A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Summary

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

Abstract

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus transmission. Hemolymph collection is the basic technology for studying virus-vector interactions. Here, we describe a novel and simple method for the quantitative collection of hemolymph from small arthropods using Laodelphax striatellus (the small brown planthopper, SBPH) as a research model, as this arthropod is the main vector of rice stripe virus (RSV). In this protocol, the process begins by gently pinching off one leg of the frozen arthropod with fine-tipped tweezers and pressing the hemolymph out of the wound. Then, a simple micropipette consisting of a capillary and a pipette bulb is used to collect the transudative hemolymph from the wound according to the principle of capillary forces. Finally, the collected hemolymph can be dissolved into a specific buffer for further study. This new method for collecting hemolymph from small arthropods is a useful and efficient tool for further research on arboviruses and vector-virus interactions.

Introduction

Both animal and plant viruses can be transmitted by arthropods, and these viruses pose a severe threat to human health and cause tremendous economic losses in agriculture^1,2,3. Importantly, the arthropod hemolymph, which serves as the circulatory system and a vital element of the immune system in arthropods, plays an important role in regulating arboviral transmission. Viruses acquired through the arthropod guts are transported to other tissues only after successfully escaping the adverse hemolymph environment^4,5....

Protocol

1. Insect rearing

1. Raise the SBPHs used in this experiment in rice seedlings (Oryza sativa cv. Nipponbare). Plant 20 rice seedlings in an incubator (65 mm x 200 mm), and grow at 25 °C under a 16 h light/8 h dark photoperiod.

2. Dissection of the SBPHs for hemolymph collection

1. Put the SBPHs into a centrifuge tube, and place them in an ice bath for 10-30 min.
   NOTE: Do not place the SBPHs in the ice bath for less.......

Discussion

Hemolymph is the medium of the circulatory system in arthropods, and arboviruses can only invade other arthropod tissues if they are able to survive the hostile hemolymph environment. Collecting a high-quality sample of hemolymph is the first step in studying the vector-virus interactions that occur in the hemolymph. It has been reported that insect hemolymph can be obtained from several sites on the insect's body, including a wound on the front leg, a minor incision in the head area, or a tear wound at the abdomen

Materials

Name	Company	Catalog Number	Comments
10% SDS-PAGE protein gel	Bio-rad	4561035	Protein separation and detection
4% paraformaldehyde	Solarbio	P1110	For fixation of the cells or tissues
Bradford dye reagent	Bio-rad	5000205	Protein concentration detection
Capillary	Hirschmann	9000101	For collecting hemolymph
Cell counting chamber	ACMEC	AYA0810	Hemocytes counting
Glass slide	Gitoglas	10127105A	For holding insects
Glass slide coated with silane	Sigma	S4651-72EA	For holding microscope samples
Gold antifade reagent with DAPI	Invitrogen	P36935	Nucleus staining
Microscope cover glass	Gitoglas	10212424C	For microscopic observation
Pipette bulb	Hirschmann	9000101	For collecting hemolymph
Prism 8.0 software	GraphPad Software	/	Statistical analyses
Stereomicroscope	Motic	SMZ-168	For insect dissection
Tweezers	Tianld	P5622	For insect dissection
Zeiss inverted microscope	Zeiss	Observer Z1	Hemocytes observation