Disruption of a horizontally transferred phytoene desaturase abolishes carotenoid accumulation and diapause in Tetranychus urticae

Abstract

Carotenoids underlie many of the vibrant yellow, orange, and red colors in animals, and are involved in processes ranging from vision to protection from stresses. Most animals acquire carotenoids from their diets because de novo synthesis of carotenoids is primarily limited to plants and some bacteria and fungi. Recently, sequencing projects in aphids and adelgids, spider mites, and gall midges identified genes with homology to fungal sequences encoding de novo carotenoid biosynthetic proteins like phytoene desaturase. The finding of horizontal gene transfers of carotenoid biosynthetic genes to three arthropod lineages was unprecedented; however, the relevance of the transfers for the arthropods that acquired them has remained largely speculative, which is especially true for spider mites that feed on plant cell contents, a known source of carotenoids. Pigmentation in spider mites results solely from carotenoids. Using a combination of genetic approaches, we show that mutations in a single horizontally transferred phytoene desaturase result in complete albinism in the two-spotted spider mite, Tetranychus urticae, as well as in the citrus red mite, Panonychus citri Further, we show that phytoene desaturase activity is essential for photoperiodic induction of diapause in an overwintering strain of T. urticae, consistent with a role for this enzyme in provisioning provitamin A carotenoids required for light perception. Carotenoid biosynthetic genes of fungal origin have therefore enabled some mites to forgo dietary carotenoids, with endogenous synthesis underlying their intense pigmentation and ability to enter diapause, a key to the global distribution of major spider mite pests of agriculture.

Keywords: bulked segregant analysis; horizontal gene transfer; spider mites; xanthophylls; β-carotene.

Fig. 1.

Carotenoids in WT and albino mutants of T. urticae. (A) Carotenoids observed in females of Tetranychus mite species as reported in earlier studies (26, 33, 34). The previously proposed pathway from presumptive dietary β-carotene to the terminal keto-carotenoid astaxanthin is shown (34); intermediates, minor carotenoid species, and carotenoid esters are not indicated. (B) Carotenoid profiles in nondiapausing (ND) and diapausing (D) WT T. urticae females, and in nondiapausing albino mutants as reported by Veerman (26) (compare with A). Plus or minus signs indicate relative levels, with the keto-carotenoids undetectable in albino mites. Schematics of WT and mutant mites are given at the top; for display, the eyes have been enlarged.

Fig. 2.

The albino pigment phenotypes of T. urticae and P. citri. Shown are (A) WT T. urticae pigmentation (strains Wasatch, MAR-AB, Foothills, and London; a representative London mite is shown), (B) albino phenotype of T. urticae (strains Alb-NL, Alb-JP, W-Alb-7, -8, -10, -11, and -14; a representative Alb-NL mite is shown), (C) WT P. citri phenotype, and (D) albino phenotype of P. citri. In all cases, adult females are shown. Arrows indicate red eye spots or red-orange in the distal front legs of WT mites that are absent in the albino mutants. The dark regions present in the mite bodies are gut contents that are visible because spider mites are partly translucent. (Scale bars: 0.1 mm.)

Fig. S1.

Diapausing WT and albino T. urticae females. Image of a diapausing (A) WT adult T. urticae female (strain Wasatch) and (B) albino adult T. urticae female (strain Alb-NL). A diapausing WT T. urticae female has a yellow to bright red-orange coloration whereas a diapausing albino T. urticae female has a uniformly white appearance. Note the absence of gut contents (dark regions in the body), reflecting cessation of feeding (compare with feeding mites shown in Fig. 2). Under inducing conditions, females of strain Alb-NL enter diapause at a very low frequency (less than ∼1 in 1,000). Arrows indicate the position of the eyes of the WT T. urticae female. (Scale bars: 0.1 mm.)

Fig. S2.

Scanning electron microscopy (SEM) images of T. urticae and P. citri eyes. SEM images of the anterior (a) and posterior (p) eyes of an (A) WT adult T. urticae female (strain London), (B) adult T. urticae albino female (strain Alb-NL), (C) WT adult P. citri female, and (D) adult P. citri female with an albino phenotype. (Scale bar: 10 μm.)

Fig. S3.

Similarity between strains as assessed with genome-wide SNP data. Values in each rectangle represent the percent similarity of pairs of strains as assessed with 488.6 thousand high quality SNPs (SI Materials and Methods) (percentage similarity for each comparison is color-coded according to the gradient at the far right). Strain names are at the left and across the top. Strains Wasatch, W-Alb-2, and W-Alb-14 are essentially identical, reflecting the inbred nature of Wasatch, and the origin of the two albino mutations on the Wasatch background (W-Alb-2 and W-Alb-14). The high level of similarity between PA1 and PA2 suggests that their progenitor Alb-NL strain was partly, but not completely, inbred at the start of the experiment (the strain had been maintained in the laboratory for more than 40 y and presumably lost genetic variation as a result of bottleneck events). Other strains show much reduced levels of identity to other strains, with Alb-NL and Alb-JP being comparatively dissimilar (reflecting an independent origin of albinism in these two strains). Although most strains are inbred or partly inbred, MAR-AB is highly outbred, potentially reflecting its apparent greater level of similarity to most other strains in the pairwise comparisons (SI Materials and Methods).

Fig. 3.

A locus on scaffold 1 underlies albinism. (A) The results for bulked segregant analysis (BSA) genetic scans for albinism in Alb-NL are shown (crosses of inbred lines derived from the Alb-NL parent to the WT strain MAR-AB). BSA scans were performed with fourfold replication (colors yellow, orange, green, and blue); changes in allele frequencies between selected (albino) and nonselected populations are shown in a sliding window analysis. Positive changes in allele frequencies denote increased fixation of alleles contributed by the Alb-NL strain in the selected albino offspring. In all cases, the maximal deviation toward Alb-NL alleles was on scaffold 1 (denoted by an asterisk at ∼5.2 Mb). The order of scaffolds is unknown; for display, scaffolds are concatenated from largest to smallest with alternating shading. (B) The minimal candidate region for albinism in strain Alb-NL as established by fine mapping (Table S1). Gene models are indicated in gray or, for the scaffold 1 carotenoid biosynthesis gene cassette, in orange. The genomic structure and annotation are from the London reference strain. (C) Other WT, nonreference strains, including Wasatch, have structural variants 3′ to the phytoene desaturase (tetur01g11270) that remove all or most of three genes (tetur01g11280, tetur01g11290, and tetur01g11300) present in the London reference (see also Fig. S4). The location of a spontaneous 6.2-kb deletion in strain Wasatch associated with the absence of any pigmentation (strains W-Alb-7, -8, -10, -11, and -14) is shown at the bottom (dashed red line, with the beginning and end of the deletion relative to the overlying Wasatch de novo assembly indicated by red arrows). In C, dark or colored block arrows indicate coding regions, with introns and untranslated regions indicated in lighter gray.

Fig. S4.

Coverage depth of sequence reads aligned to the 66.2-kb minimal candidate region for albinism. (A–F) Coverage variation across the minimal causal region for albinism in strains of T. urticae with the position and direction of transcription of genes in the region shown at bottom (G). Two carotenoid biosynthetic genes in T. urticae of fungal origin are indicated in orange. Coverage (gray) reflects the depth of overlying Illumina genomic DNA reads as aligned to the London reference genome. A region of no read coverage reflects structural variation immediately downstream from the carotenoid biosynthetic genes, including the absence of sequences present in London (this region is defined by dashed blue lines although the genomic differences are not identical across all lines: i.e., compare Wasatch with Foothills). The dashed red lines delineate an additionally deleted region in W-Alb-14, an albino line harboring a spontaneous mutation that arose in the Wasatch strain.

Fig. S5.

Alignment of phytoene desaturases. T. urticae phytoene desaturase tetur01g11270 was aligned with those of other tetranychid species, aphids, and their close relatives adelgids, fungi, and bacteria (, , , , –80). An 80% threshold was used for shading identity (black background) and similarity (gray background). Accession numbers of protein sequences are shown in parentheses. A black triangle indicates the position of the single intron in tetur01g11270. Red asterisks above the alignment indicate the substitutions in tetur01g11270 (Thr220Lys in T. urticae strain Alb-NL and Pro487Leu in T. urticae lines W-Alb-1 and W-Alb-2) that were identified in this study. A red rhombus indicates the position of the insertion into P. citri phytoene desaturase (this study). A blue circle above the alignment indicates the Glu32Lys substitution found in a phytoene desaturase of an A. pisum mutant (11) whereas brown and green circles refer to those substitutions in Phycomyces blakesleeanus (Glu426Lys, Ser444Phe and Leu446Phe, Gly482Ser) and Fusarium fujikuroi (Pro170Leu, Trp449Stop, Gly504Asp) phytoene desaturases that result in lowered desaturase activities (52, 53). The presumed carotenoid binding domain (52) is indicated with an arrow below the alignment.

Fig. 4.

Mutations in the scaffold 1 phytoene desaturase in T. urticae and P. citri albino strains. Coding exons for tetur01g11270 are represented as rectangles. WT sequences are indicated in black, with mutations shown in red (T. urticae) or blue (P. citri); where single nucleotide substitutions are observed, the impact on the coding potential is shown. For the Alb-JP mutation, boxes indicate the exon 1 splice donor in WT (at the top) with two out-of-frame splice donors created by the “T” insertion shown underneath.

Fig. 5.

Impact of inactivating mutations in tetur01g11270 on diapause incidence. The incidence of diapause under inducing conditions, as revealed by coloration (orange or green circles for WT, diapausing and nondiapausing colors, respectively, and gray for albino mites) and egg laying (y axis), in WT Wasatch and the albino W-Alb-14 and W-Alb-2 mutant strains. For egg laying, the total number per female after 11 d is shown (that is, each circle represents a single female’s total oviposition). Sample sizes are indicated at the top, and differing letter designations denote significant differences (P < 10⁻¹⁵ in pairwise comparisons, Wilcoxon rank-sum tests).