We thank Nipun Basrur, Adriana K. Rosas Villegas, Nadav Shai, and Trevor Sorrells for comments on the manuscripts, and Zhongyan Gong and Kyrollos Barsoum for technical assistance. We thank Alex Wild for photographs used in Figure 1. K.V. was supported by the Boehringer Ingelheim Fonds PhD fellowship. V.J. was supported in part by NIH T32-MH095246. This work was supported in part by a grant to The Rockefeller University from the Howard Hughes Medical Institute through the James H. Gilliam Fellowships for Advanced Study program to V.J. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. NSF DGE-1325261 to V.J. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

<ol>
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The authors have nothing to disclose.

For many laboratory applications, artificial membrane feeders offer distinct benefits compared to live hosts by allowing researchers the ability to directly manipulate the contents of the meal. Although multiple methods are available for artificial membrane feeding, the method described here offers advantages in flexibility, cost, and throughput. In comparison to other commercial membrane feeders, the Glytube assay requires a small meal volume, making it an efficient delivery mechanism for costly reagents, including drugs or pathogens, by minimizing the total volume required7,35. As both the protein-free saline and artificial blood meals promote engorgement, compounds or pathogens can be added to either meal as a high-throughput and non-invasive alternative to injections. Additionally, each component of the Glytube can easily be washed, replaced, or scaled up to deliver and quantify multiple meal types without cross-contamination of the feeding apparatus.
To quantify meal volumes consumed by mosquitoes, the fluorescence-based method enables more precise meal size quantification than weighing the mosquitoes before and after feeding. It should be noted that this method is an end-point assay. In contrast, weighing allows the mosquitoes to be kept alive for further experimentation. By using a plate reader, the fluorescence-based method can be easily scaled up for high-throughput quantification of meals consumed by hundreds of individual females.
To achieve high feeding rates, a combination of sufficient host cues must be present to activate female host-seeking behavior and attract females to the feeder. If mosquitoes are not crowding underneath the Glytube, the meal may not be properly warmed, or CO2 delivery may not be sufficient. Addition of human odor to the membrane surface reliably increases attractiveness of the artificial membrane. If mosquitoes are observed underneath the Glytube but fail to feed, the meal composition may be at fault. Females may not feed if the meal itself is not warm, the blood is too old, or if the additives to the meal are intrinsically aversive or cause an undesirable chemical reaction36. Additional ATP also reliably increases feeding rates and can be scaled up to a final concentration of 2 mM in each of the recipes provided. Females may not feed if the parafilm is not pulled taut across the Glytube cap; the parafilm should be uniformly transparent and should not buckle, as this prevents the female from being able to effectively pierce the parafilm with her stylet. If the meal leaks through the Glytube onto the mesh, the parafilm may have torn during the stretching process and should be replaced.
Changing the meal composition can also allow researchers to manipulate the length of time needed to clear the meal from the midgut as well as the subsequent host-seeking behavior. The meals presented here require 24&#8211;36 h for digestion7 similar to animal-derived blood. After feeding on any of these meals, females will suppress host-seeking during the digestion time window. Since the saline meal lacks protein, females return to host-seeking after the meal is cleared. If a faster return is desirable, researchers can choose alternate &#8220;quick clearing&#8221; saline meals that are excreted in approximately 6 h27. While the composition of the saline meal presented here is matched to directly compare results with the artificial blood meal, the &#8220;quick clearing&#8221; meal more closely matches physiological salt levels found in vertebrate blood.
The methods described here have limitations that should be considered before selecting the assay that is most suited to the researcher&#8217;s experimental goals. The fluorescein measurements described do not allow mosquitoes to be used again for additional experimentation. However, weight measurements can be taken prior to meal size quantification using the fluorescein assay. If weight and meal size are consistent across multiple trials for a given meal, weight can be used as a proxy in future experiments. Moreover, this protocol does not distinguish between deficits in host-seeking versus blood-feeding behavior; mosquitoes that show impairments in finding the membrane feeder will have a reduction in feeding rates and/or meal size. By adding a camera to record behavior throughout the assay, researchers can determine whether the females cannot find the Glytube, or whether they find the Glytube, but do not feed.
The assay described here can be adapted to explore many outstanding questions related to feeding behavior in mosquitoes. For example, the contribution of specific blood proteins can be explored by altering the ratio of constituent proteins or total protein concentration in the artificial blood meal. To evaluate meal sizes from multiple feeding events, dyes with distinct fluorescence spectra can be added to differentiate meals from unique sources37. This protocol can also be modified to separately stimulate the internal mouthparts that detect blood and that are used for ingestion (i.e., stylet), and the chemosensory appendages that contact skin (i.e., labium, legs) as the mosquito lands to begin blood feeding36. For example, if ligands are added directly to the meal, they do not contact the labium and legs, since the membrane is pierced only by the stylet. If ligands are added to the outer surface of the parafilm instead, they remain separated from the meal and may be contacted by the labium and legs36. Finally, the detailed kinetics of blood-feeding behavior are not well understood and the method presented here could be modified to combine high-resolution tracking with machine learning tools to extract behavioral readouts of locomotion, posture, and feeding dynamics38.
This protocol is aimed at being user-friendly and cost-effective, with the ability to serve researchers employing pharmacological and genetic manipulations to study mosquito blood-feeding and post-blood-feeding behavior.

We present a protocol for feeding modified blood meals to Aedes aegypti mosquitoes using an artificial membrane feeder and precisely measuring the meal volume consumed by each individual mosquito. This protocol can be flexibly adapted to alter the content of the meal or to compare the meal volume consumed by different experimental groups of mosquitoes.
The Ae. aegypti mosquito threatens global health by spreading pathogens that cause diseases including yellow fever, dengue fever, chikungunya, and Zika1,2,3,4,5. Ae. aegypti females are obligate blood-feeders; they must consume vertebrate blood to obtain the necessary protein for egg development, and each clutch of eggs requires a full blood meal from at least one host6,7,8. The female mosquito first bites her host by piercing the skin with her stylet and injecting saliva, which contains compounds that trigger the host&#8217;s immune response9. She then feeds by pumping blood through her stylet into her midgut. While consuming a blood meal from an infected host, she may ingest blood-borne pathogens6,8, which then migrate from the mosquito&#8217;s midgut to her salivary glands10. Female mosquitoes infected in this manner can spread disease by injecting pathogens along with saliva when biting subsequent hosts11,12. Understanding and quantifying the mechanisms of blood-feeding behavior are crucial steps in controlling the transmission of mosquito-borne diseases.
Many laboratory protocols for mosquito rearing and experimentation use live animals including mice, guinea pigs, or humans as a blood source13,14,15,16. The use of live animals imposes ethical concerns as well as complex requirements for personnel training, animal housing and care, and compliance with Institutional Animal Care and Use Committee (IACUC) policies. It also limits the types of compounds that can be delivered to mosquitoes, which constrains the studies that can be carried out17.
Artificial blood-feeding apparatuses, which typically use a membrane system to simulate host skin, are useful tools for studying blood-feeding behaviors that circumvent the need for the maintenance of live hosts. Whole blood can be purchased from a number of vendors and fed to mosquitoes using heated, water-jacketed artificial membrane feeders or similar devices18,19. In this protocol, we demonstrate the use of small, disposable membrane feeders termed &#8220;Glytubes&#8221;. This membrane feeder, previously published by Costa-da-Silva et al. (2013)20, can be easily assembled from standard laboratory equipment, making it ideal for delivering blood meals to moderate numbers of mosquitoes and straightforward to scale up for testing larger groups or multiple meal formulations. The Glytube is an inexpensive and efficient alternative to other commercial artificial feeders, which may require larger meal volumes and are more suitable for batch feeding large groups of mosquitoes on a single meal formulation21.
This protocol includes two sections: preparing/delivering artificial meals and quantifying consumption. In the first section, Glytubes are used as an efficient means to deliver manipulated diets. Whole blood may be substituted with an entirely artificial meal to compare the effects of blood substitutes in lieu of a blood meal. A recipe adapted from Kogan (1990)22 is presented here, although multiple artificial meal formulations have been developed23,24. Furthermore, feeding is a less invasive and less laborious method to introduce pharmacological compounds than injection. Due to the low total volume required for each meal (1&#8211;2 mL), this protocol provides an attractive delivery method to reduce the amounts of expensive reagents. Ae. aegypti females readily consume protein-free meals of saline solution with adenosine 5&#8242;-triphosphate (ATP)25,26, which provides a baseline for measuring the effects of single meal components. For example, Neuropeptide Y-like receptor 7 (NPYLR7) in Ae. aegypti is known to mediate host-seeking suppression after a protein-rich blood meal, and when NPYLR7 agonists are added to a protein-free saline meal, female mosquitoes exhibit host-seeking suppression similar to those that have consumed whole blood7.
In the second section, steps for quantifying the volume of each meal consumed by an individual female mosquito are presented. This assay is fluorescence-based and captures feeding status in higher resolution than methods in which females are classified as &#8220;fed&#8221; or &#8220;unfed&#8221; based on visual assessment of abdominal distension alone. By adding fluorescein to the meal prior to feeding, meal volumes ingested by individuals can be quantified by homogenizing each mosquito in a 96-well plate and measuring fluorescence intensity as a readout. This assay can measure differences in feeding vigor in response to variables such as meal composition or the mosquitoes&#8217; genetic background. Precise quantification is critical for intermediate meal sizes, for example when females are offered suboptimal meals containing feeding deterrents or when they consume sucrose meals of variable sizes27. If fed mosquitoes are required for subsequent behavioral assays after meal size quantification, meal size can instead be calculated by weighing anesthetized females in groups and estimating the average increased mass per individual. Although less precise than fluorescein marking, weighing still provides an aggregated estimate of meal volume and allows examination of the meal&#8217;s effect on downstream processes, such as fecundity or subsequent host attraction. While blood meal size is variable and can be influenced by a myriad of factors11,28,29, ingested meal sizes measured with the methods described here are consistent with previous quantifications7,30,31.

<table><tbody><tr><td>15 mL conical tubes</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>3 mm diameter borosilicate solid-glass bead</td><td>MilliporeSigma</td><td>Z143928</td><td>For use for bead mill homogenizer; not required if using pellet pestle grinder</td></tr><tr><td>32 oz. high-density polyethylene (HDPE) plastic cup</td><td>VWR</td><td>89009-668</td><td>Example mosquito container used for feeding assays shown; alternate options can be used</td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>96-well black polystyrene plate</td><td>ThermoFisher</td><td>12-566-09</td><td></td></tr><tr><td>96-well PCR plate sealing film</td><td>Bio-Rad</td><td>MSB1001</td><td>Alternate options can be used</td></tr><tr><td>96-well PCR plates</td><td>Bio-Rad</td><td>HSP9621</td><td>Alternate options can be used</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate (ATP) disodium salt hydrate</td><td>MilliporeSigma</td><td>A6419</td><td></td></tr><tr><td>Albumin (human serum)</td><td>MilliporeSigma</td><td>A9511</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01-213</td><td>Alternate options can be used to block light entering fluorescein container</td></tr><tr><td>Balance</td><td>Fisher Scientific</td><td>01-911</td><td>Alternate options can be used</td></tr><tr><td>Bead mill homogenizer</td><td>Qiagen</td><td>85300</td><td>Not required if using pellet pestle grinder</td></tr><tr><td>Cotton ball</td><td>Fisher Scientific</td><td>22456880</td><td>For nectar-feeding; alternate options can be used</td></tr><tr><td>Defibrinated sheep blood</td><td>Hemostat Laboratories</td><td>DSB100</td><td>Alternate options can be used</td></tr><tr><td>Drosophila CO&lt;sub&gt;2&lt;/sub&gt; fly pad</td><td>Tritech Research</td><td>MINJ-DROS-FP</td><td>Alternate options can be used</td></tr><tr><td>Fluorescein</td><td>MilliporeSigma</td><td>F6377</td><td></td></tr><tr><td>Fluorescence plate-reader</td><td>ThermoFisher</td><td>VL0000D0</td><td>Alternate options can be used</td></tr><tr><td>Gamma-globulin (human blood)</td><td>MilliporeSigma</td><td>H7379</td><td></td></tr><tr><td>Glycerol</td><td>MilliporeSigma</td><td>G7893</td><td></td></tr><tr><td>Hemoglobin (human)</td><td>MilliporeSigma</td><td>G4386</td><td></td></tr><tr><td>Laboratory wrapping film - parafilm</td><td>Fisher Scientific</td><td>13-374</td><td></td></tr><tr><td>Magnetic stirrer</td><td>Fisher Scientific</td><td>90-691</td><td>Alternate magnetic stirrers can be used</td></tr><tr><td>Microcentrifuge for 96-well plate</td><td>VWR</td><td>80094-180</td><td>Alternate options can be used</td></tr><tr><td>Microcentrifuge Tubes</td><td>MilliporeSigma</td><td>2236412</td><td>Alternate options can be used</td></tr><tr><td>Pellet pestle grinder</td><td>VWR</td><td>KT749521-1500</td><td>Not required if using bead mill homogenizer</td></tr><tr><td>Phosphate buffered solution (PBS)</td><td>Fisher Scientific</td><td>BW17-516F</td><td>Optional</td></tr><tr><td>Razor blades</td><td>Fisher Scientific</td><td>12-640</td><td>Alternate options can be used, such as a lathe for better consistency of cutting</td></tr><tr><td>Rubber bands</td><td></td><td></td><td></td></tr><tr><td>Silicone tubing</td><td>McMaster Carr</td><td></td><td>Needed if using a fly pad for CO&lt;sub&gt;2&lt;/sub&gt; delivery</td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>MilliporeSigma</td><td>S9888</td><td></td></tr><tr><td>Stir bars</td><td>Fisher Scientific</td><td>14-512</td><td>Alternate magnetic stir bars can be used</td></tr><tr><td>Translucent polypropylene storage box with removable lid</td><td></td><td></td><td>Example box used for feeding assays shown</td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>Alternate heating device may be used</td></tr><tr><td>White 0.8 mm polyester mosquito netting</td><td>American Home &amp;amp; Habit Inc.</td><td>F03A-PONO-MOSQ-M008-WT</td><td>Alternate options can be used</td></tr></tbody></table>

feeding and quantifying animal-derived blood and artificial meals in aedes aegypti mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Figure 1 presents a schematic for assembling the Glytube, whereas Figure 2 shows an overview of the experimental design to measure meal size using the fluorescence-based assay described here. Figure 3 provides representative fluorescein meal size measurements from a blood-feeding experiment. Figure 4, Figure 5, and Figure 6 illustrate a sampling of biological questions that can be addressed using this protocol. Applications of the protocol are wide-ranging and include altering blood meal composition, feeding pharmacological compounds, precisely quantifying sub-optimal blood meals or smaller nectar meals, and comparing feeding behavior across mosquito genotypes.
To generate a standard curve for meal volume calculations, fluorescence readings are plotted from the designated reference wells each containing an unfed mosquito and a known volume of the meal with 0.002% fluorescein (Figure 3A). Fluorescence readings from the remaining wells, which contain mosquitoes from either the negative control group of unfed mosquitoes or the experimental group of mosquitoes offered a meal, are compared to this standard curve to quantify the meal volume (&#181;L) consumed by each mosquito (Figure 3B). To validate the baseline readings in this assay, it should be confirmed that mosquitoes from the unfed negative control group are not assigned a positive value of &#181;L consumed (Figure 3B, left). Although all females in the experimental group were offered the blood meal, some mosquitoes fed (Figure 3B, middle) and some did not (Figure 3B, right). This result demonstrates that two types of data can be obtained from this protocol: 1) the percentage of total females that feed on a given meal, and 2) the volume ingested by the females that feed on a given meal.
This protocol can be used to deliver and quantify meals with various protein compositions. Figure 4A,B show data collected using meals with added fluorescein. The proportion of mosquitoes that fed and the meal volume they ingested, respectively, were calculated from the fluorescence readings. These readings are highly sensitive and allow for precise quantification of &#181;L, but have the limitation that mosquitoes cannot be used for future live experiments. Figure 4C,D show data collected from an independent experiment with mosquitoes that were scored as fed or unfed by eye after they were offered meals without fluorescein. Meal size was calculated as average weight/female from groups of 5 mosquitoes. Although these weight measurements are less sensitive than fluorescence measurements, they allow the females to be recovered and used for further live experimentation. The proportion of mosquitoes that feed can vary across different experimental days, as reflected in Figure 4A and Figure 4C.
Figure 5 shows the volume consumed of meals containing drugs that regulate mosquito host-seeking behavior. In these experiments, females were offered blood, saline + ATP, or saline + ATP&#160;meals with 100 &#956;M of the human NPY Y2 receptor agonist, TM30338. This drug alters host-seeking behavior through activation of Ae. aegypti NPY-like receptor 7. Measuring meal sizes is critical for the interpretation of experiments to assess the effect of this drug on post-blood-feeding behavior because it allows the researcher to calculate the dose consumed by each female.
In the previous examples, females were fed either blood or substitute blood meals, all of which resulted in 3&#8211;5 &#181;L meals (Figure 3, Figure 4, Figure 5). This fluorescence-based assay can also be used to measure smaller and/or more variable meal sizes that cannot be accurately discerned from average group weight measurements. In Figure 6, the same fluorescence quantification protocol was used to measure nectar-feeding behavior by exchanging the Glytube for a cotton ball saturated with 10% sucrose containing 0.002% fluorescein. Nectar sugars cannot be presented in the Glytube assay because females cannot detect the presence of nectar sugars with the stylet and do not initiate feeding27. These data allow the researcher to determine that sugar meals are consistently smaller than blood meals, in agreement with previous work34 (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61835/61835fig1v2.png" /> 
Figure 1: Setup of Glytube method used to feed meals to mosquitoes.&#160;(A) Schematic of a deconstructed Glytube used to feed blood and other meals to mosquitoes. (B) Schematic of a Glytube presented atop a container of mosquitoes with a mesh lid. Female mosquitoes can pierce through the mesh lid to feed. (C) Photographs of the Glytube (top), and female Aedes aegypti mosquitoes before, during, and after feeding (bottom, from left to right) on a Glytube-delivered meal. Mosquitoes are shown piercing through the mesh covering their container to access the membrane feeder. (D) Photographs showing the appearance of female Ae. aegypti mosquitoes that are unfed (left) and that have engorged on either an artificial blood meal (right, top) or a saline + ATP meal (right, bottom). The Glytube method was previously published in Costa-da-Silva et al. (2013)20. Photographs in (C) and (D) are courtesy of Alex Wild. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig1v2large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61835/61835fig2v4.png" /> 
Figure 2: Schematic of how to quantify meal size after Glytube blood-feeding protocol.&#160;(A) Mosquitoes are offered a meal with fluorescein (top, experimental group) or no meal (bottom, unfed negative control group). (B) Individual mosquitoes are added to a 96-well plate after terminating the feeding experiment. (C) Standard curve is generated using known amounts of meal containing 0.002% fluorescein. (D) Mosquitoes are homogenized to release any consumed fluorescein, and fluorescence levels in each well are quantified using a plate reader. This fluorescence quantification method is modified from Liesch et al. (2013)34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig2v4large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61835/61835fig3v2.png" /> 
Figure 3: Glytube blood-feeding experiment with fluorescein-based quantification.&#160;(A) Standard curve measurements obtained from the wells where a mosquito from the unfed control group was added to a known quantity of meal containing 0.002% fluorescein (y-axis scale = arbitrary units). (B) Meal volume calculated using fluorescence readings for females in the unfed control group (left, black, n = 40), the experimental group that fed on blood (middle, red, n = 37), and the experimental group that did not feed on blood (right, red, n = 23). Each point represents a measurement from an individual female. Data are shown as median with range. Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.01. These data were published in Jov&#233; et al. (2020)27. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig3v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61835/61835fig4v2.png" /> 
Figure 4: Quantification of meals with differing protein composition.&#160;Females were offered meals of either sheep blood (red), artificial blood with human blood proteins (Kogan (1990)22) (orange), or protein-free saline + ATP meal (aqua)7. (A) Percentage of females fed scored using fluorescence readings. Each point represents a group of 12&#8211;16 females. Data are shown as medians with ranges, n = 12. (B) Meal volume calculated using fluorescence readings. Each point represents a measurement from an individual female in a single trial from Figure 4A. Data are shown as medians with ranges, n = 12. (C) Percentage of females fully engorged after artificial membrane feeding, scored by eye. Each point represents the percent of females engorged from groups of 20&#8211;30 females. Data are shown as medians with ranges, n = 23. (D) Meal sizes scored as weight/female after feeding status was scored by eye. Weights were calculated as the average of groups of 5 mosquitoes. Data are shown as medians with ranges, n = 23. A&#8211;D: Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig4v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61835/61835fig5v2.png" /> 
Figure 5: Quantification of meals with pharmacological compounds.&#160;Females consume meals of the same size of sheep blood (red), saline + ATP (aqua), and saline + ATP + 100 &#181;M dose of human NPY Y2 receptor agonist TM30338 (dark blue). Meal volume calculated using fluorescence readings. Each point represents a measurement from an individual female. Data are shown as medians with ranges, n = 12. Letters indicate statistically distinct groups, Kruskal-Wallis test with Dunn&#8217;s multiple comparison, p&#60;0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig5v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61835/61835fig6v2.png" /> 
Figure 6: Quantification of smaller nectar meals.&#160;(A) Schematic of nectar-feeding assay. (B) Meal volume calculated using fluorescence readings for wild-type females offered meals of either water (blue, n = 36) or 10% sucrose (green, n = 53), each with 0.002% fluorescein, in the nectar-feeding assay. Each point represents a measurement from an individual female. Data are shown as medians with ranges. Letters indicate statistically distinct groups, Mann-Whitney test, p&#60;0.05. These data were published in Jov&#233; et al. (2020)27. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61835/61835fig6v5large.png" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Artificial Blood Meal</td>
		</tr>
		<tr>
			<td></td>
			<td>Concentration of Stock Solution (mg/mL)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mg/mL)</td>
		</tr>
		<tr>
			<td colspan="4">Protein Components*</td>
		</tr>
		<tr>
			<td>&#947;-Globulins</td>
			<td>50</td>
			<td>300</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Hemoglobin</td>
			<td>35</td>
			<td>230</td>
			<td>8</td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>300</td>
			<td>340</td>
			<td>102</td>
		</tr>
		<tr>
			<td>Total Protein</td>
			<td>-</td>
			<td>-</td>
			<td>125</td>
		</tr>
		<tr>
			<td colspan="4">Non-Protein Components</td>
		</tr>
		<tr>
			<td></td>
			<td>Concentration of Stock Solution (mM)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>In &#947;-globulin stock</td>
			<td>-</td>
			<td>5-10</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>In &#947;-globulin stock</td>
			<td>-</td>
			<td>120</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>125</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">*Protein components are prepared in stock solution of double-distilled water, except for &#947;-Globulins, which are dissolved in 400 mM NaHCO3 and include a variable amount of NaCl (2-4%) in the product.</td>
		</tr>
	</tbody>
</table>
Table 1: Recipe for preparing artificial blood meals (adapted from Kogan (1990)22). Artificial blood consists of protein and non-protein components regularly found in human blood and provides the option to vary the ratios of these components. Mosquitoes can produce eggs after feeding on artificial blood7,22.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Saline Meal</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Concentration of Stock Solution (mM)</td>
			<td>Volume of Stock Solution in Meal (&#956;L/mL)</td>
			<td>Final Meal Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>400</td>
			<td>300</td>
			<td>120</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>200</td>
			<td>5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>695</td>
			<td>-</td>
		</tr>
	</tbody>
</table>
Table 2: Recipe for saline meal with ATP (adapted from Duvall et al. (2019)7).&#160;Protein-free saline meals can be used to deliver compounds of interest to mosquitoes while still mimicking the abdominal distension that occurs after blood-feeding, but without triggering the egg development that occurs when proteins are ingested.

Watch this Scientific Journal Video about Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes at JoVE.com

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...

feeding-quantifying-animal-derived-blood-artificial-meals-aedes

Research

JoVE Journal

Immunology and Infection

7.7K Views.  The Rockefeller University. The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Video: Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Biology

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are delivered using a non-quantitative method. Accurate delivery of known quantities of nucleic acids or other chemicals is critical to measure the efficacy of the compound being tested and to allow reliable comparison between compounds.
Here we provide a reproducible, quantitative microinjection protocol that ensures accurate delivery of specific doses of dsRNA, reducing the mortality typically induced by injection injury. These modifications include the addition of Rhodamine B, a graduated injection needle, and an improved recovery method borrowed from Isoe and Collins. This method allows calculation of dose responses and facilitates comparisons between compounds. Versions of this method have been successfully used on three genera of mosquitoes as well as house flies to assess the reduction in fecundity resulting from gene silencing of ribosomal RNA transcripts.
This protocol provides strategies to reduce several challenges of small insect microinjection. Together, mechanical delivery of dsRNAs accompanied by visual verification, identification of effective locations for delivery, and inclusion of a post-injection recovery period ensure accurate dosing and low injury mortality. This protocol also describes an oviposition bioassay for uniform determination of effects on fecundity.

This protocol describes a microinjection methodology that we have standardized and used for several years to deliver specific quantities of nucleic acids directly to the hemolymph of mosquitoes and house flies. This protocol results in minimal injection mortality and allows dose correlated measurements of fecundity.

Synthetic dsRNAs, used to induce RNA interference, may have dose dependent phenotypic effects. These effects are difficult to define if the dsRNAs are ...

Environment

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti&#39;s&#160;following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

This methodology, which included oral feeding and intrathoracic injection infection, could effectively assess the influence of midgut and/or salivary gland barriers on arbovirus infection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public ...

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus particles are transmitted during a blood meal via the saliva of an infected mosquito. Forced salivation assays allow determining the vector potential on the basis of single mosquitoes, avoiding the use of animal experiments. The method is suitable to analyze a large number of mosquitoes in one experiment within a short period of time. Forced salivation assays were used to analyze 856 individual mosquitoes trapped in Germany, including two different Culex pipiens pipiens biotypes, Culex torrentium as well as Aedes albopictus, which were experimentally infected with Zika virus (ZIKV) and incubated at 18 &#176;C or 27 &#176;C for two and three weeks. The results indicated the lack of vector competence of the different Culex taxa for ZIKV. In contrast, Aedes albopictus was susceptible to ZIKV, but only at 27 &#176;C, with transmission rates similar to an Aedes aegypti laboratory colony tested in parallel.

For efficient control of mosquito borne virus transmission, the knowledge of the vector potential of respective mosquitoes is of particular interest. We describe forced salivation as a method to analyze vector competence of Aedes albopictus and three different Culex taxa for the transmission of Zika virus.

Vector competence is defined as the potential of a mosquito species to transmit a mosquito-borne virus (mobovirus) to a vertebrate host. Viable virus ...

A Blood-Free Diet to Rear Anopheline Mosquitoes

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable production of mosquitoes is currently inhibited by the supply of fresh vertebrate blood. Alternatives to blood are required to promote efficient control strategies for malaria and other vector borne diseases that are transmitted by blood feeding insects. With this in mind, artificial liquid diets were formulated as substitutes for fresh vertebrate blood. Herein we report a blood-free artificial liquid diet that delivers feeding rates similar to blood and mimics the physiological effects of a fresh vertebrate blood meal. The diet induces ovarian and egg maturation of Anopheles mosquitoes and also produces good larval survival and development of functional adults. The formulated blood-free liquid diet is an important advance towards sustainable mosquito breeding in captivity and will reduce the maintenance costs of mosquito colonies and eliminate the need for fresh vertebrate blood.

A protocol is presented for formulation of a blood-free artificial diet to feed Anopheles mosquitoes in captivity. This diet has a similar performance to vertebrate blood and triggers oogenesis and egg maturation and produces viable adult progeny.

Malaria research requires large-scale breeding and production conditions for mosquitoes (Anopheles spp.) in captivity. The sustainable and reliable ...

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness parameters, in particular egg production and hatch rates at the individual level, conventional methods have put a substantial burden on investigators due to high labor intensity and laboratory space requirements. Described is a simple method using 24 well tissue culture plate with agarose in each well and digital imaging of each well to determine egg numbers and hatch rates at an individual level with substantially reduced time and space requirements.

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates EAgaL Plates

Described is a time and space-saving method to count eggs and determine hatch rates of individual mosquitoes using 24 well tissue culture plates, which can substantially increase the scale and speed of fecundity and fertility assays.

Mosquitoes represent a significant public health problem as vectors of various pathogens. For those studies that require an assessment of mosquito fitness ...

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been conducted to investigate the effect of gut microbes as a whole; however, different microbes could exert distinct effects toward the host. This article provides the methodology to study the effect of each specific mosquito gut microbe and the potential mechanism.
This protocol contains two parts. The first part introduces how to dissect the mosquito midgut, isolate cultivable bacteria colonies, and identify bacteria species. The second part provides the procedure to generate antibiotic-treated mosquitoes and reintroduce one specific bacteria species.

This article presents a protocol to investigate the effect of individual mosquito gut bacteria, including isolation and identification of mosquito midgut cultivable microbes, antibiotic depletion of mosquito gut bacteria, and reintroduce one specific bacteria species.

The mosquito midgut harbors a highly dynamic microbiome that affects the host metabolism, reproduction, fitness, and vector competence. Studies have been ...

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life parameter data from genetically modified mosquitoes and comparing them to mosquitoes lacking transgenes from the same genetic background. This manuscript illustrates how to measure common life history traits in the mosquito Aedes aegypti, including fecundity, wing size and shape, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These parameters were chosen because they reflect reproductive success, are simple to measure, and are commonly reported in the literature. The representative results quantify fitness costs associated with either a gene knock-out or a single insertion of a gene drive element. Standardizing how life parameter data are collected is important because such data may be used to compare the health of transgenic mosquitoes generated across studies or to model the transgene fixation rate in a simulated wild-type mosquito population. Although this protocol is specific for transgenic Aedes aegypti, the protocol may also be used for other mosquito species or other experimental treatment conditions, with the caveat that certain biological contexts may require special adaptations.

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes

The present protocol describes how to measure common life parameter data in Aedes aegypti mosquitoes, including fecundity, wing size, fertility, sex ratio, viability, development times, male contribution, and adult longevity. These measurements can be used to assess the fitness of transgenic mosquitoes.

Transgenic mosquitoes often display fitness costs compared to their wild-type counterparts. In this regard, fitness cost studies involve collecting life ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

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Chapters in this video

Alphavirus Transducing System: Tools for Visualizing Infection in Mosquito Vectors

Protocol for Mosquito Rearing (A. gambiae)

Reproducible dsRNA Microinjection and Oviposition Bioassay in Mosquitoes and House Flies

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection

Forced Salivation As a Method to Analyze Vector Competence of Mosquitoes

A Blood-Free Diet to Rear Anopheline Mosquitoes

Improved Fecundity and Fertility Assay for Aedes aegypti using 24 Well Tissue Culture Plates (EAgaL Plates)

A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes

Quantifying Fitness Costs in Transgenic Aedes aegypti Mosquitoes