Fig 1.
Simplified overview of the flour beetle Toll signaling pathway.
(I) Pattern Recognition Receptors (PRRs) identify pathogen or danger-associated molecular patterns (PAMPs or DAMPs) triggering signal transduction through Toll. This forms an intracellular signaling scaffold consisting of MyD88, Tube, and Pelle, resulting in Cactus phosphorylation by Pelle. The subsequent degradation of Cactus allows Dif and Dorsal transcription factors to translocate into the nucleus, initiating immune effector transcription. (II) Production of AMPs from Toll signaling proceeds through a sequence of stages, starting with constitutive production before infection (A), induction upon parasite recognition (B), peak output (C), decay of transcript production, and finally resolution of the response (D). Dysfunction in negative regulators could potentially disturb signaling dynamics at any stage, leading to overproduction of immune effectors. Figure created with BioRender.com.
Fig 2.
DsRNA-mediated knockdown of cactus results in increased Toll signaling.
(a-h) The expression of cactus (a, e) and the antimicrobial peptides defensin-2 (b, f), defensin-3 (c, g) and cecropin-2 (d, h) were assayed via RT-qPCR in whole adult beetles treated with 250 ng of cactus or malE dsRNA and then septically challenged with heat-killed Bt (left) or C. albicans (right). Beetles were sacrificed before microbial challenge (hour 0), and nine additional times after challenge over 48 hours. The expression of each gene relative to the reference gene RP18s is represented on a log2 scale. Splines have been added to visualize the induction and decay dynamics from RNAi treatment using “loess” function (span 0.25) in the geom_smooth algorithm of ggplot2 in R. The * represents whether the constitutive (hour 0), induction (slope from hour 0 to peak expression), decay (slope from peak expression to 48 hours), or resolution (hour 48) windows were significantly altered by cactus depletion (α = 0.0167).
Fig 3.
Toll signaling increases functional metrics of cellular immunity and antibacterial activity.
(a) The number of circulating hemocytes in malE or cactus dsRNA-treated adult beetles three days after dsRNA exposure, before (naïve) or after subsequent exposure to a sterile saline (sham) or heat-killed Bacillus thuringiensis (hk-Bt). Groups not sharing the same letter are significantly different (Wilcoxon rank sum test and FDR correction, P < 0.05). (b) The antibacterial activity of whole-beetle homogenates was assessed by measuring the mean diameter in mm of the zone of inhibition of bacterial growth on agar plates (* indicates p < 0.05).
Fig 4.
Quantitative knockdown of cactus transcripts benefits resistance and survival during infection.
(a-d) The expression of cactus (a) and the antimicrobial peptides defensin-2 (b), defensin-3 (c) and cecropin-2 (d) were assayed via qRT-PCR in whole adult beetles treated with 250, 25, or 2.5 ng of cactus dsRNA or 250 ng of malE dsRNA and then septically challenged with heat-killed Bt. Beetles were frozen before microbial challenge, time 0, and five additional times over 48 hours. Splines have been added to visualize the induction and decay dynamics from RNAi treatment using “loess” function in the geom_smooth algorithm of ggplot2 (R). See Table I in S1 Tables for statistical analyses, which are not visualized here due to complexity. (e) Survival to an LD 50 (6.5 x 10^8/mL) Bt infection after RNAi treatment was monitored for 24 hours (N = 60–64 beetles/treatment and 29–34 per sex). (f) To measure shifts in host resistance to bacterial infection from cactus RNAi treatment, beetles were given an LD-50 dose of Bt and sacrificed seven hours later. Relative bacterial density for each individual within each dsRNA treatment was quantified via RT-qPCR and calculated as the difference between Bt-specific and host reference gene expression (RP18s) on a log2 scale.
Fig 5.
The costs of immune over-activation to fitness-associated traits.
(a) Beetle life-span after RNAi treatment was measured by monitoring survival in beetles given 250, 25, or 2.5 ng of cactus dsRNA or 250 ng of malE dsRNA for 17 days (N = 30–32 beetles/treatment and 15–16 beetles/sex). (b) Female reproductive output after RNAi treatment was measured by allowing RNAi treated virgin female beetles 24 hours to mate and counting their number of eggs laid for three days. (c) Female beetle mass after RNAi treatment was measured three to five days after cactus RNAi by pooling 3 individual beetles per measurement. Weighed beetles were discarded after measurement each day. (d) Beetle gut integrity was measured by feeding adults flour stained blue and observing whether the blue dye entered the beetle hemolymph (N = 15–21 beetles/day and 7–11 beetles/sex per day. (e) The relationship between infection survival and fecundity (y = 1 / (-8.1 + 8.1 * x) + 3.0). Survival rate to infection for each RNAi treatment was calculated as 1/ (the hazard ratio relative to MalE). Reproductive output for each RNAi treatment is the median number of eggs laid for all three days measured per female.