Cellulolytic activity in a phytophagous lepidopteran insect Philosamia ricini: The origin of the enzymes

doi:10.1016/0020-1790(89)90072-3

Insect Biochemistry

Volume 19, Issue 3, 1989, Pages 269-276

https://doi.org/10.1016/0020-1790(89)90072-3 Get rights and content

Abstract

The presence of cellulases in a phytophagous insect hitherto considered as devoid of any cellulolytic enzymes, has been reported for the first time in the phytophagous lepidopteran, the eri silkworm Philosamia ricini. Cellulose digestion in Philosamia ricini appears to occur independently of its gut flora and via enzymes synthesized by the insect itself. The failure of three wide spectrum antibiotics to induce any change in the cellulolytic activity in this insect at any stage of its development evinces the non-participation of its gut flora in cellulose digestion. Culturing the antibiotic-fed larval gut fluids in appropriate media revealed the ability of tetracycline to effect complete inactivation of all bacteria and fungi by day 4 whereas penicillin and streptomycin could achieve it only partially. The cellulolytic activity, however, in all insect groups remained unaffected. The suggests that it is the endogenous enzymes of P. ricini that catalyze cellulose hydrolysis. This has been further confirmed by long term feeding of antibiotics to the insects.

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Future research on TcEG1 will focus on localizing expression of the protein in the T. castaneum digestive tract, and evaluation of conditions affecting change in expression, such as different diets. If TcEG1 activity is critical for larval survival, it could be exploited as a potential insecticide target using RNAi, as previously demonstrated for the Cell-1 cellulase gene from Reticulitermes flavipes (Kollar) (Zhou et al., 2008), or inhibitors, as reported for A. foveicollis cellulase (Pant and Ramana, 1989). Optimization of TcEG1 via genetic engineering could enhance activity and thermostability leading to incorporation of TcEG1 in enzyme mixtures for bioreactors used to degrade lignocellulosic material for production of ethanol.
The availability of sequenced insect genomes has allowed for discovery and functional characterization of novel genes and proteins. We report use of the Tribolium castaneum (Herbst) (red flour beetle) genome to identify, clone, express, and characterize a novel endo-β-1,4-glucanase we named TcEG1 (T. castaneum endoglucanase 1). Sequence analysis of a full-length TcEG1 cDNA clone (1356 bp) revealed sequence homology to enzymes in glycosyl hydrolase family 9 (GHF9), and verified presence of a change (Gly for Ser) in the conserved catalytic domain for GHF9 cellulases. This TcEG1 cDNA clone was predicted to encode a 49.5 kDa protein with a calculated pI of 5.39. Heterologous expression of TcEG1 in Drosophila S2 cell cultures resulted in secretion of a 51-kDa protein, as determined by Western blotting. The expressed protein was used to characterize TcEG1 enzymatic activity against two cellulose substrates to determine its specificity and stability. Our data support that TcEG1 as a novel endo-β-1,4-glucanase, the first functional characterization of a cellulase enzyme derived from an insect genome with potential applications in the biofuel industry due to its high relative activity at alkaline pH.
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Additional research is needed to clone these enzymes thus facilitating cellulase identification and annotation of function. Cellulase activity reported for lepidopteran larvae have suggested increased activity with advancing larval development (Nakonieczny et al., 2006; Pant and Ramana, 1989). In contrast, we did not detect significant differences in cellulolytic activity during development of D. carolina.
Previous screening of head-derived and gut fluid extracts of Carolina grasshoppers, Dissosteira carolina (L.) revealed relatively high activity against cellulase substrates when compared to other insect groups. In this work we report on the characterization and identification of enzymes involved in cellulolytic activity in digestive fluids of D. carolina. In zymograms using carboxymethylcellulose (CMC) as substrate, we detected four distinct cellulolytic protein bands in D. carolina gut fluids, common to all developmental stages. These cellulolytic enzymes were localized to foregut and midgut regions of the D. carolina digestive tract. Cellulases were purified from D. carolina head and gut fluid extracts by liquid chromatography to obtain N-terminal amino acid sequence tags. Database searches with sequence tags from head fluids indicated high similarity with invertebrate, bacterial and plant β1,4-endoglucanases, while no homologues were identified for the gut-derived protein. Our data demonstrate the presence of cellulolytic activity in the digestive system of D. carolina and suggest that cellulases of endogenous origin are present in this organism. Considering that this grasshopper species is a pest of grasses, including switchgrass that has been suggested bioethanol feedstock, characterization of insect cellulolytic systems may aid in developing applications for plant biomass biodegradation for biofuel production.
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  1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
- 2.
  2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
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  3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
- 4.
  4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
- 5.
  5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
- 6.
  6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.
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Methods for discovery and characterization of cellulolytic enzymes from insects
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Cellulolytic activity in a phytophagous lepidopteran insect Philosamia ricini: The origin of the enzymes

Abstract

Insect Biochem.

J. Insect Physiol.

J. Insect Physiol.

Expl Parasit

Comp. Biochem. Physiol.

Insect Biochem.

Insect Biochem.

Comp. Biochem. Physiol.

Comp. Biochem. Physiol.

Insect Biochem.

Biochim. Biophys. Acta

Insect Biochem.

J. Chromat.

Digestive enzymes in the southern armyworm

J. Agric. Res.

The contribution of fungal enzymes to the digestion of leaves by Gammarus fossarum Koch

Oecologia

The mechanisms of enzymatic cellulose degradation. Purification and some properties of two different 1,4-β-glucan glucanohydrolases from Trichoderma viride

Eur. J. Biochem.

Distribution and abundance of bacteria in the gut of a soil-feeding termite Procubitermes aburiensis (Termitidae, Termitinae)

J. gen. Microbiol.

Nitrogen fixation in termites

Nature, Lond.

Symbiotic relationships between termites and their intestinal microbiots

Carbohydrate digestion by the larval midgut of Heliothis zea

Ann. ent. Soc. Am.

Ecophysiologie des Insectes Parasites des Forêts

Hydrogen ion concentration and digestive enzymes in the mature larvae of Marasmia trapezalis Guen (Pyralidae)

Indian J. Ent.

The physiological and symbiotic relationship between the intestinal protozoa of termites and their hosts, with special reference to Reticulitermes flavipes Kollar

Biol. Bull. mar. biol. Lab. Woods Hole

Digestive enzymes of mature larvae of Chilena similis Walker (Lepidoptera: Lasciocampidae)

Indian J. Ent.

Insect midgut function

Adv. Insect Physiol.

Cellulase activity in the larvae of eri silk worm, Philosamia ricini and toxic effect of malathion on it

Arogya—J. Hlth Sci.