Comparative Hematology of Wild Anguilliformes (Muraena Helena, L

Animal Biology 63 (2013) 77–92 brill.com/ab Comparative hematology of wild Anguilliformes (Muraena helena, L. 1758, Conger conger, L. 1758 and Anguilla anguilla L. 1758) Domagoj Ðikic´1,∗, Duje Lisiciˇ c´1, Sanja Matic-Skoko´ 2, Pero Tutman2, Daria Skaramuca1, Zdenko Franic´3 and Boško Skaramuca4 1 Department of Animal Physiology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia 2 Institute of Oceanography and Fisheries, Šetalište I. Meštrovica´ 63, HR 21000 Split, Croatia 3 Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10000 Zagreb, Croatia 4 Department of Aquaculture, University of Dubrovnik, Cira´ Carica´ 4, HR-2000 Dubrovnik, Croatia Submitted: November 2, 2010. Final revision received: July 16, 2012. Accepted: July 20, 2012 Abstract The objective of this study was to compare circulating blood cell counts and morphology of three eel species: Muraena helena (moray), Conger conger (European conger) and Anguilla anguilla (Euro- pean common eel). Moray and conger were collected from the Adriatic Sea at the Elaphite Islands near Dubrovnik, Croatia; common eels were collected in the Neretva River, Croatia. Hematological comparison was conducted using Natt-Harrick’s and May-Grünwald Giemsa staining methods. The hematocrit of moray and conger were similar, while common eel had higher values by 60%. Manual cell count showed that common eel had the highest erythrocyte count. Conger had a higher erythrocyte count than moray, with a higher percentage of proerythrocytes and senescent erythrocytes compared to moray and common eel. The leukocyte count was similar in common eel and moray and slightly lower in conger. The thrombocyte count was highest in conger and lowest in moray. In all three species, the neutrophil (heterophil) nuclei appeared as either circular or bi-lobed. Moray had the highest neutrophil (heterophil) percentage and a subtype with intensively basophilic cytoplasm appearing in a similar percentage as the normal type. In common eel, neutrophils (heterophils) were the only detected granulocytes. Basophils were detected in conger eels. Eosinophils were not detected in any of the sampled fish. The size of all cell types in moray was slightly larger than in other two species. In conclusion, our findings reveal major differences in the cell count and diversity in cell subtypes between three kin species of eels. ∗ ) Corresponding author; e-mail: [email protected] © Koninklijke Brill NV, Leiden, 2012 DOI 10.1163/15707563-00002395 78 D. Ðikić et al. / Animal Biology 63 (2013) 77–92 Keywords Anguilliformes; conger; common eel; granulocytes; moray; heterophils Introduction Hematology is important in fish biology and ecology as it may mirror circannual changes and provide a comparative reference for captivity-reared species in aquaculture (Larsson et al., 1976). Identification of different piscine blood cells can help to indicate physiological health status, assess the conditions that cause stress to fish (disease, parasite infections, mishandling, bioaccumulation and biomagnification of pollutants), and support explanations of phylogenetic relationships amongst different fish taxa (Kakuta and Nakai, 1992; Anderson and Zeeman, 1995; Sasal et al., 1997; van Ginneken et al., 2005; Bartoli and Gibson, 2007; Clauss et al., 2008). The basic hematological patterns in vertebrates can also be recognized in fish, though with some prominent differences. For example, the major hematopoietic organ in vertebrates is bone marrow, while in fish the major organ is the head kidney (Reavil and Roberts, 2007) with the spleen, epigonal organ and Leydig organ addi- tionally participating in granulopoiesis (Ainsworth, 1992). Erythrocytes (red blood cells, RBC) in fish are spherical, enucleated and the most numerous blood cells, and are similar to those in other vertebrates with the exception of mammals. Hema- tocrit (the percentage of the volume of whole blood made up of RBC) in teleosts ranges between 20-40%, which is less than the mammalian average, e.g., normal hematocrit is 40-45% in humans or as high as 65% in some diving marine mammals (Starck and Schuster, 2012). In fish, the mean values of hematocrit and red blood cell counts increase in the following order: rays, sharks, and teleosts. A higher proportion of juvenile erythrocyte stages can be found in fish blood than in other vertebrates. Such cells are usually smaller and differently stained than mature RBC, and their proportions in circulating blood (RBC differential) may reflect the physiological status of the fish or an adaptive response to environmental conditions. In myxinoid fish, dipnoans and some elasmobranchs, the average size of mature RBC is larger than the vertebrate average and amongst the largest vertebrate cells after amphibians (e.g. Amphiuma spp., RBC size ∼65 μm). Generally, a smaller average RBC size is recorded in active teleost fish. The nuclear size of RBC is correlated to DNA content and can be indirectly used as an estimate of ploidy of the species (Filho et al., 1992; Hrubec and Smith, 2000; Kapoor and Khanna, 2004; Campbell and Ellis, 2007; Davis et al., 2009; Arican et al., 2010; Dove et al., 2010). Different leukocyte (white blood cell, WBC) cell types and their structural het- erogeneity between fish species imply that each fish species must be analyzed separately for its distinctive WBC traits. A number of papers give a classification of fish WBC, and the difficulties concerning exact classification and nomencla- ture continue today (Ellis, 1977; Hyder et al., 1983; Parish et al., 1986; Thuvander et al., 1987; Ainsworth, 1992; Hine, 1992; Hrubec and Smith, 2000; Kapoor and Khanna, 2004; Thrall, 2004; Thrall et al., 2004; Campbell and Ellis, 2007; Dove D. Ðikić et al. / Animal Biology 63 (2013) 77–92 79 et al., 2010). These authors all agree that fish leukocytes are divided into granulocytes (neutrophils, eosinophils and baosphils) and agranulocytes (lymphocytes and monocytes) and their proportions vary from species to species. The majority of classifications claim that heterophil, neutrophil and polymorphonuclear leukocytes are constituents of the innate immune system and as the first line of defence they designate the same cell with known subtypes and synonyms differently as- signed by various authors (granulocytes – Type I, II, III, Type 1, heterophil, L2, G1, 2, 3, etc.). Although both neutrophils and heterophils are found in the same species of elasmobranchs or among teleosts in some cyprinids, only one cell type is usually present in the majority of teleosts (Ainsworth, 1992; Kapoor and Khanna, 2004; Dove et al., 2010). Some authors suggest that neutrophils and heterophils are not the same cell types (Hrubec, 2000). Confusion also arises from comparative hematology since both heterophils and neutrophils are found concurrently in reptilian and avian species or even in some rodents (rabbits and guinea pigs). Hence, some hematologists expect to find both cell types in fish. Azurophils, present in avian and reptilian species, have not been described in fish (Campbell and Ellis, 2007). Neutrophils are the most numerous granulocytes, while the occurrence of two other granulocytes (eosinophils and basophils) in teleost blood is rare, but it was confirmed that their lack is not caused by failure to preserve the granules through staining (Tavares-Dias, 2006). Numerous subtypes are also described for eosinophils (granulocytes – Type III, Type 2, Gl, G2, G3, & G4) since they occur more often than basophil. Basophil descriptions are rare and some authors even dispute their existence and function in fish (Ainsworth, 1992; Kapoor and Khanna, 2004). When eosinophils or basophils are detected, they are similar in appearance as in other vertebrate phyla with the exception of eosinophils of iguanas and some birds whose granules are pale blue (Campbell and Ellis, 2007). The absence of eosinophils and basophils can also be associated with the time of the year in which the blood of certain species is examined (Guijarro et al., 2003). Agranulocytes are more easily distinguished and classified than granulocytes and they are similar in appearance as in other vertebrate phyla (Hrubec and Smith, 2000; Kapoor and Khanna, 2004). Thrombocytes are the second most abundant blood cells after RBC, but are the least studied blood cells in fish. The expression of co- agulation factors and canalicular systems indicate their role in clotting. Granulated cytoplasm and phagocytic abilities of thrombocytes, which are not recorded in other vertebrates, have been noted in certain fish, especially elasmobranchs. Enucleated, elongated (oval, spindle, cone) and round fish thrombocytes resemble amphibian, reptilian and avian thrombocytes, though they have a different structure and origin than mammalian platelets (Kapoor and Khanna, 2004; Dove et al., 2010). Surprisingly, despite abundant descriptions of fish blood cells, there are no pub- lished data dealing with the hematology of species such as Muraena helena (moray) and Conger conger (conger) as well as other species in these genera. In a previous study, a specific neutrophil (heterophil) was found in Muraena helena (Ðikicetal.,´ 2010, 2011). Therefore, the objectives of this study were to compare whether this 80 D. Ðikić et al. / Animal Biology 63 (2013) 77–92 cell type also exists in other Anguilliform fishes and to gather information regard- ing the general hematology and physiology of eels (Fishbase, 2012). Therefore, this study aimed to fill the gaps in the basic knowledge on

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