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><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&amp;nbsp; 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&amp;amp;content-id=amzn1.sym.1ad31f34-ba12-4dca-be4b-f62f7f5bb10d%3Aamzn1.sym.1ad31f34-ba12-4dca-be4b-f62f7f5bb10d&amp;amp;crid=UXMLNC72BL0&lt;br/&gt; M&amp;amp;cv_ct_cx=cutting%2Bboards&amp;amp;keywords=cutting%2Bboards&lt;br/&gt; &amp;amp;pd_rd_i=B091118V8T&amp;amp;pd_rd_r=&lt;br/&gt; 4c48b4ad-4d4d-4b4b-8799-fc7313&lt;br/&gt; 2f8e34&amp;amp;pd_rd_w=li862&amp;amp;pd_rd_wg&lt;br/&gt; =KogbB&amp;amp;pf_rd_p=1ad31f34-ba12-4dca-be4b-f62f7f5bb10d&amp;amp;pf_rd_r=9ATJD6W&lt;br/&gt; QBF9DVRY889MP&amp;amp;qid=1673911&lt;br/&gt; 429&amp;amp;refresh=1&amp;amp;sprefix=cutting%2Bboards%2Caps%2C198&amp;amp;sr=1-2-8b2f235a-dddf-4202-bbb9-592393927392&amp;amp;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&amp;amp;keywords=&lt;br/&gt; white+12+qt+dishpan+sterilite&amp;amp;qid=1673911729&amp;amp;s=home&lt;br/&gt; -garden&amp;amp;sprefix=white+12+qr+dishpan+sterlite%2Cgarden%2C184&amp;amp;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&amp;nbsp;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&quot; Utility Knife, https://www.amazon.com/ZYLISS-Utility-Kitchen-5-5-Inch-Stainless/dp/B00421ATJK/ref=sr_1_7?crid=2U27KE1HTG5N1&amp;amp;keywords=&lt;br/&gt; fruit%2Bcutting%2Bknives&amp;amp;qid=1673911609&amp;amp;s=&lt;br/&gt; home-garden&amp;amp;sprefix=fruit%2Bcutting%2Bknives%2Cgarden%2C145&amp;amp;sr=1-7&amp;amp;th=1</td></tr><tr><td>No. 20 Mesh sieves</td><td>Hogentogler &amp;amp; Co. Inc.</td><td>4221</td><td>U.S. Standard&amp;nbsp;Testing Sieves, https://www.hogentogler.com/sieves/18-inch-sieves.asp</td></tr><tr><td>No. 45 Mesh sieves</td><td>Hogentogler &amp;amp; Co. Inc.</td><td>4226</td><td>U.S. Standard&amp;nbsp;Testing Sieves, https://www.hogentogler.com/sieves/18-inch-sieves.asp</td></tr><tr><td>No. 8 Mesh sieves</td><td>Hogentogler &amp;amp; Co. Inc.</td><td>4215</td><td>U.S. Standard&amp;nbsp;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&amp;amp;keywords=&lt;br/&gt; ziploc+freezer+bags+2+gallon&amp;amp;qid=1674504602&amp;amp;</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.

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

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

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.

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 ...

sieving-fruit-pulp-to-detect-immature-tephritid-fruit-flies

Nanyang Technological University

Research

JoVE Journal

Biology

1.6K Views.  United States Department of Agriculture (USDA). We ontwikkelen een methode om tephritid fruitvlieglarven te vinden die sneller en nauwkeuriger is dan de huidige methode om elke vrucht te snijden en visueel te inspecteren. We willen een eenvoudige techniek ontwikkelen die in het veld kan worden gebruikt om grote hoeveelheden fruit te verwerken. Een uitdaging van deze studie was het vinden van fruit met een lage besmetting, wat waarschijnlijk zou vertegenwoordigen wat we zien tijdens een nood- of uitroeiingsprogramma.Omdat we niet in ...

Video: Auteur in de schijnwerpers: fruitpulp zeven om onrijpe tefritide fruitvliegjes in het veld te detecteren  

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Environment

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Developmental Biology

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides in attempts to control its populations eliminates natural enemies and competitor flower thrips species, thereby increasing its populations. An unsustainable situation develops with concomitant resistant pest populations, secondary pest outbreaks, and environmental degradation. Integrated pest management utilizes knowledge of pest and natural enemy relationships to implement tactics that are environmentally friendly and sustainable. Minute pirate bugs are the most important worldwide predators of thrips. They can suppress and ultimately control Frankliniella species flower thrips. Flower samples taken at least weekly are needed to understand predator-prey dynamics. Shown here is the sampling of the flowers of fruiting vegetables and companion plants to estimate the densities of individual thrips and minute pirate bug species. Representative data illustrates how the protocol is used to determine the efficacy of management tactics over time and how to evaluate the benefits of predation by minute pirate bugs. The sampling protocol is similarly adaptable to sampling thrips and minute pirate bugs in other plant species hosts.

Sampling for Estimating Frankliniella Species Flower Thrips and Orius Species Predators in Field Experiments

Presented here is a protocol to determine the number of thrips and minute pirate bug predators in crops over multiple dates in field experiments. Also illustrated is how to determine the efficacy of management tactics against thrips and evaluate the benefits of predation by minute pirate bugs.

The western flower thrips, Frankliniella occidentalis (Pergande), is a polyphagous pest that has been spread worldwide. The extensive use of insecticides ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Bioengineering

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

Neuroscience

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Fruit Volatile Analysis Using an Electronic Nose

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit aroma. Maturity at harvest is one of the key factors influencing the flavor quality of fruits and vegetables1. The validation of robust methods that rapidly assess fruit maturity and aroma quality would allow improved management of advanced breeding programs, production practices and postharvest handling. 
Over the last three decades, much research has been conducted to develop so-called electronic noses, which are devices able to rapidly detect odors and flavors2-4. Currently there are several commercially available electronic noses able to perform volatile analysis, based on different technologies. The electronic nose used in our work (zNose, EST, Newbury Park, CA, USA), consists of ultra-fast gas chromatography coupled with a surface acoustic wave sensor (UFGC-SAW). This technology has already been tested for its ability to monitor quality of various commodities, including detection of deterioration in apple5; ripeness and rot evaluation in mango6; aroma profiling of thymus species7; C6 volatile compounds in grape berries8; characterization of vegetable oil9 and detection of adulterants in virgin coconut oil10. 
This system can perform the three major steps of aroma analysis: headspace sampling, separation of volatile compounds, and detection. In about one minute, the output, a chromatogram, is produced and, after a purging cycle, the instrument is ready for further analysis. The results obtained with the zNose can be compared to those of other gas-chromatographic systems by calculation of Kovats Indices (KI). Once the instrument has been tuned with an alkane standard solution, the retention times are automatically converted into KIs. However, slight changes in temperature and flow rate are expected to occur over time, causing retention times to drift. Also, depending on the polarity of the column stationary phase, the reproducibility of KI calculations can vary by several index units11. A series of programs and graphical interfaces were therefore developed to compare calculated KIs among samples in a semi-automated fashion. These programs reduce the time required for chromatogram analysis of large data sets and minimize the potential for misinterpretation of the data when chromatograms are not perfectly aligned.
We present a method for rapid volatile compound analysis in fruit. Sample preparation, data acquisition and handling procedures are also discussed.

A rapid method for volatile compound analysis in fruit is described. The volatile compounds present in the headspace of a homogenate of the sample are rapidly separated and detected with ultra-fast gas chromatography (GC) coupled with a surface acoustic wave (SAW) sensor. A procedure for data handling and analysis is also discussed.

Numerous and diverse physiological changes occur during fruit ripening, including the development of a specific volatile blend that characterizes fruit ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Immunology and Infection

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

Simple Homemade Tools to Handle Fruit Flies&#8212;Drosophila melanogaster

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in practice, as adult flies fly. Demonstrated here is how to make some simple and cost-effective tools to address difficult issues in the handling of Drosophila. Holes in foam stoppers are made and pipette tips or funnels are inserted into the holes. Flies then move only in one direction into the pipette tip/funnel assemblage, allowing efficient control of the transfer of adult Drosophila into or out of a vial. Existing protocols have been modified for cool-anesthetizing flies by chilling in crushed ice and transferring them onto a cold, hard icepack surface. The icepack is covered with a piece of medical gauze that keeps immobilized flies from the condensed water when examined under a stereomicroscope. The flies are finally euthanized for counting and sorting or discarded by microwaving. A bottle-shaped cage has also been developed for collecting eggs, as well as a labor-saving device and accompanying protocol for cleaning glass culture vials.

Described here is the use of several homemade tools to transfer, chill, and kill adult Drosophila, as well as to clean glass culture vials and collect eggs. These tools are easy to make and are rather efficient in handling Drosophila.

The fruit fly, Drosophila melanogaster, is widely used both in biological research and biology education. Handling adult flies is common but difficult in ...

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

Samenvatting

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