Dice snakes (
Aquatic environments play a vital role in the sustainable functioning of ecosystems (Walia et al., 2013). Elevated concentrations of salt in water is a recognized issue for the conservation of aquatic habitats (Crowther and Hynes, 1977; Dickman and Gochnauer, 1978; Martem'yanov, 1989; Howard and Haynes, 1993; Kjensmo, 1997; Williams et al., 2000). Some amphibians and cold-blooded amniotes can tolerate increased water salinity and some even reproduce in brackish water and seawater (Neill, 1958; Chakko, 1968; Christman, 1974; Dunson, 1977; Sillero and Ribeiro, 2010; Natchev et al., 2011). However, the effects of increased water conductivity on the populations of aquatic ectothermic tetrapods are not fully understood (Bentley and Schmidt-Nielsen, 1971; Spotila and Berman, 1976; Shoemaker et al., 1992; Gomez-Meste et al., 2004; Karraker, 2007; Ortiz-Santaliestra et al., 2010).
Some snake species live and reproduce in near-brackish and euryhaline
waters (Neill, 1958), but the
information concerning the extent of their water salinity tolerance is
somewhat limited. Dice snakes (
We used nine
Initially, all snakes were kept in fresh water for 72 h to establish
normonatremia (Yurii Kornilev and Nikolay Natchev, personal observation,
2015). Then, each animal was randomly assigned to one of three experimental
groups and exposed to 0.2 ‰ NaCl (fresh water), 16 ‰ NaCl
(Black Sea water) or 36 ‰ NaCl (World Ocean water) for 84 h. Water
of the respective salinity was added to each container (
Waters were prepared by adding solid NaCl to freshwater; the NaCl was obtained from the Black Sea at a salt extraction facility, located 10 km from Poda PS. Salinity was measured using a calibrated, hand-held, real-time conductivity measuring instrument (Testo 240, Testo AG). After the treatment, the snakes were moved in containers with fresh water for the period of 10 min prior to blood sampling.
Blood samples of 0.3–0.4 mL were obtained by cardiocentesis using a heparinized 30 G-needle and syringe (see Brischoux and Kornilev, 2014). The collected blood was transferred immediately into heparinized Eppendorf vials. Samples were placed in a cooler with ice and within 1 h the critical laboratory manipulations were performed.
Ten microlitres of blood were placed onto a clean microscope slide and a
smear was created using the bevel-edge slide technique. The slides were
air-dried at room temperature and fixed in methanol for 20 min at
4
The monolayer cells were analysed under
The assay principle is based upon the reduction of the tetrazolium salt WST-1
(Roche Applied Science) to formazan in the mitochondria of living cells. For
each individual sample, 50
All statistical tests were performed using the Statistica v.7.0 software
(StatSoft, Inc. 2004). The chosen alpha level for statistical significance
was
Various abnormalities in the form of the erythrocyte nuclei are used as
effective indicators of the negative influence of the environment on the
cells of many aquatic animals (Ateeq et al., 2002; Jiraungkoorskul et al.,
2007; Ali et al., 2008; Anbumani and Mohankumar, 2011; Walia et al., 2013;
Hayretdağ et al., 2014; Furnus et al., 2014; Asker et al., 2015). The
sublingual salt gland is the primary site of salt excretion in sea snakes
(Babonis et al., 2009), but dice snakes lack salt glands. However, these
snakes can spend long periods of time in brackish and saline waters (see
Brischoux and Kornilev, 2014). The authors measured hypernatremia up to
195.5 mmol L
Morphological changes of erythrocyte membranes or erythrocyte nuclei after treatment with waters of different salinities. Values represent the percentage of normal nuclei from all counted nuclei per treatment group, or the percentage of damaged membranes from all counted erythrocytes per treatment group.
In our experiments, the presence of lobed and notched nuclei, as well as
nuclear buds and blebs, was detected in the blood cells of the snakes from
all three treatments, albeit in very different proportions (Fig. 1, Table 1).
Erythrocytes nuclei with the correct oval shape or with slight shape
deviations were 10.2 % for the control freshwater group, 11.11 % for
Black Sea water and 20.21 % for World Ocean water. The ANOVA test
revealed marginally significant differences (
Snakes exposed to 16 and 36 ‰ salinity presented erythrocytes with
ruptured and damaged membranes of 71.16 and 45.96 % compared to only
0.37 % in the freshwater control group (Table 1). This difference was
statistically significant –
Blood cells of dice snakes after treatment with
It is possible that the occurrence of most nuclear anomalies from the fresh
water group observed in this study is related to the prolonged erythrocyte
lifespan, as the average lifespan of these cells in cold-blooded amniotes is
600–800 days (Stacy et al., 2011; Strunjak-Perovic et al., 2010). Therefore,
abnormal morphological forms of erythrocyte nuclei are not effective
indicators for assessment of the negative influence of environment onto the
formed elements of the blood in
Mitochondrial activity of blood cells (mean
The numerous cells with ruptured membranes (Fig. 2), found in blood smears of snakes exposed for a prolonged time to 16 and 36 ‰ salinity, indicate hemolysis and osmotic stress (see Madden et al., 2014). A fundamental challenge for the cell is a change in the osmolarity of the surrounding medium (Pastor et al., 2009). Osmoregulation is a major physiological challenge for numerous vertebrates that are subject to extensive changes in the salinity of their environment (Gutiérrez et al., 2011). Our data suggest that disintegration of the cell membranes was a result of the osmotic pressure gradient following prolonged dwelling in waters with increased conductivity.
The mitochondrial activity of the blood cells was estimated based on the
reduction of the tetrazolium compound (WST1) by intracellular dehydrogenases
to formazan, released in the culture medium. The mean mitochondrial
activities after removing the snakes from solutions of 16 and 36 ‰ NaCl
increased by 1.85 and 2.53 times compared to those of snakes
resident in fresh water (Fig. 3). These differences in absorbance were
statistically significant (
Our results demonstrate that the presence of abnormal morphological features of the blood cell nuclei cannot be regarded as effective indicators for an assessment of the influence of defined environmental factors in short-term studies in dice snakes. The abnormalities were found in all specimens and may be result of the pre-exposure of some physical/chemical contaminants from the sampling location – the erythrocyte turnover rate in cold-blooded amniotes is rather slow (see Stacy et al., 2011). The presence of destroyed blood cells and the increased mitochondrial activity detected in two of the snake groups used in our experiments suggest potential change in the osmotic pressure of the blood plasma and impact on the general performance and fitness of the dice snakes.
Vanya Koleva prepared the concept of the study, performed the laboratory manipulations, wrote the manuscript, prepared the images and checked the final versions of the products. Yurii Kornilev aided to the concept of the study, performed the animal handling and manipulations, wrote the manuscript, proofread the manuscript and checked the final version of the products. Ivan Telenchev performed the animal handling and manipulations and checked the final version of the products. Simeon Lukanov wrote the manuscript, performed calculations, proofread the manuscript and checked the final version of the products. Berna Hristova performed the laboratory manipulations and checked the final versions of the products. Nikolay Natchev wrote the manuscript, edited the images, proofread the manuscript and checked the final version of the products.
The authors declare that they have no conflict of interest.
This work was partly supported by the Bulgarian National Science Fund (BNSF), Grant No. RD-08-266/10.03.2015. Only materials were provided by BNSF and no personal payments were made. All animal experimentation was conducted in accordance with pertinent national statements regarding the care and use of animals (research permit number 520/23.04.2013). We thank François Brischoux for helpful comments on earlier draft of the paper. Daniel Montesinos and two anonymous reviewers provided important remarks on the manuscript. Edited by: D. Montesinos Reviewed by: S. Anbumani and one anonymous referee