A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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^4,5,6,7. The lifecycle of viruses in the arthropod hemolymph involves virus survival in the fluid plasma, entry into the hemocyte, and transport to other tissues, and various virus-vector interaction mechanisms occur in the hemolymph^8,9,10,11,12. For example, the vertical transmission of RSV by the SBPH is dependent on a molecular interaction between the SBPH vitellogenin protein and the RSV (rice stripe virus) capsid protein^13,14. Some viruses may escape the immune response of the hemolymph by binding specific vector factors^15,16,17,18. Therefore, investigating vector-virus interactions in the hemolymph of arthropods is important for developing a better understanding of arbovirus transmission.

The hemolymph of some small insects, such as planthoppers, leafhoppers, and some mosquitoes, is difficult to collect due to their size. To address this issue, several methods have been developed to collect hemolymph, including inserting a syringe needle directly into the insect body to extract a microvolume of the hemolymph, collecting exudate from the wound site with fine-tipped tweezers, and direct centrifugation. These methods have enabled the measurement of relative gene expression levels and viral titers within the hemolymph^19,20,21. However, an effective method for quantifying the hemolymph volume, which is necessary for hemocyte counting, protein quantification, and enzyme activity analysis, is currently not available for these small insects.

The SBPH (small brown planthopper) is a type of small insect vector with a body length of about 2-4 mm. The SBPH is capable of transmitting a variety of plant viruses, including RSV, maize rough dwarf virus, and rice black streaked dwarf virus^22,23,24. The interaction between the SBPH and RSV has been studied in depth over the past decade. To facilitate working with SBPHs, we developed a novel and simple method of collecting hemolymph. This method, which is based on the principle of capillary forces, uses a capillary with a scale mark to acquire the insect's hemolymph in a precise and quantifiable manner. This allows us to collect a specific volume of hemolymph from small insects efficiently and to study the hemolymph environment of small vectors in more detail.

Protokół

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 than 10 min, or the insects may revive.
2. Place a frozen SBPH on a glass slide (see Table of Materials) under a stereomicroscope (see Table of Materials) with its abdomen facing up. Adjust the focus on the six legs of the insect.
3. Prepare two high-precision tweezers (see Table of Materials) with ultra-fine tips. Use one tweezer to press down on the insect's body to keep it in place, and use the other one to carefully pull one of the legs off the insect.
   NOTE: For insects with a hard shell, an ophthalmic scalpel can be used for cutting off one of the legs.
4. Gently press the chest of the insect with a tweezer to make the hemolymph flow out through the wound.
   ​NOTE: The hemolymph presents as transparent droplets without any visible white floccule. Discard the lipid if there is any white floccule, which represents fat body contamination.

3. Hemolymph collection using micropipettes

1. Prepare a micropipette. Place a capillary tube (see Table of Materials) with a volume of 1 µL into the pipette bulb (see Table of Materials). For accurate measurements, ensure that the scale line is visible.
   NOTE: A small hole present on the top of the pipette bulb is necessary.
2. When collecting the hemolymph, hold the pipette bulb of the micropipette, and place the capillary tube close to the insect wound. Collect the hemolymph exuding from the wound by simply touching the tip of the capillary to the hemolymph.
3. Block the small hole on the top of the pipette bulb slightly with the finger to stop the absorbing process when the liquid inside the capillary tube reaches the desired scale line.
   NOTE: Collect one sample of 1 µL of hemolymph incessantly and rapidly to avoid lipid contamination and tube plugging.
4. Press the pipette bulb to discharge the collected hemolymph into 100 µL of PBS buffer (137 mmol/L NaCI, 2.7 mmol/L KCI, 4.3 mmol/L Na₂HPO₄, 1.4 mmol/L KH₂PO₄, pH 7.2-7.4).
   ​NOTE: The capillary tube is inserted into the PBS buffer.
5. Change to a new capillary tube when collecting another 1 µL sample of hemolymph.

4. Coomassie Blue staining

1. Collect 3 hemolymph samples with the same volume (1 µL) from 3rd stage larvae according to the protocols described in step 3, and discharge the collected hemolymph into the PBS buffer to make a total volume of 100 µL.
2. Add 25 µL of 5x protein loading buffer (250 mmol/L Tris-HCI [pH 6.8], 10% W/V SDS, 0.05% W/V Bromophenol Blue, 50% W/V glycerol, 5% W/V β-mercaptoethanol) into 100 µL of the diluted hemolymph samples, and heat to denature the proteins.
3. Load 20 µL of the boiled samples onto a 10% SDS-PAGE protein gel (see Table of Materials) to separate the proteins.
4. Stain the gel in a Coomassie Blue solution (mix 1 g of Coomassie Brilliant Blue R-250 with 250 mL of isopropanol, 100 mL of glacial acetic acid, and 650 mL of ddH₂O, and filter out any particles using filter paper) for 1 h at room temperature, and then decolorize the gel for 1 h with decolorizing solution (100 mL of acetic acid, 400 mL of methanol, 500 mL of ddH₂O).

5. Protein concentration determination

1. Dilute 10 mg/mL bovine serum albumin (BSA) to 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, and 0.3125 mg/mL with PBS buffer using the two-fold dilution method.
2. Aspirate 5 µL of PBS buffer and each concentration of BSA solutions in step 5.1, add 1 mL of 1x Bradford dye reagent (see Table of Materials), and incubate for 5 min at room temperature. Use PBS buffer in Bradford dye reagent as the control, and zero at OD = 595 nm. Measure the absorbance values of each of the remaining protein concentrations, and make a standard curve.
3. Add 1 µL of hemolymph from larval, female, or male SBPHs into 100 µL of PBS buffer, and then centrifugate the samples at 1,000 x g for 10 min at 4 °C to remove the hemocytes.
4. Aspirate 90 µL of supernatant to a new centrifuge tube, and measure the absorbance values of the supernatant according to the instructions in step 5.2.
5. Calculate the protein concentrations of the hemolymph samples according to the regression equation of the standard curve. Include three biological replicates with three technical replicates in the experiments.

6. Microscopic detections

1. Use micropipettes to collect hemolymph from 10-15 SBPHs at different developmental stages. Add the collected hemolymph into a tube containing 20 µL of 4% paraformaldehyde solution (see Table of Materials). Incubate the mixture at room temperature for 30 min to fix the hemolymph.
2. Pipette 20 µL of fixed hemocytes onto the center of a slide coated with silane (see Table of Materials).
3. Dry up the droplet in a slide drying rack.
   NOTE: Avoid excessive drying.
4. Cover the sample with gold antifade reagent 4',6-diamidino-2-phenylindole (DAPI) (see Table of Materials), and inspect the slide under an inverted microscope (see Table of Materials).

7. Cell quantification

1. Collect 1 µL of hemolymph from the SBPHs with a micropipette, and dissolve it into 20 µL of PBS buffer.
2. Dilute the hemolymph samples five-fold with PBS buffer.
3. Pipette 20 µL of hemolymph solution into the center of the cell counting chamber (see Table of Materials). Cover the droplet with a coverslip (see Table of Materials).
4. Count the cells in the five squares on the cell counting chamber (Supplementary Figure 1), under an inverted microscope (see Table of Materials). Include three biological replicates with three technical replicates in each of the experiments.
   ​Cells/µL = total count of five middle squares × 5 × dilution factor × 10⁴

8. Statistical analyses

1. Perform statistical analyses with the corresponding software (see Table of Materials). Create a new file, and select the column, import the data, and generate a scatter plot with a bar.
2. Calculate the mean and SD for each group of data by using column statistics, and use a one-way ANOVA tool to evaluate the significance of the differences among the groups. Determine the mean and SD from three biological replicates with three independent experiments.

Wyniki

Micropipette model and hemolymph collection
We have developed a simple micropipette whose action is based on the capillary forces of the capillary tube. The micropipette is composed of a capillary tube and a pipette bulb (Figure 1A). Capillary tubes are available in different volume sizes ranging from 1 µL to 20 µL, and the capillary tube volumes are selected according to the requirements. Capillary tubes with smaller volumes are not suggested because the ext...

Dyskusje

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

Materiały

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