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)

Samle hemolymfe fra små leddyr

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.

Forfatter Spotlight: En presis og kvantifiserbar metode for å samle hemolymfh fra små leddyr  

En presis og kvantifiserbar metode for å samle hemolymfe fra små leddyr

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)  

Innsamling og analyse av hemolymfe fra liten brun plantehopper (SBPH)

For denne studien, bruk liten brun plantehopper eller SBPHs oppvokst i risplanter. Når kulturene er klare, legg de dyrkede SBPH-ene i et sentrifugerør og legg dem i et isbad i 10 til 30 minutter. Legg deretter en frossen SBPH på et glassglass under et stereomikroskop med magen vendt opp og juster fokuset på de seks bena.Forbered to pinsett med høy presisjon med ultrafine tips. Bruk en pinsett, trykk ned på insektets kropp for å holde den på plass og trekk forsiktig av det ene benet med den andre pinsetten. Trykk deretter forsiktig på insektets bryst, slik at hemolymf kan strømme gjennom såret.For å samle hemolymfe, klargjør en mikropipette ved å plassere ett mikroliter kapillærrør i pipetpæren. Plasser kapillærrøret nær insektsåret, hold mikropipettepipetteren. Berør kapillærspissen til den utstrålende hemolymfen og samle den.Når kapillærrøret når ønsket skalalinje, blokkerer du det lille hullet på pipettepæren litt med fingeren for å stoppe absorberingsprosessen. Tøm deretter den oppsamlede hemolymfen i 100 mikroliter PBS-buffer. Nøyaktigheten av hemolymfeinnsamling ble bekreftet via SDS-PAGE, som viste et lignende proteininnhold i alle tre hemolymfeprøver, samlet separat.For larver var den totale proteinkonsentrasjonen ca. 3,7 milligram per milliliter. Hos voksne kvinnelige og mannlige SBPHer var proteinkonsentrasjonene henholdsvis ca. 3,5 og 3,6 milligram per milliliter, og viste ingen signifikant forskjell mellom de tre prøvene. Larvehemolymfen viste hemocytter på to til 20 nanometer i størrelse.I tillegg var cellekonsentrasjonen sammenlignbar i de tre innsamlede hemolymfeprøvene.