Heat Treatments for Killing Apple Maggot Fly (Diptera: Tephritidae) Puparia for Application in Disinfesting Organic Yard Waste
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Journal of Economic Entomology, XX(XX), 2022, 1–8
https://doi.org/10.1093/jee/toab269
Research
Commodity Treatment and Quarantine Entomology
Heat Treatments for Killing Apple Maggot Fly
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(Diptera: Tephritidae) Puparia for Application in
Disinfesting Organic Yard Waste
Wee L. Yee,1,4, Chad E. Kruger,2 and Tim O’Neill3
United States Department of Agriculture, Agricultural Research Service, Temperate Tree Fruit & Vegetable Research Unit, 5230
1
Konnowac Pass Road, Wapato, WA, 98951, USA, 2Washington State University, Tree Fruit Research and Extension Center and
Center for Sustaining Agriculture & Natural Resources, 1100 N Western Avenue, Wenatchee, WA, 98801, USA, 3Engineered
Compost Systems, 4220 24th Avenue W, Seattle, WA, 98199, USA, and 4Corresponding author, e-mail: wee.yee@ars.usda.gov
Subject Editor: Dong H. Cha
Received 5 October 2021; Editorial decision 20 December 2021
Abstract
Organic yard waste from western Washington, U.S. that may contain puparia of apple maggot fly, Rhagoletis
pomonella (Walsh) (Diptera: Tephritidae), had been moved to central Washington for composting, threatening
the $3 billion apple industry concentrated in that region. Heating waste to kill fly puparia before it is transported
could be a solution to this problem. Here, we report results of studies in 2016–2021 that sought to identify a
minimum heat treatment simulating that obtained using a low-pressure steam generator for maximizing kill
of R. pomonella puparia. In two experiments, puparia were exposed to temperatures ramped linearly over 6 h
from 21°C to 47.8, 51.1, 55.0, or 60.0°C in an oven. The 47.8, 51.1, and 55°C treatments did not achieve 100%
mortality, although only one adult fly from 4,000 puparia was found in the 55°C treatment, while no puparia
survived the 60°C treatment. In a third, similar experiment, no puparia out of 2,400 exposed to 55°C survived.
In a fourth and final experiment conducted over 3 years, no puparia out of 61,223 exposed to a 6-h ramp from
21°C to 55°C followed by a 1-h hold time at 55°C produced flies. In addition, all puparia in this treatment died.
Based on 42.3 to 69.8% control survival, 31,217 puparia were killed by this treatment with no survivors, for a
probit 8.7190 level of security. Results suggest that the 55°C and 1-h hold time treatment here is close to the
minimum heat regime needed for disinfesting organic waste of R. pomonella puparia.
Key words: Rhagoletis pomonella, compost, oven heat, quarantine
Organic materials comprising yard trimmings and fruit, food scraps, A problem that arose from movement of organic waste from
wood waste, and paper and paperboard products are the largest com- western to central Washington was that it spread larvae and puparia
ponent of trash in the solid waste stream in the U.S. (Environmental of the apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera:
Protection Agency 2016). Making use of these materials should be Tephritidae), into commercial apple (Malus domestica Borkhausen)-
high priority, as landfilling yard debris and other organic feedstocks growing regions (The Spokesman-Review 2015, Courtney 2016,
is an inefficient use of resources, especially as the human population Klaus 2016, Yakima County 2017, Washington State Department
and associated wastes increase (U.S. Composting Council 2012). of Agriculture 2021a). Apple production is concentrated in central
One solution to this problem is to convert the waste into compost Washington and in 2020 contributed ~$3 billion to Washington’s
for the agricultural sector (CalRecycle 2019). One example of this economy (Joyce 2020). Rhagoletis pomonella is a quarantine pest
was the movement of organic yard waste from western Washington and there is zero tolerance for it in exported apples (Wheat 2012).
State, U.S., for composting in the central portion of the state, which Unlike western Washington (Yee and Goughnour 2008), many cen-
occurred unmitigated until at least 2013 (Associated Press 2013, tral Washington apple-growing areas remain free of the fly. To date,
Clow et al. 2018). no R. pomonella has been detected in commercially packed apples
Published by Oxford University Press on behalf of Entomological Society of America 2022. This 1
work is written by (a) US Government employee(s) and is in the public domain in the US.2 Journal of Economic Entomology, 2022, Vol. XX, No. XX
in central Washington (Washington State Department of Agriculture two or four sites in western Washington from 2016 to 2020. Sites
2021b, 2016a, 2016b). However, a pest risk assessment indicated were in Vancouver (45º37′56″N, 122º37′04″W; 57 m), Woodland
that movement of organic waste is a risk for introducing the fly (45º56′26″N, 122º40′20″W; 51 m), Skamania (45º36′01″N,
into pest-free areas of central Washington (Sansford et al. 2016). 122º06′43″W; 14 m), and Centralia (46º44′38″N, 122º59′18″W;
Furthermore, losses to the Washington apple industry if there are 54 m) (Clark, Cowlitz, Skamania, and Lewis Counties, respectively).
no fly-free areas have been estimated to be $510 to $557 million Each year, 820 to 1,400 kg of apples were picked off the ground
(Galinato et al. 2018). Waste as a source of tephritid flies has pre- beneath trees in August and September. All apples were placed on
viously been studied in Florida, where composting may spread the screens suspended in tubs containing ~15 g of a sand/peat moss mix
Caribbean fruit fly Anastrepha suspensa (Loew) (Kendra et al. 2007). and held outdoors on racks covered with tarps in shade. Larvae and
Knowledge of R. pomonella biology can help identify vulner- puparia in tubs were collected every 1–4 d. Puparia were maintained
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able fly stages to target for control in yard waste. In Washington, in plastic cups with moist soil or sand at 21–23°C and 16:8 L:D
most adult R. pomonella emerge from puparia in the ground be- before being counted for tests. Over the five seasons, approximately
neath apple or hawthorn trees (Crataegus spp.) from July through 230,000 puparia were collected, with ~40% surviving long enough
August. About 2 weeks after emergence, female flies oviposit into to be tested.
fruit, where larvae develop for 2–4 weeks (Dean and Chapman
1973). Afterward, larvae leave fruit fallen on the ground in July to Heat Treatments
November and burrow to pupariate about 5 cm below the soil sur- Heat treatments for Experiments 1–3 consisted of linear temperature
face, where puparia overwinter at low temperatures that break dia- ramps over 6 h from 21°C to 47.8, 51.1, 55.0, or 60.0°C in an oven
pause, allowing adults to emerge the following season to restart the (Fig. 1A). The treatment for Experiment 4 was a 6-h linear ramp
cycle. Because larvae pupariate 2–18 h after leaving apples (Porter from 21°C to 55°C followed by a 1-h hold at 55°C (Fig. 1B). The
1928) and the puparial stage lasts 10–11 months, puparia may be the heat treatments were based on calculations of achievable rates of
major stage potentially moved in yard waste most times of the year. temperature change in moist green waste under Seattle, Washington
Killing R. pomonella puparia in waste before it is transported to ambient winter temperatures using a low-pressure steam generator
central Washington should eliminate waste movement as a threat to (T. O., unpublished). Heat treatments for all experiments were con-
the apple industry. A practical, environmentally safe, pesticide-free ducted inside a drying and heating oven with forced convection
way for doing this is to use heat. Natural biogenic heat could be used (70 cm high and wide x 84 cm long; Model FP 115, Binder GmbH,
to kill puparia in open air waste piles. Another way is to generate Tuttlingen, Germany) with no lighting. Each test day, two or three
heat using steam in a containerized or “within-vessel process” (def-
inition 41 in Chap 3.1, section 1782 in Calrecycle [2016]). This may 70
have advantages over use of natural biogenic heat, including killing A. Experiments 1 and 2 6h
65
pathogens and weed seeds, increasing composting speed, tempera- 0-h hold time
60 Target temperature
ture, and odor control, and using less land (Grüneklee 1998). 55 47.8ºC
Washington State Department of Agriculture issues special permits 51.1ºC
50 55.0ºC
for organic waste transport (WAC 16-470-124) from R. pomonella 45 60.0ºC
fly quarantine to fly-free areas (Washington State Department of 40
Agriculture 2021a). Current regulations are to treat waste at a tem- 35
perature of at least 55°C or 65°C for 2 wks or 1 wk, respectively. In 30
6-h ramp from
Temperature in ºC
enclosed composting facilities, treatments are at least 60°C for 1 wk. 25 21ºC to target
With wet heat, treatments are at least 74°C for 4 h; 80°C for 2 h; or 20 temperature
90°C for 1 h (Washington State Department of Agriculture 2021c). 15
0 60 120 180 240 300 360 420 480 540 600
Despite these regulations, no study has been conducted to determine
70
a minimum heating regime for maximizing kill of R. pomonella pu- B. Experiment 4 6h
65
paria. Protocols and permits for killing microorganisms in compost 60
state that 72 h at 55°C is lethal (Federal Register 1993). This tem- 1-h hold time
55
perature should be included in tests for killing R. pomonella puparia 50
since it is generally accepted for sanitizing compost. 45
Puparia
In this study, the objective was to evaluate heat treatments for 40 transferred
killing puparia of R. pomonella. The major goal was to identify a 35 immediately
to 21–22ºC
treatment requiring the least amount of heat and time for maxi- 30 after 7 h
mizing kill of puparia, by incrementally testing lower to higher tem- 25 6-h ramp from
peratures and then further testing the lowest temperature that may 20 21ºC to 55ºC
achieve the desired effect. The optimal temperature treatment iden- 15
tified could then be used to disinfest organic yard waste that may 0 60 120 180 240 300 360 420 480 540 600
contain live R. pomonella puparia so that the waste can be moved Minutes after placement in oven
for composting.
Fig. 1. Representative curves for temperature treatments used against
Rhagoletis pomonella puparia in a laboratory oven in heat-kill experiments:
Materials and Methods (A) Experiments 1 and 2: 6-h ramps from 21°C to 47.8, 51.1, 55.0, and 60.0°C,
no hold time, followed by gradual temperature decline to 21°C inside test
Sources of Apple Maggot Puparia
deli containers; Experiment 3 (not shown) had same ramp to 55.0°C, but
Four experiments (Experiments 1–4) were conducted using R. puparia removed from deli containers and exposed immediately to 21°C;
pomonella puparia (here, = pupae) obtained as eggs or larvae in (B) Experiment 4: 6-h ramp to 55.0°C, followed by a 1-h hold time at 55.0°C;
apples from backyard or escaped trees. Apples were collected at puparia were then exposed immediately to 21°C.Journal of Economic Entomology, 2022, Vol. XX, No. XX 3
plastic deli containers (each a replicate, described below) with pu- testing. Puparia were held at 21°C for 1 d before treatments. In all
paria were spaced ~10 cm apart in the oven and heated for 6 or 7 h, experiments, at least one control and one treatment replicate were
targeting the final temperatures stated above. tested each day.
After heat treatment and 3–42 d at 21°C (the duration at 21°C
General Experimental Procedures depending on the experiment; see following sections), puparia were
Setups and details of Experiments 1–4 are shown in Table 1, spe- chilled at 3–4°C for 4–6 mos in all experiments. After chilling ended,
cifically, the start to end dates, temperature treatments, numbers of puparia were transferred to 23–25°C and 16:8 h L:D and numbers
replicates, numbers of puparia per replicate, and total puparia tested. of emerged flies (first ones appeared 40 d later) counted every 1–3
Oven temperatures were set slightly above target temperatures be- d over 4 mos. Puparia were dissected to determine survival after
cause it was cooler inside deli containers than outside of them within treatment and before chilling (Experiment 1) and/or after fly emer-
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the oven. The corresponding control(s) for each treatment was held gence had ended (Experiments 1–4; see “Evaluation and Criteria for
simultaneously in a 21°C incubator with no lighting. A Hobo data Puparial Mortality” below). Specifics for each experiment follow
logger (Onset Computer Corp., Bourne, MA) was placed inside each (also see Table 1).
deli container to verify that targeted temperatures and 100% rela-
tive humidity (RH) were reached each test day; the parameters were Experiment 1
always reached except in a few instances, in which case the unaccept- The first experiment narrowed down temperature ranges at which
able replicates were discarded. high puparial mortality occurred, determined the relationship be-
Puparia in all experiments were tested at 14–30 d post tween temperature, days after treatment, and survival, and guided
pupariation. Pupal structures of R. pomonella are fully formed final treatment protocols. For testing, 100 control and treatment pu-
by 8 d postpupariation, in the advanced phanerocephalic condi- paria were placed on the surface of a moist paper towel (Acclaim,
tion, with no further development until after winter (Dean and Georgia-Pacific, Atlanta, GA) in a Petri dish (1.4 cm high x 8.7 cm
Chapman 1973). Thus, all puparia tested here were fully developed diameter). The dish was placed inside a 946 cm3 clear plastic deli
and likely responded similarly to environmental stresses. Only container (6.7 cm high ×16.2 cm wide × 18.4 cm long) (Genpak,
creamy tan and firm puparia that appeared alive were selected for Glen Falls, NY) sealed with a lid. The container had a 3.5 cm layer
Table 1. Setups of Experiments 1–4 testing heat treatment effects in an oven on kill of Rhagoletis pomonella puparia, 2016–2020
Year: treatment No. replicates Puparia/rep Total puparia
Experiment 1:
Ramp to target temperatures, no hold time; 8 August to 14 September 2016
2016: Control 8 100 800
47.8°C 8 100 800
2016: Control 8 100 800
51.1°C 8 100 800
2016: Control 8 100 800
55.0°C 8 100 800
2016: Control 8 100 800
60.0°C 8 100 800
Experiment 2:
Ramp to target temperatures, no hold time; 14 September to 7 October 2016 (47.8, 51.1, 55.0, 60.0°C); 22 September to 13 October 2017 (55.0°C only)
2016: Control 4 100 400
47.8°C 4 100 400
2016: Control 10 100 1,000
51.1°C 10 100 1,000
2017: Control 25 40–100 1,600
55.0°C 40 100 4,000
2016: Control 10 100 1,000
60.0°C 10 100 1,000
Experiment 3:
Ramp to 55.0°C, no hold time; 16 October to 9 November 2017
2017: Control 19 24–40 736
55.0°C 24 100 2,400
Experiment 4:
Ramp to 55.0°C, followed by a 1-h hold time at 55.0°C; 9 September to 9 October 2018; 12 August to 23 October 2019; 27 August to 28 October 2020
2018: Control 23 100 2,300
55.0°C + 1-h hold time 82 100, 49 8,149
2019: Control 17 150 2,550
55.0°C + 1-h hold time 108 150 16,200
2020: Control 38 133–200 7,317
55.0°C + 1-h hold time 192 42–200 36,874
Totals
Control 78 100–200 12,167
55.0°C + 1-h hold time 382 49–200 61,2234 Journal of Economic Entomology, 2022, Vol. XX, No. XX
of wet soil (2:1 peat moss to sand: water) that kept RH at 100% at once. Specifically, 3,000 puparia were usually heated per day (200
and had a 0.5-mm hole in the lid for ventilation. The deli container puparia per five dishes in a deli container x three deli containers).
was then placed in the oven and heated for 6 h (Table 1). Two To reduce time for mold to develop on puparia postheat treatments,
hundred puparia (100 in each of two containers in the oven) were puparia throughout Experiment 4 were held only 3–5 d at 21°C after
heat-treated each day. After target temperatures were reached, deli treatment before chilling.
containers were immediately removed from the oven and held in a
21°C incubator, resulting in gradual temperature declines inside the Evaluation and Criteria for Puparial Mortality
containers over 3 h (Fig. 1A). In Experiment 1, prechill puparial survival was evaluated at 14,
After 3 h at 21°C, the dish with puparia was removed from the 21, 28, and 42 d post-treatment by opening sampled puparia with
deli container and placed in a 473-ml plastic container with ~10 ml jeweler’s forceps, observing the pupae, and/or puncturing them under
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water on the bottom to maintain 100% RH at 21°C. The lid on the a microscope, after which inspected puparia were discarded. We de-
container had two 1-mm holes for ventilation. Prechill puparial sur- fined live pupae as being turgid and very light or shiny creamy yellow
vival and fly emergence were recorded at 14, 21, 28, and 42 d. Forty- that when punctured exuded clear fluid. Dull yellow forms that ap-
two days approximates the time between pupariation and onset of peared alive (but perhaps were dying) were recorded as alive, as were
cold in nature, during which some puparia can develop into adults gray and turgid pharates (formed adults inside puparia). Puparia
without chill (AliNiazee 1988). Prechill survival assessment con- show no obvious movement so movement could not be used as a
sisted of destructive sampling of 20–25 randomly chosen individuals criterion for being alive. Puparia were defined as dead if they were
per replicate at 14, 21, and 28 d and of three to 10 puparia at 42 d, brownish and that when punctured exuded brownish-gray fluid.
depending on the replicate and how many flies had emerged. Fifteen In Experiments 1–4, all puparia were examined for survival
remaining puparia per replicate out of the 100 starting puparia were after chilling and fly emergence had ended because not all R.
then chilled. pomonella develop and emerge as adults after one winter, with
some remaining as puparia until the following seasons (Dean and
Experiment 2 Chapman 1973). These need to be accounted for when assessing
Experiment 2 (Table 1) was designed to validate results of heat treatment efficacy. All whole, creamy tan control and heat-
Experiment 1. It followed similar procedures but differed in repli- treated puparia were opened with forceps. However, many heat-
cate sizes in addition to puparia not being dissected during the 42-d treated puparia and some control puparia were flat (dried), black
prechill period. However, any flies that emerged during the prechill or brown, or molded and rotted and did not require manipulation
period were recorded as in Experiment 1. to determine that they were dead. In Experiment 4, special atten-
tion was paid to detecting any heat-treated puparia with opened
Experiment 3 anterior ends, to ensure that no flies had started to emerge but died
Based on results of Experiments 1 and 2, Experiment 3 focused on (no such puparia were found).
the 55°C treatment (Table 1). However, to eliminate the heat associ-
ated with the gradual decline over 3 h to 21°C in those experiments, Statistics
puparia were immediately returned to 21°C by removing the dish To determine how puparial survival was related to heat treatments
with puparia after heat treatment from the deli container and pla- and days after treatment at prechill in Experiment 1, arcsine trans-
cing it into a 473-ml plastic container at 21°C. Puparia were held formed percentages of puparia alive post-treatment were analyzed
at 21°C for 21 d before chilling instead of the 42 d in Experiments using linear regression with continuous (temperature, four levels) by
1 and 2. continuous (days, four levels) interaction (SAS Institute, Inc. 2016,
IDRE 2021), correcting for control mortality (proportion survival
Experiment 4 of treated/proportion survival of control). Because of zero fly emer-
Based on results of Experiments 2 and 3, Experiment 4 was con- gence in all heat treatments except 47.8°C, regression analysis was
ducted over 3 years from 2018–2020 (Table 1) to test the hypothesis not run for numbers of flies emerged. To directly compare controls
that temperatures ramped linearly over 6 h from 21°C to 55°C fol- versus treatments within each day in Experiment 1, percent puparial
lowed by a 1-h hold time at 55°C can kill 100% of puparia. During survival and fly emergence were analyzed using Wilcoxon two-
this treatment, there was an ~5 min increase to ~55.5°C at the start sample tests (pairing controls and treatments) (Stangroom 2021a).
of the hold time (Fig. 1B) due to operational characteristics of the In Experiments 2–4, percentage fly emergence (pre- and postchill
oven. For testing, puparia were placed on the surface of moist fine combined) was analyzed using paired t-tests when data (arcsine
white aquarium sand (CaribSea Inc., Ft. Pierce, FL) in a Petri dish to transformed) were normal (Experiment 2, 47.8°C). When they were
reduce mold growth seen on paper towels in Experiments 1–3. After not, due to no variance because of zero fly emergence in treatments,
the 1-h hold time, the dish with puparia was removed from the deli the Wilcoxon signed-rank test (when control and treatment repli-
container and immediately placed in a 473-ml plastic container with cates were paired) or Mann-Whitney U test (when control and treat-
water on the bottom at 21–22°C. ment replicates were uneven) was conducted (Stangroom 2021b).
The goal of Experiment 4 was to heat treat at least 30,000 pu- Means ± sem are shown throughout although medians were ana-
paria (rounded from 29,956) (Shortenmeyer et al. 2011) and obtain lyzed when data were not normal.
no survivors to achieve a high level of quarantine security (Couey and In Experiment 4, the true survival proportion (p) of treated pu-
Chew 1986, Follett and Neven 2006) (details in Statistics, Results paria at the 95% confidence level was calculated using the value of
sections). In 2018 and 2019, deli containers holding puparia were m in Table 1 of Couey and Chew (1986) after correcting for con-
the same type as in Experiments 1–3. However, to reach the 30,000 trol mortality (value of m is dependent on the confidence level and
puparia goal, a larger deli container (11.5 cm high × 18.5 cm wide number of treatment survivors). m was then divided by numbers of
× 30 cm long; with wet soil as before to maintain 100% RH) was treatment puparia killed by heat to yield p and to determine the
used in 2020 to increase numbers of puparia that could be treated probit level (Bliss 1934, Shortenmeyer et al. 2011).Journal of Economic Entomology, 2022, Vol. XX, No. XX 5
Results In Experiment 2, flies emerged from control puparia in all treat-
ments and in the 47.8°C treatment (prechill and postchill combined)
Experiments 1 and 2
(Table 3). There was also low fly emergence from one replicate in the
In Experiment 1, survival of R. pomonella puparia decreased with 51.1°C treatment. Unlike in Experiment 1, there was one male fly
increasing temperatures and days post-treatment over the ranges found in the 55°C treatment. However, there was no fly emergence
tested, with the data strongly fitting a linear relationship, yielding from the 60°C treatment. As in Experiment 1, live puparia remained
an R2 of 0.8192 (overall analysis of variance: F = 181.30; df = 3, after the 47.8°C treatment, but not after treatment at the higher tem-
120; P < 0.0001; regression parameter estimates for temperature and peratures (Table 3).
days of –0.3734 and –0.0797, respectively; temperature: F = 489.83;
df = 1, 120; P < 0.0001; days: F = 51.96; df = 1, 120; P < 0.0001).
Experiment 3
Thus, within treatments, survival decreased as days post-treatment
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In Experiment 3 where R. pomonella puparia were exposed to a
increased. There was no significant temperature x day interaction
ramp to 55°C, 55.6 ± 7.1% (399 of 736) of control puparia pro-
(F = 2.11; df = 1, 120; P = 0.1490), as survival declined similarly
duced flies, whereas zero of 2,400 treated puparia did so (z = –5.27;
over time in 51.1, 55, and 60°C treatments, with the relatively low
P < 0.0001). In addition, 3.7% ± 1.3% (27) of control puparia after
P-value apparently caused by no survival decline in the 47.8°C
fly emergence were alive, versus zero in the treatment (z = –.92;
treatment.
P = 0.0035). Based on percent emerged control flies and live pu-
In agreement with the regression analysis, the 47.8°C treatment
paria, the 55°C treatment killed an estimated 1,389 pupae with no
in Experiment 1 had similar numbers of survivors as controls and
survivors.
produced similar numbers of flies as controls at 14–42 d; in addition,
percent fly emergence after chilling did not differ statistically (Table 2).
However, 51.1°C decreased survival at 14–42 d, and no flies emerged Experiment 4
from this temperature treatment after chilling. The 55 and 60°C treat- There was no fly emergence and all treatment puparia examined
ments further decreased survival of puparia at 14–42 d, with survival after control fly emergence had ended were dead in the 55°C and
decreasing over time and with no flies emerging (Table 2). Postfly 1-h hold time treatment (Table 4). Combining results from all
emergence, controls for 47.8, 51.1, 55, and 60°C treatments had 0, 3 years, none of the 61,223 treated puparia survived. Based on 42.3
5.7 ± 3.7, 8.3 ± 8.3, and 21.7 ± 9.5% live puparia versus 4.2 ± 2.8, to 69.8% control survival (emerged flies plus live puparia post fly
0, 0, and 0% live puparia in their respective treatments, although low emergence) (Table 4), 31,217 puparia over the 3 years were killed
sample size (n = 8) and the presence of many zeroes and tied values by the heat treatment with no survivors. At the 95% confidence
in both control and treatments prevented reliable statistical analyses. level, this translates to a true survival proportion or p = 3.00/31,217
Table 2. Experiment 1: mean percent survival of Rhagoletis pomonella puparia and fly emergence ± sem from puparia exposed to temper-
atures in an oven ramped over 6 h from 21°C to 47.8, 51.1, 55.0, or 60.0°C momentarily, no hold time
Percent puparia alive at prechill Percent fly emergencea
Days at 21°C, posttreatment Control Treatment Control Treatment
6-h ramp to 47.8°C, no hold time
14 d 89.0 ± 4.5a 82.7 ± 5.3a 6.0 ± 2.3a 2.4 ± 1.5a
21 d 93.7 ± 1.7a 87.5 ± 3.6a 1.3 ± 0.8a 0.7 ± 0.4a
28 d 94.9 ± 1.3a 91.5 ± 2.6a 1.2 ± 0.5a 0a
42 d 80.4 ± 12.2a 84.8 ± 5.1a 4.8 ± 1.8a 5.0 ± 2.2a
Percent fly emergence, postchillb -------- -------- 59.2 ± 10.5a 26.7 ± 7.8a
6-h ramp to 51.1°C, no hold time
14 d 88.4 ± 4.1a 72.6 ± 7.4b 4.7 ± 1.6a 0b
21 d 89.4 ± 4.3a 66.9 ± 4.0b 3.2 ± 1.2a 0b
28 d 94.1 ± 2.0a 59.4 ± 3.0b 1.7 ± 0.7a 0b
42 d 84.0 ± 6.9a 17.9 ± 8.2b 5.7 ± 1.5a 0b
Percent fly emergence, postchillb -------- -------- 35.2 ± 12.0a 0b
6-h ramp to 55.0°C, no hold time
14 d 93.8 ± 2.7a 51.5 ± 4.0b 4.6 ± 1.6a 0b
21 d 97.7 ± 1.2a 19.5 ± 5.2b 4.2 ± 1.5a 0b
28 d 94.0 ± 2.7a 1.0 ± 0.7b 2.0 ± 1.5a 0b
42 d 92.2 ± 6.2a 1.3 ± 1.3b 5.5 ± 1.1a 0b
Percent fly emergence, postchillb -------- -------- 57.5 ± 7.9a 0b
6-h ramp to 60.0°C, no hold time
14 d 91.4 ± 3.3a 4.0 ± 0.8b 4.5 ± 1.6a 0b
21 d 96.1 ± 2.2a 2.5 ± 1.7b 2.0 ± 0.8a 0b
28 d 99.5 ± 0.5a 0.5 ± 0.5b 4.8 ± 2.4a 0b
42 d 91.3 ± 2.9a 0b 4.5 ± 1.9a 0b
Percent fly emergence, postchillb -------- -------- 39.2 ± 10.6a 0b
See Table 1 for number of replicates and puparia/replicate. See text for criteria for live puparia.
a
Fly emergence for 14, 21, 28, and 42 d postpupariation based on n = 100, 75, 50, and 25 respectively, thus excluding puparia dissected earlier.
b
Percent fly emergence postchill based on n = 15 puparia per replicate, followed for 4 mos postremoval from chilling. Percent puparia alive and percent fly
emergence within rows between control and treatment followed by same letter are not significantly different (Wilcoxon signed-ranks test, P > 0.05).6 Journal of Economic Entomology, 2022, Vol. XX, No. XX
Table 3. Experiment 2: percent Rhagoletis pomonella fly emergence from puparia exposed to temperatures in an oven ramped over 6 h
from 21°C to 47.8, 51.1, 55.0, and 60.0°C momentarily, no hold time, combining emergence before and after 6 mos of chilling
Total puparia Total flies Mean total percent Statistic; Mean percent Statistica,
tested emerged flies emerged ± sem P-value puparia alive P-value
6-h ramp to 47.8°C, no hold time
Control 400 116 29.0 ± 14.6 t = 1.46; 0.2396 7.8 ± 5.0 t = –0.84; 0.4622
Heat treated 400 90 22.5 ± 11.5 9.0 ± 3.7
6-h ramp to 51.1°C, no hold time
Control 1,000 357 35.7 ± 7.6 z = –2.80; 0.0026 6.9 ± 2.8 W = 0;
Heat treated 1,000 10 1.0 ± 1.0 0 < .05
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6-h ramp to 55.0°C, no hold time
Control 1,600 798 48.9 ± 5.5 z = –6.75; < 0.0001 7.9 ± 2.5 z = –3.50; 0.0002
Heat treated 4,000 1 0.025 ± 0.025 0
6-h ramp to 60.0°C, no hold time
Control 1,000 477 47.7 ± 6.0 z = –2.80; 0.0051 4.6 ± 3.8b -----
Heat treated 1,000 0 0 0b
Fly emergence followed for 4 mos postremoval from chilling. See Table 1 for number of replicates and puparia/replicate. See text for criteria for live puparia.
a
Paired t-test (t), Wilcoxon signed-ranks test (W), or Mann-Whitney U test (z). Medians or means analyzed depending on analysis, although means shown.
b
Seven of 10 values tied; sample size too small to calculate Wilcoxon signed-ranks statistic.
Table 4. Experiment 4: percent Rhagoletis pomonella fly emergence from puparia and live puparia ± sem and estimated numbers of pu-
paria killed after exposure in an oven to a 6-h ramp from 21°C to 55.0°C and a 1-h hold time at 55.0°C
Estimated no.
Total Total flies Mean total percent z-score; Mean percent z-score; puparia killed
puparia tested emerged flies emerged P-value puparia alive P-value by heat treatment
Years 2018–2019
Control 2,300 1,083 47.1 ± 4.0 –7.30; < 0.0001 5.8 ± 1.9 –6.03; < 0.0001 -----
Heat treated 8,149 0 0 0 4,311
Years 2019–2020
Control 2,550 1,648 64.6 ± 3.3 –6.61; < 0.0001 5.2 ± 2.0 –4.66; < 0.0001 -----
Heat treated 16,200 0 0 0 11,308
Years 2020–2021
Control 7,317 2,981 40.0 ± 3.3 –9.73; < 0.0001 2.3 ± 0.7 –4.61; < 0.0001 -----
Heat treated 36,874 0 0 0 15,598
Adult emergence was followed for 4 mos postremoval from chilling. See Table 1 for number of replicates and puparia/replicate. See text for criteria for live pu-
paria. z-scores from Mann-Whitney U test based on medians, although means shown.
(m/n) = 0.000,096 (Couey and Chew 1986). Thus, there is 95% con- 2011), which was obtained in Experiment 4, is acceptable for some
fidence that p is ≤96 per million, equating to a probit 8.7190 level commodities by Australia, Japan, and New Zealand, countries with
of quarantine security or 99.99% treatment mortality (Couey and rigorous export requirements (Follett and Neven 2006). It is pre-
Chew 1986, Shortenmeyer et al. 2011). sumed this level of security is also acceptable for disinfesting organic
waste of R. pomonella by Washington State. Findings here indicate
that heat treatment requirements by the state for disinfesting waste
Discussion of R. pomonella exceed probit 8.7190 and are more stringent than
Results from Experiments 1 and 2 indicate that 47.8°C and 51.1°C needed, the minimum treatment being 55°C for 2 wks (Washington
treatments as conducted here cannot maximize kill of R. pomonella State Department of Agriculture 2021c).
puparia but Experiment 4 showed that 55.0°C can if it is followed Heat treatments in Experiments 1 and 2 delayed mortality of
by a 1-h hold time at that temperature. The 1-h hold time appeared R. pomonella puparia, consistent with effects of heat treatments on
critical, as unpredictably one fly was found in Experiment 2 in the mortality of other insects (Neven 2000). Specifically, some puparia
55°C -no hold time treatment, even though no fly emergence oc- exposed to 51.1 to 60°C were still alive after 28 d, based on visual
curred in Experiment 3 that tested a similar treatment. Any survival criteria used here. Measuring respiration rates of puparia (Ragland
of puparia exposed to 55°C with no hold time may be due to high et al. 2009) may have determined whether such puparia were alive
heat tolerance of only a minute segment of the fly population. Based and dying, but we did not have the setup to do this. Delayed mortality
on results of the 55°C and 1-h hold time treatment, the 60°C -no has also been reported using the fumigant methyl bromide, which
hold time treatment may also have resulted in maximum kill, but kills dipteran eggs, but over 3–20 d (Phillips et al. 2004). Lower heat
we opted to focus on 55°C because the goal was to find the lowest treatments probably denatured proteins in tissues of R. pomonella
temperature at which this occurs. puparia more slowly than higher heat treatments, causing delayed
A quarantine treatment that results in zero survivors out of tissue deterioration (perhaps explaining the changes in puparial
29,956 insects, equivalent to probit 8.7190 (Shortenmeyer et al. coloration) and kill. Results suggest that survival assessments of R.Journal of Economic Entomology, 2022, Vol. XX, No. XX 7 pomonella puparia at
8 Journal of Economic Entomology, 2022, Vol. XX, No. XX
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