First insights into the activity of major digestive enzymes in the intestine of the European catfish Silurus glanis and protective anti-enzymatic potential of its gut parasite Silurotaenia siluri.

The European catfish Silurus glanis is attracting growing interest as an object of fisheries and aquaculture, which is reinforced by the expansion of its natural range under climate change. Shaping the effective exploitation strategy for this valuable species requires detailed knowledge of its biology, including feeding and digestion processes, especially near the natural limits of the species range. Meanwhile, the digestion physiology of the European catfish remains poorly explored, including the activity of major digestive enzymes and the possible effect of intestinal parasites on this activity. In this regard, the activity of proteinases and α-amylase in the intestinal mucosa of the catfish was studied. Adult catfish were collected in the Rybinsk reservoir (Upper Volga) located close to the northern limit of the species range. It was shown that all subclasses of intestinal digestive proteinases, including serine proteinases, metalloproteases and cysteine (thiol) proteinases, function in the gut mucosa of the catfish. The mucosal levels of total proteolytic activity depended on fish size, in contrast to those of trypsin, chymotrypsin and α-amylase. The level of chymotrypsin activity was significantly higher than that of trypsin activity. It was also found that the incubation medium and extract of the cestodes Silurotaenia siluri parasitizing the catfish gut had a significant inhibitory effect on the activity of serine proteases (trypsin and chymotrypsin) operating in the intestines of the host fish.

Surviving in the fish gut: Comparative inhibitory capacities against the host proteinases in cestodes of the genus Proteocephalus.

Currently, little is known about inhibitory substances enabling tapeworms to settle in fish intestines thereby avoiding proteolysis. Contrary to previous studies with certain host-parasite pairs, this research compares the inhibitory capacities in three tapeworm species of the same genus Proteocephalus from four different fishes (P. torulosus from dace and zope, P. sagittus from stone loach and P. cernuae from ruffe). The tapeworm extracts studied significantly reduced the activity of commercial trypsin (although to a lesser degree than the synthetic inhibitor of serine proteinases PMSF), displaying clear inter-specific variation in worms' inhibitory ability. We also measured the proteolytic activity of the host intestinal mucosa exposed to tapeworm extracts which served as inhibitors. Based on per cent inhibition values, all tapeworm extracts significantly suppressed the mucosal proteolytic activity, with marked differences between certain host-parasite pairs. SDS-PAGE electrophoresis of the incubation media and extracts detected in each tapeworm species 20-36 protein bands with apparent molecular weights from 10-12 to 312.5 kDa, mostly below 50 kDa. The incubation medium and extract of each parasite shared one to six bands ranging from 12 to 35 kDa, depending on its species, with only four bands common for two or more species. The band profiles suggest that in various Proteocephalus species inhibitory capacities against host proteinases can be ensured by different proteins.

Localization of the proteinase inhibitor activity in the fish cestode Eubothrium rugosum.

The mechanisms enabling fish tapeworms to avoid proteolytic attacks by digestive enzymes of their fish host have been studied in less detail compared with mammalian cestodes. This study aimed to assess the inhibitory ability towards trypsin and chymotrypsin in Eubothrium rugosum, an intestinal parasite of burbot Lota lota, and establish its localization in the tapeworm. To this end, the worms were treated with Triton X-100 followed by differential centrifugation to isolate the tegumental brush border membrane. The protease inhibitory abilities of the worms were mostly determined by their excretory/secretory products released into the incubation medium. These inhibitory abilities proved to be linked mainly with the brush border fractions. Notably, the per cent inhibition of both studied digestive enzymes (trypsin and chymotrypsin) hardly depended on the duration of the parasite exposure in the incubation medium, probably due to intermittent glycocalyx renewal. Improved knowledge on functions of the excretory/secretory proteins produced by fish tapeworms may contribute to a better understanding of host-parasite relations and development of new approaches to the treatment and prevention of diseases caused by pathogenic helminths.

Activity of proteolytic enzymes in the intestine of bream Abramis brama infected with cestodes Caryophyllaeus laticeps (Cestoda, Caryophyllidea).

Adaptive mechanisms underlying the long-term existence of intestinal parasites in their enzymatically hostile environment are still poorly understood, particularly with regard to fish cestodes. The study describes the activity distribution of proteolytic enzymes along the gut of the bream Abramis brama infected with intestinal cestodes Caryophyllaeus laticeps and characterizes the capacity of these worms to inhibit host proteinase activity. Mucosal proteolytic activity was mainly presented by serine proteinases. The research revealed an insignificant increase in total proteolytic activity from anterior and middle to posterior part of the gut accompanied with changes in proportions of various proteinase subclasses along the intestine. The trypsin (but not chymotrypsin) activity in the posterior section was significantly higher than in the mid-section. Both the incubation medium of the worms and their extract had a significant inhibitory effect on mucosal proteolytic activity and commercial trypsin samples. In both instances, the effect was comparable with that of a synthetic serine protease inhibitor, PMSF. SDS-PAGE electrophoregrams of the incubation medium of C. laticeps and its extract revealed three common protein bands, with apparent molecular masses from 19 to 47 kDa, possibly responsible for the worms' inhibitory capacities. According to casein-zymography performed, the target host proteinases for a putative cestode inhibitor (inhibitors) have an approximate molecular weight of 28-53 kDa. A comparative test with the extracts from three other cestodes showed that each of them can suppress the proteolytic activity of the bream mucosa. The level of inhibitory activity was found to increase with protein content in the extracts of these tapeworms.