A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase

Abstract

Soldiers of some eusocial insects exhibit an altruistic self-destructive defense behavior in emergency situations when attacked by large enemies. The swarm-forming invasive millipede, Chamberlinius hualienensis, which is not classified as eusocial animal, exudes irritant chemicals such as benzoyl cyanide as a defensive secretion. Although it has been thought that this defensive chemical was converted from mandelonitrile, identification of the biocatalyst has remained unidentified for 40 years. Here, we identify the novel blood enzyme, mandelonitrile oxidase (ChuaMOX), which stoichiometrically catalyzes oxygen consumption and synthesis of benzoyl cyanide and hydrogen peroxide from mandelonitrile. Interestingly the enzymatic activity is suppressed at a blood pH of 7, and the enzyme is segregated by membranes of defensive sacs from mandelonitrile which has a pH of 4.6, the optimum pH for ChuaMOX activity. In addition, strong body muscle contractions are necessary for de novo synthesis of benzoyl cyanide. We propose that, to protect its swarm, the sacrificial millipede also applies a self-destructive defense strategy-the endogenous rupturing of the defensive sacs to mix ChuaMOX and mandelonitrile at an optimum pH. Further study of defensive systems in primitive arthropods will pave the way to elucidate the evolution of altruistic defenses in the animal kingdom.

Figure 1. Characterization of the purified ChuaMOX.

(a) Synthesis of benzoyl cyanide from racemic mandelonitrile. ChuaMOX is able to catalyze the synthesis of benzoyl cyanide at pH 5, and the retention times for benzaldehyde, benzoyl cyanide, (R)-mandelonitrile, and (S)-mandelonitrile were 5.4 min, 6.1 min, 11.4 min, and 14.3 min, respectively. Arrows indicate the peaks corresponding to benzaldehyde, benzoyl cyanide, and racemic mandelonitrile. (b) Reaction of ChuaMOX. (c) Optimum pH. (d) pH stability. (e) Optimum temperature. (f) Temperature stability. In panels (c–f), the highest mean value of the activity at pH 4 in panel c was defined as 100% to determine relative activity. Values are the means ± SD; n = 3. In panels (c,d), the symbols ◾, ▴, ⦁ and ◆ indicate citrate, phosphate, Tris-HCl, and glycine-sodium hydroxide buffers, respectively.

Figure 2. Phylogenetic analysis of ChuaMOX.

ChuaMOX is shown in bold. The accession numbers of glucose dehydrogenases and alcohol dehydrogenases appear in parentheses. Bootstrap values were determined from 1,000 replications. A bar indicates a 5% divergence.

Figure 3. Localization of ChuaMOX and its substrate.

(a) Expression of ChuaMOX. RT-PCR detects the gene expression in the paraterga, and actin expression is used as an internal control. (b) Localization of ChuaMOX. The enzyme activity was mainly detected in the blood by zymography. 1, Antenna. 2, Leg. 3, Head. 4, Integument. 5, Paraterga. 6, Fat body. 7, Gut. 8, Blood. (c) Localization of ChuaMOX substrate. (R)-Mandelonitrile is detected in the extract from the paraterga. Blue, Antenna. Light blue, Leg. Green, Head and tail. Purple, Integument. Red, Paraterga. Orange, Gut and fat body. Each extracted sample was analyzed using an HPLC equipped with a chiral column, and the inset shows a magnified view of the chromatogram for a retention time between 10 min and 15 min. Arrows indicate the peaks for benzaldehyde and (R)-mandelonitrile, respectively. Based on the calibration curves, the estimated amounts of benzaldehyde and (R)-mandelonitrile are 121 nmol and 93 nmol, respectively. (d) Whole body extract. The production of benzaldehyde, benzoyl cyanide, and benzoic acid detected by GC/MS analysis is indicated by arrows. (e) Blood extract. Cyanohydrins were not detected as substrates for ChuaMOX by GC/MS analysis.

Figure 4. Synthesis of benzoyl cyanide as a defensive secretion in vivo.

(a) Extract from a whole body. (b) Extract from a whole body after shaking. The arrows indicate peaks for benzaldehyde, benzoyl cyanide, and (R)-mandelonitrile, respectively. (c) Extract from a whole body after anesthetizing. The anesthetized animal is not able to release the defensive compounds including benzoyl cyanide. (d) Extraction from paraterga collected from the weakly anesthetized animal. The defensive sac in the paraterga was presumably ruptured by roughly snatching collection and residual blood and mandelonitrile were mixed. The isolated tissues synthesized benzoyl cyanide as non-anesthetized animals in (a,b). (e) Proposed defensive reaction of the millipede through ChuaMOX. A millipede caught by a predator strongly contracts its body muscles, and the behavior endogenously ruptures the membranes of the storage and reaction chambers. Drone blood enzyme, ChuaMOX, flows into the chambers from the hemocoel and is activated by the shift of pH from 7 to 4.6, which starts the synthesis of benzoyl cyanide from (R)-mandelonitrile.