More than a colour change: insect melanism, disease resistance and fecundity

Abstract

A 'dark morph' melanic strain of the greater wax moth, Galleria mellonella, was studied for its atypical, heightened resistance to infection with the entomopathogenic fungus, Beauveria bassiana. We show that these insects exhibit multiple intraspecific immunity and physiological traits that distinguish them from a non-melanic, fungus-susceptible morph. The melanic and non-melanic morphs were geographical variants that had evolved different, independent defence strategies. Melanic morphs exhibit a thickened cuticle, higher basal expression of immunity- and stress-management-related genes, higher numbers of circulating haemocytes, upregulated cuticle phenoloxidase (PO) activity concomitant with conidial invasion, and an enhanced capacity to encapsulate fungal particles. These insects prioritize specific augmentations to those frontline defences that are most likely to encounter invading pathogens or to sustain damage. Other immune responses that target late-stage infection, such as haemolymph lysozyme and PO activities, do not contribute to fungal tolerance. The net effect is increased larval survival times, retarded cuticular fungal penetration and a lower propensity to develop haemolymph infections when challenged naturally (topically) and by injection. In the absence of fungal infection, however, the heavy defence investments made by melanic insects result in a lower biomass, decreased longevity and lower fecundity in comparison with their non-melanic counterparts. Although melanism is clearly correlated with increased fungal resistance, the costly mechanisms enabling this protective trait constitute more than just a colour change.

Keywords: immunity; insect; insect–pathogenic fungus; melanism; resistance; stress.

Figure 1.

(a) Cuticular melanism and colour forms of G. mellonella larvae and (b) mortality rates following topical treatment with the fungus B. bassiana at a dose of 5 × 10⁶ ml⁻¹ (see the electronic supplementary material, figure S1 for responses to other doses). ***p < 0.001 compared with NM larvae (n = 80–140).

Figure 2.

(a) Mean cuticular thickness of G. mellonella larvae, showing significantly thicker cuticles of M than NM larvae ± s.e.m. (n = 20, ***p < 0.0001). (b) Cuticular PO activity in M (dark grey bars and filled circles) and NM larvae (light grey bars and open circles) topically inoculated with B. bassiana. Activity was significantly enhanced in M larvae but not NM larvae 24 h pi relative to the uninfected controls (*p < 0.05; n = 9).

Figure 3.

(a) Melanotic encapsulation response and (b) haemocyte numbers in M (solid line) and NM (dashed line) G. mellonella larvae following topical application of B. bassiana ± s.e.m. For encapsulation, melanization of each implant was assessed by image analysis and is represented by the grey value, with greater melanization being observed in M than NM larvae, n = 32–45, *p < 0.05, **p < 0.01 compared with uninfected control larvae; ***p < 0.001 compared with M larvae at 48 h. Total haemocyte counts (THCs), n = 15–27, † = p < 0.001 (comparison between M and NM larvae), *** = p < 0.001 (NM larvae compared with NM larval control), §§ = p < 0.01 (M larvae compared with M larval control), §§§ = p < 0.001 (M larvae compared with M larval control).

Figure 4.

(a) PO and (b) lysozyme-like activity in haemolymph of G. mellonella M (solid line) and NM (dashed line) larvae topically infected with B. bassiana, showing greater activity in NM than M larvae for PO (*p < 0.05) and lysozyme (*p < 0.05, **p < 0.01, ***p < 0.001) compared with uninfected control (time zero) larvae. Each graph shows the mean of at least 26 samples ± s.e.m. The PO activity in uninfected larvae was also higher in NM than M larvae (*p < 0.05).

Figure 5.

Expression of antimicrobial peptide genes and other immunity/stress-linked genes in M and NM larvae after topical fungal infection. Expression of Cecropin-D, Gallerimycin, Galiomicin, Gloverin, IMPI, Transferrin and 6-tox was assayed in fat body tissue by QRT-PCR in uninfected animals, and at 24 h and 48 h after topical infection. Expression of 18-wheeler, Hsp-90 and contigs 20595 and 704 was assayed in uninfected controls and at 48h. Basal expression in uninfected M larvae (column 1) is calculated as a fold change relative to NM uninfected larvae. Fold change in infected NM larvae (column 2) is also relative to NM uninfected larvae. Fold change in infected M larvae is relative to the M uninfected expression (column 3) and to NM uninfected baseline to indicate overall expression (column 4). The colour gradient indicates fold changes in gene expression. Basal expression in uninfected NM larvae is represented by black (1 × fold change). Increasing blue intensity indicates gene downregulation, whereas yellow and purple represents upregulation in the range 1–40-fold and 41–400-fold, respectively. The mean ΔΔC_t value of three independent blocks is reported. *p < 0.05; **p < 0.01. Further details can be found in the electronic supplementary material, §S1.12.

Figure 6.

(a) Mortality rate and (b) median LT₅₀ of M (dark grey bars, solid line) and NM larvae (light-grey bars, dahed line) after injection with 750 B. bassiana blastospores per larva. ***p < 0.001 in comparison with NM larvae.

Figure 7.

Expression of antimicrobial peptide genes and other immunity/stress-management genes in M and NM larvae 12 h after injection of fungal blastospores. Expression was assayed in fat body tissue by QRT-PCR in injected and sham-injected control animals. Expression in M controls (bar 1) is calculated as a fold change relative to NM controls. Fold changes in infected NM (bar 2) are also relative to NM controls, whereas fold changes in infected M larvae are relative to the uninfected M (bar 3) and uninfected NM to indicate overall expression (bar 4). The colour gradient indicates fold changes in expression, which in NM controls is represented by black (1 × fold change), while blue and yellow indicate downregulation and upregulation (1–40-fold), respectively. The mean ΔΔCt value of three independent experiments is reported. **p < 0.01. Further details can be found in the  electronic supplementary material, §S1.12.