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, 2 (8), e813

The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

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The Antibacterial Protein Lysozyme Identified as the Termite Egg Recognition Pheromone

Kenji Matsuura et al. PLoS One.

Abstract

Social insects rely heavily on pheromone communication to maintain their sociality. Egg protection is one of the most fundamental social behaviours in social insects. The recent discovery of the termite-egg mimicking fungus 'termite-ball' and subsequent studies on termite egg protection behaviour have shown that termites can be manipulated by using the termite egg recognition pheromone (TERP), which strongly evokes the egg-carrying and -grooming behaviours of workers. Despite the great scientific and economic importance, TERP has not been identified because of practical difficulties. Herein we identified the antibacterial protein lysozyme as the TERP. We isolated the target protein using ion-exchange and hydrophobic interaction chromatography, and the MALDI-TOF MS analysis showed a molecular size of 14.5 kDa. We found that the TERP provided antibacterial activity against a gram-positive bacterium. Among the currently known antimicrobial proteins, the molecular size of 14.5 kDa limits the target to lysozyme. Termite lysozymes obtained from eggs and salivary glands, and even hen egg lysozyme, showed a strong termite egg recognition activity. Besides eggs themselves, workers also supply lysozyme to eggs through frequent egg-grooming, by which egg surfaces are coated with saliva containing lysozyme. Reverse transcript PCR analysis showed that mRNA of termite lysozyme was expressed in both salivary glands and eggs. Western blot analysis confirmed that lysozyme production begins in immature eggs in queen ovaries. This is the first identification of proteinaceous pheromone in social insects. Researchers have focused almost exclusively on hydrocarbons when searching for recognition pheromones in social insects. The present finding of a proteinaceous pheromone represents a major step forward in, and result in the broadening of, the search for recognition pheromones. This novel function of lysozyme as a termite pheromone illuminates the profound influence of pathogenic microbes on the evolution of social behaviour in termites.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Egg protection behavior in termites.
A, Eggs in the nursery cell of Reticulitermes speratus. Termite workers always grasp the short side of oval eggs when carrying them. The short diameter provides a critical morphological cue for egg recognition. A worker is shown grooming an egg (arrow). B, Dummy-egg bioassay. Workers bring eggs and egg dummies made of 0.5-mm glass beads coated with the termite egg recognition chemical and form an egg pile in the Petri dish. C, The dummy eggs carried into nursery cells of a semi-natural colony (colony size ca. 500) in the laboratory. Dummy eggs injected in galleries were carried within 48 h. D, Dummy eggs carried into an egg pile in a mature field colony. Dummy eggs were placed in the foraging area of the mature colony. As a result, 6,098 of 31,500 dummy eggs were carried into egg piles in the nursery cells within 48 h.
Figure 2
Figure 2. Comparison of the piling rates of dummy eggs made of glass beads coated with test chemicals.
TEE: crude termite egg extract; pK: proteinase K digest of termite egg extract; BR: protein samples from the active peaks of BioRex 70 cation-exchange; Q1: UNO Q-1 anion-exchange chromatography; HIC: Methyl Hydrophobic Interaction Chromatography; HEL: hen egg lysozyme; CEL: cellulase. Data are means and standard errors from nine replicates. The chemical concentrations were 1.0 µg/bead for TEE, pK, BR, Q1 and HCI, and 4.0 µg/bead for HEL, CEL and HEL+CEL. Data for each test chemical were compared to the control using two-tailed t-tests. **: P<0.01; ***: P<0.001.
Figure 3
Figure 3. Chromatography and MALDI-TOF MS analyses.
A, UNO Q-1 anion-exchange chromatography elution patterns of the TERP. The active peak is indicated by an arrow. Lysozymes bind with coexisting anionic proteins in the Tris-HCl buffer (pH 8.2) and thus are eluted as the absorbed fraction, although lysozyme itself is a cationic protein. B, Methyl HIC column chromatography. The active peak is indicated by a red arrow. The elution pattern of hen egg lysozyme (HEL) is indicated by the blue line. C, MALDI-TOF MS spectrum of the purified TERP. Ions at m/z 14467 and 7221 (divalent ion) were identified as the target protein. Ions at m/z 11465 and 17207 were the dimer and trimer of insulin used as the internal standards (IS), respectively.
Figure 4
Figure 4. Worker and queen organs producing the egg recognition pheromone.
A, Salivary gland (SG) of a Reticulitermes speratus worker. H: head, E: oesophagus, FG: foregut, MG: midgut. B, Accessory gland (AG) and ovary of a queen. OD: oviduct, S: spermatheca. C, Egg recognition activity of the extracts from worker salivary glands (SG), queen accessory glands (AG) and immature eggs collected from a queen ovary (IME). We compared the piling rates of dummy eggs made of glass beads coated with these extracts. The chemical concentrations were 0.025 worker equivalent extracts (eq.) per bead, 0.03 queen eq. and 0.02 queen eq., respectively. Data are means and standard errors from fifteen replicates (5 replicates × 3 colonies). Data for the three colonies were pooled because there was no significant difference among the colonies. Data for each treatment were compared to the control using two-tailed t-tests. ***: P<0.001.
Figure 5
Figure 5. RT-PCR and western blotting for lysozyme gene expression analysis.
A, RT-PCR of mRNAs extracted from worker salivary glands (SG), worker legs, immature eggs dissected from queen ovaries (IME), queen accessory glands (AG) and eggs collected from termite nests. B, Western blot analysis with the anti-lysozyme polyclonal antibody performed on the protein extracts from each tissue and hen egg lysozyme (HEL) as a positive control.

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