Gene expression profiles in Malpighian tubules of the vector leafhopper Psammotettix striatus (L.) revealed regional functional diversity and heterogeneity

Abstract

Background: Many leafhoppers are known as pests and disease vectors of economically important plants. Previous studies of the physiological functions of vector leafhoppers have mainly focused on the salivary glands and the alimentary tract that are deemed to be associated with digestion, host defense and phytoplasma and/or virus transmission. By contrast, the significance of Malpighian tubules (MTs) is less studied. To clarify the physiological function of MTs of the vector leafhopper Psammotettix striatus that transmits phytoplasma triggering the wheat blue dwarf disease, we performed a transcriptome study on P. striatus MTs and compared gene expression profiles among different anatomical regions in the tubules (i.e., MT1+2, the anterior segment together with the sub-anterior segment; MT3, the inflated segment; and MT4, the distal segment).

Results: Transcriptome of P. striatus MTs generate a total of 42,815 high-quality unigenes, among which highly expressed unigenes are mainly involved in organic solute transport, detoxification and immunity in addition to osmoregulation. Region-specific comparative analyses reveal that all these MT regions have functions in osmoregulation, organic solute transport and detoxification, but each region targets different substrates. Differential expression and regional enrichment of immunity-related effector activities and molecules involved in phagocytosis and the biosynthesis of antimicrobial peptides among different regions indicate that MT1+2 and MT4 have the ability to eliminate the invading pathogens. However, in MT3 which secrets brochosomes to the integument and eggs as physical barriers, disulfide-isomerase, acidic ribosomal protein P and many other unigenes were highly expressed, which can be attractive candidate genes for future studies of the biosynthesis and the origin of brochosomes.

Conclusions: Psammotettix striatus MTs perform multiple physiological functions as versatile organs than just excretory organs with osmoregulatory function. Heterogeneity of physiological functions among different MT regions is related to organic solute transport, detoxification, immunity and brochosome biosynthesis in addition to osmoregulation, and each region targets different substrates. These functions may be helpful for P. striatus to resist pathogens from habitats and to utilize a wider range of host plants, which may assist the transmission and spread of phytoplasmas. The results provide potential molecular targets for the exploit of chemical and/or gene-silencing insecticides.

Keywords: Bioinformatics; Brochosome; Cicadellidae; Detoxification; Immunity; Organic solute transport; Osmoregulation; RNA-Seq; RT-qPCR.

Fig. 1

A schematic illustration of P. striatus and its brochosomes, alimentary tract and Malpighian tubules. CS, conical segment; FC, filter chamber; Hg, hindgut; Mg, midgut; MT1, the anterior segment of the Malpighian tubule; MT2, the sub-anterior segment of the Malpighian tubule; MT3, the inflated segment of the Malpighian tubule; MT4, the distal segment of the Malpighian tubule; Oe, oesophagus; Rc, rectum

Fig. 2

Gene expression pattern among different MT regions of P. striatus. A PCA for nine sequenced cDNA libraries, with the first two principal components (PC1 and PC2) based on transcriptomic results being shown. Each solid dot represents an individual cDNA library. B Hierarchical clustering heatmap of global gene expression based on normalized RPKM values from different regions. Red indicates higher expression, and yellow indicates lower expression. C Venn diagram exhibiting the number of common and unique unigenes between pairwise comparisons

Fig. 3

Volcano plots demonstrating the DEGs of all detected genes from the RNA-Seq dataset among different MT regions of P. striatus. DEGs are colored blue and red

Fig. 4

Schematic illustrations of molecules associated with transepithelial fluid transport in P. striatus MTs. A General catalog. B Transporters associated with transepithelial fluid transport in a cell of MT1+2. C Transporters associated with transepithelial fluid transport in a cell of MT3. D Transporters associated with transepithelial fluid transport in a cell of MT4

Fig. 5

Normalized heatmap based on RPKM values of DEGs related to detoxification of P. striatus MTs. Orange color indicates up-regulated expression, whereas blue color indicates down-regulated expression

Fig. 6

Gene expression levels of DEGs among different MT regions of P. striatus through RNA-Seq and RT-qPCR analyses

Fig. 7

Schematic illustrations of the mechanisms of resistance to the invading pathogens in different MT regions of P. striatus. A The process of immunological responses speculated (including cellular immunity and humoral immunity) in a cell of MT1+2 and MT4. B A secretory cell of MT3 secreting brochosomes