Title: Microbial insect-symbioses: the hidden fungal lifestyle

Abstract:

Mutualistic interactions between microbes and host animals are ubiquitous in nature, however, most animal-fungal symbioses remain understudied. Ambrosia beetles (Curculionidae: Scolytinae and Platypodinae) and their fungal symbionts (Ophiostomatales, Hypocreales, Polyporales, Ceratocystidaceae) represent one of the oldest and most diverse forms of mutualism in nature. These beetles rely on fungi as their sole food source and the fungi rely on their hosts for maintenance and dispersal within specialized insect transport organs termed mycangia. The laurel wilt fungal pathogen, Harringtonia lauricola (formerly Raffaelea), forms symbiotic associations with the invasive (to the US) ambrosia beetle, Xyleborus glabratus, and more recently, with native ambrosia beetle species (e.g., X. affinis, X. ferrugineus) in the Southeastern United States.  Ambrosia beetles burrow into the sapwood of trees where they construct elaborate galleries to house their brood and the fungal gardens from which they feed. These beetles are unique among fungus-farming insects in their development of specialized structures called mycangia, which act as reservoirs to house the fungi during transport and as a means for inoculating new beetle galleries with their fungal food source.. These structures have independently evolved numerous times in phylogenetically diverse beetle species and act to contain fungal symbionts on or within their bodies during dispersal to new environments, as well as to further allow for the vertical transmission of the adapted symbiotic fungi across beetle generations. Laurel wilt is a lethal disease of susceptible plants in the family Lauraceae, a group containing over 600 species including red bay, swamp bay, sassafras, and avocado. The causative agent of the disease has been shown to be H. lauricola. Upon exposure to R. lauricola, infected trees show rapid wilting of leaves and branches, leading to the death of part or all of the tree. The disease has steadily advanced throughout the Southeastern United States, largely unchecked, and is responsible for the death of over 500 million trees since its introduction less than two decades ago. We have investigated basic aspects of the physiology of H. lauricola and demonstrate that the fungus displays a unique cold tolerance as well as pH optima in terms of its growth profile. Antibiotic resistance marker profiling revealed susceptibility of H. lauricola to phosphinothricin, hygromycin, sulfonyl urea, and benomyl. Fungicide resistance profiling revealed sensitivity to a range of conazoles, dithiocarbamates, zineb (zinc fungicide), dichlorofluanid, and prochloraz. In addition, phenotypic screening using a broad spectrum (growth) substrate microarray revealed a detailed physiological profile of H. lauricola substrate utilization and chemical sensitivity. These data indicated that H. lauricola displays: (i) relatively restricted carbon utilization profile, (ii) is broadly capable of utilizing a wide range of sulfur and phosphate containing compounds, and (iii) has distinct pH and osmotic growth profile sensitivities, and (iv) that some of these latter growth limitations could be rescued by supplementation of the media with specific compounds. Growth profiling on fatty acids revealed toxicity on 10 carbon and lower (C₁₀) chain containing substrates, with active growth on C₁₂–C₁₈ fatty acids. Methods for transformation and genetic manipulation of H. lauricola as well as a range of plasmid markers were developed based on Agrobacterium and blastospore mediated approaches. Tools for the genetic manipulation of H. lauricola were developed and reporter strains expressing green and red fluorescent proteins constructed. These strains coupled to microscopic approaches were used to characterize dynamic aspects of the colonization of the beetle mycangia by H. lauricola.

Biography:

Nemat O. Keyhani, PhD, is a Professor in the Department of Microbiology and Cell Science at the University of Florida, Gainesville, FL, USA. His research involves fungal biology and genetics. Much of his career has been focused on the biological intricacies of fungal-insect interactions including entomopathogenic fungi and insect fungal mutualists. Regarding the latter, Keyhani has been applying molecular and cellular approaches to study fungal-ambrosia beetle symbioses, developing new models for understanding this unique environmental niche. Although most of these fungi are benign, they also include devasting plant pathogens that have caused tremendous losses to forest and urban trees and threatened important agricultural crops. Keyhani’s research interests also include insect communication and chemical detection. Understanding how insects perceive and respond to environmental chemicals and how they communicate to produce cooperative or self-organized behaviors is integral to increasing our knowledge concerning insect behavior and can have practical application in pest control.