by Mechas and Roberto
This is the story of how pursuing a century-old observation led to the recent discovery of the chemical ecology involved in protecting a beetle from fungal infection during larval molting. It shall come as no surprise that we would be thrilled by such a story; throughout the lifetime of the blog, we've had multiple posts on insect-microbe symbioses. There's Elio's telling of leaf-cutting ants, with their sustainably farmed fungal gardens, and Christoph's description of thistle tortoise beetles and their pectin-degrading bacterial symbionts, to name but two among many.
In the first installment on the thistle tortoise beetle, Christoph introduced us to the work of Hans-Jürgen Stammer who, along with his mentor Paul Buchner (think Buchnera aphidicola, the aphid endosymbiont described in this post by Merry), was a pioneer in the study on insect-microbe symbioses. In addition to investigating thistle tortoise beetles, Stammer did careful studies describing the life cycle and anatomy of a different group of beetles, those from the genus Lagria. In 1929 he published many of his findings, where he noted that the larvae of these small beetles (~0.5 cm in length) accumulated bacterial symbionts in three unusual dorsal compartments. Strikingly, modern-day 3-D reconstructions of the Lagria larvae beautifully confirm Stammer's original observations. We cannot help but marvel at the stunning work carried out by these early 20th century explorers of insects and their microbiomes! These bacteria-filled dorsal compartments seem to beckon loudly for further exploration. But truth is, Stammer's findings lay dormant for nearly a century.
Fortunately for science, Laura Flórez working with Martin Kaltenpoth (Max Planck Institute for Chemical Ecology, Jena and Johannes Gutenberg University, Mainz, Germany) along with several other colleagues, took notice of the Lagria symbiosis in recent years and, as the saying goes, "figured it all out" in a series of fascinating studies. They of course had good reason to begin these studies. The life cycle of these beetles, like that of all plants and animals, occurs in a sea of microbes. Going from egg to adult beetles, these animals have several developmental stages during which their protective barriers are particularly vulnerable to infections. The eggs themselves could be killed by pathogens and thus they are usually surrounded by protective bacteria. Additionally, as the larvae undergo each instar molting, they shed their "exuvia" (the melanized cuticle) and the soft tissue (unmelanized cuticle) is temporarily exposed to potential pathogens. Might bacteria-filled compartments provide a source of beneficial microbes to protect such frail larvae?
In the early days of their investigations, Flórez and Kaltenpoth discovered that the developing larvae of Lagria harbored strains of Burkholderia (very similar to free-living Burkholderia gladiola) in their dorsal compartments. In subsequent work, Flórez, Kaltenpoth and colleagues identified an antifungal polyketide – which they named lagriamide – from Lagria villosa eggs obtained from soils in Brazil, through a collaboration with Andre Rodrigues (Sao Paulo State University, Brazil). These early results set them up perfectly to address the question of whether the bacteria in the dorsal compartments could protect the larvae from fungal infections.
The team set out to determine if the ectosymbionts associated with the beetle L. villosa could protect it during its early life stages. They first evaluated if the beetle symbionts had anti-fungal activity. When exposed to the fungal pathogen Purpureocillium lilacinum, the larvae containing a natural symbiotic community survived better than aposymbiotic (symbiont-free) larvae. When tested against the generalist entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae, symbiotic larvae again survived better than larvae lacking symbionts. The symbionts also protected pupae exposed to either a mix of three fungi (P. lilacinum, B. bassiana and M. anisopliae) or to M. anisopliae conidia applied topically to pupae. From these results the investigators concluded that the natural symbiont community inhibits fungi and suggested that these symbionts have a protective role during the insect's early developmental stages.
The next step in this study was to characterize this symbiont community in L. villosa early-stage individuals collected from the field. A sequence analysis of these symbionts confirmed the predominance of Burkholderiaceae and showed that one B. gladioli strain, named Lv-StB, was the dominant strain during larval development. Fluorescence in situ hybridization (FISH) showed that strain Lv-StB fills dorsal compartments (now also called "symbiotic organs") of the larvae and covers the surface of larvae and pupae (Fig. 3, top panel). Importantly, the anti-fungal polyketide lagriamide – detected in situ by a high-resolution atmospheric pressure mass-spectrometry imaging technique known as "AP-SMALDI-MSI" – was produced during these developmental stages in the dorsal compartments and on the exoskeletons shed during molting (Fig. 3, bottom panel). Thus, these studies very nicely uncover an insect defense strategy mediated by bacterial ectosymbionts that persist during molting and larval development and protect the early and vulnerable developmental stages of the beetle L. villosa against fungal pathogens. We can only wonder about what other marvelous symbiosis will be discovered when investigators take the time and dive deeper into other observations made decades if not centuries ago!