Atypical strategies for cuticle pigmentation in the blood-feeding hemipteran Rhodnius prolixus

Abstract

Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

Keywords: Rhodnius prolixus; Chagas disease; cuticle; eye pigment; melanin; tyrosine pathway.

Fig. 1.

Cuticle pigmentation phenotypes resulting from KD of tyrosine pathway genes. a) Schematic representation of pigmentation pathways and Rhodnius prolixus loci associated with cuticle color. Loci herein investigated are shown in italic and red brown color. PAH, phenylalanine hydroxylase; TH, tyrosine hydroxylase; DDC, DOPA decarboxylase; PO, phenoloxidase; KF, kynurenine formamidase. b) Viability of adult animals injected with dsRNA. No significant differences between experimental conditions vs control were observed. Graph shows mean ± STD. The number of fifth instar nymphs injected is displayed inside the bars. c–f) Control (c, c′) and cuticle phenotypes resulting from KD for y (d, d′) t (e, e′), and aaNAT^pret (f, f′). c′–f′) High magnification of (c–f) showing details of the 3-color pattern of the R. prolixus first thoracic segment in control and KD animals. g) Cuticle darkness as quantified for black (bl), tanned (tan), and white (wh) stripes or the corresponding positions in the thorax for the different KDs. Graph shows mean ± SEM. **P < 0.01, ****P < 0.0001, Student’s t-test. h) KD efficiency for the different KDs, with expression levels for yellow, tan, and ebony, as defined by RT-qPCR. Expression levels were normalized to control dsGFP-injected animals with R. prolixus EF1α. Graphs show mean ± SEM (n = 3). *P < 0.1, **P < 0.01, 1-way ANOVA.

Fig. 2.

Double KD for tyrosine pathway genes indicates limited cuticle pigmentation function for the NBAD branch. Effects on body (a–f) and wing (g–l) pigmentation, resulting from single or double KD for tyrosine pathway loci. a, h) e KD cuticles are identical to wild-type or control (g); b, i) y KD; c, j) e + y KD; d, k) aaNAT^pret KD, and e) y + aaNAT^pret KD at 24 h after molting; f, l) y + aaNAT^pret KD at 48 h after molting. g) Wings from control KD. m) Expected outcome of the double e + y KD; n) Expected outcome of the double y + aaNAT^pret KD. (o) and (p) Cuticle darkness as quantified for black (grey circles), tanned (brown circles) and white (yellow circles) stripes in the thorax for the different KDs, as in Figure 1. (o) displays luminosity measurements for single an double KDs as in (m); (p) displays luminosity measurements for single and double KDs as in (n). Graphs show mean ± SEM. **P < 0.01, ***P < 0.0005, ****P < 0.0001, One Way ANOVA. ns: non-significant.

Fig. 3.

Loss of aaNAT function results in cuticle defects. Cuticle UV reflectance was examined in the presence (a, b) or absence (a′, b′) of ambient light for control (a) or aaNAT^pret (b) KD. Scanning EM shows the unchanged pattern of external structures in the control (c) or aaNAT^pret (d) KDs, while thin sections of the abdominal cuticle show a broad control cuticle (e) and thin aaNAT^pret KD (f) cuticle structure, as quantified in (g). exo, exocuticle; endo, endocuticle.

Fig. 4.

Eye color phenotypes resulting from the KD of genes involved in pigment production and transport. a) Phylogenetic analysis of R. prolixus ABC pigment transporters. The ABCs evolutionary history was inferred by the Maximum Likelihood method followed by 500 bootstrap replicates. Bootstrap values are displayed in nodes. The tree is drawn to scale, with branch lengths measured as the number of substitutions per site. This analysis involved 43 amino acid sequences. Adult eye color phenotypes observed following fifth instar dsRNA injections for b) control; c) ok A; d) cinnabar; e) scarlet; f) ok B; and g) white. h) Spontaneous R. prolixus red eye mutant. h′) Detail of the mutant eye and ocelli, compared to the i) wild-type. Note the absence of ocelli pigmentation (asterisk). h″) Detail of mutant abdominal connexives, with loss of red pigment in veins, present as red spots in the i′) wild-type (arrows).

Fig. 5.

Pteridine and ommochrome pigments generate color in the R. prolixus cuticle; 76 h old adults resulting from fifth instar dsRNA injections against a) GFP (control); b) ok A; c) sepia; d) ok B; e) cinnabar; f) scarlet. Details of the thorax for g) control GFP; h) ok A; i) sepia; and j) scarlet KD. No differences in eye color were observed for sepia, ok A, or ok B KDs. Note the red cuticle color in ok A, sepia, and scarlet KD (asterisks). Details of the abdominal connexives for k) control GFP; l) ok A; m) sepia; n) ok B; o) cinnabar; and p) scarlet KD. Observe the loss of red pigment spots in ok B, cinnabar, and scarlet (double arrows). q) Prospective function of loci associated with the production and transport of ommochrome and pteridine pigments in R. prolixus, based on KD phenotypes herein presented. In italic: red represents R. prolixus loci that resulted in a change in eye color compared with wild-type; pink letters or asterisks indicate loci that resulted in no visible eye pigmentation phenotype, but generated connexive pigmentation defects upon KD. Orange letters refer to functions associated with body pigmentation, particularly in the thorax and head. KF, kynurenine formamidase; PG, pigment granule.

Fig. 6.

Preferential pathways for cuticle patterning in hematophagous hemiptera. Loss-of-function analysis for pigmentation genes suggests that the blood-feeding insects R. prolixus and P. biguttattis rely preferentially on the level of melanin deposition controlled by the yellow and aaNAT^pret genes for cuticle patterning (in bold). The relative expression of these genes would define the dark and light brown hues of the insect. The plant feeding O. fasciatus utilizes all 3 tyrosine pathway branches, where the NBAD pathway (via ebony function) generates light brown/orange pigmentation. In addition to pattern based on melanin deposition, background red hues of the R. prolixus cuticle appear to result from ommochrome and pteridin pigments, transported by the action of ABC proteins.