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)

Coletando hemolinfa de pequenos artrópodes

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 em destaque: um método preciso e quantificável para coletar hemolinfa de pequenos artrópodes  

Um Método Preciso e Quantificável para Coleta de Hemolinfa de Pequenos Artrópodes

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)  

Coleta e Análise de Hemolinfa de Cigarrinha Marrom Pequena (SBPH)

Para este estudo, utilizar-se cigarrinhas marrons pequenas ou SBPHs criadas em mudas de arroz. Assim que as culturas estiverem prontas, coloque os SBPHs crescidos em um tubo de centrífuga e coloque-os em um banho de gelo por 10 a 30 minutos. Em seguida, coloque um SBPH congelado em uma lâmina de vidro sob um microscópio estéreo com o abdômen voltado para cima e ajuste o foco em suas seis pernas.Prepare duas pinças de alta precisão com pontas ultrafinas. Usando um tweezer, pressione o corpo do inseto para mantê-lo no lugar e puxe cuidadosamente uma perna com a outra tweezer. Em seguida, pressione suavemente o peito do inseto, permitindo que a hemolinfa flua através da ferida.Para coletar hemolinfa, prepare uma micropipeta colocando um tubo capilar de microlitro no bulbo da pipeta. Coloque o tubo capilar próximo à ferida do inseto, segurando o bulbo da pipeta da micropipeta. Tocar a ponta capilar na hemolinfa exsudada e coletá-la.Quando o tubo capilar atingir a linha de escala desejada, bloqueie levemente o pequeno orifício no bulbo da pipeta com o dedo para interromper o processo de absorção. Em seguida, despeje a hemolinfa coletada em 100 microlitros de tampão PBS. A acurácia da coleta de hemolinfa foi confirmada pelo SDS-PAGE, que mostrou conteúdo proteico semelhante nas três amostras de hemolinfa, coletadas separadamente.Para as larvas, a concentração de proteína total foi de aproximadamente 3,7 miligramas por mililitro. Em SBPHs adultos femininos e masculinos, as concentrações de proteína foram de cerca de 3,5 e 3,6 miligramas por mililitro, respectivamente, não mostrando diferença significativa entre as três amostras. A hemolinfa larval apresentou hemócitos de dois a 20 nanômetros de tamanho.Além disso, a concentração celular foi comparável nas três amostras de hemolinfa coletadas.