Can insects develop resistance to insect pathogenic fungi?

Abstract

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25(th) generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host's own immune responses.

Figure 1. Susceptibility of insects selected by B. bassiana to the fungal infections.

Mortality rate of selected line and non-selected line of G. mellonella larvae following topical treatment with the fungus B. bassiana (A) and M.anisopliae (B). (a-P<0.01 compared with non-selected line larvae. n  = 140–190 per line per treatment).

Figure 2. Immune function of insects selected by B. bassiana under infections.

Cuticular phenoloxidase (PO) activity (A), hemolymph phenoloxidase (B), lysozyme-like (C) activity and encapsulation responses (D) in hemolymph of G. mellonella larvae from non-selected (NS) and selected (S) lines at 24 h following topical application of B. bassiana (Bb) and M. anisopliae (Ma) (data presented as mean +/− SEM; a-P<0.05, b-P<0.01, c-P<0.001 compared with uninfected larvae from the same line; d-P<0.05 e-P<0.01 f-P<0.001 compared with NS larvae with the same treatment).

Figure 3. Defense genes expression in integuments and fat body of insects selected by B. bassiana.

Expression of antimicrobial peptide genes and other putative immunity/stress-management genes in the integument and fat body of non-selected (NS) and selected line (S) larvae, under basal (uninfected) conditions (A), 24 h after topical B. bassiana infection (B) and 24 h after M. anisopliae topical infection (C). Basal expression in uninfected S larvae (A) is illustrated as a fold change relative to NS uninfected larvae and the x-axis represents basal expression in NS larvae (i.e. 1-fold). Fold induction in infected NS larvae is also calculated relative to NS uninfected larvae (B, C). Fold induction in infected S larvae is calculated relative to the S uninfected expression (B, C). The mean ΔΔCt value of 3 independent experiments (each with a minimum of 5 insects per treatment) and the SEM are reported. *-P<0.05, **-P<0.01, compared with the corresponding induced change in NS line insects. Na = not assayed in fat body tissue.

Figure 4. Life-history traits in G. mellonella non-selected and selected lines.

Pupal weights (A) n = at least 52. Larval development time (B) (from egg hatching to onset of pupation) n = at least 52 (data presented as mean +/− SEM). a-P<0.05 compared with the NS insects.