This work was supported by the National Key R&#38;D Program of China (No. 2022YFD1401700) and by the National Science Foundation of China (No. 32090013 and No. 32072385).

1. Insect rearing
<ol>
	<li>Raise the SBPHs used in this experiment in rice seedlings (Oryza sativa cv. Nipponbare). Plant 20 rice seedlings in an incubator (65 mm x 200 mm), and grow at 25 &#176;C under a 16 h light/8 h dark photoperiod.</li>
</ol>
2. Dissection of the SBPHs for hemolymph collection
<ol>
	<li>Put the SBPHs into a centrifuge tube, and place them in an ice bath for 10-30 min. 
	NOTE: Do not place the SBPHs in the ice bath for less than 10 min, or the insects may revive.</li>
	<li>Place a frozen SBPH on a glass slide (see Table of Materials) under a stereomicroscope (see Table of Materials) with its abdomen facing up. Adjust the focus on the six legs of the insect.</li>
	<li>Prepare two high-precision tweezers (see Table of Materials) with ultra-fine tips. Use one tweezer to press down on the insect&#39;s body to keep it in place, and use the other one to carefully pull one of the legs off the insect. 
	NOTE: For insects with a hard shell, an ophthalmic scalpel can be used for cutting off one of the legs.</li>
	<li>Gently press the chest of the insect with a tweezer to make the hemolymph flow out through the wound. 
	&#8203;NOTE: The hemolymph presents as transparent droplets without any visible white floccule. Discard the lipid if there is any white floccule, which represents fat body contamination.</li>
</ol>
3. Hemolymph collection using micropipettes
<ol>
	<li>Prepare a micropipette. Place a capillary tube (see Table of Materials) with a volume of 1 &#181;L into the pipette bulb (see Table of Materials). For accurate measurements, ensure that the scale line is visible. 
	NOTE: A small hole present on the top of the pipette bulb is necessary.</li>
	<li>When collecting the hemolymph, hold the pipette bulb of the micropipette, and place the capillary tube close to the insect wound. Collect the hemolymph exuding from the wound by simply touching the tip of the capillary to the hemolymph.</li>
	<li>Block the small hole on the top of the pipette bulb slightly with the finger to stop the absorbing process when the liquid inside the capillary tube reaches the desired scale line. 
	NOTE: Collect one sample of 1 &#181;L of hemolymph incessantly and rapidly to avoid lipid contamination and tube plugging.</li>
	<li>Press the pipette bulb to discharge the collected hemolymph into 100 &#181;L of PBS buffer (137 mmol/L NaCI, 2.7 mmol/L KCI, 4.3 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, pH 7.2-7.4). 
	&#8203;NOTE: The capillary tube is inserted into the PBS buffer.</li>
	<li>Change to a new capillary tube when collecting another 1 &#181;L sample of hemolymph.</li>
</ol>
4. Coomassie Blue staining
<ol>
	<li>Collect 3 hemolymph samples with the same volume (1 &#181;L) from 3rd stage larvae according to the protocols described in step 3, and discharge the collected hemolymph into the PBS buffer to make a total volume of 100 &#181;L.</li>
	<li>Add 25 &#181;L of 5x protein loading buffer (250 mmol/L Tris-HCI [pH 6.8], 10% W/V SDS, 0.05% W/V Bromophenol Blue, 50% W/V glycerol, 5% W/V &#946;-mercaptoethanol) into 100 &#181;L of the diluted hemolymph samples, and heat to denature the proteins.</li>
	<li>Load 20 &#181;L of the boiled samples onto a 10% SDS-PAGE protein gel (see Table of Materials) to separate the proteins.</li>
	<li>Stain the gel in a Coomassie Blue solution (mix 1 g of Coomassie Brilliant Blue R-250 with 250 mL of isopropanol, 100 mL of glacial acetic acid, and 650 mL of ddH2O, and filter out any particles using filter paper) for 1 h at room temperature, and then decolorize the gel for 1 h with decolorizing solution (100 mL of acetic acid, 400 mL of methanol, 500 mL of ddH2O).</li>
</ol>
5. Protein concentration determination
<ol>
	<li>Dilute 10 mg/mL bovine serum albumin (BSA) to 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, and 0.3125 mg/mL with PBS buffer using the two-fold dilution method.</li>
	<li>Aspirate 5 &#181;L of PBS buffer and each concentration of BSA solutions in step 5.1, add 1 mL of 1x Bradford dye reagent (see Table of Materials), and incubate for 5 min at room temperature. Use PBS buffer in Bradford dye reagent as the control, and zero at OD = 595 nm. Measure the absorbance values of each of the remaining protein concentrations, and make a standard curve.</li>
	<li>Add 1 &#181;L of hemolymph from larval, female, or male SBPHs into 100 &#181;L of PBS buffer, and then centrifugate the samples at 1,000 x g for 10 min at 4 &#176;C to remove the hemocytes.</li>
	<li>Aspirate 90 &#181;L of supernatant to a new centrifuge tube, and measure the absorbance values of the supernatant according to the instructions in step 5.2.</li>
	<li>Calculate the protein concentrations of the hemolymph samples according to the regression equation of the standard curve. Include three biological replicates with three technical replicates in the experiments.</li>
</ol>
6. Microscopic detections
<ol>
	<li>Use micropipettes to collect hemolymph from 10-15 SBPHs at different developmental stages. Add the collected hemolymph into a tube containing 20 &#181;L of 4% paraformaldehyde solution (see Table of Materials). Incubate the mixture at room temperature for 30 min to fix the hemolymph.</li>
	<li>Pipette 20 &#181;L of fixed hemocytes onto the center of a slide coated with silane (see Table of Materials).</li>
	<li>Dry up the droplet in a slide drying rack. 
	NOTE: Avoid excessive drying.</li>
	<li>Cover the sample with gold antifade reagent 4&#39;,6-diamidino-2-phenylindole (DAPI) (see Table of Materials), and inspect the slide under an inverted microscope (see Table of Materials).</li>
</ol>
7. Cell quantification
<ol>
	<li>Collect 1 &#181;L of hemolymph from the SBPHs with a micropipette, and dissolve it into 20 &#181;L of PBS buffer.</li>
	<li>Dilute the hemolymph samples five-fold with PBS buffer.</li>
	<li>Pipette 20 &#181;L of hemolymph solution into the center of the cell counting chamber (see Table of Materials). Cover the droplet with a coverslip (see Table of Materials).</li>
	<li>Count the cells in the five squares on the cell counting chamber (Supplementary Figure 1), under an inverted microscope (see Table of Materials). Include three biological replicates with three technical replicates in each of the experiments. 
	&#8203;Cells/&#181;L = total count of five middle squares &#215; 5 &#215; dilution factor &#215; 104</li>
</ol>
8. Statistical analyses
<ol>
	<li>Perform statistical analyses with the corresponding software (see Table of Materials). Create a new file, and select the column, import the data, and generate a scatter plot with a bar.</li>
	<li>Calculate the mean and SD for each group of data by using column statistics, and use a one-way ANOVA tool to evaluate the significance of the differences among the groups. Determine the mean and SD from three biological replicates with three independent experiments.</li>
</ol>

Collecting Hemolymph from Small Arthropods

<ol>
	<li>Hogenhout, S. A., Ammar el, D., Whitfield, A. E., Redinbaugh, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+vector+interactions+with+persistently+transmitted+viruses.">Insect vector interactions with persistently transmitted viruses.</a> Annual Review of Phytopathology. 46, 327-359 (2008).</li><li>Ray, S., Casteel, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector-mediated+plant-virus-vector+interactions.">Effector-mediated plant-virus-vector interactions.</a> Plant Cell. 34 (5), 1514-1531 (2022).</li><li>Islam, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant-insect+vector-virus+interactions+under+environmental+change.">Plant-insect vector-virus interactions under environmental change.</a> Science of the Total Environment. 701, 135044 (2020).</li><li>Cory, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+virus+transmission:+Different+routes+to+persistence.">Insect virus transmission: Different routes to persistence.</a> Current Opinion in Insect Science. 8, 130-135 (2015).</li><li>Wang, X. W., Blanc, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+transmission+of+plant+single-stranded+DNA+viruses.">Insect transmission of plant single-stranded DNA viruses.</a> Annual Review of Entomology. 66, 389-405 (2021).</li><li>Yi, H. Y., Chowdhury, M., Huang, Y. D., Yu, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+antimicrobial+peptides+and+their+applications.">Insect antimicrobial peptides and their applications.</a> Applied Microbiology and Biotechnology. 98 (13), 5807-5822 (2014).</li><li>Liu, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+interaction+between+a+plant+virus+and+its+vector+insect+reveals+new+functions+of+hemipteran+cuticular+protein.">Proteomic analysis of interaction between a plant virus and its vector insect reveals new functions of hemipteran cuticular protein.</a> Molecular &amp; Cellular Proteomics. 14 (8), 2229-2242 (2015).</li><li>Wang, L., Van Meulebroek, L., Vanhaecke, L., Smagghe, G., Meeus, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bee+hemolymph+metabolome:+A+window+into+the+impact+of+viruses+on+bumble+bees.">The bee hemolymph metabolome: A window into the impact of viruses on bumble bees.</a> Viruses. 13 (4), 600 (2021).</li><li>Jia, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector+mediated+transmission+of+persistently+transmitted+plant+viruses.">Vector mediated transmission of persistently transmitted plant viruses.</a> Current Opinion in Virology. 28, 127-132 (2018).</li><li>Anderson, J. F., Main, A. J., Ferrandino, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Horizontal+and+vertical+transmission+of+West+Nile+Virus+by+Aedes+vexans+(Diptera:+Culicidae).">Horizontal and vertical transmission of West Nile Virus by Aedes vexans (Diptera: Culicidae).</a> Journal of Medical Entomology. 57 (5), 1614-1618 (2020).</li><li>Gadhave, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+frequency+of+horizontal+and+vertical+transmission+of+cucurbit+leaf+crumple+virus+in+whitefly+Bemisia+tabaci+Gennadius.">Low frequency of horizontal and vertical transmission of cucurbit leaf crumple virus in whitefly Bemisia tabaci Gennadius.</a> Phytopathology. 110 (6), 1235-1241 (2020).</li><li>Logan, R. A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+and+horizontal+transmission+of+cell+fusing+agent+virus+in+Aedes+aegypti.">Vertical and horizontal transmission of cell fusing agent virus in Aedes aegypti.</a> Applied and Environmental Microbiology. 88 (18), e0106222 (2022).</li><li>Huo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+transmission+of+a+plant+virus+is+mediated+by+vitellogenin+of+its+insect+vector.">Transovarial transmission of a plant virus is mediated by vitellogenin of its insect vector.</a> PLoS Pathogens. 10 (3), e1003949 (2014).</li><li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vector+development+and+vitellogenin+determine+the+transovarial+transmission+of+begomoviruses.">Vector development and vitellogenin determine the transovarial transmission of begomoviruses.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (26), 6746-6751 (2017).</li><li>Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+innate+immunity.">Toll-like receptors and innate immunity.</a> Nature Reviews Immunology. 1 (2), 135-145 (2001).</li><li>Kingsolver, M. B., Huang, Z., Hardy, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+antiviral+innate+immunity:+Pathways,+effectors,+and+connections.">Insect antiviral innate immunity: Pathways, effectors, and connections.</a> Journal of Molecular Biology. 425 (24), 4921-4936 (2013).</li><li>Pei, R. J., Chen, X. W., Lu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+hepatitis+B+virus+replication+by+interferons+and+Toll-like+receptor+signaling+pathways.">Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways.</a> World Journal of Gastroenterology. 20 (33), 11618-11629 (2014).</li><li>Kao, Y. T., Lai, M. M. C., Yu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+dengue+virus+circumvents+innate+immunity.">How dengue virus circumvents innate immunity.</a> Frontiers in Immunology. 9, 2860 (2018).</li><li>Gilliam, M., Shimanuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+of+hemolymph+of+the+larval+honey+bee+(Apis+mellifera+L).">Coagulation of hemolymph of the larval honey bee (Apis mellifera L).</a> Experientia. 26 (8), 908-909 (1970).</li><li>Huo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+tissue-specific+vitellogenin+facilitates+transmission+of+plant+virus.">Insect tissue-specific vitellogenin facilitates transmission of plant virus.</a> PLoS Pathogens. 14 (2), e1006909 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plant+virus+ensures+viral+stability+in+the+hemolymph+of+vector+insects+through+suppressing+prophenoloxidase+activation.">A plant virus ensures viral stability in the hemolymph of vector insects through suppressing prophenoloxidase activation.</a> mBio. 11 (4), e01453 (2020).</li><li>Vidano, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phases+of+maize+rough+dwarf+virus+multiplication+in+the+vector+Laodelphax+striatellus+(Fall&#233;n).">Phases of maize rough dwarf virus multiplication in the vector Laodelphax striatellus (Fall&#233;n).</a> Virology. 41 (2), 218-232 (1970).</li><li>Yu, Y. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laodelphax+striatellus+Atg8+facilitates+Rice+stripe+virus+infection+in+an+autophagy-independent+manner.">Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner.</a> Journal of Insect Science. 28 (2), 315-329 (2021).</li><li>Zhang, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+monooxygenases+CYP6AY3+and+CYP6CW1+regulate+Rice+black-streaked+dwarf+virus+replication+in+Laodelphax+striatellus+(Fallen).">Cytochrome P450 monooxygenases CYP6AY3 and CYP6CW1 regulate Rice black-streaked dwarf virus replication in Laodelphax striatellus (Fallen).</a> Viruses. 13 (8), 1576 (2021).</li><li>Ribeiro, C., Brehelin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+haemocytes:+What+type+of+cell+is+that.">Insect haemocytes: What type of cell is that.</a> Journal of Insect Physiology. 52 (5), 417-429 (2006).</li><li>Butolo, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+quality+method+for+hemolymph+collection+from+honeybee+larvae.">A high quality method for hemolymph collection from honeybee larvae.</a> PLoS One. 15 (6), e0234637 (2020).</li><li>Nesa, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+potential+of+a+ponericin-like+peptide+isolated+from+Bombyx+mori+L.+hemolymph+in+response+to+Pseudomonas+aeruginosa+infection.">Antimicrobial potential of a ponericin-like peptide isolated from Bombyx mori L. hemolymph in response to Pseudomonas aeruginosa infection.</a> Scientific Reports. 12 (1), 15493 (2022).</li><li>Mahmoud, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin-injected+Musca+domestica+larval+hemolymph:+Cecropin+upregulation+and+potential+anticancer+effect.">Curcumin-injected Musca domestica larval hemolymph: Cecropin upregulation and potential anticancer effect.</a> Molecules. 27 (5), 1570 (2022).</li><li>Patton, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=salivary+gland,+and+hemolymph+collection+from+Ixodes+scapularis+ticks.">salivary gland, and hemolymph collection from Ixodes scapularis ticks.</a> Journal of Visualized Experiments. (60), e3894 (2012).</li><li>Piyankarage, S. C., Augustin, H., Featherstone, D. E., Shippy, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+amino+acid+variations+following+behavioral+and+genetic+changes+in+individual+Drosophila+larvae.">Hemolymph amino acid variations following behavioral and genetic changes in individual Drosophila larvae.</a> Amino Acids. 38 (3), 779-788 (2010).</li><li>Fiorotti, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disclosing+hemolymph+collection+and+inoculation+of+metarhizium+blastospores+into+Rhipicephalus+microplus+ticks+towards+invertebrate+pathology+studies.">Disclosing hemolymph collection and inoculation of metarhizium blastospores into Rhipicephalus microplus ticks towards invertebrate pathology studies.</a> Journal of Visualized Experiments. (148), e59899 (2019).</li></ol>

The authors declare that they have no conflicts of interest.

Hemolymph is the medium of the circulatory system in arthropods, and arboviruses can only invade other arthropod tissues if they are able to survive the hostile hemolymph environment. Collecting a high-quality sample of hemolymph is the first step in studying the vector-virus interactions that occur in the hemolymph. It has been reported that insect hemolymph can be obtained from several sites on the insect&#39;s body, including a wound on the front leg, a minor incision in the head area, or a tear wound at the abdomen<s...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% SDS-PAGE protein gel</td>
			<td>Bio-rad</td>
			<td>4561035</td>
			<td>Protein separation and detection</td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P1110</td>
			<td>For fixation of the cells or tissues&#160;</td>
		</tr>
		<tr>
			<td>Bradford dye reagent</td>
			<td>Bio-rad</td>
			<td>5000205</td>
			<td>Protein concentration detection</td>
		</tr>
		<tr>
			<td>Capillary</td>
			<td>Hirschmann</td>
			<td>9000101</td>
			<td>For collecting hemolymph</td>
		</tr>
		<tr>
			<td>Cell counting chamber</td>
			<td>ACMEC</td>
			<td>AYA0810</td>
			<td>Hemocytes counting</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Gitoglas</td>
			<td>10127105A</td>
			<td>For holding insects</td>
		</tr>
		<tr>
			<td>Glass slide coated with silane</td>
			<td>Sigma</td>
			<td>S4651-72EA</td>
			<td>For holding microscope samples</td>
		</tr>
		<tr>
			<td>Gold antifade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>Nucleus staining</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Gitoglas</td>
			<td>10212424C</td>
			<td>For microscopic observation</td>
		</tr>
		<tr>
			<td>Pipette bulb</td>
			<td>Hirschmann</td>
			<td>9000101</td>
			<td>For collecting hemolymph</td>
		</tr>
		<tr>
			<td>Prism 8.0 software</td>
			<td>GraphPad Software</td>
			<td>/</td>
			<td>Statistical analyses</td>
		</tr>
		<tr>
			<td>Stereomicroscope&#160;</td>
			<td>Motic</td>
			<td>SMZ-168</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Tianld</td>
			<td>P5622</td>
			<td>For insect dissection</td>
		</tr>
		<tr>
			<td>Zeiss inverted microscope</td>
			<td>Zeiss</td>
			<td>Observer Z1</td>
			<td>Hemocytes observation</td>
		</tr>
	</tbody>
</table>

a precise and quantifiable method for collecting hemolymph from small arthropods

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus transmission. Hemolymph collection is the basic technology for studying virus-vector interactions. Here, we describe a novel and simple method for the quantitative collection of hemolymph from small arthropods using Laodelphax striatellus (the small brown planthopper, SBPH) as a research model, as this arthropod is the main vector of rice stripe virus (RSV). In this protocol, the process begins by gently pinching off one leg of the frozen arthropod with fine-tipped tweezers and pressing the hemolymph out of the wound. Then, a simple micropipette consisting of a capillary and a pipette bulb is used to collect the transudative hemolymph from the wound according to the principle of capillary forces. Finally, the collected hemolymph can be dissolved into a specific buffer for further study. This new method for collecting hemolymph from small arthropods is a useful and efficient tool for further research on arboviruses and vector-virus interactions.

Micropipette model and hemolymph collection 
We have developed a simple micropipette whose action is based on the capillary forces of the capillary tube. The micropipette is composed of a capillary tube and a pipette bulb (Figure 1A). Capillary tubes are available in different volume sizes ranging from 1 &#181;L to 20 &#181;L, and the capillary tube volumes are selected according to the requirements. Capillary tubes with smaller volumes are not suggested because the ext...

Watch this Scientific Journal Video about A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods at JoVE.com

Author Spotlight: A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods  

We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods

Arthropods are known to transmit a variety of viruses of medical and agricultural importance through their hemolymph, which is essential for virus ...

a-precise-quantifiable-method-for-collecting-hemolymph-from-small

Research

JoVE Journal

Biology

1.5K Views.  Chinese Academy of Sciences. We describe a method to collect quantifiable hemolymph efficiently from small arthropods for subsequent analysis.

Video: Author Spotlight: A Precise and Quantifiable Method for Collecting Hemolymph from Small Arthropods  

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds 

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.
This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1&beta;, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNF&alpha;, INF&gamma;, G-CSF, GM-CSF, MCP-1 and MIP-1&beta;, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

A technique to collect and measure surgical wound biochemical mediators at specific time points.

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. ...

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6'-acetyltransferase-Ii (AAC(6')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.

ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 &#176;C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules.

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

Introduction of small molecules to the developing Drosophila embryo offers great potential for characterizing biological activity of novel compounds, drugs, and toxins as well as for probing fundamental developmental pathways. Methods described herein outline steps that overcome natural barriers to this approach, expanding the utility of the Drosophila embryo model.

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

A Method for Extracting Pigments from Squid Doryteuthis pealeii

A protocol for the extraction of pigments from the nanostructured granules in squid Doryteuthis pealeii chromatophores is presented.

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...