Name: Collection and Analysis of Hemolymph from Small Brown Planthopper (SBPH)
Uploaded: 2023-04-28T12:00:00
Description: Watch this Scientific Journal Video about Collection and Analysis of Hemolymph from Small Brown Planthopper SBPH at JoVE.com

The video demonstrates a procedure for collecting hemolymph from small brown planthoppers (SBPHs). It involves immobilizing the insect, extracting hemolymph using a micropipette, and analyzing protein concentrations across different life stages using SDS-PAGE. The findings indicate similar protein levels in larval and adult SBPHs.

collection and analysis of hemolymph from small brown planthopper (sbph)

Collecte de l’hémolymphe sur de petits arthropodes

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 agriculture1,2,3. 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 environment4,5,6,7. 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 hemolymph8,9,10,11,12. 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 protein13,14. Some viruses may escape the immune response of the hemolymph by binding specific vector factors15,16,17,18. 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 hemolymph19,20,21. 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 virus22,23,24. 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&#39;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.

Pleins feux sur l’auteur : Une méthode précise et quantifiable pour collecter l’hémolymphe de petits arthropodes  

Une méthode précise et quantifiable pour recueillir l’hémolymphe de petits arthropodes

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 a 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 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 starting the interactions between viruses and vectors.Our upcoming research will focus on understanding the molecular mechanisms of virus transmission by vector-insects while exploring and enhancing new operational techniques.

Watch this Scientific Journal Video about Collection and Analysis of Hemolymph from Small Brown Planthopper SBPH at JoVE.com

Collection and Analysis of Hemolymph from Small Brown Planthopper (SBPH)  

Prélèvement et analyse de l’hémolymphe de la petite cicadelle brune (SBPH)

Pour cette étude, utilisez de petites cicadelles brunes ou des SBPH élevés dans des plants de riz. Une fois que les cultures sont prêtes, mettez les SBPH cultivés dans un tube à centrifuger et placez-les dans un bain de glace pendant 10 à 30 minutes. Ensuite, placez un SBPH congelé sur une lame de verre sous un stéréomicroscope avec son abdomen tourné vers le haut et ajustez la mise au point sur ses six pattes.Préparez deux pincettes de haute précision avec des pointes ultra fines. À l’aide d’une pince à épiler, appuyez sur le corps de l’insecte pour le maintenir en place et retirez soigneusement une jambe avec l’autre pince à épiler. Appuyez ensuite doucement sur la poitrine de l’insecte, permettant à l’hémolymphe de circuler dans la plaie.Pour recueillir l’hémolymphe, préparez une micropipette en plaçant un tube capillaire de microlitre dans le bulbe de pipette. Placez le tube capillaire près de la plaie de l’insecte, en tenant l’ampoule de la pipette à micropipette. Touchez l’extrémité capillaire de l’hémolymphe exsudative et recueillez-la.Une fois que le tube capillaire atteint la ligne d’échelle souhaitée, bloquez légèrement le petit trou sur l’ampoule de la pipette avec le doigt pour arrêter le processus d’absorption. Ensuite, déchargez l’hémolymphe recueillie dans 100 microlitres de tampon PBS. La précision de la collecte de l’hémolymphe a été confirmée via SDS-PAGE, qui a montré une teneur en protéines similaire dans les trois échantillons d’hémolymphe, prélevés séparément.Pour les larves, la concentration totale de protéines était d’environ 3,7 milligrammes par millilitre. Dans les SBPH femelles et mâles adultes, les concentrations de protéines étaient d’environ 3,5 et 3,6 milligrammes par millilitre respectivement, ne montrant aucune différence significative entre les trois échantillons. L’hémolymphe larvaire a montré des hémocytes de deux à 20 nanomètres.De plus, la concentration cellulaire était comparable dans les trois échantillons d’hémolymphe recueillis.