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)

Сбор гемолимфы у мелких членистоногих

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.

Автор в центре внимания: точный и поддающийся количественной оценке метод сбора гемолимфы у мелких членистоногих  

Точный и поддающийся количественной оценке метод сбора гемолимфы у мелких членистоногих

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)  

Сбор и анализ гемолимфы у малого бурого кузнечика (SBPH)

Для этого исследования используйте небольшие коричневые кузнечики или SBPH, выращенные в рассаде риса. Как только культуры будут готовы, поместите выращенные SBPH в центрифужную пробирку и поместите их на ледяную баню на 10-30 минут. Затем поместите замороженный SBPH на предметное стекло под стереомикроскоп брюшком вверх и отрегулируйте фокус на его шести ногах.Подготовьте два высокоточных пинцета с ультратонкими наконечниками. С помощью одного пинцета надавите на тело насекомого, чтобы удержать его на месте, и осторожно оттяните одну ногу другим пинцетом. Затем аккуратно надавите на грудную клетку насекомого, позволяя гемолимфе течь через ранку.Чтобы собрать гемолимфу, приготовьте микропипетку, поместив одну микролитровую капиллярную трубку в колбу пипетки. Поместите капиллярную трубку близко к ране насекомого, держась за колбу микропипетки. Прикоснитесь кончиком капилляра к источающейся гемолимфе и соберите ее.Как только капиллярная трубка достигнет желаемой линии масштабирования, слегка заблокируйте пальцем маленькое отверстие на колбе пипетки, чтобы остановить процесс всасывания. Затем выгрузите собранную гемолимфу в 100 микролитров буфера PBS. Точность сбора гемолимфы была подтверждена с помощью SDS-PAGE, который показал одинаковое содержание белка во всех трех образцах гемолимфы, собранных отдельно.Для личинки общая концентрация белка составляла примерно 3,7 миллиграмма на миллилитр. У взрослых женщин и мужчин концентрации белка составляли около 3,5 и 3,6 миллиграмма на миллилитр соответственно, не показывая существенной разницы между тремя образцами. Личиночная гемолимфа показала гемоциты размером от двух до 20 нанометров.Кроме того, концентрация клеток была сопоставима в трех собранных образцах гемолимфы.