Emil Gjurčević

The Development of the Digestive Tract in Larval European Catfish (Silurus glanis L.)

The European catfish, Silurus glanis L., has become an important aqua cultural fish in Croatia, and it is cultivated extensively in ponds in polyculture with carps. The development of the digestive tract in S. glanis was studied with the aim of improving intensive fish production. Research was carried out on S. glanis larval stadium from 1‐ to 19‐day post‐hatching (DPH). The main histological methods used were: haematoxylin and eosin staining, periodic acid Schiff staining (PAS), Alcian blue (AB) and toluidin blue staining (TB). A yolk sac was present during the first 5 days (1–5‐DPH). During the initial 3‐DPH period, there was no trace PAS and AB activity in the digestive tract. Differentiation of the digestive tract began at 3‐ to 5‐DPH. The oesophagus was positive for AB at 5‐DPH, PAS and TB after 7‐DPH. Differentiation of enterocytes began at 5‐DPG and the intestines were complete at 11‐DPH. Development of liver and pancreas was also studied. The analysis of data obtained in this study suggests that after 5‐DPH catfish larvae have morphologically completed digestive tracts.

Metacercariae of Centrocestus formosanus in goldfish (Carassius auratus L.) imported into Croatia

SummaryThe presence of metacercariae of Centrocestus formosanus was detected on the gills of goldfish (Carassius auratus L.) imported from Singapore and obtained from two pet shops. This is the first report of the parasite in Croatia. The histopathological changes observed lead to the conclusion that the infection could be one of the reasons for the death of goldfish.

Osteological Development of the Garfish (Belone belone) Larvae

Garfish, Belone belone (Linnaeus, 1761) is an elongate, slander fish inhabiting the Eastern Atlantic and Mediterranean Sea. These fish from the Belonidae (Actinopterygii) family have important commercial value for Croatian fisheries. Samples for research were collected from experimental hatching in the Novigrad Sea. Preparation techniques included fixation in buffered formalin, trypsin clearing and staining with alcian blue and alizarin red. As little is known of garfish osteology and bone morphology, the main goal of this study was to describe ossification process in garfish fry. At hatching, no skeletal structure is present. Newly‐hatched larvae also had no osteological elements. Ossification started at 7 day post‐hatching (DPH) [total length (TL) 18 mm] with head bones and vertebral neural arch. Head skeleton continued to develop mostly over the period from 7 to 10 DPH. At 21 DPH (TL 49 mm), ossification process seemed to be finished, but it was not possible to distinguish borders of all bones. The primary interest of our research was to understand the growth dynamics as well as transformation of supporting body elements from cartilage to bone. At the end, developmental characteristics and functional aspects of this formation in different fish species are discussed.

Pomphorhynchus laevis (Acanthocephala) from the Sava River basin: New insights into strain formation, mtDNA-like sequences and dynamics of infection.

Here we report the genetic variability and presence of mtDNA-like sequences of Pomphorhynchus laevis from the chub, Squalius cephalus, caught at the sampling sites along the Sava River and its tributary the Sutla River in Croatia. Sequences of the cytochrome c oxidase subunit 1 (COI) gene of the recovered P. laevis specimens were used for haplotype network construction and phylogenetic analysis. These analyses showed that some specimens contained mitochondrial-like sequences, and they uncovered the existence of a Sava River basin strain different from known strains of P. laevis. This is the first time that P. laevis has been shown to contain mtDNA-like sequences, suggesting the need to exercise caution during COI analyses of P. laevis using universal primers. Highly conserved sequences of two nuclear markers, the ITS region and 18S rRNA, were not helpful for understanding genetic variability or differentiating strains. Furthermore, analysis of the dynamics of P. laevis infections in S. cephalus from the Sava and Sutla Rivers showed decreased prevalence and abundance at sites with inferior water quality, positive association of parasite abundance with fish size, and no clear association of parasite abundance with fish condition index or sex.

Spontaneous multicentric myxoma of the dermal nerve sheaths in farmed European eels Anguilla anguilla

This report describes a peripheral nerve sheath tumour in 8 European eels Anguilla anguilla L. from a fish farm located in Croatia. The newborn tissue appeared as smooth and soft skin nodules without pronounced colour change. Nodules were dome-shaped with a pale crater and were present on different body areas. In general, nodules were located as series of differently sized protrusions extending along the lateral line on both sides of the fish, as well as sensory canals on the head. Cut sections showed a homogeneous, pale white-grey texture. Histologically, the pathological tissue was located in the dermis, occasionally intruding into the hypodermis, and pushing as a space-occupying mass against the underlying muscle tissue without any evident boundaries. The pressure also caused changes in the overlying epidermis, such as atrophy, spongiosis and erosion. In some areas, the epidermis was 1 cell thick and club and goblet cells had completely disappeared. Ultimately, these changes resulted in shallow ulceration. Tumour tissue was characterized by a scant population of spindle or stellate cells, with oval, hyperchromatic nuclei and pale cytoplasm embedded in a copious myxoid matrix. Cells were arranged in fascicles and whorls, extending in a poorly defined manner among the dermal collagen bundles. Occasionally, adipose cells were also detected, mainly in the central portion of the bulges. Myxoid areas appeared rich in metachromatic and alcianophilic mucous ground substance. Reticular fibres and collagenous connective tissue were scarce. Immunohistochemistry (IHC) using antibodies against S-100 and glial fibrillary acidic protein caused a slight positive reaction in neoplastic dendritic cells. High magnification showed the immunostaining to be cytoplasmic in all tumour cells. IHC with anti-calretinin antibody gave only negative results. Macroscopic, histological, histochemical and immunohistochemical findings were consistent with a diagnosis of multicentric myxoma of the dermal nerve sheaths, a tumour not yet reported in fish.

Morphological and histochemical study of intestine in wild and reared European eel (Anguilla anguilla L.).

Diet-related differences between the ratio of intestine length to body size and the enzymatic activity in the intestinal tract of wild and reared European eel (Anguilla anguilla L.) were studied. Compared with reared eel, wild eel showed significantly shorter relative intestine length. For the purpose of histochemical examination, different parts (anterior, middle and posterior) of intestine proper were used. Activities of non-specific esterase, alkaline and acid phosphatase, and aminopeptidase were examined in each segment. All enzymes were present in the intestines of both wild and reared European eel. Fish from both groups showed similar enzyme distribution within the enterocytes, but distribution and intensity of enzyme activity along the intestine vary depending on the group. Generally, reared European eel showed highest enzymatic activity and wider distribution of enzymes throughout all parts of the intestine. These results suggest that different diets could be one of the reasons for observed changes.

Histochemical Analyses of Digestive Enzymes in the Intestine of Adult Large‐Scaled Gurnard (Lepidotrigla Cavillone, Lacepède, 1801)

With 9 figures and 2 tables

Histological investigations of Eimeria infection in large-scaled gurnards, Lepidotrigla cavillone (Lacepède, 1801) from the Novigrad Sea, Croatia

The presence of an Eimeria sp. was found in adult large-scaled gurnards Lepidotrigla cavillone (Lacepède, 1801) from the Novigrad Sea, in Croatia. The detection of Eimeria sp. in the pyloric caeca and intestines of the large-scaled gurnards is also the first report of the parasite in this fish species. The analysis of histopathological changes in the digestive tract led to the conclusion that the parasite could be a serious pathogen in heavily infected large-scaled gurnards.

Structural changes in the rat placenta during the last third of gestation discovered by stereology

Structural changes in the rat placenta during the last third of gestation were for the first time assessed by stereology. Fischer female rats were euthanized on the day 16 or day 19 of gestation, and 35 placentas were collected. Three randomly selected placentas from each group were stereologically analyzed for the absolute volume. The proportion of the glycogenic cells and the trophoblast giant cells (TGC) in the basal part of the placenta was calculated using volume density. The absolute volume of the rat placenta on the day 16 of gestation was determined as 0.0638 cm3. The labyrinth comprised 0.0274 cm3, the basal plate 0.0271 cm3 and the decidua 0.0093 cm3. On the day 19 of gestation, the absolute volume of the placenta was 0.1627 cm3, the labyrinth occupied 0.0922 cm3, the basal plate 0.0596 cm3 and the decidua 0.0109 cm3. The volume density of trophoblast giant cells was 0.174 cm0 on the day 16 and 0.107 cm0 on the day 19 of gestation. The glycogenic cells comprised 0.379 percentage of the basal plate on the day 16 and 0.236 on the day 19 of gestation. We conclude that the absolute volume of the whole placenta and the labyrinth has increased from day 16 to the day 19 of gestation. In contrast, the volume density of glycogenic cells and trophoblast giant cells was higher on the day 16 than on the day 19 of gestation, probably due to the intensive trophoblast invasion during that time.

Reply to comment on "Structural changes in the rat placenta during the last third of gestation discovered by stereology"

Dear Editor, We are thankful to our colleagues for their interest in our paper and comments that they have provided. We could not agree more with the part of the letter regarding the method of sampling. On the basis of our 16-year-long experience in research on rat placentas, we underline sampling as one of the most important concepts in stereology, especially in the light of biological variability [1]. This variability is the reason why we approach sampling with the greatest care, as it is evident from all our published papers in the field of stereology. In addition, we consider it as one of the best methods for quantification of biological materials. Systematic random sampling, which combines both the unbiasedness of random sampling and the efficiency of a systematic sampling, was used in our research. It is based on the selection of the final sample systematically, while the first sample was selected randomly within the first sampling interval [2]. Sections were sampled at a ratio 1:10 (Section Sampling Fraction-SSF), with a random section (RS) selection from the first ten sections (in this case, the 3rd section) and then every 10th section after this initial selection. Preliminary measurements were performed for each of the experimental groups. In addition, due to its small size, it is possible to analyze the entire section of the rat placenta as a single field, which we did, as seen in Figure 2 [3]. However, this is not possible for human placenta, which, due to its larger size, cannot fit into a single section or single field for stereological analysis.