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)

Recolección de hemolinfa de pequeños artrópodos

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.

Autor destacado: Un método preciso y cuantificable para recolectar hemolinfa de pequeños artrópodos  

Un método preciso y cuantificable para recolectar hemolinfa de artrópodos pequeños

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)  

Recolección y análisis de hemolinfa de saltaplantas marrón pequeño (SBPH)

Para este estudio, use saltaplantas marrones pequeños o SBPH criados en plántulas de arroz. Una vez que los cultivos estén listos, coloque los SBPH cultivados en un tubo de centrífuga y colóquelos en un baño de hielo durante 10 a 30 minutos. Luego coloque un SBPH congelado en un portaobjetos de vidrio bajo un microscopio estereoscópico con el abdomen hacia arriba y ajuste el enfoque en sus seis patas.Prepara dos pinzas de alta precisión con puntas ultrafinas. Usando una pinza, presione el cuerpo del insecto para mantenerlo en su lugar y tire cuidadosamente de una pierna con la otra pinza. Luego presione suavemente el pecho del insecto, permitiendo que la hemolinfa fluya a través de la herida.Para recolectar hemolinfa, prepare una micropipeta colocando un tubo capilar de microlitros en el bulbo de pipeta. Coloque el tubo capilar cerca de la herida del insecto, sosteniendo el bulbo de pipeta de micropipeta. Toque la punta capilar con la hemolinfa que exuda y recógela.Una vez que el tubo capilar alcance la línea de escala deseada, bloquee ligeramente el pequeño orificio en el bulbo de pipeta con el dedo para detener el proceso de absorción. Luego descargue la hemolinfa recolectada en 100 microlitros de tampón PBS. La precisión de la recolección de hemolinfa se confirmó a través de SDS-PAGE, que mostró un contenido de proteína similar en las tres muestras de hemolinfa, recolectadas por separado.Para la larva, la concentración total de proteína fue de aproximadamente 3,7 miligramos por mililitro. En SBPH adultos femeninos y masculinos, las concentraciones de proteína fueron de aproximadamente 3.5 y 3.6 miligramos por mililitro respectivamente, sin mostrar diferencias significativas entre las tres muestras. La hemolinfa larvaria mostró hemocitos de dos a 20 nanómetros de tamaño.Además, la concentración celular fue comparable en las tres muestras de hemolinfa recogidas.