Name: dissection and immunostaining of larval salivary glands from anopheles gambiae mosquitoes
Uploaded: 2021-09-30T14:45:00
Description: Watch this Scientific Journal Video about Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes at JoVE.com

We would like to thank the Johns Hopkins Malaria Research Institute for access to and rearing of An. gambiae larvae.

1. Preparation of solutions and tools
<ol>
	<li>Preparation of dissection solution
	<ol>
		<li>To prepare dissecting solution, add 2.5 mL of 100% ethanol to 7.5 mL of distilled H2O in a 15 plastic tube. Invert the tube 3 times to mix. 
		NOTE: This solution can be stored at room temperature for several weeks.</li>
	</ol>
	</li>
	<li>Preparation of 10x phosphate-buffered saline (PBS) stock
	<ol>
		<li>To prepare 10x PBS stock, add 17.8 g Na2HPO4&#8226; 2H2</sub...

<ol>
	<li>World malaria report 2020: 20 years of global progress and challenges. World Health Organization Available from: <a target="_blank" href="https://apps.who.int/iris/handle/10665/337660">https://apps.who.int/iris/handle/10665/337660</a> (2020)</li><li>Feachem, R. G. A., 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=Malaria+eradication+within+a+generation:+ambitious,+achievable,+and+necessary.">Malaria eradication within a generation: ambitious, achievable, and necessary.</a> Lancet. 394 (10203), 1056-1112 (2019).</li><li>Adolfi, A., 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=Efficient+population+modification+gene-drive+rescue+system+in+the+malaria+mosquito+Anopheles+stephensi.">Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.</a> Nature Communications. 11, 5553 (2020).</li><li>Kyrou, K., 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+CRISPR-Cas9+gene+drive+targeting+doublesex+causes+complete+population+suppression+in+caged+Anopheles+gambiae+mosquitoes.">A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.</a> Nature Biotechnology. 36 (11), 1062-1066 (2018).</li><li>Rostami, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9+gene+drive+technology+to+control+transmission+of+vector-borne+parasitic+infections.">CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections.</a> Parasite Immunology. 42 (9), 12762 (2020).</li><li>Bhatt, 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=Coverage+and+system+efficiencies+of+insecticide-treated+nets+in+Africa+from+2000+to+2017.">Coverage and system efficiencies of insecticide-treated nets in Africa from 2000 to 2017.</a> Elife. 4, 09672 (2015).</li><li>Mellink, J. J., Vanden Bovenkamp, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+aspects+of+mosquito+salivation+in+blood+feeding+of+Aedes+aegypti.">Functional aspects of mosquito salivation in blood feeding of Aedes aegypti.</a> Mosquito News. 41 (1), 115 (1981).</li><li>Ribeiro, J. M., Rossignol, P. A., Spielman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mosquito+saliva+in+blood+vessel+location.">Role of mosquito saliva in blood vessel location.</a> Journal of Experimental Biology. 108, 1-7 (1984).</li><li>Yamamoto, D. S., Sumitani, M., Kasashima, K., Sezutsu, H., Matsuoka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+malaria+infection+in+transgenic+Anopheline+mosquitoes+lacking+salivary+gland+cells.">Inhibition of malaria infection in transgenic Anopheline mosquitoes lacking salivary gland cells.</a> PLoS Pathogens. 12 (9), 1005872 (2016).</li><li>Rishikesh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+development+of+the+salivary+glands+and+their+chromosomes+in+the+larvae+of+Anopheles+stephensi+sensu+stricto.">Morphology and development of the salivary glands and their chromosomes in the larvae of Anopheles stephensi sensu stricto.</a> Bulletin of the World Health Organization. 20 (1), 47-61 (1959).</li><li>Jensen, D. V., Jones, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+salivary+glands+in+Anopheles+albimanus+Wiedemann+(Diptera,+Culicidae).">The development of the salivary glands in Anopheles albimanus Wiedemann (Diptera, Culicidae).</a> Annals of the Entomological Society of America. 50 (5), 40824 (1957).</li><li>Chiu, M., Trigg, B., Taracena, M., Wells, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+cellular+morphologies+during+lumen+maturation+in+Anopheles+gambiae+larval+salivary+glands.">Diverse cellular morphologies during lumen maturation in Anopheles gambiae larval salivary glands.</a> Insect Molecular Biology. 30 (2), 210-230 (2021).</li><li>MR4. Methods in Anopheles research. Center for Disease Control Available from: <a target="_blank" href="https://www.beiresources.org/Portals/2/">https://www.beiresources.org/Portals/2/</a> (2015)</li><li>Wells, M. B., Andrew, D. J. <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+cellular+architecture+in+the+Asian+malaria+vector+mosquito+Anopheles+stephensi.">Salivary gland cellular architecture in the Asian malaria vector mosquito Anopheles stephensi.</a> Parasites &amp; Vectors. 8, 617 (2015).</li><li>Wells, M. B., Villamor, J., Andrew, D. J. <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+maturation+and+duct+formation+in+the+African+malaria+mosquito+Anopheles+gambiae.">Salivary gland maturation and duct formation in the African malaria mosquito Anopheles gambiae.</a> Scientific Reports. 7 (1), 601 (2017).</li><li>Wells, M. B., Andrew, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anopheles+salivary+gland+architecture+shapes+plasmodium+sporozoite+availability+for+transmission.">Anopheles salivary gland architecture shapes plasmodium sporozoite availability for transmission.</a> mBio. 10 (4), 01238 (2019).</li><li>Clements, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The physiology of mosquitoes. , (1963).</li><li>Imms, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+larval+and+pupal+stages+of+Anopheles+maculipennis,+meigen.">On the larval and pupal stages of Anopheles maculipennis, meigen.</a> Parasitology. 1 (2), 103-133 (1908).</li><li>Favia, 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=Bacteria+of+the+genus+Asaia+stably+associate+with+Anopheles+stephensi,+an+Asian+malarial+mosquito+vector.">Bacteria of the genus Asaia stably associate with Anopheles stephensi, an Asian malarial mosquito vector.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (21), 9047-9051 (2007).</li><li>Neira Oviedo, M., 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=The+salivary+transcriptome+of+Anopheles+gambiae+(Diptera:+Culicidae)+larvae:+A+microarray-based+analysis.">The salivary transcriptome of Anopheles gambiae (Diptera: Culicidae) larvae: A microarray-based analysis.</a> Insect Biochemistry and Molecular Biology. 39 (5-6), 382-394 (2009).</li><li>Linser, P. J., Smith, K. E., Seron, T. J., Neira Oviedo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbonic+anhydrases+and+anion+transport+in+mosquito+midgut+pH+regulation.">Carbonic anhydrases and anion transport in mosquito midgut pH regulation.</a> Journal of Experimental Biology. 212 (11), 1662-1671 (2009).</li><li>Linser, P. 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=Slc4-like+anion+transporters+of+the+larval+mosquito+alimentary+canal.">Slc4-like anion transporters of the larval mosquito alimentary canal.</a> Journal of Insect Physiology. 58 (4), 551-562 (2012).</li></ol>

The protocol described herein was adapted from a Drosophila SG dissection protocol and an adult mosquito dissection protocol14,15,16. However, most markers did not penetrate the basement membrane (data not shown) when using the adult dissection and SG staining methods. Adaptations of the adult protocol included dissecting the glands in a 25% EtOH solution, washing the glands with a combination of MeOH and glacial acetic acid, an...

Malaria is a major public health threat causing almost 230 million infections and an estimated 409,000 deaths in 20191. The majority of deaths are in sub-Saharan Africa and are caused by the parasite Plasmodium falciparum, whose insect vector is Anopheles gambiae, the subject of this video demonstration. Although the numbers indicate a significant drop in annual death rate since the turn of the century (&#62;300,000 fewer annual deaths), the promising decreases in disease rates observed from 2000 to 2015 are tapering, suggesting the need for new approaches to limiting disease transmission2. Among promising additional strategies for controlling and possibly eliminating malaria is targeting mosquito vector capacity using CRISPR/Cas9-based gene-editing and gene-drive3,4,5. Indeed, it is the targeting of the mosquito vector (through the expanded use of long-lasting insecticide-treated bed nets) that has had the greatest impact on reducing disease transmission6.
Female mosquitoes acquire Plasmodium gametocytes from an infected human during a blood meal. Following fertilization, maturation, midgut epithelium traversal, population expansion, and hemocoel navigation in their obligate mosquito hosts, hundreds to tens of thousands of Plasmodium sporozoites invade the mosquito SGs and fill the secretory cavities of the constituent secretory cells. Once inside the secretory cavities, the parasites have direct access to the salivary duct and are thus poised for transmission to a new vertebrate host upon the next blood meal. Because SGs are critical for the transmission of malaria-causing sporozoites to their human hosts, and laboratory studies suggest that SGs are not essential for blood-feeding, mosquito survival, or fecundity7,8,9, they represent an ideal target for transmission-blocking measures. Adult mosquito SGs form as an elaboration of &#34;duct bud&#34; remnants in the larval SGs that persist beyond early pupal SG histolysis10, making the larval SG an ideal target for interventions to limit adult-stage disease transmission.
Characterizing the larval stage of SG development can help develop not only a better understanding of its morphology and functional adaptations but can also aid in assessing new interventions that target this organ through gene editing of key SG regulators. Because all previous studies of larval salivary gland architecture predate immunostaining and modern imaging techniques10,11, we have developed a protocol for isolating and staining salivary glands with a variety of antibodies and cell markers12. This video demonstrates this approach to the extraction, fixation, and staining of larval SGs from Anopheles gambiae L4 larvae for confocal imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;KH2PO4</td>
			<td>Millipore Sigma</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Na2HPO4 &#8226; 2H2O</td>
			<td>Millipore Sigma</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;NaCl</td>
			<td>Millipore Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Millipore Sigma</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush with soft bristles</td>
			<td>Amazon (SN NJDF) Detail Paint Brush Set</td>
			<td>B08LH63D89</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips (22 x 50 mm)</td>
			<td>VWR</td>
			<td>48393-195</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (DNA)</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 proof</td>
			<td>Millipore Sigma</td>
			<td>EX0276</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman P200 Pipette</td>
			<td>Gilson</td>
			<td>P200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers forceps, Dumont No. 5</td>
			<td>Millipore Sigma</td>
			<td>F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>58221</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Millipore Sigma</td>
			<td>1414209</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Amazon (Sally Hansen)</td>
			<td>B08148YH9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Nile Red (lipid)</td>
			<td>ThermoFisher Scientific</td>
			<td>N1142</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towels/wipes</td>
			<td>ULINE</td>
			<td>S-7128</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate (to make putty plate)</td>
			<td>ThermoFisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips</td>
			<td>Gilson</td>
			<td>Tips E200ST</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Transfer Pipette</td>
			<td>Fisher Scientific</td>
			<td>S304671</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibodies (e.g., Crb, Rab11)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB); Andrew Lab</td>
			<td>Mouse anti-Crb (Cq4) or Rabbit anti-Rab11</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies with fluorescent tags (e.g., Alexa Fluor 488 Goat-anti Rabbit)</td>
			<td>ThermoFisher Scientific</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone resin and curing agent for putty plate</td>
			<td>Dow Chemicals - Ximeter Silicone</td>
			<td>PMX-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides, frosted on one end for labelling</td>
			<td>VWR&#160; 20 X 50 mm</td>
			<td>48393-195</td>
			<td></td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin</td>
			<td>ThermoFisher Scientific</td>
			<td>W834</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and immunostaining of larval salivary glands from anopheles gambiae mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Salivary glands are relatively easy to dissect from all stage 4 larvae. Male and female larvae can be distinguished at the late L4 larval stage by a red stripe along the dorsal thorax of females but not males (Figure 2). We also observe that antennal morphology is much more elaborate in male than in female L4 larvae (Figure 2), similar to the differences observed in this structure in adult mosquitoes. Along with the considerable overall growth during the L4 stag...

Watch this Scientific Journal Video about Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes at JoVE.com

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

The salivary glands of insects, are required for transmitting many human diseases. So a better understanding, of salary gland biology should help us, add to existing strategies for disease prevention. This technique will allow us, to quickly ask if mutations in the candidate regulators, affect the morphology, of the mosquito larval salivary glands, and potentially affect the transmission, of malarial parasites.The first few times you work with the mosquito larvae, they are challenging to grasp as they move quickly. The more you practice, the better you get. Getting started with the procedure, will be Marisol Luchetti, an undergraduate research assistant for my laboratory.To start the experiment, place a potty plate on the stage of a dissecting microscope, and transfer 10 mosquito L4 larvae onto the potty plate. Then place a drop of the dissection solution, onto the potty plate separate from the larvae. Use forceps to place one L4 larva, into the dissection solution drop of 25%ethanol.By holding a number five forceps in each hand, grasp the larvae with forceps just below the head, with the dominant hand, and grip the head of the larva with the non-dominant hand, then gently pull the head with minimal constant force, to detach from the rest of the body, while the glands remain attached to the head. After discarding the body portion of the larva carcass, collect the head or salivary glands, into one milliliter of PBS, in a 1.5 milliliter micro centrifuge tube, and wait for the salivary glands, to sink to the bottom of the buffer. On the next day, drain the solution to fixate the tissues, with one milliliter of cold 100%acetone for 90 seconds.After removing the acetone, gently rinse the tissues three times, with one milliliter of PBS. For immunostaining, add a primary antibody such as Rab 11, at the appropriate dilution, in 200 microliters of the total volume of PBS. Swirl the tube contents gently with a pipette tip.Incubate the primary antibodies overnight at four degrees, followed by washing three times in one milliliter of PBS. Then add the secondary antibody, Alexa Fluor 488 Goat anti-Rabbit, at the appropriate dilution, in 200 microliters of total volume. After mixing the contents with a pipette tip, incubate the tissues with dyes, such as Nile red, and Hoechst into 200 microliters of PBS, at room temperature for 60 minutes, followed by washing three times with PBS.Prepare a microscope slide, by adding 200 microliters of 100%glycerol with a pipette, and transferring up to 20 stained heads, with the attached glands and internal structures, per microscope slide using a soft brush. Spread the head samples out, to evenly distribute them along the glass slide. Viewing under a dissecting microscope, separate the larval head from the glands, using two pairs of forceps, by carefully pulling the two tissues in opposite directions.When all the samples are done, gently place a 1.5 millimeter thick cover slip, on top of the slide, avoiding the formation of air bubbles, and then seal the slide with clear nail Polish. The salivary glands are relatively easy to dissect, from stage four larvae. Male and female larvae can be distinguished, at the late L4 larval stage by a red stripe, along the dorsal thorax of females.The antennal morphology is also more elaborate, in males than in female L4 larvae as can be seen, with the white arrows pointing out, the female antennae on the left image, and the male antennae on the right. The salivary glands isolated from early L4 stage larvae, stained with Hoechst reveal the proximal and distal lobes, separated by a narrow constriction. The forming lumen was examined, in the immuno stained distal salivary gland, of a mid to late L4 larva.The apical domains of the secretory cells, surrounding the forming lumen had intense Nile red staining, suggestive of microvilli like structures. The Rab 11 staining was observed close, to the apical surface. There's only one step with the time, and that's the 90 second incubation, of the tissues in cold acetone.This protocol was only recently developed, but we expect it will be helpful, to anyone working on the mosquito salivary gland. We fully expect to use it often.

dissection-immunostaining-larval-salivary-glands-from-anopheles

Research

JoVE Journal

Biology

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Building a Better Mosquito: Identifying the Genes Enabling Malaria and Dengue Fever Resistance in A. gambiae and A. aegypti Mosquitoes

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections.   Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus infections. Explanation is given for how key refractory genes, those genes conferring resistance to vector pathogens, are identified in the mosquito and how this knowledge can be used to generate transgenic mosquitoes that are unable to carry the malaria parasite or dengue virus.

In this interview, George Dimopoulos focuses on the physiological mechanisms used by mosquitoes to combat Plasmodium falciparum and dengue virus ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...