We would like to thank Silvia Durand, Teri Allen, Jose Alegria, and Alejandra Canon for assistance in processing the guava at the University of Florida, Rick Kurashima, Jean Auth, and Bruce Inafuku for help in evaluating the artificially infested fruit in Hawaii, and Michael Stulberg for helpful comments on earlier versions of the manuscript. This project was funded in part by USDA APHIS and University of Florida Cooperative Agreement and supported in part by USDA-ARS (project 2040-22430-027-00D). The findings and conclusions in this preliminary publication have not been formally disseminated by USDA and should not be construed to represent any agency determination or policy. The 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 the USDA. The USDA is an equal opportunity provider and employer.

1. Fruit Selection
<ol>
	<li>Determine what fruit is available in the area to be surveyed.</li>
	<li>Select host fruit based on the list of known hosts for the target tephritid species.</li>
	<li>Choose soft fleshed, ripe fruit, such as mangos, papaya, and guava. Unripe or hard fleshed fruit, such as tropical almonds, should be inspected with a different method, such as fruit cutting.</li>
	<li>Select fallen, overripe fruit, or ripe fruit on trees that have signs of damage, oviposition scars, and soft spots.</li>
	<li>Process approximately 2 L of fruits at once (e.g., 5 guavas or 5 medium-sized mangos constitute adequate samples for this method). The number of fruits that can be processed at once depends on the size of the fruits (Figure 1A).</li>
</ol>
2. Mushing
<ol>
	<li>Cut the fruit into large pieces and place it into a 4 L zip lock storage bag (Figure 1B).</li>
	<li>Add water to the bag until the water covers the chopped fruit by 25-50 mm (Figure 1C).</li>
	<li>Squeeze the fruit gently by hand until all the pulp has dislodged from the peel and has a smooth consistency (i.e., no large chunks) (Figure 1D).</li>
</ol>
3. Sieving for late instar collection
<ol>
	<li>Stack the sieves. Use large sieves (457 mm diameter) for processing large amounts of fruits (~ 5 fruits at once) and smaller sieves (305 mm diameter) for individual fruit or smaller samples (&#60; 5 fruit).</li>
	<li>Stack the sieve with a large mesh (No. 8; 2.36 mm) sieve atop a small mesh (No. 20; 0.85 mm) sieve. For the detection of early instars, place a third sieve (No. 45; 0.35 mm) on the bottom of the stack (Figure 1E).</li>
	<li>Pour the pulp into the top sieve (Figure 1F).</li>
	<li>Thoroughly wash the pulp through the stack of sieves using water from a faucet, hose, or a bottle until the fine pulp has passed through the sieves (Figure 1G).</li>
	<li>Visually scan the top sieves for late instar larvae that might have been retained with the peel or any large pieces of fruit (Figure 1H).</li>
	<li>Carefully inspect the second sieve for late instar larvae. With large amounts of fine pulp, additional rinsing may be necessary.</li>
	<li>Collect larvae from the sieves with larval forceps and place them into vials with 70% EtOH.</li>
</ol>
4. Sugar floatation for early instar collection
<ol>
	<li>Premix the sugar solution by dissolving 453 g (1 box) of dark brown sugar in 2 L of tap water, which yields a Brix reading of 19&#176;10.</li>
	<li>Wash the pulp from the finer mesh sieves (e.g., No. 20 and No. 45) to the edge of the sieve with tap water, then move the material to a plastic dishpan (11 L).</li>
	<li>Add the brown sugar solution until it covers the pulp by 25-50 mm and add 2 drops of anti-foamer. Let the pulp sit in the brown sugar solution for about 5 min.</li>
	<li>Collect larvae that float to the surface of the solution with larval forceps into vials with 70% EtOH.</li>
</ol>
5. Larval curation
<ol>
	<li>Label a vial with the collection location, date, type of fruit, and collector for later examination and identification.</li>
</ol>

The Mushing, Sieving, and Floating Method for Detecting Fruit Fly Larvae

<ol>
	<li>White, I. M., Elson-Harris, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fruit Files of Economic Significance: Their Identification and Bionomics. , (1992).</li><li>Papadopoulos, N. T., Plant, R. E., Carey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+trickle+to+flood:+the+large-scale,+cryptic+invasion+of+California+by+tropical+fruit+flies.">From trickle to flood: the large-scale, cryptic invasion of California by tropical fruit flies.</a> Proceedings of the Royal Society B: Biological Sciences. 280 (1768), 20131466 (2013).</li><li>Suckling, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+tephritid+fruit+fly+pest+populations:+outcomes+and+prospects.">Eradication of tephritid fruit fly pest populations: outcomes and prospects.</a> Pest Management Science. 72 (3), 456-465 (2016).</li><li>Mcinnis, D. O., 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=Can+polyphagous+invasive+tephritid+pest+populations+escape+detection+for+years+under+favorable+climatic+and+host+conditions.">Can polyphagous invasive tephritid pest populations escape detection for years under favorable climatic and host conditions.</a> American Entomologist. 63 (2), 89-99 (2017).</li><li>Mutamiswa, R., Nyamukondiwa, C., Chikowore, G., Chidawanyika, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+oriental+fruit+fly,+Bactrocera+dorsalis+(Hendel)+(Diptera:+Tephritidae)+in+Africa:+From+invasion,+bio-ecology+to+sustainable+management.">Overview of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) in Africa: From invasion, bio-ecology to sustainable management.</a> Crop Protection. 141, 105492 (2021).</li><li>Steck, 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=Oriental+fruit+fly+eradication+in+Florida+2015-2016:+program+implementation,+unique+aspects,+and+lessons+learned.">Oriental fruit fly eradication in Florida 2015-2016: program implementation, unique aspects, and lessons learned.</a> American Entomologist. 65 (2), 108-121 (2019).</li><li>Alvarez, S., Evans, E., Hodges, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+costs+and+regional+economic+impacts+of+the+oriental+fruit+fly+(Bactrocera+dorsalis)+outbreak+in+Miami-Dade+County,+Florida.">Estimated costs and regional economic impacts of the oriental fruit fly (Bactrocera dorsalis) outbreak in Miami-Dade County, Florida.</a> University of Florida Institute of Food and Agricultural Sciences Extension. , (2016).</li><li>Gould, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probability+of+detecting+Caribbean+fruit+fly+(Diptera:+Tephritidae)+infestation+by+fruit+dissection.">Probability of detecting Caribbean fruit fly (Diptera: Tephritidae) infestation by fruit dissection.</a> Florida Entomologist. 73 (3), 502-507 (1995).</li><li>Yee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Rhagoletis+indifferens+(Diptera:+Tephritidae)+larvae+using+brown+sugar+flotation+and+hot+water+methods.">Detection of Rhagoletis indifferens (Diptera: Tephritidae) larvae using brown sugar flotation and hot water methods.</a> Journal of Applied Entomology. 136 (7), 549-560 (2012).</li><li>Yee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+brown+sugar,+hot+water,+and+salt+methods+for+detecting+western+cherry+fruit+fly+(Diptera:+Tephritidae)+larvae+in+sweet+cherry.">Comparison of the brown sugar, hot water, and salt methods for detecting western cherry fruit fly (Diptera: Tephritidae) larvae in sweet cherry.</a> Florida Entomologist. 97 (2), 422-430 (2014).</li><li>Van Timmeren, S., Diepenbrock, L. M., Bertone, M. A., Burrack, H. J., Isaacs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+filter+method+for+improved+monitoring+of+Drosophila+suzukii+(Diptera:+Drosophilidae)+larvae+in+fruit.">A filter method for improved monitoring of Drosophila suzukii (Diptera: Drosophilidae) larvae in fruit.</a> Journal of Integrated Pest Management. 8 (1), 23 (2017).</li><li>Van Timmeren, S., Davis, A. R., Isaacs, R. <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+a+larval+sampling+method+for+monitoring+Drosophila+suzukii+(Diptera:+Drosophilidae)+in+blueberries.">Optimization of a larval sampling method for monitoring Drosophila suzukii (Diptera: Drosophilidae) in blueberries.</a> Journal of Economic Entomology. 114 (4), 1690-1700 (2021).</li><li>Balagawi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+brown+sugar+flotation+for+detecting+Queensland+and+Mediterranean+fruit+fly+(Diptera:+Tephritidae)+infestation+in+Australian+cherries.">Evaluation of brown sugar flotation for detecting Queensland and Mediterranean fruit fly (Diptera: Tephritidae) infestation in Australian cherries.</a> Crop Protection. 151, 105823 (2022).</li><li>CFIA (Canadian Food Inspection Agency). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directive+D-02-04:+The+Blueberry+Certification+Program+and+domestic+phytosanitary+requirements+to+prevent+the+spread+of+blueberry+maggot+(Rhagoletis+mendax)+within+Canada.+2+Revision+10.">Directive D-02-04: The Blueberry Certification Program and domestic phytosanitary requirements to prevent the spread of blueberry maggot (Rhagoletis mendax) within Canada. 2 Revision 10.</a> CFIA (Canadian Food Inspection Agency). , (2020).</li><li>Kendra, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+chromatography+for+detection+of+citrus+infestation+by+fruit+fly+larvae+(Diptera:+Tephritidae).">Gas chromatography for detection of citrus infestation by fruit fly larvae (Diptera: Tephritidae).</a> Postharvest Biology and Technology. 59 (2), 143-149 (2011).</li><li>SAS Institute Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAS+9.4+Guide+to+Software+Updates+and+Product+Changes.">SAS 9.4 Guide to Software Updates and Product Changes.</a> SAS Institute Inc. , (2013).</li></ol>

The authors declare that they have no conflicts of interests.

Our goal was to develop an efficient and effective way to find tephritid larvae in the field. The motivation of launching an eradication program or establishing a quarantine area is the detection of mated female(s) or larvae6, which indicates a breeding population. The current method of cutting and visually searching fruit is inefficient in finding larvae as there are usually many more host fruits present than can be individually inspected. In addition, the populations of the tephritids are likely low in an area of new invasion, making the chances of finding larvae in a large amount of fruit incredibly difficult. For example, in the 2015 Bactrocera dorsalis eradication program in Florida, 54 different host species were identified, and more than 4,000 fruits were cut. In this eradication program, only a few larvae were found in mango, and no other hosts were found to be infested6. We found that the MSF/MS method was both more sensitive and faster in detecting A. suspensa and B. dorsalis larvae when processing fruits that had a large amount of pulp (mangos, guava, and papaya) in bulk compared to fruit cutting. The larger amount of host fruits that it is possible to inspect using the mushing and sieving method, combined with the increase of detection of a rare larva, could increase the probability that an infestation would be found early. The early detection of a breeding population could increase the likelihood of eradication and reduce the costs of the program.
Our experiments showed that the number of larvae detected by workers cutting and visually inspecting fruits varied considerably. Workers cutting fruit missed 50% and 75% of the B. dorsalis larvae placed in mangos and papaya, respectively. In contrast, only 5% and 15% of the larvae were missed using the MS method for processing mango and papaya fruit, respectively. Similarly, a study evaluating fruit cutting at ports of entry showed there was considerable variation in the number of infested fruits and larvae found by the inspectors8. The study showed that experienced port inspectors missed 64%-99% of A. suspensa larvae and 16%-82% of the infested fruit when fruit was cut and visually inspected8. Our results suggest that the mushing and sieving method could decrease the likelihood that a worker would miss detecting an infested fruit.
Sugar and hot water floatation are accepted protocols in a systems approach method for ensuring cherries and blueberries are free of fruit flies14. A subset of a shipment is crushed into the solution, whereupon an inspector visually screens the surface of the sugar solution for the presence of eggs and larvae. Although a larger number of fruits can be processed compared to cutting individual fruit, the probability of finding larvae using these techniques is still affected by the ability of the inspector, the stage and number of larvae present, and the type of fruit8. We found that, like other tephritids, B. dorsalis and A. suspensa become dislodged from the fruit pulp and float to the surface. Interestingly, we found that with larger late instar larvae, which are the target in emergency and eradication programs as they can be identified morphologically, including sugar floatation did not increase the accuracy of the method. In fact, adding the floatation method increased the processing time by 90% for papaya and by 48% for mango. Increased processing time plus the additional materials (i.e., water, bins, sugar, etc.) do not operationally support adding this step when searching for large instars in the field. The sugar floatation method may be appropriate when the goal is to detect all stages including early instars, such as at ports of entry and packing houses. Filtering the sugar solution with a fine mesh sieve would most likely provide the most accurate detection of eggs and early larval instars11,12.
The MS and MSF techniques work well with fruit that can easily be mushed and have a large volume of pulp. Tephritid larvae tend to burrow into fruit pulp, which makes visual detection difficult. A critical aspect of the MS and MSF methods is separating the larvae from the pulp. The sieving process removes the pulp, thus exposing the larvae on sieve screens. Similarly, the sugar water method separates the larvae from the pulp by making the larvae float, while the pulp sinks to the bottom of the pan. Larvae separated from the pulp by the MS or MSF methods are readily observed moving on the sieve screen or water surface. Although the mushing, sieving, and optionally floating method greatly improved the speed and accuracy of detecting tephritid larvae in important host fruit, the process may not be appropriate for all fruits. For example, host fruit with hard pulp, such as green avocados or fruit with a large seed/pit and relatively small amount of pulp, such as tropical almonds, may be easier to process by hand cutting and visual inspection.
We found that the MS and MSF methods were faster when a relatively small number of fruit (5-10) were processed. The difference would likely be greater if larger amounts of fruits were processed, which might be necessary and typical of emergency fruit fly programs. Removing the floatation step further increased the detection speed without compromising the accuracy of finding large tephritid larvae (&#62;3 mm). We showed that these techniques could be taken to the field, which simulated the conditions experienced by workers during an emergency fruit fly program. Our studies indicate that the MS methods may allow for a timelier detection of late instar larvae and subsequent eradication of tephritid breeding populations. MSF could be used to detect eggs and early instars currently not targeted by eradication programs.

Tephritid fruit flies are among the most destructive agricultural pests, with the genera Anastrepha, Bactrocera, and Ceratitis posing the greatest risk1. Many areas are at high risk for exotic fruit fly establishment, based on 1) historical incursions and associated delimitation and eradication programs, 2) the high arrival rate of fruit fly host material at ports of entry, and 3) climatic conditions favorable for the establishment of reproducing populations. The state of California experiences multiple incursions and detections of tephritids annually2. There have been more than 200 incursions and eradication programs against tephritids globally over the last century, and this has accelerated significantly in recent decades3. Though the vast majority of these programs are successful in eradicating the invading fruit fly3,4, the economic and environmental burden of these invasions remains still high, and the possibility of establishment is always present; a recent catastrophic example is the infection of Bactrocera dorsalis in the African continent5.
During emergency fruit fly programs, a concerted effort is made to detect and control breeding populations of the invading species. For example, the state of Florida responds to tephritid incursions by applying soil drenches (under the dripline of fruit-bearing host plants) and removing host fruit in a 200 m radius around sites where mated females and/or larvae are found6. These actions and tactics serve to kill larvae and pupae in the soil and remove any eggs and larvae from fruit within the area. In some eradication programs, a significant amount of host fruit is removed. In 2015, over 100,000 kg of fruit was destroyed during the B. dorsalis eradication program in Florida6. The economic losses to growers and associated industries in the quarantined area alone were estimated to be over $10.7 million7.
To find tephritid larvae in the quarantine areas, a small team of entomologists collect host fruits in a 200 m radius around a female fly detection area and cut and visually inspect each fruit for larvae6. With limited staff resources and hundreds of possible hosts, the task becomes difficult, particularly in the areas where plant diversity in both commercial production areas and residential yards is high. In addition, larvae may be missed when cutting host fruits. In a study evaluating fruit cutting at the ports of entry, cutting fruit was found not to be as effective in detecting A. suspensa when compared to holding the infested fruits for several weeks and counting the larvae and pupae found in the pupation substrate8.
There are&#160;alternatives to&#160;fruit cutting for detecting an infestation9,10,11,12,13. For example, a brown sugar floatation and a hot water method are both accepted procedures used to detect western cherry fruit flies in harvested cherries9,10. The brown sugar method involves placing crushed fruit in sugar water solution and collecting larvae that float to the top. The brown sugar floatation method was developed specifically to meet regulatory rules for exported cherries, which require packing houses to monitor for quarantine fruit fly pests. There is also an approved US-Canada blueberry certification program that includes brown sugar water floatation, salt water floatation, or boiling to support phytosanitation14. When testing the accuracy of sugar and hot water floatation, researchers used the sieving method to determine how many larvae are missed9,10,11,12,13. A study showed that mixing crushed blueberries in a salt solution and filtering the solution through a reusable coffee filter was four times better in detecting Drosophila suzukii larvae than visually inspecting the surface of salt and sugar solutions14. In addition, gas chromatography was used for the detection of A. suspensa larvae in citrus15. These approaches have not been tested for applicability in field surveys.
Our goal was to develop and test a method to find tephritid larvae in the field using sieving and sugar water floatation. This method allows the more efficient detection of immature fruit flies than the traditional fruit cutting method, supporting the timely control of breeding populations during fruit fly eradication programs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti foamer</td>
			<td>MicroLubrol</td>
			<td>ML200-50-4</td>
			<td>MicroLubrol 2000 Fluid Pure Silicone Oil, https://www.microlubrol.com</td>
		</tr>
		<tr>
			<td>Brown Sugar</td>
			<td>Dominos</td>
			<td></td>
			<td>1 lb Box&#160; Dark Brown Sugar Crystals, https://www.dominosugar.com/products/dark-brown-sugar</td>
		</tr>
		<tr>
			<td>Cutting Boards</td>
			<td>KitchenAid</td>
			<td>KE703NOSMGA</td>
			<td>KitchenAid Classic Nonslip Plastic Cutting Board, 12x18-Inch, https://www.amazon.com/KitchenAid-Classic-Nonslip-Plastic-11x14-Inch/dp/B09117L774/ref=sxin_24_ac_d_mf_brs?ac_md=2-1-S2l0Y2hlbkFpZA%3D%3D-ac_d_mf_brs_brs&#38;content-id=amzn1.sym.1ad31f34-ba12-4dca-be4b-f62f7f5bb10d%3Aamzn1.sym.1ad31f34-ba12-4dca-be4b-f62f7f5bb10d&#38;crid=UXMLNC72BL0 
			M&#38;cv_ct_cx=cutting%2Bboards&#38;keywords=cutting%2Bboards 
			&#38;pd_rd_i=B091118V8T&#38;pd_rd_r= 
			4c48b4ad-4d4d-4b4b-8799-fc7313 
			2f8e34&#38;pd_rd_w=li862&#38;pd_rd_wg 
			=KogbB&#38;pf_rd_p=1ad31f34-ba12-4dca-be4b-f62f7f5bb10d&#38;pf_rd_r=9ATJD6W 
			QBF9DVRY889MP&#38;qid=1673911 
			429&#38;refresh=1&#38;sprefix=cutting%2Bboards%2Caps%2C198&#38;sr=1-2-8b2f235a-dddf-4202-bbb9-592393927392&#38;th=1</td>
		</tr>
		<tr>
			<td>Dish Pans</td>
			<td>Sterilite</td>
			<td>06578012</td>
			<td>White 12 qrt Dishpan, https://www.amazon.com/STERILITE-06578012-Sterilite-White-Dishpan/dp/B0039V2G5E/ref=sr_1_1?crid=2SMBMLFJF18U&#38;keywords= 
			white+12+qt+dishpan+sterilite&#38;qid=1673911729&#38;s=home 
			-garden&#38;sprefix=white+12+qr+dishpan+sterlite%2Cgarden%2C184&#38;sr=1-1</td>
		</tr>
		<tr>
			<td>EthOH</td>
			<td>Fisher Scientific</td>
			<td>BP8202500</td>
			<td>Ethanol Solution 96%, Molecular Biology Grade, https://www.fishersci.com/shop/products/ethanol-solution-96-molecular-biology-grade-fisher-bioreagents/BP8202500</td>
		</tr>
		<tr>
			<td>Glass Vials</td>
			<td>Fisher Scientific</td>
			<td>0333921H</td>
			<td>Fisherbrand&#160;Class B Clear Glass Threaded Vials With Closures, https://www.fishersci.com/shop/products/class-b-clear-glass-threaded-vials-with-closures-packaged-separately/0333921H</td>
		</tr>
		<tr>
			<td>Knives</td>
			<td>Zyliss</td>
			<td>31380</td>
			<td>5.25&#34; Utility Knife, https://www.amazon.com/ZYLISS-Utility-Kitchen-5-5-Inch-Stainless/dp/B00421ATJK/ref=sr_1_7?crid=2U27KE1HTG5N1&#38;keywords= 
			fruit%2Bcutting%2Bknives&#38;qid=1673911609&#38;s= 
			home-garden&#38;sprefix=fruit%2Bcutting%2Bknives%2Cgarden%2C145&#38;sr=1-7&#38;th=1</td>
		</tr>
		<tr>
			<td>No. 20 Mesh sieves</td>
			<td>Hogentogler &#38; Co. Inc.</td>
			<td>4221</td>
			<td>U.S. Standard&#160;Testing Sieves, https://www.hogentogler.com/sieves/18-inch-sieves.asp</td>
		</tr>
		<tr>
			<td>No. 45 Mesh sieves</td>
			<td>Hogentogler &#38; Co. Inc.</td>
			<td>4226</td>
			<td>U.S. Standard&#160;Testing Sieves, https://www.hogentogler.com/sieves/18-inch-sieves.asp</td>
		</tr>
		<tr>
			<td>No. 8 Mesh sieves</td>
			<td>Hogentogler &#38; Co. Inc.</td>
			<td>4215</td>
			<td>U.S. Standard&#160;Testing Sieves, https://www.hogentogler.com/sieves/18-inch-sieves.asp</td>
		</tr>
		<tr>
			<td>Soft Forceps</td>
			<td>DR Instruments</td>
			<td>DRENTF01</td>
			<td>DR Instruments Featherweight Entomology Forceps, https://www.amazon.com/DR-Instruments-DRENTF01-Featherweight-Entomology/dp/B008RBLO8Q</td>
		</tr>
		<tr>
			<td>Zipper Lock Storage Bags</td>
			<td>Ziploc</td>
			<td>682254</td>
			<td>Ziploc brand 2 gal Clear Freezer Bags, https://www.amazon.com/Ziploc-Freezer-Bag-Gallon-100/dp/B01NCDWR8A/ref=sr_1_1_sspa?crid=3SQFBT64Z76ES&#38;keywords= 
			ziploc+freezer+bags+2+gallon&#38;qid=1674504602&#38;</td>
		</tr>
	</tbody>
</table>

sieving fruit pulp to detect immature tephritid fruit flies in the field

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive eradication programs to eliminate incipient populations. During eradication programs, a concerted effort is made to detect larvae, as this strongly indicates a breeding population and helps establish the spatial extent of the infestation. The detection of immature life stages triggers additional control and regulatory actions to contain and prevent any further spread of the pest. Traditionally, larval detection is accomplished by cutting individual host fruits and examining them visually. This method is labor intensive, as only a limited number of fruit can be processed, and the probability of missing a larva is high. An extraction technique that combines i) mushing host fruit in a plastic bag, ii) straining pulp through a series of sieves, iii) placing retained pulp in a brown sugar water solution, and iv) collecting larvae that float to the surface was tested. The method was evaluated in Florida with field-collected guava naturally infested by Anastrepha suspensa. To mimic low populations more representative of a fruit fly eradication program, mangos and papaya in Hawaii were infested with a known, low number of Bactrocera dorsalis larvae. The applicability of the method was tested in the field on guava naturally infested by B. dorsalis to evaluate the method under conditions experienced by workers during an emergency fruit fly program. In both field and laboratory trials, mushing and sieving the pulp was more efficient (required less time) and more sensitive (more larvae found) than cutting fruit. Floating the pulp in brown sugar water solution helped detect earlier instar larvae. Mushing and sieving fruit pulp of important tephritid hosts may increase the probability of detecting larvae during emergency programs.

Author Spotlight: Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field  

Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field

We are developing a method to find tephritid fruit fly larvae that is faster and more accurate than the current method of cutting and visually inspecting each fruit. We want to develop a simple technique that can be used in the field to process large amounts of fruit. A challenge of this study was finding fruit with a low infestation, which would likely represent what we see during an emergency or eradication program.As we cannot see into the fruit, we use methods to artificially infest key hosts with a low number of larvae. In both field and laboratory trials, mushing and sieving the pulp require less time, and more larvae were found than cutting fruit. Floating the pulp in a brown sugar water solution helped detect earlier instar larvae.Mushing and sieving fruit pulp of important tephritid hosts may increase the probability of detecting larvae during emergency programs. We'll continue to optimize the protocol to determine the optimal sieve size for different species of regulated fruit flies and establish criteria such as the type of fruit, the quantity of fruit, et cetera, that will help decide whether the use of the larval destruction method or standard fruit cutting is warranted.

Watch this Scientific Journal Video about Sieving Fruit Pulp to Detect Immature Tephritid Fruit Flies in the Field at JoVE.com

Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection.

Fruit flies of the Tephritidae family are among the most destructive and invasive agricultural pests in the world. Many countries undertake expensive ...

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Research

JoVE Journal

Biology

Mushing, Sieving, and Sugar Floatation for Fruit Fly Larvae Collection

To begin, select fallen, overripe, or ripe fruit with signs of damage, over position scars and soft spots. Cut the fruit into large pieces and place them into a four-liter Ziploc storage bag. Add water to the bag until the water covers the chopped fruit by 25 to 50 millimeters.Squeeze the fruit gently by hand until all the pulp has dislodged from the peel and has a smooth consistency. Use large sieves to process around five fruits at once. Stack the sieve with a large mesh sieve atop a small mesh sieve, and place a third sieve on the bottom of the stack to detect early instars.Pour the pulp into the top sieve, and thoroughly wash the pulp through the stack of sieves using water from a hose, faucet or a bottle until the fine pulp has passed through the sieves. Visually scan the top sieves for late instar larvae that might have been retained with the peel or any large pieces of fruit. And carefully inspect the second sieve for late instar larvae.Collect larvae from the sieves with larval forceps and place them into vials with 70%ethanol. Premix the sugar solution by dissolving 453 grams of dark brown sugar in two liters of tap water. Wash the pulp from the finer mesh sieves to the edge of the sieve with tap water, and then move the material to an 11-liter plastic dish pan.Add the brown sugar solution until it covers the pulp by 25 to 50 millimeters, and add two drops of anti-foamer. After five minutes, collect larvae that float to the surface of the solution with larval forceps into vials with 70%ethanol. Label a vial with the collection location, date, type of fruit, and collector for later examination and identification.The mean number of Anastrepha suspensa larvae and the mean number of larvae collected per minute from five guavas and processed by cutting and by mushing, sieving and floating or MSF method are shown here. The result shows that the MSF method yielded greater numbers of larvae and more larvae per minute than hand cutting. This MSF method was validated using manual infestation of mango and papaya to simulate low Bactrocera dorsalis infestation.More larvae were found using the MSF method for both papaya and mango when compared to the fruit cutting method. For both mangoes and papayas, the full MSF method resulted in higher numbers of larval detections and was faster than fruit cutting.

1.1K Views.  United States Department of Agriculture (USDA).  Increasing the detection of immature tephritid fruit flies in the field can trigger timely efforts to eliminate populations of these destructive pests. Detecting late instar larvae is faster and more accurate when mushing host fruit in a bag and passing the pulp through a series of sieves than hand cutting and visual inspection.

The Mushing, Sieving, and Floating Method for Detecting Fruit Fly Larvae

Summary