Symbiont-mediated chemical defense in the invasive ladybird Harmonia axyridis

Abstract

The volatile alkylpyrazines methyl- and methoxypyrazines (MPs) present in the reflex bleeds of coccinellid beetles such as the harlequin ladybird beetle Harmonia axyridis are important semiochemicals that function in antipredatory defense behavior. Pyrazines have also been coadapted from a primarily defensive role into pheromones that function in intraspecific communication, attraction, and aggregation behavior. However, the biosynthesis of MPs in ladybird beetles is poorly understood. Here, we tested the hypothesis that MPs could be produced by microbial symbionts in H. axyridis, which generates four different MPs. The evaluation of tissue-specific MP production showed that MP concentrations were highest in the gut tissue and hemolymph of the beetles rather than the fat body tissue as the presumed site of MP biosynthesis. Furthermore, manipulation of gut microbiota by antibiotic-containing diets resulted in a lower MP content in adult beetles. The analysis of the bacterial community of the digestive tract revealed the presence of bacteria of the genera Serratia and Lactococcus which are reportedly able to produce MPs. In line with the known diet-dependent production of MP in H. axyridis, we determined that the presence or relative abundance of some of the potential MP producers (Enterococcus and Staphylococcus) is also diet-dependent. We hypothesize a potential role of the microbiota in MP production in H. axyridis as a possible example for outsourcing the synthesis of ecologically important semiochemicals to its gut bacteria.

Keywords: Harmonia axyridis; Lactococcus lactis; chemical defense; gut microbiota; methoxypyrazines (MPs); symbiosis.

Figure 1

Methoxypyrazine (MP) content in life stages of the ladybird beetle H. axyridis. (a) Total MP content. (c) IPMP (2‐isopropyl‐3‐methoxypyrazine) content. (d) IBMP (2‐isobutyl‐3‐methoxypyrazine) content. Gray: data for MP pg/mg fresh weight (fw); dark gray: data for MP pg/sample. Life stages of H. axyridis: eggs, last larval instars (L4), beetles post hatching without food intake (beetle p.h.), mature beetles (adult beetle), beetles in diapause (beetle diapause). Statistical significant differences in values *p < 0.05

Figure 2

Influence of diets on total MP content in mature beetles. Quantification of MP content in gut and residual body (body) of male and female H. axyrids. Gray: data for MP pg/mg fresh weight (fw); dark gray: data for MP pg/sample. The following feeding assays were performed for 10 days: (a) Aphid diet on Petri dishes. (b) Grape diet. (c) Honey syrup diet. (d) Honey syrup/S. cerealella eggs diet. Statistical significant differences in values *p < 0.05; **p < 0.01.

Figure 3

Microbiome analysis of gut of Harmonia axyridis. (a) Micrograph of bacteria in the lumen of the hindgut of a female H. axyridis. b: bacteria; hc: hemocoel, hg: hindgut; mu: muscle layer; mp: Malpighian tubules. (b) Heatmap showing relative proportion of bacteria summarized at genus level for male and female samples (gut tissue and residual body tissue) for adult beetles fed on aphid and grape diets. ap: aphid diet; bod: residual body tissue; M: males; F: females; gr: grape diet. Bacteria highlighted in red are presumed to produce MPs

Figure 4

Influence of antibiotics on total MP content of H. axyridis. A Total MP concentration (pg/mg) fresh weight of guts and residual bodies (body) of female and male H. axyridis fed for 10 days with Sitotroga eggs (in honey) (gray) and Sitotroga eggs mixed with honey and antibiotics (dark spotted). (b) Total MP contents in pg/sample after same procedure as shown in 4A. (c) Total MP (pg/mg) fresh weight of whole individuals, dissected guts (gut) and residual bodies (body) from L4 larval instars and newly hatched beetles (beetle p.h.) under Sitotroga eggs (gray) and Sitotroga eggs‐antibiotic mix (dark spotted). (d) Total MP pg/sample of experiment presented in C. Statistical significant differences in values *p < 0.05; **p < 0.01