Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.4 (2024)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus
Submit

Research Article

Indian Journal of Animal Research, volume 54 issue 1 (january 2020) : 31-35

Respiratory Metabolism, Energy Utilization and Biochemical Responses of Juvenile Common Cuttlefish (Sepiella maindroni) to Hypoxia

S.J. Qian1, Y. Zhang1, K. Wang1, F. Yin1,*, W.W. Song1,*
1Key Laboratory of Applied Marine Biotechnology, Ministry of Education; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture; School of Marine Sci, Ningbo Univ, Ningbo 315211, P.R. China. School of Marine Sciences, Ningbo University, Ningbo 315832, P.R.China.
Cite article:- Qian S.J., Zhang Y., Wang K., Yin F., Song W.W. (2019). Respiratory Metabolism, Energy Utilization and Biochemical Responses of Juvenile Common Cuttlefish (Sepiella maindroni) to Hypoxia . Indian Journal of Animal Research. 54(1): 31-35. doi: 10.18805/ijar.B-1177.

ABSTRACT

To evaluate respiratory metabolism, energy utilization and enzymatic responses in common Chinese cuttlefish (Sepiella maindroni) to hypoxia, juvenile Chinese cuttlefish (26.66 ± 2.52 g) were exposed to a 1.4-L airtight respiration chamber until suffocation for 83.4 min. The results showed that dissolved oxygen (DO) was mainly consumed in the closed chamber and the rates of oxygen consumption and ammonia excretion decreased significantly with decreasing DO content. The suffocation point for the cuttlefish was 1.16 ± 0.10 mg·L-1. The O:N ratio increased significantly with decreasing DO content. The TP content in the hemolymph at 83.4 min was significantly lower than that at other time points; however, the highest GLU content was noted at 83.4 min, the highest AST and ALP activities occurred at 60 min and 83.4 min, respectively. These results indicated that although hypoxia inhibits respiratory metabolism, the cuttlefish demonstrated a capacity for energy mobilization under hypoxic conditions.

INTRODUCTION

The common Chinese cuttlefish (Sepiella maindroni) is an important aquatic species harvested in Asia. Considering its successful artificial propagation, S. maindroni is becoming an important component of the aquaculture industry. Sepiella maindroni has a characteristically rapid growth rate and a high oxygen consumption rate (Xia et al., 2009). As a major constraint to cuttlefish farming, acute hypoxia resulting from a high stocking density, increased feed and misapplied culture system is harmful to the S. maindroni aquaculture industry (Wang et al., 2008).
        
Dissolved oxygen (DO) is a major variable limiting water quality of intensive aquaculture. Hypoxia has been shown to increase in aquatic species subjected to metabolic disturbances and significant stress (Tripp-Valdez et al., 2017). Such a stress results in histopathological changes as well as reduced food consumption and growth rates (Li et al., 2006; Paschke et al., 2010) and even death (Wang et al., 2008; Zhang et al., 2010). To cope with the stress, animals may alter their metabolic activities to adapt to the physiological changes. Respiratory metabolism is one of the most important aspects in bioenergetics (Paschke et al., 2010) and oxygen consumption and ammonia-N excretion are widely considered to be critical factors for evaluating the physiological responses of aquatic organisms at different levels of DO (Valverde et al., 2006). Hemolymph plays a role in the transport of nutrients, temperature regulation, immune defense and wound healing in invertebrates. In addition to respiratory metabolism, other biochemical factors, such as protein, glucose, aminotransferase and phosphatase in hemolymph, play a role in the metabolism (Boutet et al., 2005; Silkin and Silkina, 2005; Zhang et al., 2010; Parisi et al., 2017). Therefore, this study intends to explore whether hypoxia causes metabolic disorders in cuttlefish and identify the changes that will occur in cuttlefish metabolism.

MATERIALS AND METHODS

Cuttlefish and rearing conditions
 
Healthy juvenile S. maindroni cuttlefish were obtained from Ningde Aquatic Product Sci-Tech Co. Ltd. (Fujian Province, China), transferred to 3 × 4 × 1 mm3 rectangular blue fiberglass tanks in an aquaculture laboratory and acclimated with a salinity of 23.0 % ± 1.0 % for two weeks at 23.0°C ± 1.0°C prior to the experiment. The light intensity (1,000 lx) and photoperiod (12:12 light: dark cycle) were controlled by natural light and sunlight lamp. Non-ionic ammonia or inorganic nitrogen concentrations were 0.02 mg/L or 0.2 mg/L, respectively. Sand-filtered seawater was changed daily. The cuttlefish were fed to apparent satiation four times daily (at 07:00, 11:00, 14:00 and 18:00) with frozen shrimp Penaeus vannamei (body length=4–5 cm, protein content  = 220-235 g/kg, lipid content = 14-16 g/kg).
 
Oxygen consumption, ammonia-N excretion, O:N ratio and suffocation point
 
Eighteen cuttlefish (26.66 ± 2.52 g) were captured after two weeks of acclimation. Cuttlefish were starved for one day prior to grouping and randomly placed in 18 cylindrical plastic respiration chambers (1.4 L, r = 5.65 cm, h = 14 cm), which were aerated for at least two hour to reach oxygen saturation prior to measurements. The 18 chambers were randomly and averagely divided into three groups (A, B and C) and the other six blank chambers with no cuttlefish were used as the control. After the chambers were closed, R and U were measured at 30 (group A), 60 (group B) and 83.4 min (group C, suffocate time).
        
Before the cuttlefish were captured, the seawater in the plastic respiration chambers was siphoned into a 125-mL brown glass bottle to measure the oxygen concentration by the Winkler method. In addition, water samples of 50 mL were obtained from the treatment and control chambers to measure the concentration of ammonia-N by the phenol-hypochlorite method.
        
The suffocation point (SP) was determined as previously described (Wang et al., 2008). In brief, when the first cuttlefish in group C released ink, the oxygen concentration in the water of this group was defined as the SP, which is the minimum oxygen concentration (mg/L) below which the cuttlefish used in the experiment suffocated owing to oxygen deficiency.
        
The O:N atomic ratio was calculated as the quotient R/U for each individual. This ratio was used to estimate the amounts of proteins, lipids and carbohydrates that were used as an energy source for the organisms under different experimental conditions. Ratios of 3–16 indicated protein oxidation. Ratios between 50 and 60 indicated catabolism of similar proportions of proteins and lipids. High O:N values corresponded to increases in the lipid and carbohydrate catabolism (Mayzaud and Conover, 1988).
 
Hemolymph chemical analysis
 
Anesthesia induction and hemolymph collection were performed using a previously described method with slight modification (Collins and Nyholm, 2010). Briefly, cuttlefish were captured from each treatment group after exposure to hypoxic conditions for 30, 60 and 83.4 ± 8.4 min and control (0 min) and immediately transferred to a tank filled with anesthetic agent (2% solution of ethanol in seawater). The cuttlefish were placed in the tank for approximately 10 min. The cuttlefish ceased swimming and did not respond actively to any tactile stimulus. Hemolymph was withdrawn from the cephalic blood vessel using a sterile 1-mL syringe (4.5-gauge needle) and was centrifuged at 1,776 ×g for 10 min at 4°C to collect plasma, which was then stored at –80°C for subsequent analysis.
        
The TP of the cuttlefish was determined by the Bradford method, with bovine serum albumin as the standard. The glucose (GLU) content was determined using a commercially available kit purchased from Koichi Tanaka, Shimadzu Corp. (Kyoto, Japan) according to the manufacturer’s instructions. AST and ALP activities were measured with assay kits (developed by the Nanjing Jiancheng Bioengineering Institute, China) according to the protocol supplied with the kits.
 
Statistical analysis
 
One-way ANOVA was used to compare the R, U, SP, O:N ratio, hemolymph TP and GLU contents, AST and ALP activities of the cuttlefish. All the variables were tested for normality and homogeneity prior to conducting one-way ANOVA. Duncan’s multiple comparison procedure was applied to compare different means among the treatments. Statistical analyses were performed using the SPSS11.5. The relationships between the exposure duration and DO content were described by linear equations having reflective points. The differences between linear regressions were tested by analysis of covariance (ANCOVA).

RESULTS AND DISCUSSION

Respiratory metabolism
 
The oxygen consumption rate (R) reflects the ingested energy and physiologically useful energy (Farias et al., 2009); the ammonia excretion rate (U) is a major factor in metabolic waste accumulation (Fernandes and Tanner, 2008). The DO content is a factor influencing R and U. Rosas et al., (1999) indicated that the decrease in R and U is directly proportional to the decrease in the DO content at 15-% in unfed white shrimp (Penaeus setiferus) (Rosas et al., 1999). Xia et al., (1999) indicated that the R of S. maindroni ranged from 0.52 mg/(g.h) to 0.67 mg/(g.h) and U ranged from 62 μg/(g.h) to 71 μg/(g.h) (Xia et al., 2009). In the current study, oxygen was consumed gradually and the DO content (Fig 1), R (Fig 2) and U (Fig 3) in the closed chamber decreased significantly (P < 0.05). A low DO content inhibited respiratory metabolism of Sepia officinalis (Wachter et al., 1988). Harris et al., reported that decreased R under extreme hypoxia is correlated with histopathological changes in gills (Harris et al., 1998) and may result in impeded oxygen diffusion across gills (Van Heerden et al., 2004). Rosas et al., (1999) summarized that ammonia excretion is diminished as a consequence of metabolic depression wherein oxygen is the limiting factor (Rosas et al., 1999).
 

Fig 1: The relationships between exposure duration and dissolved oxygen (DO) content in Sepiella maindroni. Variables were expressed by linear equations.


 

Fig 2: Oxygen consumption rate (R) of Sepiella maindroni under hypoxic conditions.


 

Fig 3: Ammonia excretion rate (U) of Sepiella maindroni under hypoxic conditions.


 
Extreme hypoxia may lead to asphyxia in animals. The SP is an indicator for evaluation of tolerance to low DO content. In this study, the suffocation duration and SP for the cuttlefish were 83.4 min and 1.16 ± 0.10 mg·L-1 (Fig 1) respectively, which is higher than those for many other aquatic organisms, such as Scylla paramamosain (0.716 ± 0.017 mg/L, 1.00 ± 0.22 g) (Wang et al., 2010), Barbus capito (0.18 mg/L, 24.78 g) (Geng et al., 2012) and Hemifusus tuba (0.43 mg/L, 22°C) (Luo et al., 2008).
 
Energy utilization
 
Protein is the primary source of energy for cephalopods. Variations in the DO content can influence the protein levels in hemolymph, causing a change in the protein metabolism (Paschke et al., 2010). In this study, the lowest hemolymph TP content occurred at 83.4 min, the time period needed to reach the SP (Fig 4). Paschke et al., (2010) found that reduced blood protein and oxyhemocyanin levels under hypoxic conditions demonstrate a reduction in the ingestion rate (Paschke et al., 2010). The decreased protein catabolism of juvenile cuttlefish exposed to low levels of DO was supported by high O:N ratios (Fig 5).
 

Fig 4: Variations of total protein (TP) content in the hemolymph of Sepiella maindroni under hypoxic conditions.


 

Fig 5: O:N ratio of Sepiella maindroni kept under hypoxic conditions.


        
The O:N atomic ratio is also linked to the availability of energy storage and utilization of body protein compared with carbohydrates and lipids, which are catabolized by the organism (Mayzaud and Conover, 1988; Farias et al., 2009). In this study, the O:N atomic ratio increased significantly (P < 0.05) from 43.94 ± 1.09 to 56.47 ± 1.95, with prolonged hypoxia (Fig 5) and it was higher than those (8.39–9.44) under normoxia in S. maindroni (Xia et al., 2009). These ratios might indicate decreased protein catabolism. However, Sepiella maindroni, similar to other cephalopods, have low lipid stores (Song et al., 2009). Thus, prospective research should be conducted to assess whether lipid is used under hypoxia.
        
The GLU content increases in many aquatic organisms when they are exposed to severe hypoxia (Zou et al., 1996; Silkin and Silkina, 2005). In this study, the highest GLU content occurred at 83.4 min (P < 0.05) and no significant (P > 0.05) differences were found among the control (0 min), group A and group B (Fig 6). Therefore, the increase in the hemolymph GLU level in response to hypoxia may be owing to a hormonal rearrangement with respect to metabolism (Silkin and Silkina, 2005). The elevation in the hemolymph GLU level is a physiological adaptation for high substrate demands of fermentation and a strategy to prepare for anoxia (Zou et al., 1996). Energy storage, under stressful conditions of low O2 availability, might be mobilized as a source of fuel for anaerobic metabolism (Qiu et al., 2011) and glycogenolysis in the tissues and delivery of carbohydrates into the hemolymph circulation may be enhanced (Silkin and Silkina, 2005).
 

Fig 6: Variations of glucose (GLU) content in the hemolymph of Sepiella maindroni under hypoxic conditions.


 
Enzymatic responses
 
Levels of AST and ALP can be assessed to investigate the biological effects of hypoxia on tissue injuries and metabolic disturbances (Boutet et al., 2005). Metabolic processes are involved during hypoxic periods that act to enhance the ATP level. In marine invertebrates, cytosolic AST acts in the first phase of ATP production using aspartate as the substrate. In the present study, the AST activity increased significantly from 0 min (the control group) to 60 min (group B) and then decreased significantly (P < 0.05) (Fig 7). An increase in AST activity was observed and was associated with an increase in ammonia levels and a shift in the nitrogen metabolism toward the synthesis of urea and glutamine (Boutet et al., 2005). This study showed that the ammonia-N levels (172.63–181.70 μg) accumulated gradually with prolonged hypoxia but U decreased (Fig 3).
 

Fig 7: Variation of aspartate aminotransferase (AST) activity in the hemolymph of Sepiella maindroni under hypoxic conditions.


        
ALP activity increased significantly with decreasing DO and peaked at 83.4 min (P < 0.05) (Fig 8). These findings indicate that under extremely anoxic conditions, especially when the cuttlefish suffocate, ATP generation is highly reduced because oxidative phosphorylation by mitochondria cannot proceed without oxygen (Storey and Storey, 2007; Zhang et al., 2010; Dawson and Storey, 2011). This phenomenon is supported by the decreased AST activity at 83.4 min (Fig 7).
 

Fig 8: Variation of alkaline phosphatase (ALP) activity in the hemolymph of Sepiella maindroni under hypoxic conditions

ACKNOWLEDGMENT

This study was supported by the National Natural Science Foundation of China (Grant No. 41206114) and the K. C. Wong Magna Fund in Ningbo University.
 

REFERENCES


  1. Boutet I, Meistertzheim AL, Tanguy A, Thebault MT and Moraga D (2005). Molecular characterization and expression of the gene encoding aspartate aminotransferase from the Pacific oyster Crassostrea gigas exposed to environmental stressors. Comparative Biochemistry and Physiology C. 140(1): 69-78.

  2. Collins AJ and Nyholm SV (2010). Obtaining hemocytes from the Hawaiian bobtail squid Euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria. JOVE-Journal of Visualized Experiments. 36: e1714.

  3. Dawson NJ and Storey KB (2011). Regulation of tail muscle arginine kinase by reversible phosphorylation in an anoxia-tolerant crayfish. Journal of Comparative Physiology. B: Biochemical, Systemic and Environmental Physiology. 181(7): 851-59.

  4. Farias A, Uriarte I, Hernandez J, Pino S, Pascual C, Caamal C, Domingues P and Rosas C (2009). How size relates to oxygen consumption, ammonia excretion and ingestion rates in cold (Enteroctopus megalocyathus) and tropical (Octopus maya) octopus species. Marine Biology. 156(8): 1547-58.

  5. Fernandes M and Tanner J (2008). Modelling of nitrogen loads from the farming of yellowtail kingfish Seriola laiandi (Valenciennes, 1833). Aquaculture Research. 39(12): 1328-38.

  6. Geng LW, Xu W, Li CT and Jin GX (2012). Study on oxygen consumption rate and suffocation point of Barbus capito. Journal of Shanghai Ocean University. 21(3): 363-67.

  7. Harris JO, Maguire GB, Edwards S and Hindrum SM (1998). Effect of ammonia on the growth rate and oxygen consumption of juvenile greenlip abalone, Haliotis laevigata Donovan. Aquaculture. 160(3-4): 259-72.

  8. Li YQ, Li J and Wang QY (2006). The effects of dissolved oxygen concentration and stocking density on growth and non-specific immunity factors in Chinese shrimp, Fenneropenaeus chinensis. Aquaculture. 256(1-4): 608-16.

  9. Luo J, Liu CW, Li F and Tang HC (2008). Suffocation point and diurnal metabolism pattern of Hemifusus tuba (Gmelin). Chinese Journal of Applied Ecology. 19(9): 2092-96.

  10. Mayzaud P and Conover RJ (1988). O:N atomic ratio as a tool to describe zooplankton metabolism. Marine Ecology Progress Series. 45(3): 289-302.

  11. Parisi MG, Mauro M, Sarà G and Cammarata M (2017). Temperature increases, hypoxia and changes in food availability affect immunological biomarkers in the marine mussel Mytilus galloprovincialis. Journal of Comparative Physiology. B: Biochemical, Systemic and Environmental Physiology. 187(8): 1117-26.

  12. Paschke K, Pablo Cumillaf J, Loyola S, Gebauer P, Urbina M, Eugenia Chimal M, Pascual C and Rosas C (2010). Effect of dissolved oxygen level on respiratory metabolism, nutritional physiology and immune condition of southern king crab Lithodes santolla (Molina, 1782) (Decapoda, Lithodidae). Marine Biology. 157(1): 7-18.

  13. Qiu RJ, Cheng YX, Huang XX, Wu XG, Yang XZ and Tong R (2011). Effect of hypoxia on immunological, physiological response and hepatopancreatic metabolism of juvenile Chinese mitten crab Eriocheir sinensis. Aquaculture International. 19(2): 283-99.

  14. Rosas C, Martinez E, Gaxiola G, Brito R, Sanchez A and Soto LA (1999). The effect of dissolved oxygen and salinity on oxygen consumption, ammonia excretion and osmotic pressure of Penaeus setiferus (Linnaeus) juveniles. Journal of Experimental Marine Biology and Ecology. 234(1): 41-57.

  15. Silkin YA and Silkina EN (2005). Effect of hypoxia on physiological-biochemical blood parameters in some marine fish. Journal of Evolutionary Biochemistry and Physiology. 41(5): 527-32.

  16. Song CX, Wang CL, Shao YW, Wang JW and Fan XX (2009). The nutritional compositions and evaluation of muscle between wild and cultivated Sepiella maindroni. Acta Nutrimenta Sinica. 31: 301-03.

  17. Storey KB and Storey JM (2007). Tribute to P. L. Lutz: putting life on ‘pause’ - molecular regulation of hypometabolism. Journal of Experimental Biology. 210(10): 1700-14.

  18. Tripp-Valdez MA, Bock C, Lucassen M, Lluch-Cota SE, Teresa Sicard M, Lannig G and Poertner HO (2017). Metabolic response and thermal tolerance of green abalone juveniles (Haliotis fulgens: Gastropoda) under acute hypoxia and hypercapnia. Journal of Experimental Marine Biology and Ecology. 497: 11-18.

  19. Valverde JC, Lopez FJM and Garcia BG (2006). Oxygen consumption and ventilatory frequency responses to gradual hypoxia in common dentex (Dentex dentex): Basis for suitable oxygen level estimations. Aquaculture. 256(1-4): 542-51.

  20. Van Heerden D, Vosloo A and Nikinmaa M (2004). Effects of short-term copper exposure on gill structure, metallothionein and hypoxia-inducible factor-1 alpha (HIF-1 alpha) levels in rainbow trout (Oncorhynchus mykiss). Aquatic Toxicology. 69(3): 271-80.

  21. Wachter BD, Wolf G, Richard A and Decleir W (1988). Regulation of respiration during juvenile development of Sepia officinalis (Mollusca: Cephalopoda). Marine Biology. 97(3): 365-71.

  22. Wang CL, Wu DH, Dong TY and Jiang XM (2008). Oxygen consumption rate and effects of hypoxia stress on enzyme activities of Sepiella maindron. Chinese Journal of Applied Ecology. 19(11): 2420-27.

  23. Wang JG, Tang BJ, Qiao ZG and Zou X (2010). Preliminary study on the oxygen consumption rate and suffocation point of juvenile crab Scylla paramamosian. Marine Fishery. 32(3): 345-50.

  24. Xia LM, Fan ZJ, Wu CW, Lv ZM and Xu YT (2009). Study on oxygen consumption and ammonia excretion rate of Sepiella maindroni juvenile and its affecting factors. Fishery Modernization. 36: 42-45.

  25. Zhang XM, Wang CL, Li LG and Chen W (2010). Oxygen consumption rate and effect of hypoxia stress on emzyme activity of Octopus variabilis. Journal of Hydroecology. 3: 72-79.

  26. Zou EM, Du NS and Lai W (1996). The effects of severe hypoxia on lactate and glucose concentrations in the blood of the Chinese freshwater crab Eriocheir sinensis (Crustacea: Decapoda). Comparative Biochemistry and Physiology A. 114(2): 105-09. 
Reviewed By

Download Article
In this Article
    Contact us
    Follow us

    Editorial Board

    View all (0)