Abolfazl Masoudi

Ambrosia beetles rely on obligate fungal partners for survival, and these associations are emerging as model systems for examining the development and evol

Fungal‐animal mutualisms remain significantly understudied, yet they represent some of the most successful partnerships known in nature. Fungal farming ambrosia beetles cultivate a consortium of fungal partners that include obligate filamentous members and yeasts. These fungi are maintained in highly specialised insect organs, termed mycangia, and are cultivated as food along the beetle galleries elaborated within host trees. Here, we isolated fungi from the mycangia of Xyleborus affinis ambrosia beetles using both standard ethanol‐wash and ethanol‐free protocols. Omitting the ethanol wash significantly increased fungal recovery and diversity. We identify two previously described filamentous species, Raffaelea arxii and R. fusca , and the yeast, Ambrosiozyma monospora , together with two new filamentous fungi, Neocosmospora affinis and Graphium ambrosium , and two novel yeasts, Alloascoidea xylebori and Wickerhamomyces ambrosius , from both gallery walls and beetle mycangia. To meet Koch‐like postulates, using mycangial colonisation assays, we demonstrate that all seven fungal species were individually competent at colonising aposymbiotic X. affinis mycangia, demonstrating each as viable symbionts. Our results show that ethanol protocols can bias recovery of mycangial fungi, leading to underestimation of fungal diversity associated with ambrosia beetles. These findings provide a framework for improved characterisation of ambrosia beetle‐fungal mutualisms and experimental validation of fungal symbionts.

Fungal-animal mutualisms remain significantly understudied, yet they represent some of the most successful partnerships known in nature. Fungal farming ambrosia beetles cultivate a consortium of fungal partners that include obligate filamentous members and yeasts. These fungi are maintained in highly specialized insect organs, termed mycangia, and are cultivated as food along the beetle galleries elaborated within host trees. Here, we isolated fungi from the mycangia of Xyleborus affinis ambrosia beetles using both standard ethanol-wash and ethanol-free protocols. Omitting the ethanol wash significantly increased fungal recovery and diversity. We identify two previously described filamentous species, Raffaelea arxii and R. fusca , and the yeast, Ambrosiozyma monospora , together with two new filamentous fungi, Neocosmospora affinis and Graphium ambrosium , and two novel yeasts, Alloascoidea xylebori and Wickerhamomyces ambrosius , from both gallery walls and beetle mycangia. To meet Koch-like postulates, using mycangial colonization assays, we demonstrate that all seven fungal species were individually competent at colonizing aposymbiotic X. affinis mycangia, demonstrating each as viable symbionts. Our results show that ethanol-protocols can bias recovery of mycangial fungi, leading to underestimation of fungal diversity associated with ambrosia beetles. These findings provide a framework for improved characterization of ambrosia beetle-fungal mutualisms and experimental validation of fungal symbionts.

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 have evolved specialized structures termed “mycangia”, which are used to house and transport partner 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, although the roles of these secondary partners remain poorly understood. Beetles in the genus Xyleborus possess a set of two pre-oral mycangial structures located within the head on either side of the mouth parts/mandibles. These mycangia develop in pupae, with newly emerged adults acquiring partners from the environment. However, information concerning the cellular structure and function of the Xyleborus mycangia remains limited. We show that the mycangia are lined with a thick layer of striated dense material, followed by layers of insect epithelial cells, with different types of spine-like structures directed into the mycangia. Larger (5-10 m m) Ross projections were concentrated within and near the entrance of the mycangia, with smaller tubules and filaments (4-8 m m) within the mycangia itself. In addition, rows of “eyelash” structures were seen lining the inside of mycangia, with the fungal cells apparently free-floating within the mycangial enclosure or in close association with projections. Serial sections revealed nearby mandible articulations, eyes and antennae, and mandibular, pharyngeal, and labial muscles, along with the mycangial entry/exit channel leading into the pre-oral cavity. Sheets of comb-like large spines at the mycangial entrance and opposite the mycangia attached to the roof of the labrum or epipharynx were noted that may serve as an interlocking mechanism for opening/closing the mycangia and for guiding fungal cells into entry/exit channels. In addition, mandibular fibra (muscle tissue) potentially enervating and affecting the mechanism of mycangial functioning, i.e., acting to fill/empty the mycangia were noted. These data support the hypothesis that the twin pre-oral mycangia in Xyleborus beetles are dynamic organs with evolved specialized cellular structures that mediate their function in housing and dispensing partner 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.