Mannitol oxidase catalyses the oxidation of D-mannitol to produce the sugar mannose, releasing hydrogen peroxide. This enzyme was detected in the digestive gland and digestive tract of some herbivorous gastropods, suggesting a role in metabolization of mannitol contained in their foodstuff [1, 2, 3]. Mannitol is a 6 carbon polyalcohol present in algae, fungi and plants. Therefore, it seems that herbivorous gastropods get nutritional benefits from the ingestion of polyalcohols present in algae and plants due to enzymes that convert these compounds into sugars [1]. Cell fractionation studies revealed that in the digestive gland of terrestrial gastropods mannitol oxidase is associated with a special kind of tubules with a diameter of 40 nm [3]. But, so far, most studies about mannitol oxidase were performed with terrestrial gastropods.To extend existing knowledge, this enzyme was investigated in the digestive gland of the marine gastropods Aplysia depilans, Bulla striata, Siphonaria pectinata and Onchidella celtica, as well as in the garden snail Cornu aspersum (=Helix aspersa) and the land slug Lehmannia valentiana. For histochemical location of mannitol oxidase digestive gland samples were frozen in liquid nitrogen. Cryostat sections 12 µm thick on glass slides coated with APES were air dried for about 1 hour before being introduced in reaction medium. This medium adapted from previously published methods [1] contained 0.5 mg/ml 3-3'-diaminobenzidine (DAB), 0.25 mg/ml horseradish peroxidase (50 U/ml) and 50 mM D-mannitol in 50 mM phosphate buffer pH 7.5. Control sections were incubated in medium without mannitol. Sections were incubated for about 2 hours at 30ºC. Mannitol oxidase activity was also assessed by biochemical methods. For ultrastructural studies digestive gland samples were fixed with 2.5% glutaraldehyde and 4% formaldehyde in cacodylate buffer, postfixed with osmium tetroxide, dehydrated and embedded in Epon. In cryostat sections, digestive gland cells containing mannitol oxidase were stained brown due to DAB oxidation ( Fig. 1 A-C).In control sections reaction was not observed in digestive gland cells (Fig. 1 D), but in other tissues unspecific reactions can occur. Biochemical assays also showed that all these six herbivorous species contain mannitol oxidase in the digestive gland. Enzyme activity was lower in B. striata and S. pectinata, being much higher in O. celtica and L. valentiana, and presented an intermediate value in the digestive gland of A. depilans and C. aspersum. The tubular structures typically associated with mannitol oxidase [3] were found in digestive gland cells of A. depilans, B. striata, C. aspersum and L. valentiana ( Fig. 1 E-F). The tubules are located inside rough endoplasmic reticulum cisternae in A. depilans, while in B. striata, C. aspersum and L. valentiana they are found within smooth membrane cisternae (Fig. 1 E). On the other hand, in S. pectinata and O. celtica these tubules were not observed. Thus, at least in marine gastropods, mannitol oxidase is not always asso...
Mannitol oxidase and polyol dehydrogenases are enzymes that convert polyalcohols into sugars. Mannitol oxidase was previously investigated in terrestrial snails and slugs, being also present in a few aquatic gastropods. However, the overall distribution of this enzyme in the Gastropoda was not known. Polyol dehydrogenases are also poorly studied in gastropods and other mollusks. In this study, polyalcohol oxidase and dehydrogenase activities were assayed in the digestive gland of 26 species of gastropods, representing the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, including herbivores and carnivores were analyzed. Ultrastructural observations were undertake in species possessing mannitol oxidase, in order to investigate the correlation between this enzyme and the presence of tubular structures known to be associated with it. Mannitol oxidase activity was detected in the digestive gland of herbivores from the clades Caenogastropoda and Heterobranchia, but not in any carnivores or in herbivores from the clades Patellogastropoda, Neritimorpha and Vetigastropoda. In most of the species used in this study, dehydrogenase activities were detected using both D-mannitol and D-sorbitol as substrates. Nevertheless, in some carnivores these activities were not detected with both polyalcohols. Ultrastructural observations revealed tubular structures in digestive gland cells of some species having mannitol oxidase activity, but they were not observed in others. Based on our results, we suggest that mannitol oxidase first occurred in a herbivorous or omnivorous ancestor of Apogastropoda, the clade formed by caenogastropods and heterobranchs, being subsequently lost in those species that shifted towards a carnivorous diet.
Alcohol oxidases and dehydrogenases are poorly studied in the Mollusca, the second largest phylum of metazoans. In order to obtain an overview of the distribution of aromatic alcohols and ethanol-oxidizing enzymes in the gastropod phylogenetic tree, we investigated the activity of these enzymes in the digestive gland of 26 gastropod species in the clades Patellogastropoda, Neritimorpha, Vetigastropoda, Caenogastropoda and Heterobranchia. Marine, freshwater and terrestrial species, as well as herbivores and carnivores, were sampled so that gastropods varying widely in habitat and diet were included in the study. An aromatic alcohol oxidase, which was previously reported in herbivorous terrestrial gastropods, was detected in 25 of the studied species. The activity of a cinnamyl alcohol dehydrogenase was detected for the first time in gastropods and this enzyme was found to be present in all the species that were studied. Our study, thus, demonstrates that alcohol oxidases and dehydrogenases are ubiquitous enzymes among gastropods; these enzymes are found across the gastropod phylogenetic tree and across species varying widely in habitat and diet. The enzymes that catalyze the oxidation or dehydrogenation of cinnamyl alcohol must be involved in the metabolism of aromatic alcohols of very different dietary origins and conceivably have a detoxification function. Oxidase or dehydrogenase activities involving ethanol as a substrate were detected only in a few species, mostly those belonging to the Panpulmonata. This suggests that for many gastropods ethanol may not be metabolically relevant.
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