Exhibit 4b, #33 STORED-PRODUCT AND QUARANTINE ENTOMOLOGY Hot Water Immersion to Ensure Quarantine Security for spp. (: ) in and Exported from

PETER A. FOLLETT AND SUZANNE S. SANXTER

U.S. PaciÞc Basin Agricultural Research Center, USDAÐARS, P.O. Box 4459, Hilo, HI 96720

J. Econ. Entomol. 94(5): 1292Ð1295 (2001) ABSTRACT We determined whether immersion in 49ЊC water for 20 min, a quarantine treatment developed for disinfestation of fruit ßies in lychee, Litchi chinensis Sonn., and longan, Dimocarpus longan (Lourd.) Steud., exported from Hawaii, would also disinfest fruit of two species of Crypto- phlebia. The pattern of tolerance to heat in Cryptophlebia illepida (Butler) was generally eggs Ͻ neonates Ͻ early instars ϭ lateinstars Ͻ pupae. No C. illepida survived immersion for 16 or 20 min. Late fourth and Þfth instars were determined to be the most tolerant stage that occurs in harvested fruit. Lateinstars of Cryptophlebia ombrodelta (Lower) were more tolerant of hot-water immersion than thoseof C. illepida, but no C. ombrodelta late instars survived immersion for 16 or 20 min. The hot water immersion quarantine treatment for fruit ßies should effectively disinfest and of any Cryptophlebia.

KEY WORDS Cryptophlebia, quarantine pest, heat treatment, disinfestations

HOT WATER IMMERSION at 49ЊC for 20 min is approved by four varieties of longan harvested from orchards in the U.S. Department of Agriculture, Plant Hawaii over a two-year period, Cryptophlebia infes- Health Inspection Service, as a quarantine treatment tation rates were 1.5 and 0.14%, respectively (G. T. for HawaiiÕs tephritid fruit ßy pests attacking lychee, McQuate, USDA, Hilo, HI, personal communication). Litchi chinensis Sonn., and longan, Dimocarpus longan Current regulations for hot-water immersion quar- (Lour.) Steud. (Federal Register 1997). In addition to antine treatment of Hawaii-grown lychee stipulate fruit ßies, two species of Cryptophlebia arealso pests that exported fruits must be found free of Cryptophle- of quarantine concern in sapindaceous fruits (lychee, bia spp. (Federal Register 1997). Thus, the presence longan, and ) exported from Hawaii. Cryp- of Cryptophlebia can prevent export of lychee from tophlebia illepida (Butler), the koa seedworm, is en- Hawaii. The quarantine treatment used for lychee has demic to Hawaii, and C. ombrodelta (Lower), the been proposed for longan also exported from Hawaii litchi fruit , is nativeto . Cryptophlebia (P.A.F., unpublished data). If the accepted hot-water spp. are the most commonly intercepted pests immersion quarantine treatment for pest fruit ßies is in sapindaceous fruits from Hawaii carried in passen- also effective against C. illepida and C. ombrodelta, gerbaggageand arriving in themail on theU.S. main- these species could be added to the regulation, pre- land (USDAÐAPHISÐPPQ 1997). venting potential interruption of shipments because of Cryptophlebia spp. lay eggs singly on the fruit sur- the presence of these . The objectives of the face, and neonates bore through the skin and feed at studies with Cryptophlebia reported here were to ex- the skinÐpulp interface. Although many eggs may be amine the effectiveness of the hot-water immersion laid on thefruit surface,typically only onelarva is treatment against various stages of Cryptophlebia, and found feeding in a fruit. Cryptophlebia spp. arepoten- thereby determine if the approved quarantine treat- tially multivoltine in Hawaii (Jones et al. 1997). Lab- ment provides quarantine security against these pests. oratory tests and Þeld surveys in Hawaii indicate that lycheeandlongan arepoor hosts for Cryptophlebia Materials and Methods (McQuate et al. 2000; P.A.F., unpublished data). Ac- tual damage from larval feeding is often minimal, and Insect Rearing. A colony of C. illepida was started larval survival to pupation is rarewhenfruit areon the using larvae( Ϸ1,000) collected from nuts tree. However, when a neonate enters the fruit at the from an orchard in Pahala, HI. Because of difÞculties calyx or stem end, it may bore through woody tissue collecting C. ombrodelta in theÞeld, C. ombrodelta to the seed and feed there, improving the chance for pupae(108) wereobtainedfrom a laboratory colony survival to pupation. Successful larval development in maintained at the Maroochy Experiment Station in fruit also may improveoncefruit areharvested Queensland, Australia, to start a colony; the Maroochy (P.A.F., unpublished data). In a study of Ͼ36,000 ma- colony had been maintained in the laboratory for 3 yr ture fruits of six varieties of lychee and 9,700 fruit of and wild from infested macadamia were intro- October 2001 FOLLETT AND SANXTER:HOT WATER IMMERSION TO CONTROL Cryptophlebia 1293

caps had TeßonÐsilicone septa to permit insertion of temperature probes. All hot-water immersion treat- ments were done in a 75-liter circulating bath heated by two electric heaters (immersion circulator model 73, PolyScience, Preston Industries, Niles, IL) to 49 Ϯ 0.2ЊC. Bath temperature was veriÞed before and after each run using a mercury thermometer with 0.1ЊC gradations. During treatment, temperature was mon- itored in eight vials at the surface and center of the diet,and at theinsidewall of theglass vial abovethe diet, at 30-s intervals using temperature probes and a data logger (model 5160-64-A, Omni International, Logan, UT) calibrated to a mercury thermometer. Fig. 1. Temperature proÞles of diet in vials and of lychee Vials with individual larvaeor pairs of pupaewere Њ fruit during hot water immersion at 49 C for 20 min followed placed in wire baskets (1 cm mesh) and submersed to by immersion in ambient temperature water for 20 min. the bottom of the tank for the desired treatment time. For larval treatment, baskets with vials were tapped on duced annually to maintain genetic variation. For both a tableto dislodgeinsectsfrom thewall of theglass vial species, adults were placed in 3.8-liter plastic jars with onto the diet surface before submersion. For egg treat- ventilated lids for mating and provided with a 5% ment, gravid females were conÞned to 29-ml plastic honey water solution through a wick. Eggs were laid cups overnight, and open cups with eggs were placed on theinsidesurfaceof thejar. Emergingneonates in wire cages and treated as above (i.e., eggs were in were transferred daily to 29-ml diet cups with labo- direct contact with the water.) Regardless of treat- ratory diet. The laboratory diet was modiÞed from ment time, the basket with vials or cups and insects Sinclair (1974) as described in Follett and Lower was transferred to a 75-liter ambient-temperature Ϸ Њ (2000). As larvae approached pupation, diet cups ( 22Ð24 C) cooling bath for 20 min after heat treat- (with larvae) with lids removed were placed in 3.8- ment. (Although not part of the ofÞcial treatment, a liter plastic tubs (Rubbermaid, Wooster, OH) with 20-min cooling bath is recommended after hot water 30 g of sand covering the bottoms to a depth of 0.25 cm. immersion of lychee fruits.) Larval mortality was mea- C. illepida larvaetypically leavethediettrays to pu- sured after 24 h by prodding larvae with a camels-hair patein thesand, whereas C. ombrodelta typically pu- brush for signs of any movement. Pupae were held pate in the diet. Emerging moths were cooled for after treatment in 29-ml plastic cups at 25ЊCfor2wk handling and transferred to the plastic mating jars. for adult emergence. Treated eggs attached to plastic Rearing conditions were 25 Ϯ 2ЊC and a photoperiod cups were held for 1 wk at 25ЊC for neonate emer- of 10:14 (L:D) h for the duration of the experiments. gence. For stage-speciÞc tests, larvae were placed into cate- The two-year survey mentioned earlier that exam- gories (L1, L2/3, and L4/5 corresponding to instars) ined the fruit pests in lychee and longan in Hawaii based on size and known developmental rates (un- showed mean fruit weights of 17.2 g (SD ϭ 4.8, n ϭ published data). 36,351) and 9.4 g (SD ϭ 2.3, n ϭ 9,700), respectively Hot Water Immersion Treatment. Cryptophlebia (G. T. McQuate, personal communication). In pre- are difÞcult to rear on fresh lychee and longan; there- liminary studies, the heating rate at the seed surface of fore, all heat treatments were applied to Cryptophlebia lycheefruitof typical size(17Ð19 g) rangedfrom 3.1 Ϫ in 29-ml plastic cups (eggs) or 25-ml glass vials con- to 3.3ЊC min 1 during theÞrst 7 min of thetreatment taining 10 g of artiÞcial diet (larvae and pupae). Direct (thetimeperiodwhentherateof heatingis thegreat- exposure to hot water of eggs attached to the inner est). Diet was heated so that the thermal proÞle at the surfaceof cups is similar to exposureonthesurfaceof surfaceof thedietin vials mimickedtheproÞlefor the fruit. The amount of diet required in vials was deter- seed surface of a typical lychee fruit (Fig. 1). The mined by comparing thermal diffusion proÞles of dif- heating rate at the diet surface in our tests averaged ferent amounts of diet with that of lychee fruit. Vial 3.3ЊC minϪ1 during the Þrst 7 min after immersion. The

Table 1. Mean ؎ SEM percentage mortality of C. illepida after immersion in water at 49°C

a Immersion Mortality time, min Eggs (1 day old) Eggs (3 d old) L1 L2/3 L4/5 Pupae 0 586 13.7 Ϯ 3.8 231 19.8 Ϯ 9.1 97 13.8 Ϯ 5.2 94 0.0 Ϯ 0.0 111 1.0 Ϯ 1.0 118 5.5 Ϯ 3.1 4 585 100.0 Ϯ 0.0 569 100.0 Ϯ 0.0 157 68.2 Ϯ 4.8 116 16.2 Ϯ 5.7 123 4.2 Ϯ 1.8 162 17.4 Ϯ 2.3 8 624 100.0 Ϯ 0.0 466 100.0 Ϯ 0.0 152 97.4 Ϯ 2.0 114 51.4 Ϯ 12.5 124 35.6 Ϯ 6.2 158 16.1 Ϯ 4.5 12 686 100.0 Ϯ 0.0 452 100.0 Ϯ 0.0 137 100.0 Ϯ 0.0 115 96.7 Ϯ 3.3 121 98.3 Ϯ 1.7 160 95.3 Ϯ 3.0 16 522 100.0 Ϯ 0.0 426 100.0 Ϯ 0.0 121 100.0 Ϯ 0.0 101 100.0 Ϯ 0.0 106 100.0 Ϯ 0.0 155 100.0 Ϯ 0.0 20 698 100.0 Ϯ 0.0 378 100.0 Ϯ 0.0 114 100.0 Ϯ 0.0 78 100.0 Ϯ 0.0 99 100.0 Ϯ 0.0 149 100.0 Ϯ 0.0

a Mortality of each life stage is preceded by the number of test subjects. Larval stages: L1, neonates; L2/3, early instars; L4/5, late instars. 1294 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 94, no. 5

Table 2. Linear regressions of percentage mortality against immersion time for C. illepida larvae immersed in 49°C water

Predicted time (95% CL) Stagea n Intercept Ϯ SE Slope Ϯ SE R2 for 100% mortality, min L1 446 49.8 Ϯ 19.2 4.6 Ϯ 2.2 0.81 10.9b L2/3 446 Ϫ8.1 Ϯ 17.1 7.4 Ϯ 1.6 0.92 14.6 (10.5 Ϫ 59.8) L4/5 474 Ϫ28.0 Ϯ 22.0 8.8 Ϯ 2.0 0.90 14.6 (10.2 Ϫ 400.1) Pupae635 Ϫ31.9 Ϯ 31.9 8.7 Ϯ 2.9 0.82 15.2b

Linear regression, y ϭ b ϩ mx, where b is the intercept, m is the slope, x is immersion time (min), and y is percentage mortality. a Larval stages are L1 ϭ neonates, L2/3 ϭ early instars, L4/5 ϭ lateinstars. b 95% CL not calculablebecauseofa poor Þt. heatingrateof a particularly large(31 g) lycheefruit gression was used because of the limited number of was 2.7ЊC minϪ1 during theÞrst 7 min of thetreatment doses tested and the better Þt to the data (Jandel (Fig. 1). ScientiÞc 1994, Robertson et al. 1994). Untransformed All hot water immersion tests used laboratory- data from the comparative study between C. illepida reared Cryptophlebia. C. illepida eggs (1 d old, 3 d old), and C. ombrodelta were subjected to analysis of vari- larvae (neonates, secondÐthird instars, and fourthÐ ance (ANOVA), and means were separated using a Þfth instars), and pupae (7Ð10 d old) were treated at t-test(SAS Institute2000). Data from thecomparative 49ЊC for 0, 4, 8, 12, 16, or 20 min in a factorial design, study were Þtted to a logistic rather than linear model and percentage mortality was determined. Although because of the better Þt to the data (Jandel ScientiÞc thepupal stageof Cryptophlebia is not found in fruits, 1994). For all models, predicted time to 100% mortality we tested the effects of heat against this stage for the was calculated to estimate the time required at 49ЊCto purpose of comparison. Pupal development to the completely eliminate live insects from fruit. adult stage is 11 d under our rearing conditions (25 Ϯ 2ЊC). For each egg, larval, or pupal ageÐstage, four to Results six replicates were treated on different dates, and a control group in each replicate was handled in the Noneof the1-d-old or 3-d-old C. illepida eggs same way as treated groups except they were im- treated at 49ЊC for Ն4 min survived (Table 1). Control mersed in ambient rather than heated water. The mortality was 13.7 Ϯ 3.8% (mean ϩ SEM) and 19.8 Ϯ number of insects per replicate was Ϸ25 larvaeand 9.1% in 1-d-old and 3-d-old eggs, respectively. Neonate pupaeand 100 eggs. mortality was 97.4 Ϯ 2.0% after 8 min, and no indi- To determine whether C. illepida or C. ombrodelta viduals survived immersion for Ն12 min. Mortality of was more heat tolerant, late (fourth and Þfth) instars early instars (L2/3) was 96.7 Ϯ 3.3% after 12 min, and of each species were treated as above for 0, 8, 10, 12, no individuals survived immersion for 16 or 20 min. 16, and 20 min at 49ЊC, and percentage mortality was Mortality of lateinstars (L4/5) was lowerfor the4 and determined. Each treatment was replicated four to 8 min treatments than for L2/3, but similar at 12 min; seven times, and a control group in each replicate was none survived treatment for 16 or 20 min (Table 1). handled in the same way as treated groups except they Thepupal stagehad thelowestmortality at 8 min. were immersed only in ambient rather than heated (16.1 Ϯ 4.5%) among the life stages. However, pupal water. Extensive conÞrmatory testing of late instars in mortality was high at 12 min (95.3 Ϯ 3.0%), and no glass vials treated at 49ЊC for 20 min as abovewas pupae survived immersion for 16 or 20 min. Control conducted for both species. mortality for L1, L2/3, L4/5, and pupaewas 13.8, 0.0, Data Analysis. Simple linear regressions were done 1.0, and 5.5%, respectively. The predicted immersion on untransformed data from studies of life stage and timeat 49 ЊC to achieve 100% mortality ranged from species-speciÞc tolerance of hot water immersion 10.9 min for L1 to 15.2 min for pupae(Table2). (SAS Institute 2000). For each life stage, data used in Fourth- and Þfth-instar larvaewerechosenfor the the linear regression model included any hot-water species tolerance comparison tests because of their immersion dose causing mortality between 0 and higher survival at 4 and 8 min, relative to L2/3s, in the 100%, and thelowestdosecausing 100% mortality. doseÐresponse tests. C. ombrodelta fourth-Þfth instars Mortality values Ͻ100% were adjusted for control were more tolerant of hot water immersion compared mortality using AbbottÕs formula (Abbott 1925). After with thoseof C. illepida (Table 3). Mean percentage comparison with probit and logit models, linear re- mortality was higher for C. illepida compared with

Table 3. Mean ؎ SEM percentage mortality of late instar C. illepida and C. ombrodelta immersed in 49°C water

Immersion time, min Species 0810121620 C. illepida 0.2 ϩ 0.2a 35.4 ϩ 8.2a 49.1 ϩ 8.5a 99.5 ϩ 0.3a 100.0 ϩ 0.0a 100.0 ϩ 0.0a C. ombrodelta 0.5 ϩ 0.3a 28.6 ϩ 6.7a 25.0 ϩ 4.4a 74.0 ϩ 7.9b 100.0 ϩ 0.0a 100.0 ϩ 0.0a

Means Ϯ SEM within thesamecolumn followedby thesameletterarenot signiÞcantly different( P ϭ 0.05) by t-test. October 2001 FOLLETT AND SANXTER:HOT WATER IMMERSION TO CONTROL Cryptophlebia 1295

Table 4. Logistic analysis of percentage mortality against immersion time of late-instar C. illepida and C. ombrodelta immersed in 49°C water

Model parameters (ϮSE) Predicted time (95% CL) Species n R2 abc for 100% mortality, min C. illepida 1,539 109.9 Ϯ 32.0 9.6 Ϯ 1.5 Ϫ5.8 Ϯ 4.8 0.91 14.7 (11.8Ð232.2) C. ombrodelta 1,510 130.2 Ϯ 109.8 12.0 Ϯ 5.7 Ϫ4.4 Ϯ 4.9 0.99 15.7 (14.8Ð16.9)

Logistic doseresponsemodel,y ϭ a/[1 ϩ (x/b)c].

C. ombrodelta after immersion for 8, 10, and 12 min at Acknowledgments 49ЊC, but differences were signiÞcant only for 12 min ϭ ϭ ϭ We thank Geoffrey Waite at the Maroochy Experiment (t 3.2, df 10, P 0.01); no individuals of either Station in Queensland, Australia, for graciously providing C. species survived immersion for 16 or 20 min. The ombrodelta to start our colony; Alan Yamaguchi of Kau Agri- predicted time to 100% mortality was 14.7 min for business for pointing us to a heavily infested macadamia C. illepida and 15.7 min for C. ombrodelta using the orchard in Pahala, HI, to collect C. illepida; John Brown, logistic model (Table 4). USDA Systematic Entomology Laboratory, Beltsville, MD, Extensive testing of late instars conÞrmed the efÞ- for conÞrming Cryptophlebia species identiÞcations; and Bob cacy of the approved fruit ßy quarantine treatment Lower, Zona Gabbard, Shannon DeLuz, Camille Mehau, Chris Johnson, and Malia Laber for assisting with mainte- against Cryptophlebia. Noneof the4,434 late-instar C. nanceof the Cryptophlebia colonies. Permission to import ombrodelta and 4,546 C. illepida survived immersion in C. ombrodelta was granted by the Hawaii Department of 49ЊC water for 20 min. Agriculture(PermitNo. 99-11-h-I23) and USDAÐAPHIS (Permit No. 9398878).

References Cited Discussion Abbott, W. S. 1925. A method for computing the effective- No C. illepida and C. ombrodelta immersed for 16 or ness of an insecticide. J. Econ. Entomol. 18: 265Ð267. Њ Federal Register. 1997. Papaya, carambola, and litchi from 20 min in 49 C water survived in any of our tests. Hawaii. 62 (132): 36967Ð36976, July 10, 1997, Rules and Therefore, the hot-water immersion quarantine treat- Regulations. ment for HawaiiÕs lychees and longans should effec- Follett, P. A., and R. A. Lower. 2000. Irradiation to ensure tively disinfest fruit of any Cryptophlebia. quarantinesecurityfor Cryptophlebia spp. (Lepidoptera: Other heat treatments (heated air or water) have Tortricidae) in sapindaceous fruits from Hawaii. J. Econ. been approved by USDA-APHIS for internal and ex- Entomol. 93: 1848Ð1854. ternal pests on various commodities exported to the Jandel Scientific. 1994. TableCurve 2D Windows v2.0. Jan- del ScientiÞc, San Rafael, CA. mainland United States (USDA-APHIS-PPQ 1998). Jones, V. P., C.H.M. Tome, and L. C. Caprio. 1997. Lifetable The only other hot water immersion treatment ap- for the koa seedworm (Lepidoptera: Tortricidae) based proved for internal pests is for fruit ßies in mangoes. on degree-day demography. J. Econ. Entomol. 90: 1291Ð The hot-water immersion treatment for lychee is 1298. unique in that it does not require measurement of fruit McQuate, G. T., P. A. Follett, and J. M. Yoshimoto. 2000. temperature during treatment (unlike all heated air Field infestation of rambutan fruits by internal-feeding treatments), and does not specify any treatment ad- pests in Hawaii. J. Econ. Entomol. 93: 846Ð851. Robertson, J. L., H. K. Preisler, E. R. Frampton, and J. W. justments for different fruit sizes (unlike the hot- Armstrong. 1994. Statistical analyses to estimate efÞcacy water immersion treatment for mangoes). In our of disinfestations treatments, pp. 47Ð65. In J. L. Sharp and study, treating larvae in vials with diet was not a G. J. Hallman [eds.], Quarantine treatments for pests of perfect representation of a natural situation with in- food plants. Westview, San Francisco, CA. sects in fruit, but was necessary because of the poor SAS Institute. 2000. JMP userÕs guide. SAS Institute, Cary, host status of lychee for Cryptophlebia. Mortality of NC. larvaein lycheefruitwill bea function of thesizeof Sinclair, E. R. 1974. A lifesystemstudy of Cryptophlebia ombrodelta (Lower) (Lepidoptera: Tortricidae) in thefruit and thelocation of thelarvaein thefruit. southwest Queensland. Ph.D. dissertation, University of Survival of larvae may be higher than predicted when Queensland, Brisbane, Australia. the heating rate during treatment is slower, as would (USDA–APHIS–PPQ). 1997. Pest risk assessments: Hawai- bethecasewith larvaefeedingneartheseedin large ian fruits. U.S. Dep. Agric., Animal Plant Health Inspec- fruit. However, treatment at 49ЊC for 20 min provides tion Service, Plant Protection Quarantine, Riverdale, a substantial level of overkill and therefore should be MD. effective over a wide range of lychee fruit sizes. Lon- (USDA–APHIS–PPQ). 1998. Treatment manual: interim edition. U.S. Dep. Agric., Animal Plant Health Inspection gan is a smaller fruit than lychee, so we assume that Service, Plant Protection Quarantine, Riverdale, MD. heat penetration will be more rapid and larvae will be more susceptible to heat treatment in longan than in Received for publication 14 December 2000; accepted 24 lychee. March 2001.