Fungi That Live Within Animals: Application of Cell Cytometry to Examine Fungal Colonization of Ambrosia Beetle (Xyleborus sp.) Mycangia

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 <i>Xyleborus glabratus</i> has vectored its fungal partner, <i>Harringtonia lauricola</i>, 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 <i>X. affinis</i> ambrosia beetle by <i>H. lauricola</i>, and the native fungal species, <i>H. aguacate</i> and <i>Raffaelea arxii</i>. While <i>X. affinis</i> was a good host for <i>H. lauricola</i>, the related ambrosia beetle, <i>X. ferrugineus</i>, was only marginally colonized by <i>H. lauricola</i>. <i>X. affinis</i> beetles neither fed on, nor were colonized by, the distantly related fungus, <i>Magnaporthe oryzae</i>. 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. <i>X. affinis</i> 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 <i>H. lauricola</i> 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.