We thank Drs. R.-D. Xue, C. Bibbs, W. Qualls, and V. Aryaprema of the Anastasia Mosquito Control District, St. Augustine, Florida, for partnership in developing the SIT program and expert insight on effective operational release of sterile male Aedes aegypti. This research was supported by the USDA-ARS and the Florida Department of Agriculture and Consumer Services (FDACS). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by USDA or FDACS.

<ol>
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A., Hendrichs, J., Robinson, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+the+sterile+insect+technique.">History of the sterile insect technique.</a> Sterile insect technique: principles and practice in area-wide integrated pest management. , 3-36 (2005).</li><li>Alphey, 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=Sterile-insect+methods+for+control+of+mosquito-borne+diseases:+an+analysis.">Sterile-insect methods for control of mosquito-borne diseases: an analysis.</a> Vector Borne and Zoonotic Diseases. 10 (3), 295-311 (2010).</li><li>Dame, D. A., Curtis, C. F., Benedict, M. Q., Robinson, A. S., Knols, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+applications+of+induced+sterilisation+in+field+populations+of+mosquitoes.">Historical applications of induced sterilisation in field populations of mosquitoes.</a> Malaria Journal. 8, (2009).</li><li>Bond, J. 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=Optimization+of+irradiation+dose+to+Aedes+aegypti+and+Ae.+albopictus+in+a+sterile+insect+technique+program.">Optimization of irradiation dose to Aedes aegypti and Ae. albopictus in a sterile insect technique program.</a> PloS One. 14 (2), 0212520 (2019).</li><li>Bourtzis, K., Lees, R. S., Hendrichs, J., Vreysen, M. J. 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Methods in Aedes Research Available from: <a target="_blank" href="https://www.beiresources.org/Portals/2/VectorResources/Methods%20in%20Aedes%20Research%202016.pdf">https://www.beiresources.org/Portals/2/VectorResources/Methods%20in%20Aedes%20Research%202016.pdf</a> (2016)</li><li>Focks, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+separator+for+the+developmental+stages,+sexes,+and+species+of+mosquitoes+(Diptera:+Culicidae).">An improved separator for the developmental stages, sexes, and species of mosquitoes (Diptera: Culicidae).</a> Journal of Medical Entomology. 17 (6), 567-568 (1980).</li><li>International Atomic Energy Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of Dosimetry in Radiotherapy. Technical Reports Series No. 110. , (1970).</li><li>Aldridge, R. 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=Gamma-irradiation+reduces+survivorship,+feeding+behavior,+and+oviposition+of+female+Aedes+aegypti.">Gamma-irradiation reduces survivorship, feeding behavior, and oviposition of female Aedes aegypti.</a> Journal of the American Mosquito Control Association. 36 (3), 152-160 (2020).</li><li>Cianci, 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=Estimating+mosquito+population+size+from+mark-release-recapture+data.">Estimating mosquito population size from mark-release-recapture data.</a> Journal of Medical Entomology. 50 (3), 533-542 (2013).</li><li>Knipling, E. 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All authors have declared no conflicts of interest.

Initiation of a control program featuring SIT that uses radiation requires the establishment of a local strain of Aedes aegypti. This step is critical and can allow SIT to truly distinguish itself from similar control technologies. By developing the project from a local strain of mosquito, the males generated will likely have behaviors that allow them to adapt to environmental changes and cues and to locate and mate with wild females in the vicinity. Furthermore, the release of irradiated local males may not generate negative public opinion compared to, say, release of a non-local strain of genetically modified mosquitoes that could, for example, introduce novel alleles into the local mosquito population.
Expending substantial resources to rear vast quantities of mosquitoes only to be able to use about half of them for control purposes is a limitation of the Aedes aegypti SIT program. Refinements should be made to the rearing protocol to condense the maturation of larvae into more defined timeframes when the pupae will be ready. This would allow more pupae to be collected at the optimal time of separation. However, additional pupae to process increases the risk of more females pupating when the pupae are collected and therefore increasing the likelihood of females ending up in Petri dishes with males and possibly being released. Although the lifespan, bloodfeeding behavior, and oviposition behavior in irradiated female Aedes aegypti pupae are reduced in adults, it is not a good strategy to release females incidentally alongside irradiated males22. Therefore, it should remain a priority to minimize the number of females inadvertently separated, irradiated, marked, and released with males.
Success of an SIT program ultimately relies on successful mate competition by colony-reared, irradiated males. Preserving male competitiveness relies on exhaustive experimentally derived selection of the dose and maximizing the estimated ratio of sterile:wild males in the population. Dose selection is determined by several key factors that include longevity, fertility, fecundity, and pupal mortality. It has been observed that male mosquitoes will exhibit an asymptotic fertility curve that approaches zero as radiation increases (KJL, RLA, SCB unpublished data). Simultaneously, male mosquito longevity and activity levels diminish exponentially as the radiation dose increases (KJL, RLA, SCB unpublished data). Therefore, rather than identifying a dose that yields 99.9% sterility in males, it is preferable to focus on a lower sterility percentage while supporting survivorship. Once a dose range is identified that does not differentiate longevity or pupal mortality of irradiated males from that of non-irradiated males, additional assessments on fertility should be conducted to identify a dose that renders males overwhelmingly sterile, yet competitive.
Simultaneously, it is critical to compare the number of male mosquitoes in the population to that of released irradiated males. This can be accomplished by collecting males from various locations in and around the target release area repeatedly from the same location and before, during, and after initiation of the SIT program. A mark, release, recapture study should be conducted to assess the ratio of wild male mosquitoes to released mosquitoes. A mark, release, recapture study relies on the release of a known number of marked mosquitoes from a specific point and their later recapture at points in close proximity surrounding the initial release point. By comparing the number of recaptured males and wild males at distances from the release point, it is possible to estimate the general wild population of males in the area so that competitive ratios of sterile males can be released23. Maximizing the ratio of sterile:wild males can be achieved by releasing more sterile males and/or by reducing the wild population by classical control means such as source reduction, immature control, or adulticide treatments.
To gauge the effectiveness of sterile male releases, adult collections can be compared chronologically against a non-intervention area. As sterile males are released and the number of collected males and females in an area diminishes in relation to a comparable non-intervention area, then it may be hypothesized that it is due to the released sterile males successfully outcompeting local fertile males. This effect can also be observed in oviposition trap cups deployed in both the intervention and non-intervention sites. Eggs may still be produced in the intervention site, but if fewer hatch than those from the non-intervention site, it may be hypothesized they are not fertilized because of females mating with sterile males. More and more oviposition of unfertilized eggs could eventually lead to reduced oviposition due to non-replacement of females in the intervention site8,24.
Future directions of SIT technology and programs naturally expand into additional medically important mosquito species. For instance, this technology may be readily adapted to control Aedes albopictus, given the very similar bionomics of Aedes aegypti and Aedes albopictus. Other disease vector mosquito species of interest include Culex quinquefasciatus, Culex tarsalis, and&#160;various Anopheles species. Improving the efficacy of this technology depends on increasing the capacity of male pupae produced at a given time, which could be achieved through genetic manipulation or artificial selection, and improving male competitiveness, which could be achieved by increasing virility, fertility, or longevity.
Ultimately, SIT programs are not a silver bullet for controlling mosquitoes. They are instead a tool in a suite of other control techniques, such as IVM programs, that cross-compensate for weaknesses among techniques. For example, whereas chemical control offers rapid and cheap control, it also fosters the development of resistance and non-target mortality; and whereas SIT is species-specific and is not likely to generate resistance, SIT males must be produced and released in perpetuity to control immigrating populations from outside the vector control district.

Transmission of mosquito-borne pathogens to humans causes millions of cases of disease and deaths each year worldwide. In the absence of effective, approved vaccines for mosquito-borne diseases, such as Zika or dengue fever, one of the most effective ways to reduce transmission is to reduce disease-vector mosquito populations. Vexingly, an increasing number of mosquito species, traditionally targeted by pesticides, are displaying increasing levels of pesticide resistance1. Simultaneously, government agencies have aggressively deregistered or banned previously approved pesticides, and few new, effective chemical control measures are being developed2,3. This constellation of obstacles to mosquito control has motivated the exploration of alternative non-chemical techniques to reduce mosquito populations.
Certain mosquito species present challenges to control issues of resistance and pesticide registration. Aedes aegypti (L.) is a prominent disease-vector mosquito that is extremely difficult to control through traditional integrated vector management due to the cryptic peridomestic habitat exploited by this species for immature development and adult resting4,5. Challenges related to the exploitation of the cryptic habitat around residences include the difficulty of reaching these locations with pesticide spray techniques as well as the potential lack of acceptance by the public for repeated access to private property for public health vector control agencies to conduct the intensive surveillance and control activities crucial to effective integrated vector management (IVM) for this species.
Fortunately, SIT, an approach proven successful for enduring control of other highly challenging insect species6, is being applied to the Aedes aegypti problem in a groundbreaking series of experiments and operational trials based in St. Augustine, Florida (KJL, RLA, SCB unpublished data). SIT has been applied to a range of insect species, including mosquitoes, and has been reviewed in depth7,8. SIT leverages the mass release of colony-reared males sterilized, for example, by exposure to ionizing radiation or chemicals to overwhelm the mate choice of natural populations of females. Sterilized males that mate with wild females render the eggs infertile due to damage suffered by male gametes, and if present in sufficient numbers, can theoretically crash the natural Aedes aegypti population.
An SIT program was initiated to attempt to reduce populations of Aedes aegypti in an urban area in Atlantic coastal Florida where this species recently re-colonized and is expanding and presenting a public health risk for transmission of viruses such as Zika, dengue, or chikungunya. To maximize the potential for compatibility with wild females, a new colony was established using wild-caught Aedes aegypti from the target population to produce males for the program9. This was based on the hypothesis that locally derived, colony-reared males would be more likely to be competitive with local wild males for mating with local wild females. For the SIT to be effective, not only do overwhelming numbers of sterile males need to be present in the target area, but they also have to be capable of effectively courting and mating with local wild female mosquitoes.
A series of experiments was conducted to determine the optimal number of sterile males to release (KJL, RLA, SCB unpublished data) as well as optimal doses of radiation that would render the males infertile without interfering with survival, behavior, or acceptance by wild females (KJL, RLA, SCB unpublished data). These data are forthcoming in allied publications from this group, but some of these findings are captured in this protocol as well and could be used as a starting point for new SIT Aedes aegypti control programs elsewhere. This species is constantly expanding its range, and SIT programs show great promise to be cost-effective, long-term solutions to control this population. The objective of this protocol is to produce sterilized, male, colony-reared Aedes aegypti mosquitoes for systematic release into outdoor areas to disrupt the natural reproductive cycles of local Aedes aegypti populations in an operational public health vector control program.
While similar protocols and workflows have been published for the production of transgenic Aedes aegypti males and production workflows for Aedes SIT, or Wolbachia-based incompatibility programs have been published elsewhere, this protocol illustrates how existing protocols have been adapted for Aedes aegypti production, separation and irradiation of male pupae, marking and packaging adult males, and shipment to the release site for this program9,10,11,12,13,14,15,16,17,18. The marking component of this protocol may not be required in a mature operational SIT program; however, it has been included here because it is one way to monitor the efficacy and control the quality of the entire process in the early years of establishing the SIT program. Mosquito control programs are typically run by local authorities, so they can vary widely in many aspects of their organization from size and funding base to tuning control tactics to maximize local success. Thus, the protocol described herein should be evaluated for compatibility with available resources.

<table><tbody><tr><td>1-1/8&quot; wrench (1&quot; (1 inch) = 2.54 cm)</td><td>Craftsman</td><td>CMMT44707</td><td></td></tr><tr><td>1/2 pint cardstock cup (1/2 pint = 236.5 mL)</td><td>Science Supplies WLE corp</td><td>1/2 pint</td><td></td></tr><tr><td>1/4&quot; tubing - tygon</td><td>Hudson Extrusions</td><td>LLDPE1/8 X 1/4 BLK</td><td>to attach to CO2 gas regulator</td></tr><tr><td>1/8&quot; brass barb w/ MIP connection</td><td>B&amp;amp;K</td><td>BHB-85NLB</td><td>to attach to CO2 gas regulator</td></tr><tr><td>1000 mL graduated plastic beakers with handle</td><td>Thermo Scientific</td><td>1223-1000</td><td></td></tr><tr><td>3000 mL graduated plastic beakers with handle</td><td>Thermo Scientific</td><td>1223-3000</td><td></td></tr><tr><td>Adult large cage</td><td>Bioquip</td><td>1450D</td><td></td></tr><tr><td>Aspirator vials</td><td>Bioquip</td><td>2809V</td><td></td></tr><tr><td>Bovine liver powder</td><td>MP Biomedicals</td><td>290039601</td><td></td></tr><tr><td>Brewers yeast</td><td>MP Biomericals</td><td>02903312-CF</td><td></td></tr><tr><td>CO2 regulator</td><td>Randor</td><td>64003038</td><td></td></tr><tr><td>CO2 tank (20# canister)</td><td>Praxair</td><td>CDBEVCARB20</td><td></td></tr><tr><td>Collection basin</td><td>Treasure Gurus</td><td>KI-ENAMELBOWL</td><td>for separator</td></tr><tr><td>Cotton balls - large</td><td>Fisher Scientific</td><td>22-456-883</td><td></td></tr><tr><td>Deli cups w/lids - 470 mL</td><td>Pactiv DELItainer</td><td>PCTYSD2516</td><td></td></tr><tr><td>Deli cups w/lids - 1900 mL</td><td>Berry Global</td><td>T60764</td><td></td></tr><tr><td>DoseReader 4</td><td></td><td></td><td>ND0.5 and ND1.0 QA Filter Set standards</td></tr><tr><td>Dosimetry film</td><td>Far West Technology, Inc.</td><td></td><td></td></tr><tr><td>Filter paper</td><td>Millipore</td><td>AP10045S0</td><td></td></tr><tr><td>Flashlight aspirator</td><td>Bioquip</td><td>2809D</td><td></td></tr><tr><td>Forceps - fine featherweight</td><td>Bioquip</td><td>4748 or 4750</td><td>featherweight</td></tr><tr><td>GAFchromic</td><td></td><td></td><td>radiochromic film</td></tr><tr><td>Gammator M</td><td>Radiation Machinery Corporation, Parsippany, NJ</td><td></td><td>Cesium-137 irradiator</td></tr><tr><td>Hand held mechanical aspirator</td><td>Clarke Mosquito</td><td>13500</td><td></td></tr><tr><td>Lambskin condoms</td><td>Trojan</td><td>Naturalamb</td><td></td></tr><tr><td>Large CO2 chamber</td><td>Sterilite</td><td>Walmart #&amp;nbsp;568789514</td><td></td></tr><tr><td>Larval rearing pans</td><td>Blue Ridge Thermoforming</td><td>&amp;nbsp;01-FG-400-3N-ABS</td><td>Dimensions: 22.375 x 17.5 x 3 (inches)</td></tr><tr><td>Magnets - 20# pull</td><td>Master magnetics</td><td>MHHH20BX</td><td></td></tr><tr><td>Marking dye</td><td>Dayglo</td><td>ECO-11</td><td>Aurora Pink</td></tr><tr><td>Marking dye</td><td>Dayglo</td><td>ECO-17</td><td>Saturn Yellow</td></tr><tr><td>Mesh</td><td>Falk</td><td></td><td>T301</td></tr><tr><td>Pasture pipettes</td><td>Thermo Scientific</td><td>02-708-006</td><td></td></tr><tr><td>Petri dishes - large</td><td>VWR International</td><td>25384-090 CS</td><td></td></tr><tr><td>Petri dishes - small (60 mm x 15 mm)</td><td>Fisher Brand</td><td>FB0875713A</td><td></td></tr><tr><td>Pupa separator</td><td>J.W. Hock</td><td>1512</td><td></td></tr><tr><td>Red rubber hose</td><td>Welch</td><td>331040-5</td><td></td></tr><tr><td>Release containers</td><td>Science Supplies WLE corp</td><td>1 gallon</td><td></td></tr><tr><td>Rubber bands - cross #19</td><td>Alliance</td><td>ALL37196</td><td></td></tr><tr><td>Rubber bands - latitude #64</td><td>Skillcraft</td><td>NSN0589974</td><td></td></tr><tr><td>Scale</td><td>Ohaus</td><td>H-4737</td><td></td></tr><tr><td>Seed germination paper - Heavy stock 76#</td><td>Anchor Paper</td><td>#76</td><td></td></tr><tr><td>Shipping coolers- 16 x 13 x 12.5&quot;</td><td>MrBoxonline.com</td><td>Husky Foam Cooler kit</td><td></td></tr><tr><td>Sieve #20</td><td>Advantech</td><td>20BS8F</td><td></td></tr><tr><td>Sieve #30</td><td>Advantech</td><td>30BS8F</td><td></td></tr><tr><td>Small cage - Bug Dorm</td><td>MegaView</td><td>Bug Dorm-1</td><td></td></tr><tr><td>Small CO2 chamber</td><td>Mainstays</td><td>Walmart #&amp;nbsp;562922221</td><td></td></tr><tr><td>Souffle cup lid</td><td>SOLO</td><td>41165277456</td><td></td></tr><tr><td>Souffle cups - 4 oz (1 oz = 29.6 mL)</td><td>SOLO</td><td>41165024104</td><td></td></tr><tr><td>Sponge</td><td>ocelo</td><td>MMM7274FD</td><td></td></tr><tr><td>Squeeze bottle</td><td>Dynalon</td><td>3UUP6</td><td></td></tr><tr><td>Stereoscope</td><td>Meiji Techno</td><td>EMZ-5</td><td></td></tr><tr><td>Stockinette</td><td>BSN Medical</td><td>30-1006</td><td></td></tr><tr><td>Styrofoam</td><td></td><td></td><td>extruded polystyrene foam</td></tr><tr><td>Tropical fish flake food</td><td>Tetra</td><td>4.52 pound</td><td></td></tr><tr><td>Vaccum chamber - desiccator</td><td>BelArt</td><td>T9FB892757</td><td></td></tr><tr><td>Weigh boats</td><td>Globe Scientific</td><td>3621</td><td></td></tr></tbody></table>

preparing irradiated and marked male aedes aegypti mosquitoes for release in an operational sterile insect technique program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no prevention. Control of mosquito vectors can rely on chemicals applied to the immature and adult stages, which can contribute to the mortality of non-targets and more importantly, lead to insecticide resistance in the vector. The sterile insect technique (SIT) is a method of controlling populations of pests through the release of sterilized adult males that mate with wild females to produce non-viable offspring. This paper describes the process of producing sterile males for use in an operational SIT program for the control of Aedes aegypti mosquitoes. Outlined here are the steps used in the program including rearing and maintaining a colony, separating male and female pupae, irradiating and marking adult males, and shipping Aedes aegypti males to the release site. Also discussed are procedural caveats, program limitations, and future objectives.

Vigilant and adequate mosquito rearing consists of well-balanced availability of males and females in colony cages, maintenance of fresh sucrose solution and honey, and consistent high-quality blood feeding. These conditions will provide for densely packed egg sheets optimal for use in SIT larval rearing pans. Proper storage and usage of dried egg sheets, such as systematic labeling to facilitate use from oldest to newest, will support uniform hatching across all pans. Filling all larval rearing pans with water prior to hatching can diminish the time for which the egg sheets are in hatch containers and promote healthy development. Maintenance of larval pans from hatch to pupation requires careful engagement by colony personnel as some pans may need more or less food or additional water depending on development stages and environmental variables. If there are issues with the stage of development by the scheduled day of pupal sex separation, adjustments should be made earlier in the process such as hatching earlier or later, adjusting food, or changing the incubator temperature.
The rearing process in this protocol does not render all eggs hatched in time to develop into pupae that can be irradiated and used for control purposes. Between 20 and 50% of the colony-reared mosquitoes will still be larvae by the time the pupae need to be separated. However, these larvae are not squandered, but allowed to mature for 24 h to render additional pupae that can be combined with female pupae from the previous day&#39;s separation and recycled back into colony cages. In the colony cages, pupae will be allowed to mature into adults, mate, bloodfeed, and produce eggs that sustain the SIT project.
Separating pupae, pouring pupae into Petri dishes, irradiation, and placement into adult holding cages after irradiation must happen in one day; hence, adequate time should be allotted to process all steps comfortably. The assembly and preparation of release containers should be done prior to the marking process. When shipping boxes are returned from the release site, release containers should be inspected and prepared for their next use. Discarding wet cotton balls, airing out wet release containers, cleaning Petri dishes, replacing mesh, and removing elastic bands from the container, while not in use, will greatly prolong the life of the release containers.
Given the worldwide reality of the COVID-19 viral pandemic, this protocol that is typically a multi-person operation has been modified to be tractable by one person working alone in a lab for each step. The steps in the process that are hindered most by a one-person scenario are the sexing, marking, weighing, and colony-rearing maintenance steps. Separating pupae by sex by one person should be sufficient if there are multiple separators operating simultaneously in different rooms. In a pandemic situation wherein social distancing occurs in the workplace, equipping multiple stations is required to complete steps from sexing to packing. Depending on the speed of the operator, it takes one person ~4 h to sex 15,000 mosquitoes and then another 1-2 h to mark, weigh, and package them. A two-person scenario diminishes the time during which mosquitoes are anesthetized for marking and reduces the overall work time. Yet, even in a two-person scenario, allocating the full 2.0 g of mosquitoes per release cage can be challenging due to limited work time while the mosquitoes are sedated. Although the process of cleaning and preparing larval and adult rearing materials is extremely time-consuming and labor-intensive, it can be partitioned such that individual operators can work independently and safely during a pandemic.
Releasing adult, marked, irradiated Aedes aegypti males is outside the scope of this protocol but is presented here in brief. The process of releasing marked, irradiated, male mosquitoes starts by determining a uniform release distribution of the release containers based on weights (and thus, inferred numbers of sterile males), as reported in Table 3. After shipments are delivered to the vector control district, the boxes are opened, and the release containers evaluated for any issues with mortality or condition of the release containers. Mosquitoes in the release containers are then allowed to acclimate to ambient temperature and humidity for 1-2 h prior to transport to the treatment area. Release sites in the treatment area are identified after intensive surveillance for hot spots of wild populations of Aedes aegypti. The timing, frequency, and density of releases is balanced by the bionomics of the species as well as meteorology, public support, and laboratory-rearing capabilities.
As specific release containers are matched to particular release sites, the label must be cross-checked before the release container is opened by cutting the mesh on the top, allowing the operator to deform the mesh so that a portion of the males may escape. This fractional release method is repeated at each assigned release point for the container until all freely flying males have been released. This process is then repeated for each release container at their respective assigned release location until all containers have been processed. Optionally, after the mosquitoes have been released, any dead or disabled mosquitoes that did not leave freely can be collected into Petri dishes and labeled to be counted by hand or weighed to correct the estimated number released. Ongoing and pervasive surveillance of adult, egg, and immature stages of wild Aedes aegypti in the target area, and possibly in non-intervention control sites, is conducted to assess the efficacy of the SIT operation.

Watch this Scientific Journal Video about Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program at JoVE.com

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The sterile insect technique (SIT) is used to control specific, medically important mosquito populations that may be resistant to chemical controls. Here, we describe a method of mass rearing and preparation of sterile male mosquitoes for release in an operational SIT program targeting the Aedes aegypti mosquito.

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

The control of such human diseases as dengue, Zika, and chikungunya relies on the control of their vector, the Aedes aegypti mosquito, because there is no ...

preparing-irradiated-marked-male-aedes-aegypti-mosquitoes-for-release

Research

JoVE Journal

Biology

3.6K Views.  Agricultural Research Service Center for Medical, Agricultural, & Veterinary Entomology. De unieke waarde van dit protocol is dat twee personeelsleden die direct beschikbare apparatuur in kleine laboratoriumruimtes gebruiken, voldoende steriele malmuggen kunnen produceren om een operationeel SIT-programma van 50 hectare te ondersteunen. De efficiëntie en lage kosten van deze techniek maken het toegankelijk, onhandelbaar voor zelfs kleine muggenbestrijdingsdistricten om hun geïntegreerde vectorcontroleprogramma uit te breiden. De procedure wordt gedemonstreerd door mijzelf en Ba...

Video: Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the development of new and innovative methods of mosquito control based around the Sterile Insect Technique (SIT)1-3. A control method known as RIDL&#160;(Release of Insects carrying a Dominant Lethal)4, is based around SIT, but uses genetic methods to remove the need for radiation-sterilization5-8. A RIDL&#160;strain of Ae. aegypti was successfully tested in the field in Grand Cayman9,10; further field use is planned or in progress in other countries around the world.
Mass rearing of insects has been established in several insect species and to levels of billions a week. However, in mosquitoes, rearing has generally been performed on a much smaller scale, with most large scale rearing being performed in the 1970s and 80s. For a RIDL&#160;program it is desirable to release as few females as possible as they bite and transmit disease. In a mass rearing program&#160;there are several stages to produce the males to be released: egg production, rearing eggs until pupation, and then sorting males from females before release. These males are then used for a RIDL&#160;control program,&#160;released as either pupae or adults11,12.
To suppress a mosquito population using RIDL&#160;a large number of high quality male adults need to be reared13,14. The following describes the methods for the mass rearing of OX513A, a RIDL&#160;strain of Ae. aegypti 8, for release and covers the techniques required for the production of eggs and mass rearing RIDL&#160;males for a control program.

Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil

To achieve population suppression of Aedes aegypti using the RIDL&#174; (Release of Insects carrying a Dominant Lethal) system, large numbers of male mosquitoes need to be released. This requires the use of mass rearing techniques and technology to provide reliable systems to obtain the maximum number of high quality male mosquitoes.

New techniques and methods are being sought to try to win the battle against mosquitoes. Recent advances in molecular techniques have led to the ...

Environment

Microinjection of A. aegypti Embryos to Obtain Transgenic Mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever.  The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, dessicating embryos, and performing microinjection are demonstrated.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue ...

Protocol for Dengue Infections in Mosquitoes (A. aegypti) and Infection Phenotype Determination

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of the virus in the mosquito tissues. This protocol is routinely used for studying mosquito-virus interactions, especially for identification of novel host factors that are able to determine vector competence. The entire experiment must be conducted in a BSL2 laboratory. Similar to Plasmodium falciparum infections, proper attire including gloves and lab coat must be worn at all times. After the experiment, all the materials that came in contact with the virus need to be treated with 75% ethanol and bleached before proceeding with normal washing. All other materials need to be autoclaved before discarding them.

Protocol for Dengue Infections in Mosquitoes A. aegypti and Infection Phenotype Determination

Once a gene is identified as potentially refractory for the dengue virus, it must be evaluated for it's role in preventing viral infections within the mosquito. This protocol illustrates how the extent of dengue infections of mosquitoes can be assayed. The techniques for growing up the virus in culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

The purpose of this procedure is to infect the Aedes mosquito with dengue virus in a laboratory condition and examine the infection level and dynamic of ...

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 ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Immunology and Infection

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

Genetics

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 ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

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 ...

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain&#8217;s fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.&#160;
Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.&#160;

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes

Here, we present a protocol for studying mating competitiveness of male Aedes aegypti using fluorescent dye as a marker. Female mosquitoes are exposed to both marked and unmarked males for copulation. Post mating, their spermathecae are examined under a fluorescence microscope to determine their mating partner.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for ...

Preparing Irradiated and Marked Male Aedes aegypti Mosquitoes for Release in an Operational Sterile Insect Technique Program

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