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Many insects live in symbiosis with certain species of bacteria. These bacteria make important contributions to their hosts' nutrition, digestion, detoxification, reproduction and defense. Due to their close coexistence with insects, symbionts often lose genes for metabolic products that are also provided by their hosts over the course of their coevolution. One such example is the symbionts of reed beetles, which retain a very small genome containing only genes important for beetle development.

Interestingly, the beetle larvae and adults have different diets: the larvae suck amino acid-poor root sap, while the adults eat leaves and flowers with difficult-to-digest cell walls. The symbiotic bacteria support the larvae by producing amino acids, thereby supplementing their diet. They also support the adult beetles by producing an enzyme that breaks down plant cell walls. However, not all symbionts benefit both life stages.

Variable symbiotic services provided by bacteria

The Department of Insect Symbiosis, headed by Martin Kaltenpoth, conducted a study of symbionts of reed beetles in more detail. All reed beetles harbor the same symbiont. However, the research team found that in some cases, this symbiont had lost the ability to produce enzymes for breaking down the hard-to-digest plant cell wall. The scientists hypothesized that the production of these enzymes is only beneficial for adult beetles.

The paper is in the journal EMBO Reports.

"There are two types of symbiotic association within reed beetles. On the one hand, there are beetle species in which the symbiont benefits both life stages; on the other hand, there are those in which only the larvae benefit directly from the symbiont. Initially, we wanted to understand whether symbiont gene expression would corroborate this hypothesis, and whether symbiont regulation of gene expression would differ between species with one or two benefits—and, given the small total genome size, whether such regulation is even possible," says first author Ana Carvalho, summarizing the initial focus of her study.

Gene activity of reed beetle symbionts is adapted to the needs of the beetles at different stages of their development.

Carvalho and her colleagues used RNA sequencing, enzymatic activity assays and Fluorescence In Situ Hybridization (FISH) to examine gene expression, digestive activity in beetles and their symbionts, as well as the localization and cell shape of symbionts in four species of reed beetles in different stages of development.

"We found that the symbiont consistently enhanced the expression of genes for amino acid biosynthesis during the larval stage across four species of reed beetles. We also observed coordination between the expression of plant cell wall digestive enzymes from the host and the symbiont during the adult stage of the host, highlighting how fine-tuning symbiont gene expression may optimize symbiont benefit provision," says Carvalho.

The team was not only able to detect altered gene expression in different stages of beetle development, but also used imaging techniques to demonstrate that the symbiont changes its cell shape during its life cycle. This may be connected to its altered metabolic function during the larval and adult stages of the host beetle.

Plasticity in gene expression even under altered environmental conditions

As the focus of the investigations was on whether the symbiont can regulate its gene expression despite its small genome, the research team also examined possible differences in response to temperature fluctuations experienced by reed beetles during their life cycle. To this end, the researchers exposed reed beetle larvae to two different temperature cycles for one month, with fluctuations between 12 °C and 8 °C, and between 22 °C and 14 °C respectively.

Despite the greatly reduced genome and regulatory apparatus, a clear temperature-dependent gene expression pattern emerged. The symbiont was able to activate different genes depending on the temperature. In cold conditions, for instance, the symbiont activated a stress mechanism that typically responds to heat in free-living bacteria but appears to have evolved to respond to low temperature stress in this case.

While the study answers many questions, it also raises new ones. What functions do the remaining symbiont gene switches (transcription factors) have, and how are certain genes controlled in their absence? Why do the symbionts change shape, and what benefit does this bring to them and their host? Further experiments with reed beetles or easier-to-study insect-bacteria models are needed to clarify this.

"Our findings reveal that small symbiont genomes can regulate some very important processes, demonstrating that a regulated metabolism can be maintained with a minimal set of genes. We would now like to gain a more fundamental understanding of exactly how the metabolic coordination between host and symbiont works," summarizes Kaltenpoth.