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ICES Journal of Marine Science: Journal du Conseil Advance Access originally published online on October 31, 2007
ICES Journal of Marine Science: Journal du Conseil 2007 64(9):1743-1748; doi:10.1093/icesjms/fsm154
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© 2007 International Council for the Exploration of the Sea. Published by Oxford Journals. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Aggregata octopiana (Protista: Apicomplexa): a dangerous pathogen during commercial Octopus vulgaris ongrowing

Camino Gestal, Angel Guerra and Santiago Pascual

Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), Eduardo Cabello 6, 36208 Vigo, Spain

Correspondence to S. Pascual: tel: +34 986 291330; fax: +34 986 292762; e-mail: spascual{at}iim.csic.es

Gestal, C., Guerra, A., and Pascual, S. 2007. Aggregata octopiana (Protista: Apicomplexa): a dangerous pathogen during commercial Octopus vulgaris ongrowing. – ICES Journal of Marine Science, 64.

The haemolymph parameters for Octopus vulgaris and the condition index at molecular level were analysed using RNA/DNA and RNA/protein ratios on animals reared in floating cage systems and naturally infected by the coccidian Aggregata octopiana. Statistical analysis showed that as A. octopiana infection increased, there was a decrease in most inorganic elements and/or in haemocyte concentrations in cellular and/or plasma fractions. Also, the protein concentrations in the haemolymph plasma fraction dropped significantly as the coccidian infection increased. Results suggest that the severity of the infection by A. octopiana is a limiting factor during commercial ongrowing of the common octopus, at least in floating cage systems. Control measurements are needed in aquaculture to avoid this environmental stressor.

Keywords: Aggregata octopiana, culture, Octopus vulgaris, pathogen

Received 2 July 2007; accepted 28 September 2007; advance access publication 31 October 2007.


   Introduction
Top
 Introduction
Material and methods
 Results
 Discussion
 References
 
The common octopus Octopus vulgaris (Cephalopoda: Octopodidae) is an important species in world fisheries (Globefish, 2007). It represents a major protein resource in most fish-eating countries, and is of commercial and social importance in Galicia, northwest Spain (Otero et al., 2005). Interest in its commercial culture has risen in recent years (Iglesias et al., 2007), and mortality during its ongrowing in tanks and in floating cages in Galician and Portuguese waters (Sendao, 1997; Chapela et al., 2006; Iglesias et al., 2007) has encouraged studies to be made on the effects of pathogens.

The eimeriorin coccidian Aggregata (Protozoa: Apicomplexa) has been recorded widely as the dominant epizootiological agent in wild and cultured octopus stocks from European waters (Pascual et al., 1996; Gestal, 2000). Coccidians within the genus Aggregata are intracellular parasites with a two-host life history, and they are transmitted through the foodweb. Sexual stages are found in the digestive tract of cephalopods, the definitive host. Asexual stages infect the digestive tract of crustaceans, the intermediate hosts (Hochberg, 1990). Gestal et al. (2002a) described the histopathological effects of A. octopiana in the digestive tract of O. vulgaris. The lesions associated with infected A. octopiana included host cell hypertrophy with nuclear displacement, inflammation, phagocytosis, ulceration, and partial destruction of the organ architecture. Additionally, a spectrophotometric analysis of the enzymes involved in the absorption process revealed a significant decrease with increasing A. octopiana infection. As a consequence, gastrointestinal function is detrimentally impacted, and this is induced by a decrease or malfunction of absorption enzymes (Gestal et al., 2002b). Moreover, although enteritic coccidiosis is not believed to be a primary cause of death, it is likely that the malabsorption syndrome impairs octopus development and growth (Gestal et al., 2002b).

Haemolymph and more specifically the haemocytes are crucial in physiological functions such as nutrition, oxygen transport, and detoxification. Further, mollusc haemocytes play a major role in cellular defence against pathogens (Cheng, 1981). Although determination of the role of the haemolymph and haemocytes in physiological functions and the immune system of bivalve molluscs has been the objective of many studies, few data have been published yet on cephalopods (Lee, 1994; Malham et al., 1998, 2002; Novoa et al., 2002).

Our aim was to investigate the impact of A. octopiana infection on the haemolymph and some growth indicators of commercially reared O. vulgaris, specifically investigating the carrier’s well-being.


   Material and methods
Top
 Introduction
 Material and methods
Results
 Discussion
 References
 
Sampling
In all, 28 live O. vulgaris (14 males and 14 females) with an average body weight (BW) of 1137.7 ± 442.1 g (Table 1) were collected randomly off an ongrowing floating cage system in the Ría de Aldán, Galicia, Spain (NE Atlantic: 42°15'N 8°48'W). The floating culture system consisted of 12 cylindrical cages of 10 m3 each (1.6 x 5 m on top), with 250 PVC dens holding 200 animals per cage simultaneously. The culture conditions allowed easy sampling, made it possible to standardize the feeding parameters, and provided a sample of animals grown in similar environmental conditions (12–17°C, 35.5 psu). After collection, and to minimize physiological stress, all animals were maintained at the laboratory in culture tanks for 24 h before experimentation. The animals were first anaesthetized in a 70% ethanol:seawater (1:5) mix for 1.5–2.5 min (AG, unpublished). To avoid damage to the animals, the flesh was not cut open at all. The branchial blood vessels were exposed by turning the mantle gently. Haemolymph (1 ml) was withdrawn from the branchial blood vessel of each animal with a disposable syringe and diluted in marine anticoagulant (MAS, modified Alsever solution, 1:2; Bachère et al., 1998). All animals recovered normal respiratory rhythm (25 inspirations per minute) after 5 min post-anaesthesia.


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  		Table 1. Data from the 28 Octopus vulgaris sampled, and biochemical and haemolymph parameters.

 
Haemolymph analysis
The number of haemocytes was determined in a Neubauer count chamber. The haemolymph suspension was centrifuged (750g for 10 min at 4°C) and the cells resuspended in 5 ml of distilled water for further RNA extraction and element analysis. Plasma and resuspended cells were then frozen at –80°C separately until further processing.

Analysis of haemolymph elements was carried out on defrosted samples as follows: 1 ml each of plasma and burst cells was submitted to complete microwave digestion with HNO3 (Suprapur, Merk) inside a Telflon-lined bomb. The concentrations (mg ml–1) of some haemolymph elements (Fe, Cu, Na, K, Ca, Mg) were determined using a flame atomic absorption spectrometer (Varian Spectra 250 Plus), and a graphite furnace atomic absorption spectrometer (Varian Spectra AA-800 Plus) with aqueous calibration standards (Merck, Chemicals). To determine the concentrations of elements in the plasma, the concentration of each in the used anticoagulant (MAS) was measured and used as a control. Element concentration was expressed as the total element concentration minus the MAS concentration.

The protein concentrations of plasma and the cellular fraction were determined according to the method of Bradford (1976), and the protein content of the crude tissue extracts by the Lowry modified method (Smith et al., 1985). The sample was diluted 1:7, digested by NaOH 1M at 30°C for 14 h, and 2 ml of bicinchoninic acid and 4% Cu SO4 in proportion 50:1 added to 100 µl of the diluted sample. That mixture was maintained at 60°C for 30 min, then the absorbance was measured at 560 nm. Protein concentration was calculated using bovine serum albumin as standard, and values were expressed as mg protein per mg tissue.

Muscular DNA, RNA, and protein concentrations
DNA and RNA concentrations were calculated from samples of muscular tissue sealed in aluminium foil and stored frozen at –80°C until assay. Tissue samples were homogenized in an electric homogenizer. Aliquots of total tissue extracts were frozen to –80°C until processing. Extraction and division of the nucleic acids was carried out following the method of Schmidt and Thannhauser (1945), as modified by Munro and Fleck (1966). Basically, the method archives nucleic acids, proteins, and other macromolecule precipitate by requiring 1 ml of 0.6 M perchloric acid to be added to 2 ml of homogenate tissue. After centrifugation in a Beckman J2–21M refrigerated centrifuge to 8300g at 4°C for 10 min, the precipitate is resuspended in 3 ml of 0.2 M perchloric acid with the aid of an ultrasound bath. This process is repeated three times. The final precipitate obtained is resuspended in 2 ml MilliQ water, then 0.3 ml of 3 M NaOH is added to produce alkaline digestion, which is archived after incubation at 37°C for 1 h, followed by acidification of the sample using 0.666 ml 20% perchloric acid (Fleck and Munro, 1962). After centrifugation as above, the soluble RNA is liberated in the supernatant, and the DNA and proteins stay in the precipitate. RNA concentration is estimated by ultraviolet absorption at 232 and 260 nm, and the precipitate resuspended in 2 ml of 0.2 M perchloric acid and recentrifuged. Soluble DNA and the precipitated proteins can be extracted from the resulting pellet by resuspension in 3 ml 0.6 M perchloric acid incubated for 30 min at 70°C. DNA concentration is then estimated after centrifugation at 8300g at 4°C for 10 min, and measured by ultraviolet absorption at 232 and 260 nm.

DNA and RNA concentrations were obtained from the equations:


Formula

where Vf is the supernatant volume following the protocol (3.666 ml for RNA and 3 ml for DNA), and Vi is the total initial volume divided by the volume used for RNA and DNA measurement (2 ml).

Finally, the concentrations of proteins were estimated as described above in the haemolymph analyses.

Parasite counts
The caecum was dissected after removing the digestive tract, weighed, and vigorously washed in homogenization phosphate buffer 10 mM, pH 7.0. To obtain the levels of infection, tissue extracts were prepared by homogenization in an electric tissue grinder (IKA-UltraTurrax T-25), following the standard procedure described by Gestal et al. (1999). The number of Aggregata sporocysts infecting the caecum was enumerated in a Neubauer count chamber. The number of sporocysts was expressed as the total number of sporocysts in the whole caecum (TS), but the number of sporocysts per gramme of caecum tissue (S g–1) was also deduced.

Statistical analyses
Data on haemolymph element concentration, proteins, and nucleic acids were compared with infection values measured at an infrapopulation level (i.e. the total sporocyst counts, TS; Bush et al., 1997). Linear regression models were fitted to log-transformed [log10(n+1)] data. Analysis of variance (one-way ANOVA) was used to test the significance of the regressions (Zar, 1999). The limit of statistical significance was set at p < 0.05. Data analysis was performed using the SPSSWIN 10.0 statistical software.


   Results
Top
 Introduction
 Material and methods
 Results
Discussion
 References
 
General parameters
Table 1 lists the results of the biochemical analysis (for RNA, DNA, and proteins) and the values of the ratios RNA/DNA and RNA/protein. It also lists the haemolymph parameters, including the total number of haemocytes and the number of haemocytes per millilitre. Table 2 shows the concentrations (ppm) of elements and proteins analysed from the plasma and cellular fractions.


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  		Table 2. Element (ppm) and protein (µg mg–1) concentrations in the plasma and cellular fractions of haemolymph of 28 O. vulgaris.

 
Haemolymph vs. infection
Parameters of the regression analysis between TS and the concentration of each element are summarized in Table 3. The regression patterns obtained show that an increase in infection level causes a significant decline in K, Cu, Fe, and protein concentrations in the plasma fraction, and also a significant drop in Ca, Mg, Fe, and haemocyte concentrations in the cellular fraction. Moreover, the protein concentrations in the haemolymph plasma fraction diminished as TS increased (Table 3).


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  		Table 3. Linear regression analyses of log-transformed data (log10 n+1) for the relationship between Octopus vulgaris haemolymph and muscular biochemical parameters plotted against the total number of sporocysts in the caecum (TS).

 
Biochemistry of the muscle vs. infection
Results of comparing the biochemical parameters and the severity of Aggregata infection related to TS were statistically significant (Table 3). The statistical regression patterns indicated that an increasing TS caused decreases in individual RNA and protein concentrations. Further, an increasing TS in the caecum of heavily infected animals gave rise to a decrease in the condition indices RNA/DNA and RNA/protein. Regressions indicate that 39.98% and 30.72% of the variability found in RNA and protein contents, respectively, as well as the 3.73% and 37.78% of RNA/DNA and RNA/protein ratios, can be attributed to the severity of infection (Table 3).


   Discussion
Top
 Introduction
 Material and methods
 Results
 Discussion
References
 
Changes in the haemolymph or haemocyte composition of marine invertebrates can be used as indicators of physiological condition (Feng, 1965), especially of pathological damage (Ford et al., 1986). A constant equilibrium state between plasma protein (amino acids present in the haemolymph) and tissue protein should exist in normal physiological behaviour of healthy animals (Barnes, 1984). The decreases in haemocytes and plasma protein concentration found here do not preclude the possibility that some of the variability in the number of circulating haemocytes was due to stress during handling, as previously indicated by Malham et al. (2002) for the octopus Eledone cirrhosa exposed to air for 5 min. However, the decrease together with an increasing infection of Aggregata may also indicate an imbalance caused by infection. This may be due to tissue damage and a reduction in enzyme activity and hence amino acid absorption in the caecum. This malabsorption syndrome, described for cephalopods by Gestal et al. (2002b), was also reported with fish coccidiosis (Steinhagen et al., 1997). The absorption failure causes decreasing plasma protein concentration as a consequence of the deficiency in amino acid absorption and general protein synthesis. Castro et al. (1993) indicated that restricting the diet of cultured cuttlefish Sepia officinalis produced a decrease in the amount of copper (Cu) joined to the respiratory pigment haemocyanine in the haemolymph. Similarly, the decrease in Cu concentration observed in the haemolymph in this study and related to the degree of Aggregata infection (Table 3) likely originates in the absorption deficiency and the general decrease in plasma protein. Inadequate oxygenation may induce tissue hypoxia, necrosis, and atrophy of the infected area, by decreasing the oxygen-carrying capacity of the blood. Such changes could impact negatively on development and growth, and therefore lower the condition of the animal.

Declines in blood cell and plasma protein concentration as a consequence of Eimeria infection have also been demonstrated for vertebrates (Long, 1973). The Aggregata infection demonstrated here for octopuses is similar, perhaps a consequence of the general decrease in protein synthesis attributable to lessening of amino acid absorption, or to a displacement of blood cells to the tissue infected by Aggregata. Cephalopods acquire most of their salt requirement from seawater (Lee, 1994).

Growth measurements usually expressed as RNA and muscular protein concentration or as ratios of nucleic acids have been shown to be reliable and useful as indicators of condition. Such biochemical indices are well established for studies of fish growth and are also reliable indicators for growth studies on cephalopods (Clarke et al., 1989; Houlihan et al., 1990; Castro and Lee, 1994; Domingues et al., 2006). Steinhart and Eckmann (1992) showed the severity of parasitic infection to be related directly to protein concentration and the RNA/DNA ratio in muscular tissue of larval whitefish Coregonus spp., similar to the negative impact on the growth rate of infected octopus expressed in terms of decreasing RNA/DNA and RNA/protein ratios noted here. Although the r2 values for the fit of linear regressions did not account for 100% of the intraspecific variance in condition measurements, the impact of the Aggregata infection could explain as much (up to 40%) as all other environmental factors combined. Further, mechanical tissue damage (Gestal et al., 2002a) together with a malabsorption syndrome produced by the parasite’s sporogonial development (Gestal et al., 2002b) may lead to impairment of the balance between the rates of protein synthesis and protein degradation. Therefore, and because the intake of food was similar in all octopuses sampled, it is thought that the infection decreases muscle protein concentration. The increasing sporocyst number explained up to 40% of the variance in the RNA and RNA/protein ratios, whereas the infection values accounted for just 30% of the variance in the muscular protein concentration of infected octopuses. With the general rationale that the quantity of food eaten affects ribosome concentration in somatic tissues and hence protein synthesis and growth (Houlihan et al., 1990), it follows that the Aggregata infection may influence the synthesis of RNA in muscle tissue cells, probably through the rRNA, as suggested by Kikuchi et al. (1986) and Reeds (1987) for mammalian systems. This interpretation mirrors that of reduced K-Fulton condition values in heavily infected octopuses (Gestal et al., 2002b).

In conclusion, this study has shown that the elements and protein balance of an octopus’ haemolymph, as well as its muscle protein synthesis and general growth, are influenced by the level of infection of the Aggregata octopiana. Taking into account the total results from a series of condition analyses carried out us on O. vulgaris populations at individual, tissue, cellular, and here molecular level, A. octopiana should be considered to be a dangerous pathogen. Control measurements are clearly needed in aquaculture to avoid this coccidian infection, because it is definitely an octopus health-limiting factor during commercial ongrowing in floating cage systems.


   Acknowledgements
 
We thank Pulpos Atlántico for providing the samples used in these analyses and the anonymous reviewers for their constructive comments.


   References
Top
 Introduction
 Material and methods
 Results
 Discussion
 References
 

    Bachère E., Chagot D., Grizel H. Separation of Crassostrea gigas hemocytes by density gradient centrifugation and counterflow centrifugal elutriation. Developmental and Comparative Immunology (1998) 12:549–559.

    Barnes R. D. Zoología de los Invertebrados. (1984) 4th edn. Mexico City: Interamericana. 1157.

    Bradford M. A. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle protein-dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]

    Bush A. O., Lafferty K. D., Lotz J., Shostak A. W. Parasitology meets ecology on its own terms: Margolis et al. revised. Journal of Parasitology (1997) 83:575–583.[CrossRef][Medline]

    Castro B. G., Di Marco F. P., De Rusha R. H., Lee P. G. The effects of surimi and pelleted diets on the laboratory survival, growth, and feeding rate of the cuttlefish Sepia officinalis L. Journal of Experimental Marine Biology and Ecology (1993) 170:241–252.[CrossRef][Web of Science]

    Castro B. G., Lee P. G. The effects of semi-purified diets on growth and condition of Sepia officinalis L. (Mollusca: Cephalopoda). Comparative Biochemistry and Physiology (1994) 109:1007–1016.

    Chapela A., González A. F., Dawe E. G., Rocha F., Guerra A. Growth of common octopus (Octopus vulgaris) in cages suspended from rafts. Scientia Marina (2006) 70:121–129.[Web of Science]

    Cheng T. C. Bivalves. In: Invertebrate Blood Cells—Ratcliffe N. A., Rowley A. F., eds. (1981) London: Academic Press. 235–300.

    Clarke A., Rodhouse P. G., Holmes L. J., Pascoe P. L. Growth rate and nucleic acid ratio in cultured cuttlefish Sepia officinalis (Mollusca: Cephalopoda). Journal of Experimental Marine Biology and Ecology (1989) 133:229–240.[Web of Science]

    Domingues P., Bettencourt V., Guerra A. Growth of Sepia officinalis in captivity and in nature. Vie et Milieu (2006) 56:109–120.

    Feng S. Y. Heart rate and leucocyte circulation in Crassostrea virginica (Gmelin). Biological Bulletin (1965) 128:198–210.[Abstract/Free Full Text]

    Fleck A., Munro H. N. The precision of the ultraviolet absorption measurements in the Schmidt–Thannhauser procedure for nucleic acid estimation. Biochemical and Biophysical Acta (1962) 55:571–583.[CrossRef]

    Ford L. A., Alexander S. K., Cooper K. M., Hanlon R. T. Bacterial populations of normal ulcerated mantle tissue of the squids, Lolliguncula brevis. Journal of Invertebrate Pathology (1986) 48:13–26.[CrossRef][Web of Science][Medline]

    Gestal C. Epidemiología y patología de las coccidiosis en cefalópodos. Tesis doctoral, Universidad de Vigo. (2000) 157.

    Gestal C., Abollo E., Pascual S. Evaluation of a method for isolation and purification of sporocysts of the cephalopod coccidian parasite Aggregata Frenzel, 1885 (Apicomplexa: Aggregatidae). Iberus (1999) 17:115–121.

    Gestal C., Abollo E., Pascual S. Observations on associated histopathology with Aggregata octopiana infection (Protista: Apicomplexa) in Octopus vulgaris. Diseases of Aquatic Organisms (2002) a 50:45–49.[CrossRef][Web of Science][Medline]

    Gestal C., Páez de la Cadena M., Pascual S. Malabsorption syndrome observed in the common octopus Octopus vulgaris infected with Aggregata octopiana (Protista: Apicomplexa). Diseases of Aquatic Organisms (2002) b 51:61–65.[CrossRef][Web of Science][Medline]

    Globefish. Octopus Market. (2007) Report for June 2007.

    Hochberg F. G. Diseases of Mollusca: Cephalopoda. In Diseases of Marine Animals. 3. In: Cephalopoda to Urochordata—Kinne O., ed. (1990) Hamburg, Germany: Biologische Anstalt Helgoland. 47–227.

    Houlihan D. F., McMillan D. N., Agnisola C., Trara Genoino I., Foti L. Protein synthesis and growth in Octopus vulgaris. Marine Biology (1990) 106:251–259.[CrossRef]

    Iglesias J., Sanchez F. J., Bersano J. G. F., Carrasco J. F., Dhont J., Fuentes L., Linares F., et al. Rearing of Octopus vulgaris paralarvae: present status, bottlenecks and trends. Aquaculture (2007) 266:1–15.[CrossRef][Web of Science]

    Kikuchi T., Okamoto H., Chiku K., Natori Y. Effects of glucose and amino acid depletions on protein synthetic parameters in liver and skeletal muscle of rats during parental nutrition. Journal of Nutritional Science and Vitaminology (1986) 32:601–612.[Medline]

    Lee P. G. Nutrition of cephalopods: fuelling the system. Pörtner H. O., O'Dor R. K., Macmillan D. L., eds. (1994) Australia: Gordon and Breach Publishers. 35–51. Physiology of Cephalopod Molluscs.

    Long P. L. Pathology and pathogenicity of coccidial infections. Hammond D. M., Long P. L., eds. (1973) Baltimore: University Park Press. 253–294. The Coccidia.

    Malham S.K., Coulson C.L., Runham N.W. Effects of repeated sampling on the haemocytes and haemolymph of Eledone cirrhosa (Lam.). Comparative Biochemistry and Physiology (1998) 121:431–440.

    Malham S. K., Lacoste A., Gélébart F., Cueff A., Poulet S. A first insight into stress-induced neuroendocrine and immune changes in the octopus Eledone cirrhosa. Aquatic Living Resources (2002) 15:187–192.[CrossRef][Web of Science]

    Munro H. N., Fleck A. The determination of nucleic acids. In Methods of Biochemical Analysis. Glick D., ed. (1966) 14. New York: Interscience Publishers. 113–176.

    Novoa B., Tafalla C., Guerra A., Figueras A. Cellular immunological parameters of the octopus, Octopus vulgaris. Journal of Shellfish Research (2002) 21:243–248.[Web of Science]

    Otero J., Rocha F., González A. F., García J., Guerra A. Modelling artisanal coastal fisheries of Galicia (NW Spain) based on data obtained from fishers: the case of Octopus vulgaris. Scientia Marina (2005) 69:577–585.[Web of Science]

    Pascual S., Gestal C., Estévez J., Rodríguez H., Soto M., Abollo E., Arias C. Parasites in commercially-exploited cephalopods (Mollusca, Cephalopoda) in Spain: an update perspective. Aquaculture (1996) 142:1–10.[CrossRef][Web of Science]

    Reeds P. J. Metabolic control and future opportunities for growth regulation. Animal Production (1987) 45:149–169.[Web of Science]

    Schmidt G., Thannhauser S. J. A method for determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. Journal of Biological Chemistry (1945) 161:83–89.[Free Full Text]

    Sendao J. O polvo comun (Octopus vulgaris), um candidato para aquacultura?: alguns aspectos relacionados com o seu crescimento e mercado. Relatório de estágio do curso de biología marinha e pesca. Universidade do Algarve, Portugal. (1997) 84.

    Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gatner F. H., Provenzano M. D., Fujimoto E. K., et al. Measurement of protein using bicinchonimic acid. Analytical Biochemistry, (1985) 150:76–85.

    Steinhagen D., Oesterreich B., Körting W. Carp coccidiosis: clinical and hematological observations of carp infected with Goussia carpelli. Diseases of Aquatic Organisms (1997) 30:137–143.[CrossRef][Web of Science]

    Steinhart M., Eckmann R. Evaluating the nutritional condition of individual whitefish Coregonus spp. larvae by the RNA/DNA ratio. Journal of Fish Biology (1992) 40:791–799.[CrossRef][Web of Science]

    Zar J. H. Biostatistical Analysis. (1999) 2nd edn. NJ: Prentice Hall International, Englewood cliffs. 633.


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