Zvonimir Kozarić

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.

Yersinia ruckeri septicaemia in experimentally infected carp (Cyprinus carpio L.) fingerlings.

The presence of Yersinia ruckeri, the causal agent of enteric redmouth disease (ERM) in salmonids and a few other freshwater fish, has so far been reported from a variety of sources including the intestine of healthy carp. Since there are no data on the pathogenicity of this bacterium for carp, 15 fingerlings were experimentally infected by intraperitoneal injection of about 5 x 10(5) cells. Thirteen injected fish were moribund or died within 4 days with septicaemic lesions. Two survivors were sampled on Day 28 after infection. Yersinia ruckeri was reisolated from the internal organs of all experimental fish. By histopathological examination moribund fish had generalised bacteriaemia with inflammation, degeneration and necrotic foci in kidney, liver and spleen, corresponding to findings described previously in ERM of rainbow trout. Survivors of challenge on Day 28 had a chronic disease characterised by prominent peritonitis and enteritis, exhaustion of the erythroid, granuloid and lymphoid components in haematopoietic kidney tissue as well as focal degeneration and necrosis in organs. These data indicate a high sensitivity of carp to intraperitoneal infection with a relatively low dose of Y. ruckeri.

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.

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 distribution of digestive enzymes in the intestine of the common two-banded seabream, Diplodus vulgaris, Geoffroy St-Hilaire 1817.

The histochemical localization of non‐specific esterase, alkaline and acid phosphatase as well as aminopeptidase in the intestine of the free‐living common two‐banded sea bream (Diplodus vulgaris) was investigated. Fish were caught near the town of Zadar (Adriatic Sea, Croatia). Samples of pyloric caeca and three parts of the intestine proper (anterior, middle and posterior) were used for the description of non‐specific esterase, alkaline and acid phosphatase as well as aminopeptidase. Non‐specific esterase activity was found in the cytoplasm of enterocytes in pyloric caeca and in all investigated intestinal segments. The activity was stronger in the anterior and posterior part of the intestine than in the pyloric caeca and middle segment of the intestine. Intestinal alkaline phosphatase was detected in brush border of enterocytes of all investigated intestinal segments. Enzymatic activity gradually decreased in a posterior direction. Acid phosphatase activity was observed as a fine granular reaction product in the supranuclear region of enterocytes. This activity was almost equal in pyloric caeca as well as in the anterior intestinal segment, while it was stronger in the middle and posterior intestinal segment. Aminopeptidase was present along the intestinal epithelium brush border in all investigated parts of the digestive tube. The intensity of aminopeptidase increased posteriorly. The possible role of investigated enzymes in intracellular digestion and transport is discussed.

Zeolite clinoptilolite as a dietary supplement and remedy for honeybee (Apis mellifera L.) colonies

Control of the nosema disease poses a major challenge, and therefore, treatment of this serious para - sitic disease using natural preparations could be of great benefit. The aim of this study was to test the performance of zeolite clinoptilolite as a curative measure against honeybee colonies ( Apis mellifera L.) naturally infected by Nosema ceranae. The histopathological structure, and the content and distribution of mucosubstances and his - tochemical activity of aminopeptidase and non-specific esterase in the midgut mucosa of honeybees originating from colonies fed sugar syrup supplemented with zeolite minerals was studied. A decline in the number of spores in honeybees fed with zeolite clinoliptolite was observed on the first sampling day (Day 10; 53.25 15.15 million spores/bee), though a statistically lower number of spores in comparison to the control was confirmed on Day 20 (41.08 ± 9.4 million spores/bee), Day 30 (28.42 ± 7.79 million spores/bee) and Day 40 (24 ± 6.25 million spores/bee). The possibility of using natural zeolites as a dietary supplement for honeybee colonies as a preventative measure and for the reduction of the deleterious effects of nosemosis is discussed.

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.

Whale Poaching Detection Based on Microscopic Characteristics of Bottlenose Dolphins’ (Tursiops truncatus) Bone Fragments

Although many countries prohibit whaling, it remains a significant cause of whale population decline. The meal of whales, likely killed during illegal whaling or caught accidentally, can ap-pear in a fish meal as a contaminant detectable by microscopic examination of bone fragments. In order to provide a rigorous basis for such a detection, microscopic characterization of bone fragments of 10 female and 10 male, less than a year to 21 years old bottlenose dolphins (Tur-siops truncatus), a widespread cetacean species herein used as a whale model, was performed and compared to the reference bone fragments of fish, ruminant, poultry and pig. The processing of bones mimicked that used in the production of meat and bone meals, while their description was based on qualitative characteristics, i.e. the shape of a bone fragment ; the shape, density and distribution of osteocyte lacunae, and the distribution and density of canaliculae. Bottlenose dol-phin bone fragments are smoothly contoured ; their elliptical osteocyte lacunae are clearly visible, while the canaliculae radiate from the lacunae in all directions. The comparison of these qualita-tive bone characteristics with that of other vertebrata under study revealed that bottlenose dol-phin bone fragments can definitely be differentiated from that of fish, may be differentiated from that of poultry and pig, but cannot be differentiated from that of ruminants. Measurements of the osteocyte lacunae showed the lacunar length to be strongly associated with animal’s age and lacunar shape, while their width was strongly associated with animal’s gender lacunar shape. The results indicate the possibility of detecting a whale meal admixture in a fish meal using light microscopy, which should be followed by PCR to enable the identification of the admixture source.