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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38513077
202402869
10.1073/pnas.2402869121
commCommentaryevolutionEvolution418
437
Commentary
Biological Sciences
Evolution
Bugs promote immune priming via gut-breaching bacteria
Ravenscraft Alison a
Hunter Martha S. mhunter@arizona.edu
b 1 https://orcid.org/0000-0002-6342-675X

aDepartment of Biology, University of Texas at Arlington, Arlington, TX 76019
bDepartment of Entomology, The University of Arizona, Tucson, AZ 85721
1To whom correspondence may be addressed. Email: mhunter@arizona.edu.
21 3 2024
2 4 2024
21 9 2024
121 14 e2402869121Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

USDA | National Institute of Food and Agriculture (NIFA) 100005825 2023-67013-39897 Alison RavenscraftMartha (Molly) S Hunter USDA | National Institute of Food and Agriculture (NIFA) 100005825 2023-67013-39897 Alison RavenscraftMartha (Molly) S Hunter
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pmcAn astonishing diversity of microbes colonize animal guts. We expect these beneficial gut bacteria to have molecular conversations with their host from behind protective layers of gut epithelia. In fact, we assume bacteria that breach those protective barriers to be marauders, wreaking havoc and causing disease. In the extraordinary findings of Jang et al. (1), however, the opposite is true: A lineage of ingested bacteria in the Burkholderiaceae crosses the midgut epithelium of a host bug and triggers a systemic immune response. Far from costly, the invasion instead confers general protection against pathogens. There is no doubt that the host is firmly in control of this astonishing phenomenon. Before the breach, the bacterial cell membranes are compromised by host antimicrobial peptides (AMPs), and after the breach, the bacteria are readily engulfed by phagocytosis, such that continual ingestion of the bacteria is required for maintenance of the immunity. Thus, Jang et al. (1) elucidate a remarkable mechanism for immune priming in an invertebrate.

Insects lack adaptive immunity, but their innate immune system is sophisticated and highly effective (2). When a fruit fly swallows bacteria, the first lines of defense are secretion of microbicidal chemicals into the gut lumen, including AMPs. Midgut cells are also physically protected by the peritrophic membrane, which prevents contact with gut contents. If the bacteria manage to penetrate the gut lining and invade the hemolymph, systemic cellular and humoral defense systems are activated. For cellular defense, phagocytic immune cells patrol the body cavity, engulfing and lysing intruding bacteria. AMPs are the major weapons of humoral immunity. Upon detection of an intruder, AMPs are secreted by the insect fat body into the hemolymph (insect blood), where they rapidly kill bacterial invaders.

This understanding of insect immunity is based on a few model insects (fruit flies, mosquitos, caterpillars, and bees) that all belong to the group of species that undergo complete metamorphosis (holometabolous insects). We know much less about the immune system of hemimetabolous species, those whose young look much like wingless adults—insects like grasshoppers, roaches, dragonflies, and “true” bugs, the last an especially species-rich group that includes aphids and the bean bug investigated by Jang et al. What data exist suggest that immune defenses vary and immune pathways assumed to be universally conserved may, in fact, operate differently in fruit flies versus aphids (3). Most notably, many hemimetabolous insects lack a peritrophic membrane (4), meaning that ingested bacteria have direct access to gut epithelial cells.

Insects also show a responsive immunity called immune priming. Multiple studies have shown that previous exposure to a nonlethal dose or compromised pathogen can result in increased survival of a subsequent pathogen challenge (5). The underlying mechanisms are poorly understood. A few possible mechanisms include a general upregulation of immune activity following pathogen exposure, upregulation of specific AMPs, or epigenetic modifications (5). Jang et al. (1) suggest an astonishing additional mechanism of immune priming.

In the bean bug, Riptortus pedestris, as in many bug species, the midgut is elaborated into several sections (Fig. 1). Young bug nymphs ingest nutrients and microbes from the soybean host plant and the soil, and a diverse assemblage of bacteria accumulate in the midgut 3 (M3) section. The arrival of these bacteria triggers local gut immune responses and secretion of AMPs. All of this might be expected. However, here is where the story gets wild. The authors noticed that other organ systems, including fat body and distal parts of the gut where the bacteria were not present, also showed upregulation of immunity, evidence of a systemic response. How do bacteria within the M3 region of the gut trigger that response in distant body parts? It turns out that some bacteria, members of the Betaproteobacterial family Burkholderiaceae, migrate between or through cells of the gut epithelium into the body cavity, as the authors’ beautiful fluorescence and electron micrographs clearly show. No protective peritrophic membrane lines the gut in these bugs to prevent this direct interaction.

Fig. 1. The bean bug Riptortus pedestris ingests a complex microbial community from the soil. Most ingested bacteria are retained in the midgut 3 (M3) region, but Jang et al. (1) show that certain lineages in the Burkholderiaceae breach the gut wall and enter the body cavity. These bacteria appear to inflict no cost on the bug and instead prime systemic immunity so that injected pathogens are neutralized. This occurs anterior to a symbiotic organ, the midgut 4 (M4) region. While not the focus of the current study, previous research has shown bacteria in the Caballeronia genus (also in the Burkholderiaceae) colonize the M4 through a highly selective constricted region between the M3 and M4. Once Caballeronia is through, it forms a virtually monocultural population in crypts in the M4, providing nutritional benefits to the bug throughout its life. Illustration: Aya Yakota, Peter Mergaert, and Seonghan Jang.

Jang et al. elucidate a remarkable mechanism for immune priming in an invertebrate.

When the Burkholderia bacteria get into the body cavity, the cells are phagocytosed and cleared by the immune system. However, this represents no ordinary invasion of a pathogen. Jang et al. (1) show there is no fitness cost to having these bacteria circulate in the body cavity until cleared; bugs that ingested the Burkholderia achieved equivalent survival, size, and fecundity as control bugs. The harmlessness of the invading Burkholderia is likely because they were already compromised, presumably by AMPs, in the M3 prior to migrating into the body cavity. While the same Burkholderia strain grown in culture and injected into the bugs caused mortality, bacteria isolated from the M3 and then injected caused none.

The apparent similarity of bugs with gut-breaching bacteria to control bugs vanished when the insects were challenged by injection of a pathogenic bacterium, Pseudomonas entomophila, into the body cavity. The Burkholderia bugs with primed systemic immunity survived the challenge, while control bugs did not. In an additional ingenious test to determine the mechanism of Burkholderia-triggered protection against P. entomophila, the authors knocked out the cellular response (overwhelming blood cells with beads), the humoral response (via RNAi knockdown of the key humoral immunity gene relish), or both, in advance of the pathogen challenge. The results clearly showed that the bug’s survival when challenged by the pathogen was due to the immune response induced by Burkholderia. Knockdown of either humoral or cellular immunity in these bugs reduced survival, but the humoral response was more important. Loss of just humoral immunity resulted in abysmal bug survival, indistinguishable from knockdown of both cellular and humoral immune systems.

How long the systemic immune priming effect lasts is tied to ingestion of these gut-breaching bacteria. While bacteria are entering the M3 and crossing the gut wall to be removed in the body cavity, systemic immunity persists. In an experiment in which the authors withdrew Burkholderia after an initial 48 h of feeding, the titer of Burkholderia dropped, and with it the survivorship of these bugs when challenged with P. entomophila. Thus, when the flow of bacteria stops, these bugs are as vulnerable as insects that never ingested Burkholderia.

What bacteria are able or allowed to penetrate the M3 epithelium? It seems many bacterial species in the Burkholderiaceae can do it, so a bug exposed to soil, where these lineages are common, would likely have a steady diet of beneficial trespassers. However, a bacterial strain in a lineage a bit farther out, Achromobacter, in the order Burkholderiales, did not breach the gut or trigger systemic immunity. Another bacterial lineage that did not cross the gut or trigger a systemic response is Caballeronia, a genus recently removed from Burkholderia. Caballeronia is the nutritional symbiont that colonizes the distal midgut 4 (M4) region and makes it a nearly monocultural symbiotic organ (more details on this below). The specificity of different roles for this allied group of bacteria in different sections of the gut suggests the bugs are in a sense masterful curators—filtering the complex soil microbiota to allow a subset into the gut, then selecting certain bacterial lineages to colonize and populate the M4, detaining others in the M3, and attenuating others before admitting them through the M3 epithelium into the body cavity where they prime systemic immunity against pathogens.

The findings of Jang et al. (1) reveal another chapter of host–microbe interactions in a system that is already a model for understanding environmental acquisition of nutritional symbionts. Specialized beneficial relationships with environmentally-acquired, free-living bacteria are uncommon in terrestrial arthropods, with nutritional symbionts more likely to be intracellular, inherited from mother to offspring, and engaged in long-term coevolutionary associations. Kikuchi et al. discovered an ancient exception in at least seven families of true bugs, comprising thousands of insect species, in which young nymphs ingest free-living Caballeronia bacteria from the soil and these cells then squeeze through a narrow passage to colonize the M4 [Fig. 1; (6)]. The Caballeronia take up residence and provide nutrition to the bug (7). That the gut-breaching Burkholderia are closely related to the nutritional symbiont could be a coincidence or perhaps is an outcome of an evolved ability of the bug to recognize and control these bacteria. That they are not interchangeable—Caballeronia do not breach the gut, and the gut-breaching Burkholderiaceae do not colonize the M4 (1)—shows the specificity of these interactions.

One of the most exciting aspects of this study is how many questions it raises and the multiple research avenues it opens. How general is the phenomenon of immune priming via gut-breaching bacteria? Is it common to the thousands of species of true bugs that associate with Caballeronia as a nutritional symbiont? Is it more common still? In light of these results, is the absence of the peritrophic membrane in true bugs not an ancestral constraint but an adaptive feature that allows interactions with microbial friends and enemies that are not possible when it is present? We might also ask why a bug would rely on continual ingestion of microbes to maintain systemic immunity when that immunity appears to be cost-free? One might expect that under stressful field conditions, systemic immunity may indeed have some cost, and in these circumstances, immune priming might be most helpful when pathogen encounters are most likely, e.g., when bugs are in contact with microbially rich soil. Lastly, has dependence on a nutritional symbiont acquired from soil preadapted these insects to seek out and ingest, not avoid, complex microbial mixtures, and in so doing filter and harness that complexity for their own benefit? The marvelous findings of Jang et al. (1) require that we deepen our appreciation of both insect immunity, and of the countless ways animals interact with microbes.

The authors’ research is supported by a USDA NIFA grant (2023-67013-39897) to M.S.H. and A.R.

Author contributions

A.R. and M.S.H. wrote the paper.

Competing interests

The authors declare no competing interest.

See companion article, “Ingested soil bacteria breach gut epithelia and prime systemic immunity in an insect,” 10.1073/pnas.2315540121.
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