Sinergasilus polycolpus, a new copepod species in the ichthyoparasitofauna of Serbia and Montenegro

The parasitic copepod Sinergasilus polycolpus was identified on the gills of bighead carp Aristichthys nobilis from 2 localities (Kladovo and Slankamen) in the Serbian part of the River Danube. This parasite is species-specific for 2 Chinese carp, the bighead carp and the silver carp Hypophthalmichthys molitrix. It was accidentally introduced into Serbia and Montenegro together with fry of these herbivorous carp intended for aquaculture and control of phytoplankton blooms. There is no record in the available literature of this parasite for European freshwaters. Our identification of S. polycolpus signals the possible spread of the infectious disease sinergasilosis in natural freshwaters and in fishponds, similar to bothriocephalosis, caused by Bothriocephalus opsariichthydis, which was introduced with the fry of various herbivorous species from the Amour River basin (USSR) into almost all countries throughout the world.     

Unchanged beta-adrenergic stimulation of cardiac L-type calcium channels in Ca v 1.2 phosphorylation site S1928A mutant mice

Phosphorylation of serine 1928 (Ser(1928)) of the cardiac Ca(v)1.2 subunit of L-type Ca(2+) channels has been proposed as the mechanism for regulation of L-type Ca(2+) channels by protein kinase A (PKA). To test this directly in vivo, we generated a knock-in mouse with targeted mutation of Ser(1928) to alanine. This mutation did not affect basal L-type current characteristics or regulation of the L-type current by PKA and the beta-adrenergic receptor, whereas the mutation abolished phosphorylation of Ca(v)1.2 by PKA. Therefore, our data show that PKA phosphorylation of Ser(1928) of Ca(v)1.2 is not functionally involved in beta-adrenergic stimulation of Ca(v)1.2-mediated Ca(2+) influx into the cardiomyocyte.     

Non-native and translocated fish species in Serbia and their impact on the native ichthyofauna

A total of 22 fish species have been introduced into the inland waters of Serbia, either intentionally or accidentally. This paper provides a summary of data concerning time and reason of introduction, mode of expansion, degree of acclimatization, impact on native fish and estimated area of recent distribution. Four of the non-native fish species currently occupy more than 51% of Serbian territory while 5 of them occupy between 21–50% of territory. This paper reviews impacts of introduced freshwater fish in Serbia based on collected data.     

Comparative analysis of morphometric characters of juvenile sterlet Acipenser ruthenus L. from natural population and aquaculture

A comparative analysis was performed on sixteen morphometric characters, in three different juvenile sterlet Acipenser ruthenus L. populations. Specimens were collected from a wild population in the Serbian part of the Danube River (n = 46), from aquaculture stocks in the Czech Republic originating from Russia (n = 40), and aquaculture stocks in the Slovak Republic originating from Slovakian part of the Danube River (n = 28). Average values for total length were 29·9 ± 3·9 cm, 29·1 ± 3·7 cm and 27·3 ± 7·7 cm for Serbia, Czech Republic and Slovak Republic, respectively. Populations were compared using t‐test and sequential Bonferroni correction for multiple comparisons was applied in order to determine significant differences between them. Results of analysis showed that all three populations differed in prebarbel length, interocular distance and maximum head width. Although all these characters are head‐related, head length itself was very uniform among all populations. The natural population from the Serbian part of the Danube River differed from the populations reared in aquaculture in seven morphometric characters. The two populations reared in aquaculture consistently showed lower morphological variability than the wild population, even though they had different genetic backgrounds (Russia and Slovakia). Future genetic studies will show if this tendency is caused by a reduction in genetic variability.     

Assessment of Extinction Risk and Reasons for Decline in Sturgeon

Sturgeon populations in the Danube River have been affected by a combination of hydropower development, over-harvesting, habitat degradation from agricultural and industrial practices and from urbanization. The effects of these changes have been monitored on six sturgeon species inhabiting the Danube River. Two of them are resident species, while the other four migrate to the river for spawning. Atlantic sturgeon (Acipenser sturio) has completely disappeared from this region. Ship sturgeon (Acipenser nudiventris) is very rare in professional fishing catches. Beluga (Huso huso), Russian sturgeon (Acipenser gueldenstaedtii), stellate sturgeon (Acipenser stellatus) and sterlet (Acipenser ruthenus) are endangered with different levels of extinction risk. Here, we model the time dependence of the beluga and Russian sturgeon catch in the Serbian part of the Danube River. Predicted extinction of Russian sturgeon was estimated to fall around the middle of the century, and for beluga approximately at middle of the millennium. Suggestions for sturgeon conservation measures on a national level and coordination of all relevant institutions in Serbia are also presented.     

Seasonal reproductive cycle of pike, Esox lucius L., from the River Danube

Seasonal changes in ovaries and testes of the pike, sampled in the Danube (at 1163 and 1175 km) were analysed in this research. According to the presence of developing and resting oocytes in histological sections of pike ovaries sampled during August, it is possible to determine which female will be sexually mature the following spring. Active vitellogenesis starts in September (with oocyte diameters of 514 ± 54 μm and 453 ± 68 μm, at the upstream and downstream locality, respectively), at which time the differences between the developing and resting oocytes are even more obvious. Active spermatogenesis starts in September and lasts for 2 months, and by November primarily spermatozoa can be seen in histological sections. The reproductive score points to differences in samples from the two localities that can be explained by the differing characteristics of the habitat. Values of absolute fecundity (AF) ranged from 524 to 123 896 eggs, and the mean value of relative fecundity (RF) was 40.4 ± 12.5.     

Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites

Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.     

Seasonal changes in some blood chemistry parameters and in relative liver and gonad weights of pike (Esox lucius L.) from the River Danube

Samples from a natural population of pike (Esox lucius L.) from the River Danube were used in a 12-month study to determine seasonal variations in biochemical parameters of pike blood sera, hepatosomatic index (HSI) and gonadosomatic index (GSI). The ranges of enzyme activities for sample means were: aspartate aminotransferase (ASAT) 252.0–583.8 U 1⁻¹, alanine aminotransferase (ALAT) 4.9–11 -2 U1 and alkaline phosphatase (AP) 39.5–91.8 U1⁻¹. The ranges of other parameters analysed in serum were total protein 27.7–40.1 g 1 ¹, urea 0.57–l.52 mmol 1 ¹ and creatinine 21.2–118.6 μmol 1 ¹. The range of sample means for HSI and GSI were 1.28–4.16 and 0.07–20.2 respectively. Temperature ranged from 4.5 to 23.5°C. The activity of serum AP was positively correlated to water temperature in males only, while urea and creatinine showed a positive correlation to water temperature in individuals of both sexes. GSI was correlated significantly with HSI in females. Total protein reached the lowest values during the spawning period, while creatinine levels depended on both the sex and season.     

Pros and cons of external swabbing of amphibians for genetic analyses

Non-invasive DNA sampling is an important tool in amphibian conservation. Buccal swabs are nowadays replacing the wounding toe-clipping method. Skin and cloaca swabbing are even less invasive and easier to handle than buccal swabbing, but could result in contaminations of genetic material. Therefore, we test if external skin and cloaca swabs are as reliable as buccal swabs for genetic analysis of amphibians. We analysed eight microsatellite loci for the common frog (Rana temporaria, Linnaeus 1758) and compared genotyping results for buccal, skin and cloaca swabs regarding allelic dropouts and false alleles. Furthermore, we compared two DNA extraction methods regarding efficiency and cost. DNA quality and quantity (amplification success, genotyping error rate, in nanogram per microlitre) were comparable among DNA sources and extraction methods. However, skin and cloaca samples exhibited high degrees of contamination with foreign individuals, which was due to sample collection during mating season. Here, we established a simple low budget procedure to receive DNA of amphibians avoiding stressful buccal swabbing or harmful toe clipping. However, the possibility of contaminations of external swabs has to be considered.