Two-layered Membrane Sandwich Method for Laodelphax striatellus Saliva Collection

Summary

The present protocol describes a method to collect sufficient saliva from piercing-sucking insects using an artificial medium. This is a convenient method for collecting insect saliva and studying salivary function on insect feeding behavior and vector-borne virus transmission.

Abstract

Rice stripe virus (RSV), which causes significant economic loss of agriculture in East Asia, entirely depends on insect vectors for its effective transmission among host rice. Laodelphax striatellus (small brown planthopper, SBPH) is the primary insect vector that horizontally transmits RSV while sucking sap from the phloem. Saliva plays a significant role in insects' feeding behavior. A convenient method that will be useful for research on insects' saliva with piercing-sucking feeding behavior is described here. In this method, insects were allowed to feed on an artificial diet sandwiched between two stretched paraffin film layers. The diet containing the saliva was collected each day, filtered, and concentrated for further analysis. Finally, the quality of collected saliva was examined by protein staining and immunoblotting. This method was exemplified by detecting the presence of RSV and a mucin-like protein in the saliva of SBPH. These artificial feeding and saliva collection method will lay a foundation for further research on factors in insect saliva related to feeding behavior and virus transmission.

Introduction

Rice stripe virus (RSV), a negative-stranded RNA virus in the genus Tenuivirus, causes severe diseases in rice production in East Asia^1,2,3. Transmission of RSV from infected rice plants to healthy ones depends on insect vectors, mainly Laodelphax striatellus, which transmits RSV in a persistent-propagative manner. SBPH acquires the virus after feeding on RSV-infected plants. Once inside the insect, RSV infects the midgut epithelial cell one day after feeding and then passes through the midgut barrier to penetrate the hemolymph. Subsequently, RSV spreads int....

Protocol

1. SBPH maintenance

1. Rear the viruliferous and RSV-free SBPH individuals in a glass incubator (65 x 200 mm) with 5-6 rice (Oryza sativa cv. Nipponbare) seedlings per glass chamber in the laboratory. Grow the rice plants at 25 °C under a 16 h light / 8 h dark photoperiod.
   NOTE: The viruliferous and RSV-free SBPH individuals were initially caught in Jiangsu Province, China.
2. Detect RSV in SBPH by dot-enzyme-linked immunosorbent assay (dot-ELISA) with a rabbit RSV-specific polyclonal antibody (see Table of Materials) raised against the RSV ribonucleoproteins (RNPs).
   NOTE: For ensuring high offspring infecti....

Results

Schematics of artificial feeding installation and saliva collection
Figure 1A depicts the glass cylinder (15 cm x 2.5 cm) used as a feeding chamber to collect the saliva. Firstly, the SBPH larvae were starved for several hours to improve the collection efficiency and then immobilized by chilling for 5 min. After the insects were transferred into the glass cylinder, both open ends of the chamber were covered with stretched Paraffin membrane. At one end, 200 µL of 5% sucro.......

Discussion

Successful rearing of insects on artificial diets was first reported in 1962 when Mittler and Dadd described the Paraffin membrane technique to hold an artificial diet^29,30. And this method has been explored in many aspects of insect biology and behavior, for example, nutrient supplement, dsRNA feeding, and virus acquisition. Based on the requirements of saliva analysis, 5% sucrose is used as the general artificial diet to collect saliva of SBPH in this study. Fo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-KD centrifugal filter	Merck Millipore	R5PA83496	For concentration
10x Protein Transfer Buffer(wet)	macGENE	MP008	Transfer buffer for western blotting
10x TBST buffer	Coolaber	SL1328-500mL×10	Wash buffer for western blotting
Azure c600 biosystems	Azure Biosystems	Azure c600	Imaging system for western blotting and silver staining
Color Prestained protein ladder	GenStar	M221-01	Protein marker for western blotting
ECL western blotting detection reagents	GE Healthcare	RPN2209	Western blotting detection
Enchanced HRP-DAB Chromogenic Kit	TIANGEN	#PA110	Chromogenic reaction
Horseradish peroxidase-conjugated goat anti-rabbit antibodies	Sigma	401393-2ML	Polyclonal secondary antibody for western blotting
Immobilon(R)-P Polyvinylidene difluoride membrane	Merck Millipore	IPVH00010	Transfer membrane for western blotting
iTaq Universal SYBR Green Supermix	Bio-Rad	1725125	For quantitative real-time PCR (qRT-PCR)
KIT,iSCRIPT cDNA SYNTHES	Bio-Rad	1708891	For Reverse-transcriptional PCR (RT-PCR)
Millex-GP Filter, 0.22 µm	Merck Millipore	SLGP033RB	For filtration
Mini-PROTEAB TGX Gels	Bio-Rad	4561043	For SDS-PAGE
NanoDrop One	Thermo Scientific	ND-ONEC-W	Detection of protein concentration
Nylon membrane	PALL	T42754	Membrane for dot-ELISA
Parafilm M Membrane	Sigma	P7793-1EA	Making artifical diet sandwichs
Rabbit anti-LssgMP polyclonal antibody against LssgMP peptides	Genstript		Rabbit primary anti-LssgMP polyclonal antibody for western blotting
Rabbit anti-RSV polyclonal antibody	Genstript		Rabbit primary anti-RSV polyclonal antibody for western blotting and dot-ELISA
RNAprep pure Micro Kit	TIANGEN	DP420	For RNA Extraction