Ohrid Trout Research Articles

Distribution and Genotyping of Infectious Hematopoietic Necrosis Virus in Farmed Rainbow Trout and Autochthonous Salmonids in North Macedonia

Abstract Infectious hematopoietic necrosis (IHN) is a common disease in the intensive production of salmonids. The IHN virus (IHNV) was isolated for the first time in North Macedonia in 2018. This study aimed to investigate the distribution and genotype of IHN in farmed rainbow trout and autochthonous salmonid fish in North Macedonia following the first detection. The samples were collected from 47 trout farms. Trout fry with or without clinical signs of IHN were selected as individual samples. Kidney, spleen, and heart were taken from each fish during the dissection. Three pooled samples were collected from each farm. A total of 141 pooled samples were collected: Rainbow trout (Oncorhynchus mykiss) n=127, Macedonian trout (Salmo macedonicus) n=11, and Ohrid trout (Salmo letnica) n=3. The virus was detected in 43 samples (30.50%): rainbow trout (n=40), Macedonian trout (n=2), and Ohrid trout (n=1). There were 18 (38.30%) positive fish farms. The MAKIHNV1 isolate from 2018 (MN641902) and the newly isolated virus shared a similarity of >99 and were placed in clade E-1 of European genogroup E. The IHN has spread throughout the country and is also present in the autochthonous salmonids.

A Combined Approach of Principal Component Analysis and Support Vector Machine for Early Development Phase Modeling of Ohrid Trout (Salmo Letnica)

Ohrid trout (Salamo letnica) is an endemic species of fish found in Lake Ohrid in the Former Yugoslav Republic of Macedonia (FYROM). The growth of Ohrid trout was examined in a controlled environment for a certain period, thereafter released into the lake to grow their natural population. The external features of the fish were measured regularly during the cultivation period in the laboratory to monitor their growth. The data mining methods-based computational model can be used for fast, accurate, reliable, automatic, and improved growth monitoring procedures and classification of Ohrid trout. With this motivation, a combined approach of principal component analysis (PCA) and support vector machine (SVM) has been implemented for the visual discrimination and quantitative classification of Ohrid trout of the experimental and natural breeding and their growth stages. The PCA results in better discrimination of breeding categories of Ohrid trout at different development phases while the maximum classification accuracy of 98.33% was achieved using the combination of PCA and SVM. The classification performance of the combination of PCA and SVM has been compared to combinations of PCA and other classification methods (multilayer perceptron, naïve Bayes, random committee, decision stump, random forest, and random tree). Besides, the classification accuracy of multilayer perceptron using the original features has been studied.

Changes in copper load in hepatocytes of Ohrid trout in relation to the ovarian maturation cycle

The main aim of this study was to histochemically estimate the relative copper content in the liver during the breeding cycle of female Ohrid trout, a wild population endemic to the Ohrid Lake. The histology of the oogenetic cycle in this species was documented for the first time. Histochemistry with semi-quantification revealed positive correlations between the gonadosomatic indices and copper load. Uptake of the metal increases in the liver as the ovaries increase in size, with the highest hepatic copper load occurring postspawning. The plasmatic levels of 17β-oestradiol were also quantified, but no correlations to copper load were found, so there is no direct estrogenic control on the uptake and release of copper from the bloodstream to either organ. Therefore, when interpreting field studies or assays on hepatic copper pollution and toxicity, breeding-related seasonal changes in Ohrid trout must be taken into account.

Heavy metals risk assessment in Salmo letnica from Lake Ohrid in Albania

The Ohrid trout (Salmo letnica) is an endemic species of trout found only in Lake Ohrid, in the Balkan republics of North Macedonia and Albania, where it is a species of significant economic and nutritional interest. Considering the importance of chemical risk assessment in fish for human consumption set out by European and international legislation and the risk of extinction of the Ohrid trout, the aim of the present study was to evaluate the safety of Ohrid trout flesh for human consumption by determining the heavy metal concentration in different organs through atomic absorption spectrometry (AAS). Heavy metal concentrations differed significantly in different organs (P<0.001). The mean and range of Pb and Cr concentrations in muscle were lower than the permissible limits recommended for human consumption according to the European Union (EU), as well as the Food and Agriculture Organization of the United Nations (FAO) and World Health Organization, whereas the mean concentration and range of Cd in muscle tissue were above the critical limits for human consumption set by the EU and FAO, revealing inspection-related problems. Accordingly, S. letnica is not completely safe for human consumption. Therefore, the long-term assessment of heavy metal concentrations in Ohrid trout from Lake Ohrid is an important safety measure for fish consumers and will help assess the status of exploitation of this biological resource.

HISTOCHEMICAL EVALUATION OF IRON CONTENT IN THE LIVER OF WILD FEMALE OHRID TROUT (SALMO LETNICA KAR.) ALONG THE BREEDING CYCLE

Iron is an essential micronutrient for the normal metabolism of all cells. Intracellular iron varies within normality ranges, and depletion or overload of brings serious consequences. As such, iron can also be deleterious when appearing as an environmental toxicant. The aim of this study was to estimate semi-quantitatively the liver iron content in female Ohrid trout, from the Lake Ohrid, during the breeding cycle. Seasonal variations in hepatocytic iron were observed. The maximum content occurred at post-spawning, which was significantly higher than loads detected at pre- and early-vitellogenesis. Positive correlations existed between iron amounts and stages of ovary maturation. By the contrary, there were no correlations between iron contents and plasma oestradiol levels. The new uncovered "iron-breeding associa-tion" strongly endorses that normal seasonal variations in hepatocytic iron content should be taken into consideration in field studies investigating and monitoring the metal burden and toxicological effects of metals on wild fish.

DO SPLEEN MACROPHAGE AGGREGATES UNDERGO SEASON-DEPENDENT CHANGES? A STEREOMICROSCOPIC INVESTIGATION IN WILD OHRID TROUT (SALMO LETNICA KAR.)

In past decades macrophage aggregates (MACs) were being used in many monitoring studies as useful immu-nological biomarkers of environmental pollution and they are still encountered in the so-called “future biomarkers”, since quantitative studies in aquaculture and/or field of their normal fluctuations as a result of the influence of natural factors are extremely rare. Our investigation was designed to quantitatively evaluate the influence of the seasons on MAC's relative volume, number and diameter in the Ohrid trout (Salmo letnica Kar.) spleen. Data showed that all investigated parameters had significantly higher values in fish collected during spring and winter. Correlation anal-yses revealed that the increasing of the relative volume of MACs is a result of the increasing of the relative volume of melanin loaded MACs, as well as the increasing of their number and diameter. These results confirmed that the sea-son can affect MACs variations and should be taken into consideration, especially when spleen MACs are used as pollution biomarkers in monitoring studies.

Seasonal changes in hepatocytic lipid droplets, glycogen deposits, and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)-A semiquantitative ultrastructural study.

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cell's structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late-vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well-studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700-706, 2016. © 2016 Wiley Periodicals, Inc.

Risks to stocks of native trout of the genus Salmo (Actinopterygii: Salmoniformes: Salmonidae) of Serbia and management for their recovery

Background. Insufficiently controlled stocking compromises the high diversity of wild trout stocks of Serbia. Native brown trout, Salmo cf. trutta Linnaeus, 1758, and Macedonian trout, Salmo macedonicus (Karaman, 1924), reveal remarkable diversity assessed using the mtDNA molecular markers, with the eight exclusive and several more widely spread haplotypes found in them. Four alien trout species and strains and one strain of Macedonian trout were introduced into the home areas of the native wild trout stocks in Serbia. In addition to them, wild trout stocks were also affected by farmed rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), and brook trout Salvelinus fontinalis (Mitchill, 1815), that regularly escape to streams, and from Ohrid trout, Salmo letnica (Karaman, 1924), and Arctic charr, Salvelinus alpinus (Linnaeus, 1758), stocked into streams and reservoirs. Risk of invasiveness that wild trout stocks are exposed to and their restoration were driving forces for this study. Materials and methods. Trout specimens from the Jerma River sampled in 2013 were additionally analysed for their mtDNA haplotype. The invasiveness potential of eleven alien trout species and strains introduced by stocking into wild brown- and Macedonian trout stocks in Serbia were assessed with the Fish Invasiveness Screening Kit (FISK). Results. Five of introduced trout species and strains were classified as having a high risk (sensu lato) and two of them as having a high risk (sensu stricto) of being (or becoming) invasive. Conclusion. Progressively rising and insufficiently controlled fishing and management with stocking of non-indigenous trout in wild brown trout stocks are the main current threats to the original diversity. Alien brown trout strains cross breed with native brown trout and incorporate into their stocks. Currently, feral rainbow-, brook-, and Ohrid trout reveal great invasive potential by naturalization in waters heavily stocked with them. The fisheries measures aiming to control and/or to eradicate alien strains of brown trout involve the restriction of stocking, landing of trout suspected as of alien strain or species, as well as the stringent control of stocking material used for the restorative stocking. They are mandatory, regarding the conservational dependence of wild brown trout stocks.

Risks to stocks of native trout of the genus Salmo (Actinopterygii: Salmoniformes: Salmonidae) of Serbia and management for their recovery

Background. Insufficiently controlled stocking compromises the high diversity of wild trout stocks of Serbia. Native brown trout, Salmo cf. trutta Linnaeus, 1758, and Macedonian trout, Salmo macedonicus (Karaman, 1924), reveal remarkable diversity assessed using the mtDNA molecular markers, with the eight exclusive and several more widely spread haplotypes found in them. Four alien trout species and strains and one strain of Macedonian trout were introduced into the home areas of the native wild trout stocks in Serbia. In addition to them, wild trout stocks were also affected by farmed rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), and brook trout Salvelinus fontinalis (Mitchill, 1815), that regularly escape to streams, and from Ohrid trout, Salmo letnica (Karaman, 1924), and Arctic charr, Salvelinus alpinus (Linnaeus, 1758), stocked into streams and reservoirs. Risk of invasiveness that wild trout stocks are exposed to and their restoration were driving forces for this study. Materials and methods. Trout specimens from the Jerma River sampled in 2013 were additionally analysed for their mtDNA haplotype. The invasiveness potential of eleven alien trout species and strains introduced by stocking into wild brown- and Macedonian trout stocks in Serbia were assessed with the Fish Invasiveness Screening Kit (FISK). Results. Five of introduced trout species and strains were classified as having a high risk (sensu lato) and two of them as having a high risk (sensu stricto) of being (or becoming) invasive. Conclusion. Progressively rising and insufficiently controlled fishing and management with stocking of non-indigenous trout in wild brown trout stocks are the main current threats to the original diversity. Alien brown trout strains cross breed with native brown trout and incorporate into their stocks. Currently, feral rainbow-, brook-, and Ohrid trout reveal great invasive potential by naturalization in waters heavily stocked with them. The fisheries measures aiming to control and/or to eradicate alien strains of brown trout involve the restriction of stocking, landing of trout suspected as of alien strain or species, as well as the stringent control of stocking material used for the restorative stocking. They are mandatory, regarding the conservational dependence of wild brown trout stocks.

Variations in the volumes of parenchyma and stroma of the liver and in the cytology of hepatocytes are associated with gonadal stages in female Ohrid trout (Salmo letnica)

Quantitative microscopic studies of season- and breeding-related changes in the liver of farm-raised fish are very scarce, and those of wild fish populations are virtually nonexistent. Moreover, none of the available studies investigated breeding-related changes in hepatic stroma, although it is already known that changes might exist in the parenchyma. To the best of our knowledge, ours is the first study using wild adult Ohrid trout females to evaluate quantitative influences of the breeding cycle on the volumes of the two liver compartments—parenchyma and stroma. Quantitative microscopy (stereology) using light microscopy was supplemented with semi-quantitative and quantitative analyses of hepatocyte cytology to express in numbers the seasonal changes in the extent of vacuolated versus basophilic cytoplasm. The liver volume and that of each compartment changed from that at the time of pre-vitellogenesis to the end of the spawning season. The changes in total volumes of parenchyma and stroma—increasing from pre-vitellogenesis to late vitellogenesis—followed the changes in total liver volume. Despite all the changes in total volumes, no differences existed in the relative proportions between the two major compartments. After spawning, the stromal volume continued to increase while that of the parenchyma reduced despite no detectable statistically significant impacts on the liver volume. Changes in both the liver and parenchyma volumes were correlated with the gonadosomatic index and plasma oestradiol levels. With this study, we first establish that fish liver remodelling might also occur in the stroma. When comparing Ohrid trout with other fish species, we noted interspecies similarities and differences with regard to the hepatocyte cytology during the breeding cycle. In our study, the semi-quantitative and stereological studies revealed that, from pre-vitellogenesis to post-spawning, although cytoplasmic vacuolation of the hepatocytes decreased, the basophilia increased. Overall, these cytological changes were only partially in agreement with the data from other trout studies. We believe that this is because of intrinsic interspecies differences in association with natural conditions. Thus, establishing normal patterns is crucial, at least for flagship species, namely to support interpretation of histopathological changes in biomonitoring.

Changes in the amount of kidney pigmented macrophage aggregates throughout the breeding cycle of female Ohrid trout, Salmo letnica Kar. (Teleostei, Salmonidae)

Changes in fish macrophages (Macs) are useful indicators of environmental pressures, but responses due to chemical and nonchemical stresses may be confounded by natural sources of variability. These may include sex and gonadal stage. In this study, we start addressing the following question: is the seasonally dependent ovary stage a factor to consider when using kidney Macs as biomarkers? To tackle this problem, the relative amount of pigmented Macs in kidney (head, trunk, and tail portions) was stereologically estimated in Ohrid trout, and related with the breeding status. The amount of Macs significantly increased from pre vitellogenesis to late vitellogenesis and showed a decreasing trend then after, with lower values noted after spawning in the head (1.9% versus 7.5% versus 2.0%), trunk (1.8% versus 7.5% versus 2.5%), and tail (2.5% versus 6.7% versus 2.9%) kidney. The decrease seen at spawning was significant in head and trunk kidney, and at post spawning it was significant for all kidney portions. The amounts of Macs were positively correlated with the ovary relative weights and plasma estradiol levels. We proved for the first time that fish kidney pigmented Macs can vary in amount during the breeding cycle. Our data, combined with literature, strongly support that the sex-steroid profile and kidney status-seasonal remodeling both influence the Macs pool; likely not only in female trout. So, while increases in Macs may warn of ecosystem problems, we show that using kidney Macs for biomonitoring should also take into account seasonally, particularly that related with ovary maturation.

Spleen histology in the female Ohrid trout, Salmo letnica (Kar.) (Teleostei, Salmonidae) during the reproductive cycle

The Ohrid trout is an endemic autochthonous species dating from the Tertiary period with an unique ecological and commercial value. We still lack the basic knowledge of normal histology of many visceral organs in this species, including the spleen. To tackle this limitation, the investigation was focused on a histological description of the spleen and the influence of the breeding cycle on some aspects of the spleen (the relative volumes of the red and white pulp). Examination by light microscopy showed the same basic structural features of the salmonid spleen: red and white pulp with randomly distributed melanomacrophage centers, surrounded by a fibrous capsule. White pulp comprises about 35-40% of the volume of the parenchyma, 2-fold less than red pulp. During the breeding cycle, we observed a significant increase of white pulp and decrease of red pulp in the spawning stage compared to earlier stages. After spawning, the relative volumes of white and red pulp exhibit a tendency to decrease and increase, respectively. Based on correlations between the amount of white and red pulp and the ovary somatic ratio, we assumed there could be a connection between the sex steroid status and the pulp content in the spleen.

Introgression of distant mitochondria into the genome of Gyrodactylus salaris: Nuclear and mitochondrial markers are necessary to identify parasite strains

AbstractNovel combinations of mitochondrial DNA (CO1) and internal transcribed spacers of nuclear ribosomal DNA (ITS) were detected among Gyrodactylus parasites on brown trout (Salmo trutta L.), rainbow trout (Oncorhynchus mykiss (Walbaum)), and Ohrid trout (Salmo letnica (Karaman)) from salmonid farms in Poland and Macedonia. Some clones differed from standard ITS only by ≤ 4 nucleotides, but they belonged to a mtDNA clade that differed from the Northern European lineages of G. salaris by d MCL = 0.266 ± 0.108 (maximum composite likelihood distance). The divergence of d MCL = 0.013 ± 0.005 within the alien mtDNA clade suggested introgression from an unknown maternal ancestor into the G. salaris Malmberg genome 137 to 57 kyr ago (or, less probably, repeated introgression). A comparable modern hybrid was detected based on permanently heterozygous ITS (28 bp/1264 = 2.2%) in a clone that is widespread throughout Finnish rainbow trout farms. This was a F1 hybrid of maternal G. pomeraniae Kuusela, Ziętara et Lumme (on roach, Rutilus rutilus (L.)) and G. lavareti Malmberg (on whitefish, Coregonus lavaretus (L.)). The mtDNA of the parental species differed by d MCL = 0.290 ± 0.130. The observations emphasize that both nuclear and maternally-inherited DNA markers are necessary to characterize sexually-produced lineages or clones of Gyrodactylus. The hybridization events demonstrated predict incongruence of mitochondrial vs. nuclear gene trees, i.e., reticulate evolution in species trees.

Crystalline inclusions in hepatocytes and associated interhepatocytic macrophages from female Ohrid trout ( Salmo letnica Kar.)

When characterizing the liver ultrastructure in Ohrid trout, we noticed that cells often displayed unusual cytoplasmic crystalline inclusions. Their morphology varied much, concerning the size, shape and electron density, showing aspects never reported in fish liver. In hepatocytes, the inclusions existed in close topographical relationship with dense bodies (often within them). Such “crystals” inclusions also existed in macrophages and in biliary passages lumina. Data revealed seasonal/breeding-related changes of the dense bodies and crystalline inclusions within hepatocytes; decreasing from the earliest to the more advanced ovary maturation stages. Additionally, based on the negative correlations between the amounts of dense bodies and of “crystals” and the ovary somatic ratio, we suggested there might be connections between the sex steroids status and the inclusions formation. A positive correlation between the quantities of “crystals” and of dense bodies further suggested that the inclusions might derive from normal processes, e.g., the females’ cyclic liver changes, that involves dense bodies’ turnover. However, a toxicological aetiology cannot be excluded. Additionally, multiple mechanisms can contribute to the “crystals” genesis. Facing literature reports and because the inclusions content seemed washed out during tissue processing, they are likely lipid in nature, but their exact composition and genesis require further analyses.

A qualitative and quantitative study of the hepatic pigmented macrophage aggregates during the breeding cycle of ohrid trout, Salmo letnica Kar. (Teloestei, Salmonidae)

It is well known that factors such as aging and water quality may influence pigmented macrophages (PMacs) in fishes, but it is not established yet if PMacs undergo seasonal and breeding dependent variations. This study explored this caveat and reports qualitative histological and stereological data on liver PMacs from wild female Ohrid trout, Salmo letnica, during the annual breeding cycle. Data showed that a minority of PMacs contained melanin and that the vast majority of them contained only hemosiderin or hemosiderin and lipofuscin/ceroid pigment. It was suggested that this is the normal scenario for the species. One remarkable result was the demonstration of a striking increase, after spawning, of the relative and total volumes of the hepatic macrophages, both parenchyma and stroma located. Because the melano PMacs did not vary, those changes were due to fluctuations in the hemosiderin-laden PMacs. We concluded that Ohrid trout presented a pigment composition in liver macrophages that differed from other fish, including salmonids, where most liver phagocytes essentially display melanin. Our quantitative data support interspecies differences in the amount of liver macrophages and also that after spawning expansion of the macrophage pool is crucial and most likely connected with the needs of liver remodeling (leading to a decrease in hepatic mass). So, we suggested that the hormonal (sex steroidal) constellation influenced the liver macrophage pool. Additionally, we proved that the use of fish liver macrophages for biomonitoring should take into account the considerable natural breeding/seasonal dependent variations that are expected to occur.

A quantitative study of the hepatic eosinophilic granule cells and rodlet cells during the breeding cycle of Ohrid trout, Salmo letnica Kar. (Teloestei, Salmonidae)

Fundamental questions remain on the nature and role of fish eosinophilic granule cells (EGCs) and rodlet cells (RCs). A poorly studied aspect is their suspected seasonality of occurrence under normal circumstances. In the liver of female Ohrid trout, Salmo letnica, we detected EGCs and RCs associated with stromal components. Both cell types could be considered a normal liver component in that wild fish population. A stereological study was performed to investigate eventual seasonality / breeding-related alterations in the amount (relative and total volumes) of EGCs and RCs. Differences only existed in the quantity of RCs; increasing from the earliest stages to the more advanced stages of ovarian maturation. EGCs made a greater pool than RCs only at early- and late vitellogenesis. Based on the breeding related fluctuations we suggest there is a hormonal regulation, by sex steroids, of the RCs liver content. Moreover, our data endorses the idea that, when using changes in the RCs pool as a biomarker, the fish breeding status must be considered as a potential source of changes in the RCs pool.

Historical demography of brown trout ( Salmo trutta) in the Adriatic drainage including the putative S. letnica endemic to Lake Ohrid

We explore the historical demography of the Adriatic lineage of brown trout and more explicitly the colonization and phylogenetic placement of Ohrid trout, based on variation at 12 microsatellite loci and the mtDNA control region. All Adriatic basin haplotypes reside in derived positions in a network that represents the entire lineage. The central presumably most ancestral haplotype in this network is restricted to the Iberian Peninsula, where it is very common, supporting a Western Mediterranean origin for the lineage. The expansion statistic R2, Bayesian based estimates of demographic parameters, and star-like genealogies support expansions on several geographic scales, whereas application of pairwise mismatch analysis was somewhat ambiguous. The estimated time since expansion (155,000 years ago) for the Adriatic lineage was supported by a narrow confidence interval compared to previous studies. Based on microsatellite and mtDNA sequence variation, the endemic Ohrid trout represents a monophyletic lineage isolated from other Adriatic basin populations, but nonetheless most likely evolving from within the Adriatic lineage of brown trout. Our results do not support the existence of population structuring within Lake Ohrid, even though samples included two putative intra-lacustrine forms. In the interests of protecting the unique biodiversity of this ancient ecosystem, we recommend retaining the taxonomic epithet Salmo letnica for the endemic Ohrid trout.

Genetic and morphological characterization of a Lake Ohrid endemic, Salmo (Acantholingua) ohridanus with a comparison to sympatric Salmo trutta

Analysis of both uni‐(two mtDNA gene sequences) and bi‐parentally (seven microsatellite loci) inherited genetic markers, together with analysis of 40 morphological characters, described Salmo ohridanus as a highly divergent member of the genus Salmo. Based on comparative substitution rate differences at the cytochrome b gene, and a rough estimated age of the Salmo trutta complex (i.e. at least 2 million years), the S. ohridanus and Salmo obtusirostris clade probably split from a common ancestor of brown trout Salmo trutta >4 million years ago, overlapping with minimum age estimates of the formation of Europe's oldest freshwater habitat, Lake Ohrid. Comparative analysis with Lake Ohrid brown trout (known regionally as Salmo letnica), supported the notion that these fish have more recently colonized the lake and phylogenetically belong to the Adriatic lineage of brown trout. It is further suggested that species‐specific saturation in the mtDNA control region underestimated the divergence between S. ohridanus and S. trutta. Evidence of rare hybridization between S. ohridanus and Lake Ohrid brown trout was seen at both mtDNA and microsatellite markers, but there was no support for extensive introgression.

Early development of the chondrocranium in Salmo letnica(Karaman, 1924)(Teleostei: Salmonidae)

The ontogenetic development of the chondrocranium of Ohrid trout Salmo letnica was studied from hatching until 92 days post‐hatching (dph). Most of the samples were in toto trypsin cleared and stained, some specimens were used for serial histological sectioning. The serial histological sections of fish specimens at the age of 92 dph were used for a graphical reconstruction of the cartilaginous neurocranium. A chronological evaluation of the formation of the cartilaginous skull in the early development of S. letnica was performed. In order to investigate to what degree the ontogeny of the Ohrid trout is unique, the results were compared with data of the development of other salmonids, as well as some non‐salmonid teleosts. The development of the cartilaginous structures of the Ohrid trout was found to be similar to that of other salmonids. Most of the cartilage structures of the neurocranium and the viscerocranium are present at the moment of hatching of this species. A fully developed chondrocranium was observed at the age of 92 dph, when the first signs of cartilage resorption could also be observed.

Mitochondrial DNA differentiation between two forms of trout Salmo letnica, endemic to the Balkan Lake Ohrid, reflects their reproductive isolation

Mitochondrial haplotype diversity in sympatric populations of Ohrid trout, Salmo letnica was investigated by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mtDNA control region and ND1, ND3/4, ND5/6 segments. A 310 bp fragment at the 5' end, and a 340-572 bp fragment at the 3' end of the control region were sequenced from representatives of the populations studied. Based on pairwise comparison of the sequences, five new haplotypes were identified plus one identical with the brown trout Andalusian haplotype from the southern Iberian Peninsula. The combination of both RFLP and sequence data sets yielded a total of 10 composite haplotypes. A high degree of genetic subdivision between S. letnica typicus and S. letnica aestivalis populations was observed. The notion of a sympatric origin for the two morphs is discussed. Length variation of the mtDNA control region due to the presence of an 82 bp unit, tandemly repeated one to four times, in the region between the conserved sequence block-3 (CSB-3) and the gene for phenylalanine tRNA is reported. Further, we demonstrate that a single duplication of the approximately 82 bp repeat unit is a common element of the salmonid mitochondrial control region. The unique genetic structure of Ohrid trout represents a highly valuable genetic resource that deserves appropriate management and conservation.