Genome wide gene-expression analysis of facultative reproductive diapause in the two-spotted spider mite Tetranychus urticae

Abstract

Background: Diapause or developmental arrest, is one of the major adaptations that allows mites and insects to survive unfavorable conditions. Diapause evokes a number of physiological, morphological and molecular modifications. In general, diapause is characterized by a suppression of the metabolism, change in behavior, increased stress tolerance and often by the synthesis of cryoprotectants. At the molecular level, diapause is less studied but characterized by a complex and regulated change in gene-expression. The spider mite Tetranychus urticae is a serious polyphagous pest that exhibits a reproductive facultative diapause, which allows it to survive winter conditions. Diapausing mites turn deeply orange in color, stop feeding and do not lay eggs.

Results: We investigated essential physiological processes in diapausing mites by studying genome-wide expression changes, using a custom built microarray. Analysis of this dataset showed that a remarkable number, 11% of the total number of predicted T. urticae genes, were differentially expressed. Gene Ontology analysis revealed that many metabolic pathways were affected in diapausing females. Genes related to digestion and detoxification, cryoprotection, carotenoid synthesis and the organization of the cytoskeleton were profoundly influenced by the state of diapause. Furthermore, we identified and analyzed an unique class of putative antifreeze proteins that were highly upregulated in diapausing females. We also further confirmed the involvement of horizontally transferred carotenoid synthesis genes in diapause and different color morphs of T. urticae.

Conclusions: This study offers the first in-depth analysis of genome-wide gene-expression patterns related to diapause in a member of the Chelicerata, and further adds to our understanding of the overall strategies of diapause in arthropods.

Figure 1

Different types of body coloration of adult T. urticae females. (A) Green morphs (LS-VL strain), (B) red morphs (Tu-SB9 strain) and (C) diapausing forms of green morphs displaying the typical orange color (LS-VL strain).

Figure 2

Volcano plot of differentially expressed genes in diapausing T. urticae females identified by microarray analysis. The log₁₀(FDR corrected p-values) were plotted against the log₂(FC) in gene expression. Genes upregulated (n = 916) by twofold or more and with a FDR corrected p-value < 0.05 are depicted as red dots, genes that were downregulated (n = 1078) by twofold or more and with a FDR corrected p-value < 0.05 are shown with green dots. All other genes in the array that were not found to be differentially expressed are depicted as grey dots.

Figure 3

Gene Ontology (GO) categories with a significant difference between the number of up- and downregulated genes in diapausing T. urticae females. Red bars show the number of downregulated T. urticae genes while blue bars show the number of upregulated T. urticae genes for each GO category. The number of upregulated and downregulated genes within each GO category were considered as statistically different when the Fisher Exact test FDR corrected p-value was less than 0.01.

Figure 4

T. urticae enzymes involved in inositol phosphate metabolism and their expression in diapausing T. urticae females. The inositol phosphate metabolism pathway was derived from the KEGG metabolic pathway database for D. melanogaster (http://www.genome.jp/kegg-bin/show_pathway?ko00562). The name and E.C. number of each enzyme involved in the IPM pathway is listed next to the arrows while accession numbers of T. urticae homologues of D. melanogaster enzymes involved in IPM are mentioned between brackets (see Additional file 11). T. urticae homologues presented with a green font where significantly upregulated (FDR < 0.05, |FC| ≥ 2) in diapausing T. urticae females while those shown with a red font were significantly downregulated (FDR < 0.05, |FC| ≥ 2). Abbreviations: INPP1 = Inositol polyphosphate 1-phosphatase, I3K = inositol 3-kinase, IMPA = inositol monophosphatase 3, MIOX = inositol oxygenase, INPP5 = inositol-1,4,5-trisphosphate 5-phosphatase, PLC = phosphatidylinositol phospholipase C.

Figure 5

Ribbon illustration of a putative T. urticae antifreeze protein (AFP). Side (A) and end-on (B) view of the predicted 3D-structure of a putative spider mite AFP (tetur22g03033) with β-sheets indicated by blue arrows. Asn, Asp and Thr side-chains are indicated in orange while Cys side-chains are indicated in yellow. The N-and C-terminus of the T. urticae AFP structure are indicated with the letters N and C, respectively. The 3D-model for tetur22g03033 was created using the Phyre² server (with pdb-model: d1ezga) [48] and further edited with Swiss PDB viewer [49].

Figure 6

Expression levels of genes coding for putative T. urticae antifreeze proteins. qPCR quantification of expression levels of putative AFP genes in T. urticae. Green, orange and blue bars represent the relative mean expression in non-diapausing spider mites at 17°C, diapausing spider mites at 17°C and non-diapausing spider mites at 5°C, respectively, relative to expression in non-diapausing mites of the LS-VL strain at 24°C. Error bars represent the standard error of the calculated mean based on three biological replicates. Asterisks indicate significantly differential expressed genes (random reallocation test) compared to the reference condition (green, non-diapausing LS-VL strain at 24°C).

Figure 7

Expression levels of carotenoid biosynthesis genes in T. urticae. qPCR quantification of expression levels of carotenoid biosynthesis genes in T. urticae. Green, light red, dark red and orange bars represent the relative mean expression in non-diapausing spider mites of the London strain (green morph), non-diapausing spider mites of the MR-VL strain (red morph), non-diapausing red spider mites of the Tu-SB9 strain (red morph) and diapausing spider mites, respectively, relative to expression in non-diapausing forms of the LS-VL strain (green morph). Error bars represent the standard error of the calculated mean based on three biological replicates. Asterisks indicate significant different expression compared to the reference condition (green, non-diapausing LS-VL strain at 24°C).