Changes in termites feeding diets for gut micro-organisms selection and further cultivation
Bauwens Julien1, Tarayre Cédric2, Mattéotti Christel3, Brasseur Catherine4, Destain Jacqueline2, Vandenbol
Micheline3, Portetelle Daniel3, Thonart Philippe2, De Pauw Edwin4, Haubruge Eric1 and Francis Frédéric1.
1 : Functional and evolutionary entomology unit – University of Liege Gembloux Agro-Bio Tech
2: Bio-Industries unit – University of Liege Gembloux Agro-Bio Tech
3: Animal and microbial biology unit – University of Liege Gembloux Agro-Bio Tech
4: Mass spectrometry laboratory – University of Liege
julien.bauwens@ulg.ac.be
Introduction
Symbiotic interactions in termites are a possible source of efficient key enzymes for bioethanol production. Bacterial, fungal and protist species are involved
in lower termites. From this highly diversified system, only few symbiotic species have been cultivated yet from termites gut. The aim of this work was to
establish relatively progressive changes using termites as first bioreactor for further proteomic analyses and microbial cultivation.
• Enzymatic assays
Basic model of symbiotic interactions in lower termites
U (nmolglucoseh-1.µg prot-1)
CMC diet
Xylan diet
Glucose diet
350
Total Cellulase
300
250
200
150
Endo-β-1,4glucanase
100
50
0
Hindgut Hindgut Hindgut Hindgut Hindgut Hindgut
Wall Content
Wall Content Wall Content
Hemicellulase
• Proteomic analysis
Termite gut proteome was investigated following two different approaches:
• 2D SDS-PAGE coupled with MALDI-TOF/MS: mostly prokaryotic identifications.
Artificial diets
Lower termites (Reticulitermes santonensis (Feytaud)) were reared on
several agar-based diets including various carbon sources, such as
carboxymethyl-cellulose, microcrystalline cellulose, xylan, cellobiose or
glucose.
Cellulose
Glucose
Xylan
Taxonomic assignment of prokaryotic
identification by 2DE coupled with MALDI-TOF/MS
Cellulose + Lignin
2% 2% 2%
4%
Proteobacteria
6%
Actinobacteria
Firmicutes
6%
44%
Bacteroidetes
Archea
11%
Spirochaete
Digestive tracts were dissected in order to extract proteins and to cultivate
microorganisms.
Cellulose
Verrucomicrobia
23%
• ESI-LC/MS-MS: mostly eukaryotic identifications in NCBI “all entries” database.
Results
• Microscopic observations
Wood
Fusobacteria
Glucose
Taxonomic assignment of non redundant peptides
identified from total hindgut proteome by 2D-LC/MSMS
2%
1%
3% 3%
5%
14%
15%
24%
Major changes:
• Strong diminution or total loss of most large flagellates, especially
Dinenympha sp., Trichonymphea sp. and Spirotrichonymphea sp..
• Some flagellates seemed to be favored by specific diets.
28%
5%
Metazoa
Arthropods
Insecta
Fungi/Yeast
Parabasalia
Oxymonadida
Other Protists
Archaea
Plants
Bacteria
Proteins assigned to protists from “control termites” mostly consisted
in tubulins, but also included some glycosyl hydrolases family 7
sequences and actins. Proteins assigned to insects, other arthropods
or other metazoans were almost exclusively actins.
Discussion
Termites gut may overcome important dietary perturbations, initial diversity acting as key point buffering effects on host, although termites possess their own
enzymatic system. Some artificial diets permitted a simplification of the lower termites gut symbiotic system, which could be used as first step in symbionts
isolation and cultivation. Preliminary assay of cultivation actually gave encouraging results. Proteomic proved to be suitable tool to investigate such a complex
system. Nevertheless, for some symbionts very few genes are sequenced, which should lead to more targeted proteomic studies. Protein chromatography
will allow to split up the proteome and more accurate analysis.