Endogenous cellulase enzymes in the stick insect (Phasmatodea) gut

Area of Science:

• Insect biochemistry
• Molecular biology
• Evolutionary biology

Background:

• Cellulase activity is essential for herbivores to digest plant cellulose.
• The presence and origin of cellulase in insects outside of termites and cockroaches are not well understood.
• Phasmids (walking sticks) are leaf-feeding insects whose ability to digest cellulose has been questioned.

Purpose of the Study:

• To investigate the presence and origin of cellulase activity in the walking stick Eurycantha calcarata.
• To identify the specific genes encoding cellulase in E. calcarata.
• To explore the evolutionary distribution of cellulase genes within the Phasmatodea order.

Main Methods:

• Enzyme isolation and mass spectrometry analysis of midgut contents.
• Reverse transcription polymerase chain reaction (RT-PCR) to identify endoglucanase genes.
• Sequence homology analysis and immunological assays (antigency) using anti-serum.

Main Results:

• High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of E. calcarata.
• Two endoglucanase genes, EcEG1 and EcEG2, were identified; the purified enzyme is the product of EcEG1.
• Phasmid cellulases show high homology to insect glycoside hydrolase family 9 (GH9) endoglucanases, particularly from termites and cockroaches, and exhibit antigency against termite cellulase anti-serum, suggesting an endogenous origin.

Conclusions:

• The walking stick E. calcarata possesses an endogenous cellulase enzyme, likely derived from gene EcEG1.
• Cellulase genes are likely distributed broadly within phasmids, as indicated by homologous cDNA isolation from Entoria okinawaensis.
• This finding suggests that cellulose may serve as a nutritive component for leaf-feeding phasmids, challenging previous assumptions based on other herbivorous insects like Lepidoptera.