Enzymatic activities in the digestive tract of spirostreptid and spirobolid millipedes (Diplopoda: Spirostreptida and Spirobolida)

Highlights

• Enzyme activities were measured in gut compartments of two species of millipedes.

• Amylase, laminarinase, cellulase, chitinases and disaccharidases were found.

• Microapocrine or merocrine secretion were found in midgut, merocrine in hindgut.

• Most of enzymes were active mainly in content and tissues of the more acid midgut.

• Dissacharidases showed less midgut-hindgut differences and more alkaline activity.

Abstract

Millipedes represent a model for the study of organic matter transformation, animal-microbial interactions, and compartmentalisation of digestion. The activity of saccharidases (amylase, laminarinase, cellulase, xylanase, chitinase, maltase, cellobiase, and trehalase) and protease were measured in the midgut and hindgut contents and walls of the millipedes Archispirostreptus gigas and Epibolus pulchripes. Assays done at pH 4 and 7 confirmed activities of all enzymes except xylanase. Hydrolysing of starch and laminarin prevailed. The hindgut of E. pulchripes was shorter, less differentiated. Micro-apocrine secretion was observed only in the midgut of A. gigas. Merocrine secretion was present in midgut and hindgut of E. pulchripes, and in the pyloric valve and anterior hindgut of A. gigas. Alpha-polysaccharidases were mostly active in the midgut content and walls, with higher activity at pH 4. The low activity of amylase (A. gigas) and laminarinase (E. pulchripes) in midgut tissue may indicate their synthesis in salivary glands. Cellulases were found in midgut. Chitinases, found in midgut content and tissue (E. pulchripes) or concentrated in the midgut wall (A. gigas), were more active at an acidic pH. Polysaccharidases were low in hindguts. Protease shows midgut origin and alkaline activity extending to the hindgut in E. pulchripes, whereas in A. gigas it is of salivary gland origin and acid activity restricted to the midgut. Some disaccharidases, with more alkaline activity, showed less apparent midgut-hindgut differences. It may indicate an axial separating of the primary and secondary digestion along the intestinal pH gradient or the presence of enzymes of hindgut parasites.

Introduction

Tropical millipedes of the orders Spirostreptida and Spirobolida belong to the largest arthropods on the Earth. They are important decomposers of dead organic matter in their biotopes (Dangerfield, 1990; Dangerfield and Milner, 1996; Dangerfield and Telford, 1996; Mugao Kathambi, 2016) and represent a source of methane (Hackstein and Stumm, 2006; Šustr and Šimek, 2009). A rich community of prokaryotes, protozoans, and eukaryotic commensals or parasites was reported in their digestive tract (Byzov, 2006; Hackstein and Graaf, 2010; Hausman, 1958). Therefore, they represent a suitable model for the investigation of soil organic matter transformation and animal-microbial interactions.

Enzymatic activity was studied in the gut of 29 different millipede species (Beck and Friebe, 1981; Marcuzzi and Turchetto Lafisca, 1976; Nielsen, 1962; Nunez and Crawford, 1976; Shukla and Shukla, 1980, 1986; Shukla, 1984; Šustr, 1999; Taylor, 1982; Urbášek and Tajovský, 1991). Enzymatic cleavage of 28 different substrates was confirmed: 11 polysaccharides, 15 oligosaccharides, 1 polypeptide, and 1 lipid substrate.

Millipede food rich in cellulose, anoxia in the digestive tract (Šustr et al. in prep.) and presence of methanogenesis (Hackstein and Stumm, 2006; Šustr et al., 2014) suggest cellulose degradation in the intestine. On the other hand, the assimilation of highly recalcitrant food components is not needed to reach the relatively low assimilation efficiency level (~30%) reported in millipedes (summary in Hopkin and Read (1992), because the content of cellulose in freshly fallen leaves might reach about 45%, and the content of lignocellulose may range from 19 to 74% (Striganova, 1971; Zimmer, 1999).

However, the utilisation of the cellulose by millipedes was confirmed by comparison of cellulose content in the food and excrements (Striganova, 1971) and by assimilation of ¹⁴C labeled cellulose (Bignell, 1989; Taylor, 1982). We currently do not have enough quantitative data to consider the importance of cellulose digestion for millipede metabolism.

Cellulolytic or xylanolytic activities were confirmed in millipedes by Beck and Friebe (1981), Cazemier et al. (1997), Nielsen (1962), Nunez and Crawford (1976), Striganova (1971), Taylor (1982), Urbášek and Tajovský (1991) and Šustr (1999). There is no detailed information about the origin of these enzymes, although some sequences corresponding to putative cellulolytic enzymes may be found in transcriptomic data of several millipede species (Rehm et al., 2014).

Anatomical distribution of enzyme activity in the millipede digestive tract was studied sporadically (Nunez and Crawford, 1976; Taylor, 1982). The compartmentalisation of enzymatic digestion in the midgut was described in insects. The selective permeability of the peritrophic membrane and fluid movements inside the gut play an important role in compartmentalisation (Terra and Ferreira, 1994). Water and minerals reabsorption is considered as the main physiological role of the hindgut (Terra, 1988). In millipedes, the midgut is regarded as the main site of enzymatic digestion, and the peritrophic membrane is described (Hopkin and Read, 1992).

Some xylophagous insects, nutritionally similar to leaf-litter consuming millipedes, have hindguts modified into fermentation chambers harboring symbiotic microorganisms that may assist in the cellulose digestion (Terra and Ferreira, 1994). Fermentation products of the hindgut microorganisms may be absorbed by these host insects (Breznak and Brune, 1994). To the contrary, the digestive tract of millipedes looks like a straight tube (Hopkin and Read, 1992) without morphological modifications of the hindgut. Reabsorbing of water from the hindgut content was mentioned as a main function of the fluid-secretion hindgut epithelium in millipedes (Hopkin and Read, 1992). Regardless of the presumed importance of the microorganism in digestion (Byzov, 2006), there is no evidence of a true resident symbiotic microflora in millipedes (Hopkin and Read, 1992). However, they can use microbial enzymes to digest recalcitrant molecules.

In this study, the tropical millipedes Archispirostreptus gigas (Peters, 1855) (Spirostreptida) and Epibolus pulchripes (Gerstäcker, 1873) (Spirobolida) were used as model objects to study compartmentalisation of enzymes in the digestive tract of leaf-litter eating arthropods. Specific activities of saccharolytic enzymes in sections of the digestive tract (midgut and hindgut) and compartments (gut content and gut wall) were compared. The large body size of the giant millipedes enables us to measure several enzymatic activities from the same sample at two different pH levels. We made histological observations of midgut and hindgut epithelium to examine the functional organisation of the intestine from the morphological point of view. All the obtained data were discussed concerning the existence of compartmentalisation of digestion in millipedes and origin of their digestive enzymes.