Stuart M. Linton

A review of feeding and nutrition of herbivorous land crabs: adaptations to low quality plant diets

This paper reviews the nutritional ecology, the digestive physiology, and biochemistry of herbivorous land crabs and the adaptations that they possess towards a diet of plant material. Land crab species that breathe air and forage out of water can be divided into three feeding specialisations: primarily carnivorous, deposit feeders feeding on micro-organisms and organic matter in the sediment, and herbivores consuming mainly plant material and its detritus. The last forms the focus of this review. The diets of the herbivores are low in nitrogen and high in carbon, are difficult to digest since they contain cellulose and hemicellulose, and may disrupt digestion due to the presence of tannins. Herbivorous crustaceans are able to efficiently utilise plant material as their primary nutrient source and are indeed able to meet their nitrogen requirements from it. Herbivorous land crabs display a range of adaptations towards a low nitrogen intake and these are discussed in this review. They also appear to endogenously produce cellulase and hemicellulase enzymes for the digestion of cellulose and hemicellulose. Generalised and specific adaptations allow them to inhibit the potentially negative digestive effects of tannins. To digest plant material, they possess a plastic digestive strategy of high food intake, short retention time, high assimilation of cell contents, and substantial digestion of cellulose and hemicellulose.

Presence and properties of cellulase and hemicellulase enzymes of the gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes.

SUMMARY Digestive juice from the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes exhibited total cellulase activity and activities of two cellulase enzymes; endo-β-1,4-glucanase and β-1,4-glucosidase. These enzymes hydrolysed native cellulose to glucose. The digestive juice of both species also contained laminarinase, licheninase and xylanase, which hydrolysed laminarin, lichenin and xylan, respectively, to component sugars. The pH optima of β-1,4-glucosidase, endo-β-1,4-glucanase and total cellulase from G. natalis were 4–5.5, 5.5 and 5.5–7, respectively. In the digestive juice from D. hirtipes, the corresponding values were 4–7, 5.5–7 and 4–9, respectively. The pH of the digestive juice was 6.69±0.03 for G. natalis and 6.03±0.04 for D. hirtipes and it is likely that the cellulases operate near maximally in vivo. In G. natalis, total cellulase activity and endo-β-1,4-glucanase activity were higher than in D. hirtipes, and the former species can thus hydrolyse cellulose more rapidly. β-1,4-glucosidase from G. natalis was inhibited less by glucono-d-lactone (Ki=11.12 mmol l-1) than was theβ -1,4-glucosidase from D. hirtipes (Ki=4.53 mmol l-1). The greater resistance to inhibition by theβ -1,4-glucosidase from G. natalis may contribute to the efficiency of the cellulase system in vivo by counteracting the effects of product inhibition and possibly dietary tannins. The activity ofβ -1,4-glucosidase in the digestive juice of D. hirtipes was higher than that of G. natalis.

Purification and characterisation of endo-β-1,4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor

SUMMARY Laminarinase and endo-β-1,4-glucanase were purified and characterised from the midgut gland of the herbivorous land crab Gecarcoidea natalis and the crayfish Cherax destructor. The laminarinase isolated from G. natalis was estimated to have a molecular mass of 41 kDa by SDS-PAGE and 71 kDa by gel filtration chromatography. A similar discrepancy was noted for C. destructor. Possible reasons for this are discussed. Laminarinase (EC 3.2.1.6) from G. natalis had a Vmax of 42.0 μmol reducing sugars produced min–1 mg protein–1, a Km of 0.126% (w/v) and an optimum pH range of 5.5–7, and hydrolysed mainlyβ -1,3-glycosidic bonds. In addition to the hydrolysis ofβ -1,3-glycosidic bonds, laminarinase (EC 3.2.1.39) from C. destructor was capable of significant hydrolysis of β-1,4-glycosidic bonds. It had a Vmax of 19.6 μmol reducing sugars produced min–1 mg protein–1, a Km of 0.059% (w/v) and an optimum pH of 5.5. Laminarinase from both species produced glucose and other short oligomers from the hydrolysis of laminarin. Endo-β-1,4-glucanase (EC 3.2.1.4) from G. natalis had a molecular mass of 52 kDa and an optimum pH of 4–7. It mainly hydrolysed β-1,4-glycosidic bonds, but was also capable of significant hydrolysis of β-1,3-glycosidic bonds. Two endo-β-1,4-glucanases, termed 1 and 2, with respective molecular masses of 53±3 and 52 kDa, were purified from C. destructor. Endo-β-1,4-glucanase 1 was only capable of hydrolysingβ -1,4-glycosidic bonds and had an optimum pH of 5.5. Endo-β-1,4-glucanases from both species produced some glucose, cellobiose and other short oligomers from the hydrolysis of carboxymethyl cellulose.

Intracellular purine deposits in the gecarcinid land crab Gecarcoidea natalis

The terrestrial crab Gecarcoidea natalis stores large amounts of purine in the body. The major component of the purine deposits is urate (85% of the total purines). The other 15% is comprised of hypoxanthine, guanine, and xanthine. Microscopy studies reveal that these urate deposits are located intracellularly in spongy connective tissue cells throughout the body. Urate exists as numerous membrane‐bound crystals 1 μm in diameter. Vesicles thought to represent urate vesicles at various stages of development are also present in the cytoplasm of the cell. Few organelles are visible in the urate storage cells, and it is unlikely that the urate is synthesized on site. Crabs (N = 2) fed a high‐nitrogen diet have greater numbers of urate storage cells at more connective tissue sites, and the cells are larger (36.3 ± 1.8 μm (mean ± SE) and 44.0 ± 1.4 μm (mean ± SE)) and contain more urate than urate storage cells in animals collected from the field (N = 3) or maintained in the laboratory on a low‐nitrogen diet (N = 1). The mean diameter of urate storage cells in animals fed a diet low in nitrogen and field‐collected animals ranges from (13.5 ± 0.5 μm (SE) ‐ 22.3 ± 1.0 μm (SE)). This histological study supports a strong correlation between purine accumulation and the nitrogen content of the diet. J. Morphol. 231:101‐110, 1997.

The last piece in the cellulase puzzle: the characterisation of β-glucosidase from the herbivorous gecarcinid land crab Gecarcoidea natalis

SUMMARY A 160 kDa enzyme with β-glucosidase activity was purified from the midgut gland of the land crab Gecarcoidea natalis. The enzyme was capable of releasing glucose progressively from cellobiose, cellotriose or cellotetraose. Although β-glucosidases (EC 3.2.1.21) have some activity towards substrates longer than cellobiose, the enzyme was classified as a glucohydrolase (EC 3.2.1.74) as it had a preference for larger substrates (cellobiose<cellotriose=cellotetraose). It was able to synthesise some cellotetraose by the transglycosylation of smaller substrates – another common feature of glucohydrolases. The interaction between the glucohydrolase described here and the endo-β-1,4-glucanases described previously for G. natalis provides a complete model for cellulose hydrolysis in crustaceans and possibly in other invertebrates. After mechanical fragmentation by the gastric mill, multiple endo-β-1,4-glucanases would initially cleave β-1,4-glycosidic bonds within native cellulose, releasing small oligomers, including cellobiose, cellotriose and cellotetraose. The glucohydrolase would then attach to these oligomers, progressively releasing glucose. The glucohydrolase might also attach directly to crystalline cellulose to release glucose from free chain ends. This two-enzyme system differs from the traditional model, which suggests that total cellulose hydrolysis requires the presence an endo-β-1,4-glucanse, a cellobiohydrolase and a β-glucosidase.

Functional morphology of the gastric mills of carnivorous, omnivorous, and herbivorous land crabs

Terrestrial decapods consume a wide variety of plant and animal material. The potential adaptations of carnivorous, omnivorous, and herbivorous terrestrial crustaceans were studied by examining the functional morphology of the gastric mill. Two closely related species from each feeding preference group were examined to identify which features of the mill were due to phylogeny and which were due to adaptation. The morphology of the gastric mill matched the diet well; the gastric mills of the carnivorous species (Geograpsus grayi and Geograpsus crinipes) possessed a blunt, rounded medial tooth and flattened lateral teeth with a longitudinal grinding groove. These features make them well suited to a carnivorous diet of soft animal tissue as well as hard material, such as arthropod exoskeleton. In contrast, the mill of the herbivorous gecarcinids (Gecarcoidea natalis and Discoplax hirtipes) consisted of a medial tooth with sharp transverse ridges and lateral teeth with sharp interlocking cusps and ridges and no grinding surface. These features would efficiently shred fibrous plant material. The morphology of the mill of the omnivorous coenobitids (Coenobita perlatus and Birgus latro) was more generalized toward a mixed diet. However, the mill of B. latro was more adapted to deal with highly nutritious food items, such as nuts and heavily calcified decapods. Its mill possessed lateral teeth with extended ridges, which sat close to the calcified cardiopyloric valve to form a flattened floor. Hard items trapped in the mill would be crushed against this surface by the medial tooth. J. Morphol. 2010.

Accumulation and excretion of morphine by Calliphora stygia, an Australian blow fly species of forensic importance.

This study examined the ability of the forensically important blow fly, Calliphora stygia to actively excrete morphine, thereby maintaining a low morphine level within its body when fed on a diet containing morphine at low (7pmolg(-1)) and high (17.5pmolg(-1)) concentrations. Morphine was accumulated within the bodies of maggots (≈70% within the tissues) at concentrations which were lower than that of the meat (3-24%). The morphine content of the initial developing stages (second and third instar maggots) maintained on the high morphine diet was higher than those on the low morphine diet. Morphine was cleared from the body with negatively exponential kinetics (High morphine group: Morphine (pmolg(-1) wet weight)=8425e(-0.014t). Low morphine group: Morphine (pmolg(-1) wet weight)=2180e(-0.010t)). Clearance constants for morphine by animals in both groups were similar and thus both groups had a similar ability to excrete morphine. The Malpighian tubules of maggots were able to actively secrete morphine using a transport mechanism that transports small type II organic cations, such as morphine and quinine. The rate of morphine secretion by the Malpighian tubules could explain the clearance of the drug by the maggots. As the morphine was transported across the Malpighian tubules cells, a significant proportion was metabolised into a compound that is yet to be fully characterised.

Enzymes of urate synthesis and catabolism in the Gecarcinid land crab Gecarcoidea natalis

This study investigated the sites of urate synthesis and catabolism in the gecarcinid land crabGecarcoidea natalis by assaying spongy connective tissue, midgut gland, muscle and gill for xanthine oxidoreductase, the last enzyme involved in urate synthesis, and uricase and urease, the first and last enzymes involved in urate catabolism. The spongy connective tissue and midgut gland of theG. natalis contained activities of xanthine oxidoreductase and were considered to be sites of urate synthesis. The midgut gland had a high activity of xanthine oxidoreductase and were considered to be sites of urate synthesis.The midgut gland had a high activity of xanthine oxidoreductase [(58.87±4.6 (SE) nmol urate produced g-1 wet wt. tissue min-1], 2.7 times the xanthine oxidoreductase activity contained within the spongy connective tissue, and was thought to be the main site of urate synthesis. Xanthine dehydrogenase (EC 1.1.1.204) was the only form of xanthine oxidoreductase detected within the tissues. Its presence means that the cost of synthesising urate de novo is relatively small (between 1 and 3 ATP). Uricase (EC 1.7.3.3) and urease (EC 3.5.1.5) activities were present in the tissues ofG. natalis. Spongy connective tissue contained the highest activities of urease [48.44±4.29 (SE) nmol urate consumed g-1 wet wt. tissue min-1] while the highest activities of urease [365.31±3721 (SE) nmol urate consumed g-1 wet wt tissue minmin-1] were contained within the gills. From this evidence it is clear thatG. natalis possesses the uricolytic pathway and hence the ability to catabolise urate, and urate catabolism is begun at the site of urate storage, the spongy connective tissue, and is completed at the gills. As the gills are the site of ammonia excretion in this species the ammonia produced from the catabolism of urate is probably excreted. The urate deposits within the body ofG. natalis may be involved in temporary storage of nitrogenous wastes.

Role of the Midgut Gland in Purine Excretion in the Robber Crab, Birgus latro (Anomura: Coenobitidae)

White fecal strands of Birgus latro are composed of small spherules of uric acid with a mean diameter of 1.6 ± 0.6 μm. Large numbers of membrane‐bound spherules with concentric lamellae are present in the R cells of the midgut gland, so we suggest that lengths of white feces are produced by coordinated secretion of these spherules into the lumen of the midgut gland tubules. There are four cell types in the tubules with embryonic (E) cells at the distal tip, B cells in a narrow band at the distal end and R cells making up the bulk of the tubules and gland. F cells are sparsely scattered among the R cells. Midgut gland tissue was assayed for activities of xanthine dehydrogenase and xanthine oxidase, the two forms of xanthine oxidoreductase. Contrary to previous reports, we found that the midgut gland of B. latro contains only high activities of xanthine dehydrogenase. If proteinase inhibitors were omitted from the assays, however, significant activity of xanthine oxidase was measured, a result we regard as an artifact attributable to the partial conversion of xanthine dehydrogenase to xanthine oxidase by endogenous proteinases. R cells were demonstrated to contain peroxisomes, which may be involved in lipid metabolism rather than synthesis of uric acid. J. Morphol. 241:227–235, 1999

Food utilisation and digestive ability of aquatic and semi-terrestrial crayfishes, Cherax destructor and Engaeus sericatus (Astacidae, Parastacidae)

Both Engaeus sericatus and Cherax destructor are omnivorous crayfishes consuming a variety of food items. Materials identified in the faeces of both E. sericatus and C. destructor consisted of mainly plant material with minor amounts of arthropod animals, algae and fungi. The morphology of the gastric mill of C. destructor suggests that it is mainly involved in crushing of food material while the gastric mill of E. sericatus appears to be better suited to cutting of food material. Given this, the gastric mill of E. sericatus may be better able to cut the cellulose and hemicellulose fibres associated with fibrous plant material. In contrast, the gastric mill of C. destructor appears to be more efficient in grinding soft materials such as animal protein and algae. Both species accumulated high amounts of lipids in their midgut glands (about 60% of the dry mass) which were dominated by triacylglycerols (81–82% of total lipids). The dominating fatty acids were 16:0, 16:1(n-7), 18:1(n-9), 18:2(n-6), and 18:3(n-3). The two latter fatty acids can only be synthesised by plants, and are thus indicative of the consumption of terrestrial plants by the crayfishes. The similarity analysis of the fatty acid patterns showed three distinct clusters of plants and each of the crayfish species. The complement of digestive enzymes, proteinases, total cellulase, endo-β-1,4-glucanase, β-glucosidase, laminarinase and xylanase within midgut gland suggests that both C. destructor and E. sericatus are capable of hydrolysing a variety of substrates associated with an omnivorous diet. Higher activities of total cellulase, endo-β-1,4-glucanase and β-glucosidase indicate that E. sericatus is better able to hydrolyse cellulose within plant material than C. destructor. In contrast to E. sericatus, higher total protease and N-acetyl-β-d-glucosaminidase activity in the midgut gland of C. destructor suggests that this species is better able to digest animal materials in the form of arthropods. Differences in total cellulase and gastric mill morphology suggest that E. sericatus is more efficient at digesting plant material than C. destructor. However, the contents of faecal pellets and the fatty acid compositions seem to indicate that both species opportunistically feed on the most abundant and easily accessible food items.