments (4 replicates each): 1 Non-enriched Artemia (Art -P-L) 2 Lipid emulsion-enriched Artemia (Art -P+L) 3 PHB-enriched Artemia (Art +P-L) 4 PHB + Lipid emulsion-enriched Artemia (Art +P+L)
Feed was administered daily at 9:00 and 16:00 h ad libitum and the feeding trial lasted for 21 days.
The survival of postlarvae was determined at the end of the feeding trial by counting the number of surviving postlarvae in each tank. The percentage survival at the end of the experimental trial was computed following the formula of Thompson & Bergersen (1991): Survival (%) = (X/N-A)*100, where X is the number of postlarvae present at the end of the feeding trial, N is the number of postlarvae at stocking (day 0) and A is the total number of postlarvae sacrificed for larval stage index determination. The mean weight within each replicate tank was determined by randomly collecting 20 postlarvae. These were sacrificed by immersion in ice-cold seawater, blotted dry to remove adhering water and weighed individually on an analytical balance with 0.0001 g precision. Next, the mean weight for each treatment at the end of the experimental trial was computed as the mean of the average weights from the replicate tanks within each treatment. The larval development was assessed as the larval stage index (LSI) determined at different time points. Ten postlarvae per experimental tank were randomly collected each day for the first 13 days, and the larval stage of each sampled larva was determined according to the description of Motoh (1985) and assigned with a value: mysis 1 = 1, mysis II = 2, . . . until postlarva PL3 = 6 (Millamena & Bangcaya 2001). The larval stage index (LSI) was calculated as follows:

LSI

¼

ðtotal

number

of

mysis 1

Â

1

þ

total

number of mysis II Â 2 þ Á Á Á Total number of larvae

þ

total

number

of

postlarvae

PL3

Â

6Þ

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Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

The survival of the postlarvae following exposure to a lethal dose of ammonia was evaluated at the end of the experiment. A preliminary experiment (three replicates) was performed with 50 untreated postlarvae of the same original batch to establish the concentration of NH4+-N causing death to 50% of the test animals after 24 h (LD50) in seawater (with an average pH and water temperature of 8.2 and 30 °C, respectively). The LD50 was computed using the method of Reed & Muench (1938). Preparation of ammonia test solution was carried out according to the methods described by Najdegerami et al. (2015).
The computed LD50 of 143 mg LÀ1 NH4+-N was used in the ammonia stress test that was carried out according to the method of Alcaraz et al. (1999) with slight modifications. From 4 experimental tanks per treatment, 50 postlarvae were randomly collected and transferred to four containers containing 2-L UV-sterilized seawater and exposed for 24 h to 143 mg LÀ1 NH4+-N. Air was provided into each container using an air stone diffuser. Test animals were not fed 12 h prior to the test and during the exposure period. The number of dead postlarvae was recorded after 24 h of exposure. Death was assumed when larvae were immobile and showed no response to external stimulus such as picking or touching with forceps.
The survival of the postlarvae following exposure to a pathogenic challenge with Vibrio campbellii LMG 21363 was evaluated. At the end of the feeding trial, 50 postlarvae were randomly collected from each tank replicate and subjected to a sublethal concentration of NH4+-N (100 mg LÀ1) for 24 h. Each tank was individually aerated by means of an air stone diffuser. After 24 h, postlarvae were gently collected and transferred to a new tank containing 107 cells mLÀ1 of the pathogenic V. campbellii LMG 21363. Preparation of the bacterial strain was done according to the method described by Defoirdt et al. (2007). Postlarvae were given supplementary feeds all throughout the exposure period; however, no water exchange was administered. Cumulative mortality (%) was recorded every 24 h for 15 days.

emulsion) as well as any interactions among them. When interaction was significant, a one-way ANOVA followed by a Duncan’s multiple range post-hoc test was used to compare means between different groups. A P < 0.05 was chosen as the significance level. Data on percentages were arcsinetransformed. All statistical analyses were performed using SPSS Statistical software v. 11.5.
The tests performed on WSSV and IHHNV on the P. monodon postlarvae indicated that the postlarvae were not infected with any of the viruses or that the viral load was lower than the detection limit. The larvae were also found negative for MBV occlusion bodies, and muscular deformities were not detected. These results indicated that the mysis used in this experiment were physically fit for the experiment.
The average weight (mg) and survival (%) of the postlarvae after the 21-day feeding trial are shown in Table 1. Results from the 2 9 2 factorial design showed that the interaction between PHB and lipid did not significantly affect the weight of the postlarvae (F = 2.560, P = 0.141). Similarly, PHB (F = 1.528, P = 0.245) and lipid treatments (F = 2.757, P = 0.128) alone did not significantly influence the weight of the postlarvae at the end of the feeding trial. The interaction between PHB and lipid on the survival of the postlarvae was not statistically significant (F = 1.586, P = 0.240). However, PHB treatment (F = 4.993, P = 0.046) significantly influenced the survival of the postlarvae with a mean survival of 41.0 Æ 2.7% in postlarvae fed PHB-enriched Artemia while this was 31.2 Æ 3.4% for postlarvae fed non-PHB-enriched Artemia. Lipid treatment did not contribute to the survival of the postlarvae (F = .076, P = 0.797). The development of the postlarvae fed the different dietary treatments is shown in Fig. 1. There was no significant interaction between PHB and lipid (F = .860, P = 0.372), and the PHB (F = .158, P = 0.698) or the lipid treatment (F = .860, P = 0.372) alone did also not significantly affect the development of the larvae.

All data were analysed using two-way analysis of variance (two-way ANOVA) to identify statistically significant differences among the variables tested (PHB and lipid

The protective effect of PHB and lipid supplementation against ammonia stress after 24 h immersion is shown in
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Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

Table 1 Weight (mg) and survival (%) of P. monodon postlarvae fed with differentially enriched Artemia after 21 days of feeding. P-value in bold indicates significant difference (P < 0.05)

Non-PHB enriched

PHB enriched

P-value

Parameters

Non-lipid enriched (Art-P-L)

Lipid enriched (Art-P+L)

Non-lipid enriched (Art+P-L)

Lipid enriched (Art+P+L)

PHB

Lipid

PHB 9 Lipid

Mean weight Survival

3.0 Æ 0.2 29.0 Æ 4.5

4.2 Æ 0.6 33.4 Æ 5.6

3.1 Æ 0.2 44.4 Æ 2.5

3.2 Æ 0.4 37.6 Æ 4.4

0.245 0.046

0.128 0.797

0.141 0.240

Values represent means Æ SEM (n = 4).

Figure 1 Larval stage index (LSI) of P. monodon postlarvae fed nonenriched (Art-P-L) and enriched Artemia (lipid emulsion enriched (Art-P+L); PHB enriched (Art+P-L); and PHB+lipid emulsion enriched (Art+P+L)) during the 21-day feeding trial. Values represent means Æ SEM (n = 4). No significant differences were detected.

Larval stage index

10.00

Art -P-L (control) ............... Art -P+L

8.00 Art +P-L

Art +P+L 6204....00000000 ......................................................................................................... ............................................................................................ ........................................................................................................ ............................................................................................................ .................................................................................................................... ................................................................................................... .................................................................................................................................................... .................................................................................................................................................... .............................................................................................................................................................................................. ................................................................................................................................................................ ........................................................................................................................................................................
1 2 3 4 5 6 7 8 9 11 13
Days after stocking

Fig. 2. There was a significant interaction between PHB and lipid affecting the survival of the postlarvae (F = 5.261, P = 0.043). In treatments without PHB, lipid supplementation did not significantly enhance the survival of the postlarvae (P = 0.178). PHB treatment alone, on the contrary, caused a significant increase in the survival of the larvae (P = 0.018). An additional lipid treatment nullified this effect.

Survival (%)

100.00 80.00 60.00 40.00 20.00

Non-lipid enriched Lipid enriched
a
a

b a

Figure 3 shows the 15-day cumulative mortality (%) of P. monodon postlarvae after 24 h immersion in a sublethal concentration of ammonia followed by bath challenge with V. campbellii. The combined exposure to ammonia-N followed by exposure to pathogenic Vibrio had a direct effect on the postlarvae as seen by the number of dead postlarvae after 24 h postchallenge. Results from the 2 9 2 factorial design showed that the cumulative mortality of the postlarvae at 15 days postchallenge was significantly affected by the interaction between PHB and lipid (F = 9.597, P = 0.015). PHB treatment significantly lowered the mortality of the postlarvae. However, in the presence of lipids, this effect was nullified. Lipid treatment alone did not influence the resistance of the postlarvae against the combined effects of sublethal ammonia and pathogen bacteria (P = 0.262).
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0.00 Non-PHB enriched

PHB enriched

Dietary treatments

Figure 2 Survival (%) of P. monodon postlarvae fed differentially enriched Artemia for 21 days and subsequently exposed for 24 h to a lethal dose of ammonia. Bars represent means Æ SEM (n = 4). Bars with different letters are significantly different (P < 0.05). Interaction between PHB and lipid was statistically significant (F = 5.261, P = 0.043) by which the PHB effect was nullified in combination with lipid enrichment.

In various crustaceans and fish species, the application of the bacterial storage compound poly-beta-hydroxybutyrate (PHB) as an alternative disease control approach has been tested (Defoirdt et al. 2007, 2009). Here, the potential of

Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

100.00

Cummulative mortality (%)

80.00

60.00

40.00

Art -P-L (control)

20.00

Art -P+L Art +P-L

Art +P+L 0.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Days after challenge

Figure 3 Cumulative mortality (%) of P. monodon postlarvae fed non-enriched (Art-P-L) and enriched Artemia [lipid emulsion enriched (Art-P+L); PHB enriched (Art+P-L); and PHB+ lipid emulsion enriched (Art+P+L)] followed by exposure to a sublethal dose of ammonia for 24 h and subsequent challenge with pathogenic V. campbellii. Values represent means Æ SEM (n = 4). Statistical analysis performed at 15 days after challenge showed a significant interaction between PHB and lipid (F = 9.597, P = 0.015). PHB effect was nullified when combined with lipid enrichment.

PHB encapsulated in Artemia nauplii in a short-term enrichment strategy was demonstrated. Our findings indicated that although PHB supplementation could not be used to stimulate growth in P. monodon postlarvae, it could effectively enhance the survival of the postlarvae. Most importantly, supplementation of PHB to the diet of P. monodon postlarvae resulted in a higher chance of surviving under adverse environmental conditions.
Supplementing lipid emulsion in addition to PHB in a short-term enrichment strategy did not exhibit growth-promoting effects in the postlarval P. monodon based on the comparable mean weight between postlarvae fed PHBenriched and postlarvae fed PHB + lipid-enriched Artemia. This was also the case for the LSI. These findings are not in correspondence with the finding of Nhan et al. (2010) who showed that the LSI of Macrobrachium rosenbergii was significantly increased when larvae were fed 24 henriched Artemia using a combination of PHB and lipid. Supplementation with PHB or lipid emulsion alone did not lead to a significant increase in growth and larval development either. Findings obtained by Sui et al. (2014) revealed that larval development of Chinese mitten crab Eriocheir sinensis larvae fed PHB-enriched Artemia was significantly improved. This indicates that the growth-promoting effects of PHB could be species dependent. Alternatively, the different gut microbial communities associated with different

species could have caused this effect. Further research should be performed to investigate these aspects as well as to focus on the supplementation of different doses of PHB to P. monodon postlarvae including the quantification of the actual amount of PHB encapsulated in the Artemia and as such passed on to the next level in the food chain.
PHB supplementation alone brought significant increase in the survival of the postlarvae. This is in accordance with the studies conducted on other crustacean species (Nhan et al. 2010; Sui et al. 2014). Prolonged survival was also observed for PHB-fed Artemia (Defoirdt et al. 2007). These observations suggest that PHB and/or its degradation product b-HB can be used as an energy source by crustaceans. The mechanism behind the energy delivering effects of PHB in the crustaceans, however, is not yet fully understood.
Lipid supplementation did not enhance the survival of the P. monodon postlarvae. This result is not in accordance with the findings in literature. Bengtson et al. (1991) showed that feeding n-3 HUFA-enriched Artemia resulted in increased larval survival and growth in several Penaeus spp. and Macrobrachium rosenbergii. Leger & Sorgeloos (1992) also demonstrated that in penaied shrimp, feeding n-3 HUFA-rich Artemia during zoeal stages resulted in a better survival and growth in the later stages. While most Artemia enrichment trials employed long-term enrichment duration (e.g 24–48 h), the short-term lipid enrichment strategy used in this study may not have been sufficient to incorporate the lipids in the tissue of the Artemia resulting in low quality as larval food for the P. monodon. Dhont et al. (1991) stated that gradual and long-term enrichment has an advantage in that Artemia are able to accumulate lipids in their tissue in addition to the lipids accumulated in their gut. Lipids accumulated in the gut are more likely excreted before the Artemia is predated upon (Dhont, pers. comm.). The short-term enrichment with PHB in combination with lipids did not enhance the survival of the postlarval P. monodon either. It is hypothesized that supplementing lipids in addition to PHB during the short-term enrichment strategy decreased PHB uptake by the Artemia either due to a dilution effect by the lipids or the preference of the Artemia to take up one of two enrichment products. As Makridis & Vadstein (1999) have reported food size selectivity of Artemia franciscana metanauplii, further research should be conducted to investigate preferential uptake by Artemia between PHB and lipid enrichment.
Results on the ammonia stress test showed that PHB supplementation alone increased the survival of the P. monodon
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Aquaculture Nutrition ª 2016 John Wiley & Sons Ltd

postlarvae, indicating that PHB could enhance its robustness against adverse environmental condition. Lipid supplementation containing high levels of HUFA did not contribute to the robustness of the postlarvae. This is not in accordance with the findings of Martins et al. (2006) who reported that feeding n-3 HUFA-enriched Artemia to Farfantepenaues paulensis larvae increased its survival and tolerance to ammonia. Cavalli et al. (2000) also showed higher ammonia tolerance in Macrobrachium larvae fed Artemia enriched with n-3 HUFA. These above-mentioned studies employed 24-h Artemia enrichment. As it was previously mentioned, the short-term enrichment strategy used in this study may not be sufficient to incorporate the lipids in the tissue of the Artemia resulting in low quality food for postlarval P. monodon. Remarkably, the combined enrichment with PHB and lipids seemed to nullify the effect of PHB. This result supports our earlier statement that lipid enrichment could have potentially competed with the PHB uptake.
The resistance-enhancing effect of PHB against sublethal ammonia concentration and subsequent exposure to pathogenic bacteria was also investigated. This approach was used as Gomez-Gil et al. (1996) stated that Vibrio spp. in general are opportunistic bacteria and mainly become a threat when the natural defence mechanisms are suppressed. The significant reduction in mortality of postlarvae fed PHB-enriched Artemia confirms earlier results in literature on PHB as potential antimicrobial agent. PHB, according to Defoirdt et al. (2007), may protect the host against infection by indirectly improving its overall fitness and directly, by inhibiting the growth of the pathogens. De Schryver et al. (2010) have seen lower gut pH in European sea bass Dicentrarchus labrax juveniles given high levels of dietary PHB suggesting an increase in the production of short-chain fatty acids in the gut. PHB was also found to protect the zoea of the Chinese mitten crab E. sinensis against pathogenic Vibrios (Sui et al. 2012). Suguna et al. (2014) also demonstrated a protective capacity of PHB against Aeromonas hydrophila in tilapia Oreochromis mossambicus. Protection against virulent pathogenic Vibrio was also obtained using PHB-accumulating bacteria isolated from activated sludge (Halet et al. 2007). PHB-accumulating bacteria either supplied through formulated shrimp diets (Laranja et al. 2014) or bioencapsulated in Artemia (Thai et al. 2014) protected the postlarvae of P. monodon and larvae of M. rosenbergii, respectively. Lipid supplementation alone or the combined supplementation of lipids and PHB did not result in an increased resistance of the postlarvae. The same explanations as given above
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(short-term enrichment and competition between PHB and lipids for uptake) are hypothesized to be the basis of these observations. It is evident that the substantial influence of lipids on the effect of PHB during a short-term enrichment strategy should be further investigated.
In conclusion, efficient delivery of PHB via Artemia was achieved by a short-term enrichment strategy and this significantly improved the survival, robustness and resistance of the P. monodon postlarvae but not the growth. Supplementing dietary nutrients in the form of lipid emulsion in addition to PHB in a short-term enrichment strategy did not have an additional benefit. Therefore, our suggested strategy for future application would be long-term enrichment of Artemia nauplii with dietary nutrients rich in highly unsaturated fatty acids (HUFAs) to improve its nutritional content followed by a PHB treatment during the last hours of the enrichment process to assure efficient delivery of PHB to the larval predator.
This study was funded by the Flemish Interuniversity Council (VLIR). Peter DS is supported as a postdoctoral research fellow of the Research Fund – Flanders (FWO, Belgium).
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