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Water acidification activates the innate immune system of Senegalese sole Solea senegalensis

Marina Machado1, 2*, Francisco Arenas1, Rita Azeredo1, 3, Renata Serradeiro4 and Benjamín Costas1, 2
  • 1 Centro Interdisciplinar de Investigação Marinha e Ambienta, CIIMAR, Portugal
  • 2 Instituto de Ciências Biomédicas Abel Salazar (ICBAS-UP), Produção Aquática, Portugal
  • 3 Faculdade de Ciências da Universidade do Porto (FCUP), Departamento de Biologia, Portugal
  • 4 Sea8- Aquacria Piscícolas, SA, Portugal

-Introduction Senegalese sole, Solea senegalensis (Kaup), is currently reared in recirculation aquaculture systems (RAS). High production densities together with the minimal water change are features of intensive RAS production [1] and may lead to a gradual decrease of pH by the accumulation of water CO2 [2]. The effects of hypercapnia rest on CO2 concentration and duration of fish exposure to acidic water [3] and, while being quoted as a stress factor, hypercapnia can be responsible for the deviation of energy [4]. Fish immunity is very sensitive to environmental variables. Thus, this study intended to assess the effects of acute and prolonged exposure to low pH levels on the innate immune status of Senegalese sole. -Material and Methods The trial was performed at the Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR; Porto, Portugal). Senegalese sole juveniles (± 82 g) were obtained from a commercial fish farm and were acclimated to the CIIMAR facilities for one week. After being weighed, fish were distributed in four independent recirculating seawater systems (n = 6) comprised by a header tank and 6 flat-bottomed tanks (0.05 m2; Temp.: 19±1°C; Salinity: 24 ppm; O2 saturation: 80%). A pH-stat system (Aqua Medic®, AT Control) was used to handle the flux of CO2 from a reservoir into the water of the header tank, establishing three pH levels: 7.9, 7.6 and 7.3. These levels were controlled by solenoid valves and were activated when 0.1 pH units above the predetermined value were registered (down regulation). The pH values of all header tanks were constantly monitored and registered with Aqua Medic pH electrodes and a data-logger. A control pH comprising the normal seawater pH (pH 8.1) was established and kept in a fourth system. One fish per tank was sampled 4 and 24 hours after the beginning of the trial in order to assess the acute effect of a decrease in pH. The remaining fish were fed with a commercial diet and were weighed and sampled after a four-week period for the assessment of the prolonged water acidification. At all sampling times, fish were anaesthetized and sampled for evaluation of the haematological profile and plasma humoral immune parameters. Head-kidney was also collected for gene expression analysis. -Results The majority of the tested cellular and humoral parameters increased at 4 weeks compared to the other sampling times. Regarding overall water acidification effects, pH 7.3 increased total circulating leucocytes compared to all other treatments (Figure 1), together with higher concentration of all leucocyte types compared to both pH 7.9 and control (pH 8.1). The prolonged exposure (4 weeks) to pH 7.3 led to leucocytosis (Figure 1) and thrombocytosis (results not shown) compared to pH 7.9 and 8.1. Furthermore, water acidification augmented leucocyte concentration at 4 weeks compared to 4 and 24 h (Figure 1). Interestingly, an acute increase of the plasma bactericidal and antiproteases activities was observed for pH 7.3 at 4h and 24h, respectively (results not shown). Figure 1. Peripheral blood leucocytes in Senegalese sole (Solea senegalensis) submitted to different pH levels for 4 h, 24 h and 4 weeks. Values are presented as means ± SD. P-values from two-way ANOVA (p ≥ 0.05). If interaction was significant, one-way ANOVA was performed. Different lowercase letters stand for significant differences between pH for the same time whereas symbols display differences between times for the same pH. Different capital letters indicate differences among pH regardless time. -Discussion An increase in water CO2 can induce hypercapnia, which can be associated with the disturbance of the acid-base balance [5], and may explain the reduced growth found in some fish species. Despite the relatively short-time exposure period from this study, Senegalese sole weight was negatively affected by increased water acidification (data not shown). Cech and Crocker [4] suggested that exposure to high CO2 levels can be stressful. The present results support such hypothesis, with acute activation of some humoral immune responses at 4 and 24 h, as well as the recruitment of peripheral immune cells upon chronic exposure to the lowest pH level. Regardless preliminary results from the present study, the general scientific opinion defends that cellular responses are pH-sensitive, being diminished in acidic environments and inhibiting immune functions [6]. Head-kidney immune related gene expression may bring further insights about the immune modulation upon differential exposure to several hypercapnia levels.

Figure 1
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Acknowledgements

This work was partially funded by projects AQLARA (31-03-05-FEP-0051) supported by PROMAR Program (Portugal) and PEst-C/MAR/LA0015/2013 by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through Foundation for Science and Technology (FCT). M. Machado,R. Azeredo and B. Costas were supported by FCT through grants SFRH/BD/108243/2015, SFRH/BD/89457/2012 and SFRH/BPD/111111/2015, respectively.

References

1. Martins, C.I.M., Pistrin, M.G., Ende, S.S.W., Eding, E.H., and Verreth, J.A.J. The accumulation of substances in Recirculating Aquaculture Systems (RAS) affects embryonic and larval development in common carp Cyprinus carpio. Aquaculture, 2009. 291(1-2): p. 65-73.
2. Summerfelt, S.T., Vinci, B.J., and Piedrahita, R.H. Oxygenation and carbon dioxide control in water reuse systems. Aquacultural Engineering, 2000. 22(1-2): p. 87-108.
3. Santos, G.A., Schrama, J.W., Capelle, J., Rombout, J.H.W.M., and Verreth, J.A.J. Effects of dissolved carbon dioxide on energy metabolism and stress responses in European seabass (Dicentrarchus labrax). Aquaculture Research, 2013. 44(9): p. 1370-1382.
4. Cech, J.J. and Crocker, C.E. Physiology of sturgeon: effects of hypoxia and hypercapnia. Journal of Applied Ichthyology, 2002. 18(4-6): p. 320-324.
5. Crocker, C.E. and Cech, J.J. The effects of hypercapnia on the growth of juvenile white sturgeon, Acipenser transmontanus. Aquaculture, 1996. 147(3-4): p. 293-299.
6. Lardner, A. The effects of extracellular pH on immune function. Journal of Leukocyte Biology, 2001. 69(4): p. 522-530.

Keywords: Immune System, Senegalese Sole, Water acidification, RAS system, Gene Expression

Conference: IMMR | International Meeting on Marine Research 2016, Peniche, Portugal, 14 Jul - 15 Jul, 2016.

Presentation Type: Oral presentation

Topic: Aquaculture

Citation: Machado M, Arenas F, Azeredo R, Serradeiro R and Costas B (2016). Water acidification activates the innate immune system of Senegalese sole Solea senegalensis. Front. Mar. Sci. Conference Abstract: IMMR | International Meeting on Marine Research 2016. doi: 10.3389/conf.FMARS.2016.04.00120

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Received: 29 Apr 2016; Published Online: 13 Jul 2016.

* Correspondence: Miss. Marina Machado, Centro Interdisciplinar de Investigação Marinha e Ambienta, CIIMAR, Porto, Porto, 4050-123, Portugal, mcasimiro@ciimar.up.pt