Sex pheromone component produced by microbial associates of the forest pest Megaplatypus mutatus

Abstract

Megaplatypus mutatus (Chapuis) (Coleoptera: Platypodidae) is an ambrosia beetle native to South America that causes economic loss and was recently introduced to Italy, where it attacks and damages live poplar trees. Sulcatol and sulcatone are male‐produced pheromone components involved in the mating process of M. mutatus. Their relative proportions are highly variable among insects, although the temporal pattern shows that initially only sulcatol is present, and sulcatone increases with time, until they are finally both depleted. Sulcatol and sulcatone may be produced de novo by the beetles, they may be produced by fungi, or both pathways may contribute to their production. Sulcatol is stored in the males’ hindgut but sulcatone is only present in emissions, so there is an oxidation process to transform the alcohol to the ketone before or during pheromone release. It is our hypothesis that fungi associated with M. mutatus are responsible for this process. In this work, we studied a possible contribution of associated microorganisms in the conversion of sulcatol into sulcatone and its consequent role in the temporal release pattern of these sex pheromone components observed in male insects. Moreover, we inhibited the postulated enzymes involved in this pheromone conversion process – 3‐hydroxy‐3‐methyl‐glutatyl‐CoA reductase (HMGR) and P450 enzymes of a fungal strain – and added an antibiotic and a fungicide to the homogenate during sulcatol‐sulcatone conversion. Among the fungal species, particular interest was given to Graphium basitruncatum (Matsush.) Seifert & Okada (Microascales), as it is present in male but not in female exoskeletons and in insect gallery samples, suggesting a possible different role in pherome production, as the male is the pheromone‐producing sex. Several isolated strains were able to convert sulcatol to sulcatone, whereas the fungus G. basitruncatum showed the highest production of this ketone. Additionally, inhibition of P450 enzymes and HMGR from G. basitruncatum on this alcohol‐ketone conversion demonstrated that HMGR is involved in sulcatone generation using sulcatol as precursor, and that P450 enzymes are not. Finally, sulcatone production diminished significantly in homogenized tissues of male and female M. mutatus following addition of an antibiotic and a fungicide. The results suggest that fungi associated with M. mutatus are involved in pheromone production.

Male swarming aggregation pheromones increase female attraction and mating success among multiple African malaria vector mosquito species

“…Sulcatone is the most widespread norterpene in insects 38 functioning as pheromone in approximately 60 species predominantly in the orders Hymeneoptera and Coleoptera 39 . Sulcatone could be biosynthesized either by insects or by their microbial symbionts de novo through the mevalonate pathway from a few possible intermediates 40,41 or from a prenyl unit and an acetoacetate unit 38 . Acetoin is a well-known product from microbial metabolism and is widespread in nature 42 .…”

Volatile organic compounds emitted by Megaplatypus mutatus associated fungi: chemical identification and temperature-modulated responses by the ambrosial beetle

“…Fungal strains were cultivated according to the conditions and culture media outlined by Slodowicz et al (2019). In brief, fungal strains were grown in 50-ml glass vials on 10 ml of 2% solid malt extract agar (MEA) in the dark at 20 ºC for 15 days.…”

“…Samples were separated on both a DB-5MS (J&W Scientific, CA, USA), column (30 m × 0.25 mm × 0.25 μm) and a DB-WAX J&W Scientific column (30 m × 0.25 mm × 0.32 μm). Desorption of DB-5MS and DB-WAX columns was conducted following the method described by Slodowicz et al (2019). To determine the chromatographic retention indices (RI) under the applied conditions, a mixture of normal alkanes ranging from C7 to C20 was injected.…”

“…Taking into account Additional file 1, the putative roles for each species have been suggested: a potential phytopathogenic role for FSSC and a gut symbiotic association for C. insectalens. Meanwhile, G. basitruncatum has the capacity to contribute to the biosynthesis of a male sex pheromone component that attracts the female (Slodowicz et al 2019) and it is the first fungal species to colonize new tree host tissues. Finally, Raffaelea species have been extensively studied among other ambrosia interactions and are considered a key nutritional symbiont in these interactions.…”

Symbionts in insect biology and pest control – an introduction

“…Slodowicz et al. () demonstrate that several fungal strains isolated from the beetle's exoskeleton are able to perform the conversion from sulcatol to sulcatone. The fungus Graphium basitruncatum (Matsush.)…”