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)

Hemolymfe verzamelen van kleine geleedpotigen

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.

Auteur Spotlight: een nauwkeurige en kwantificeerbare methode voor het verzamelen van hemolymfe van kleine geleedpotigen  

Een nauwkeurige en kwantificeerbare methode voor het verzamelen van hemolymfe van kleine geleedpotigen

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)  

Verzameling en analyse van hemolymfe van Small Brown Planthopper (SBPH)

Gebruik voor deze studie kleine bruine planthopper of SBPHs die zijn grootgebracht in rijstzaailingen. Zodra de culturen klaar zijn, doe je de gekweekte SBPHs in een centrifugebuis en plaats je ze 10 tot 30 minuten in een ijsbad. Plaats vervolgens een bevroren SBPH op een glazen dia onder een stereomicroscoop met zijn buik naar boven gericht en pas de focus aan op zijn zes poten.Bereid twee uiterst nauwkeurige pincetten met ultrafijne uiteinden. Druk met één pincet op het lichaam van het insect om het op zijn plaats te houden en trek voorzichtig één poot af met het andere pincet. Druk vervolgens zachtjes op de borst van het insect, waardoor hemolymfe door de wond kan stromen.Om hemolymfe te verzamelen, bereidt u een micropipette voor door een microliter capillaire buis in de pipetlamp te plaatsen. Plaats de capillaire buis dicht bij de insectenwond en houd de micropipetpipetbol vast. Raak de capillaire punt aan op de exsuderende hemolymfe en verzamel deze.Zodra de capillaire buis de gewenste schaallijn heeft bereikt, blokkeert u het kleine gaatje op de pipetbol enigszins met de vinger om het absorptieproces te stoppen. Loost vervolgens de verzamelde hemolymfe in 100 microliter PBS-buffer. De nauwkeurigheid van de hemolymfeverzameling werd bevestigd via SDS-PAGE, dat een vergelijkbaar eiwitgehalte toonde in alle drie de hemolymfemonsters, afzonderlijk verzameld.Voor larven was de totale eiwitconcentratie ongeveer 3,7 milligram per milliliter. Bij volwassen vrouwelijke en mannelijke SBPHs waren de eiwitconcentraties respectievelijk ongeveer 3,5 en 3,6 milligram per milliliter, wat geen significant verschil tussen de drie monsters liet zien. De larvale hemolymfe vertoonde hemocyten van twee tot 20 nanometer groot.Bovendien was de celconcentratie vergelijkbaar in de drie verzamelde hemolymfemonsters.