Our research focuses on arboviruses, insects, and plant interactions. Obtaining hemolymph samples from small arthropods presents a technical challenge for research on insect-borne diseases. This protocol shows our precise and quantifiable technique to counter this issue.
Recent research developments in the transmission of arboviruses have discovered that viruses can manipulate several important vector factors to break through the host's barrier. This protocol is a simple, cost-effective, and valuable technique that allows for precise quantification of the hemolymph. This method also offers valuable technology for studying the interactions between viruses and vectors.
Our upcoming research will focus on understanding the molecular mechanisms of various transmission by vector insects. We are exploring and enhancing new operational techniques. 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 pipette 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 2 to 20 nanometers in size. In addition, the cell concentration was comparable in the three collected hemolymph samples.
