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Ambrosia beetles are social, fungal-farming insects that nest within tree xylem. Their close living conditions make them potentially vulnerable to microbial infectious diseases. We show that the insect pathogenic fungus Metarhizium anisopliae effectively infects and kills Xyleborus affinis adults, even within sawdust-based colony habitats. Healthy beetles did not avoid infected nestmates, and increased contact led to higher mortality and reduced offspring; however, larvae and pupae were still produced, even when colonies began with only infected beetles. Diseased individuals and Metarhizium CFUs were concentrated in the upper third of the nest, while surviving adults and brood were found in the middle/lower areas. A beetle symbiotic fungus, Neocosmospora sp. Xa1 was identified, which inhibited Metarhizium growth, potentially aiding in defense. Our findings suggest spatial structuring and microbial interactions within the nest help protect vulnerable brood to support colony persistence, revealing colony-level mechanisms that buffer against spread of infectious diseases, favoring offspring survival. 

Ambrosia beetles have evolved specialized structures termed “mycangia”, which house and transport symbiotic microbes. Microbial partners include at least one obligate mutualistic filamentous fungus used as food for larvae and adults, and potentially secondary filamentous fungi, yeasts, and bacteria. Beetles in the genus Xyleborus possess paired pre-oral mycangial structures located within the head on either side of the mouth parts. Mycangia develop in pupae, with newly emerged adults acquiring partners from the environment. However, information concerning the cellular structure and function of Xyleborus mycangia remains limited. We show that in X. affinis, mycangia are lined with a layer of striated dense material, enclosing layers of insect epithelial cells, with diverse spine-like structures. Larger (5–10 μm) projections were concentrated within and near the entrance of mycangia, with smaller filaments (4–8 μm) within the mycangia itself. Rows of “eyelash” structures lined the inside of mycangia, with fungal cells free-floating or in close association with these projections. Serial sections revealed mandibular articulations, and mandibular, pharyngeal, and labial muscles, along with the mycangial entry/exit channel. Sheets of comb-like spines at the mycangial entrance and opposite the mycangia attached to the roof of the labrum or epipharynx may serve as an interlocking mechanism for opening/closing the mycangia and guiding fungal cells into entry/exit channels. Additionally, mandibular fibra (muscle tissue) potentially enervating and affecting the mechanism of mycangial functioning were noted. These data add crucial mechanistic detail to the model of pre-oral mycangia in Xyleborus beetles, their cellular structures, and how they house and dispense microbial symbionts. 

Ambrosia beetles bore into trees, excavating galleries where they farm fungi as their sole source of nutrition. These mutualistic fungi typically do not cause significant damage to host trees; however, since their invasion into the U.S., the beetle Xyleborus glabratus has vectored its fungal partner, Harringtonia lauricola, which has acted as a devastating plant pathogen resulting in the deaths of over 500 million trees. Here, we show differences in the mycangial colonization of the indigenous X. affinis ambrosia beetle by H. lauricola, and the native fungal species, H. aguacate and Raffaelea arxii. While X. affinis was a good host for H. lauricola, the related ambrosia beetle, X. ferrugineus, was only marginally colonized by H. lauricola. X. affinis beetles neither fed on, nor were colonized by, the distantly related fungus, Magnaporthe oryzae. Mycangial colonization was affected by the nutritional state of the fungus. A novel method for direct quantification of mycangial contents based on image cell cytometry was developed and validated. The method was used to confirm mycangial colonization and demonstrate alternating fungal partner switching, which showed significant variation and dynamic turnover. X. affinis pre-oral mycangial pouches were visualized using fluorescent and light microscopy, revealing that newly emerged pupae displayed uncolonized mycangia prior to feeding, whereas beetles fed H. lauricola contained single-celled fungi within 6 h post-feeding. Mixed populations of fungal cells were seen in the mycangia of beetles following alternating colonization. Nuclear counter-staining revealed insect cells surrounding the mycangia. These data highlight variation and specificity in ambrosia beetle–fungal pairings and provide a facile method for direct quantification of mycangial contents.