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)

작은 절지동물에서 혈림프 수집

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.

저자 스포트라이트: 작은 절지동물에서 혈림프를 수집하기 위한 정확하고 정량화 가능한 방법  

작은 절지동물에서 체림프를 수집하기 위한 정확하고 정량화 가능한 방법

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)  

Small Brown Planthopper(SBPH)의 체액 림프 수집 및 분석

이 연구에서는 벼 묘목에서 자란 작은 갈색 화분 호퍼 또는 SBPH를 사용합니다. 배양이 준비되면 성장한 SBPH를 원심분리기 튜브에 넣고 10-30분 동안 얼음 욕조에 넣습니다. 그런 다음 실체 현미경으로 유리 슬라이드에 얼어붙은 SBPH를 놓고 복부가 위를 향하도록 하고 6개의 다리에 초점을 맞춥니다.초미세 팁이 있는 고정밀 핀셋 2개를 준비합니다. 핀셋 하나를 사용하여 곤충의 몸을 눌러 제자리에 고정하고 다른 핀셋으로 한쪽 다리를 조심스럽게 잡아당깁니다. 그런 다음 곤충의 가슴을 부드럽게 눌러 혈림프가 상처를 통해 흐르도록합니다.체액 림프를 수집하려면 하나의 마이크로 리터 모세관 튜브를 피펫 전구에 넣어 마이크로 피펫을 준비하십시오. 모세관을 곤충 상처 가까이에 놓고 마이크로피펫 피펫 전구를 잡습니다. 모세 혈관 끝을 삼출 혈 림프에 대고 수집하십시오.모세관이 원하는 스케일 라인에 도달하면 손가락으로 피펫 전구의 작은 구멍을 약간 막아 흡수 과정을 중지합니다. 이어서, 수집된 체액림프를 100 마이크로리터의 PBS 완충액으로 배출한다. SDS-PAGE를 통해 체액 채취의 정확성을 확인하였으며, 별도로 채취한 3개의 체액 림프 샘플 모두에서 유사한 단백질 함량을 보였다.유충의 경우 총 단백질 농도는 밀리리터당 약 3.7mg이었습니다. 성인 여성 및 남성 SBPH에서, 단백질 농도는 각각 밀리리터당 약 3.5 및 3.6 밀리그램이었고, 3개의 샘플 간에 유의한 차이를 나타내지 않았다. 유충 혈림프는 2 내지 20 나노미터 크기의 혈구를 나타내었다.또한, 세포 농도는 3개의 수집된 혈림프 샘플에서 비슷하였다.