For this study, use small brown planthopper or SBPHs raised in rice seedlings. Once the cultures are ready, put the grown SBPHs into a centrifuge tube and place them in an ice bath for 10 to 30 minutes. Then place a frozen SBPH on a glass slide under a stereo microscope with its abdomen facing up and adjust the focus on its six legs.
Prepare two high precision tweezers with ultra fine tips. Using one tweezer, press down on the insect's body to keep it in place and carefully pull off one leg with the other tweezer. Then gently press the insect's chest, allowing hemolymph to flow through the wound.
To collect hemolymph, prepare a micropipette by placing one microliter capillary tube into the pipet bulb. Place the capillary tube close to the insect wound, holding the micropipette pipette bulb. Touch the capillary tip to the exuding hemolymph and collect it.
Once the capillary tube reaches the desired scale line, slightly block the small hole on the pipette bulb with the finger to stop the absorbing process. Then discharge the collected hemolymph into 100 microliters of PBS buffer. The accuracy of hemolymph collection was confirmed via SDS-PAGE, which showed a similar protein content in all three hemolymph samples, collected separately.
For larva, the total protein concentration was approximately 3.7 milligrams per milliliter. In adult female and male SBPHs, the protein concentrations were about 3.5 and 3.6 milligrams per milliliter respectively, showing no significant difference between the three samples. The larval hemolymph showed hemocytes of two to 20 nanometers in size.
In addition, the cell concentration was comparable in the three collected hemolymph samples.
