Location: Beneficial Insects Introduction Research Unit
Project Number: 8010-30400-001-023-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 3, 2025
End Date: Sep 29, 2026
Objective:
The proposed research aims to continue to rear and identify a genetic line of the introduced parasitoid Spathius agrili from established sites in Tennessee (or other sites in the southern U.S.) that have been adapted to a warmer climate and may be thus effective for field
releases against EAB in the southern U.S.
Specific objectives are:
1. Continue to receive and rear Spathius parasitoids from established sites in Tennessee andconfirm their identity as S. agrili.
2. Mix newly collected Tennessee S. agrili with the existing Tennessee colony of S. agrili (established in FY24) for rearing and send the expanded colonies to the USDA APHIS EAB Biocontrol Rearing Facility for mass production and field releases against EAB.
3. Evaluate the effect of temperature and photoperiod on diapause of the Tennessee S. agrili population.
Approach:
All parasitoid rearing will be conducted in reach-in environmental chambers under normal rearing conditions (~25C, 65% RH, and long day photoperiod – 16:8 hrs L:D). To rear EAB parasitoids (Spathius agrili) collected from different sites or regions, we will first need
to rear late instar EAB larvae on tropical ash bolts (diam ~1.5 cm x length ~ 15 cm) infested with EAB eggs. Newly emerging female and male wasps of S. agrili will be first hosted together in rearing cages for a week and then exposed to EAB-infested tropical ash bolts for another week. After parasitoid exposure, exposed ash bolts will be moved to clean containers and incubated under normal rearing conditions for adult emergence (normally 4 – 6 weeks post exposure). For Objective 1, voucher specimens (5-10) of newly emerged Spathius wasps from each site will be collected and preserved in molecular alcohol and sent to USDA APHIS Forest Pest Methods
Laboratory (FPML) for morphological and DNA identification. Once authoritative identification is confirmed, the above rearing procedure will be repeated for at least three generations. For Objective 2, newly emerged S. agrili wasps (F2) will be mixed with the previously established colony of Tennessee S. agrili (F5 – 10) and then exposed to suitable stages of EAB larvae to produce a mixed colony of S agrili. The progeny of this mixed S. agrili (F3 – 11) will then be sent to the USDA APHIS EAB Biocontrol Rearing Facility for mass-production for field releases against EAB. For Objective 3, two photoperiods, representing long (16:8 hr. L:D) and short (8:16 hr. L:D)
days, and two temperatures, representing cold (17°C) and warmer (25°C) seasons will be tested for their effect on diapause initiation, development, and termination. Four (2x2) exposure treatments will be set up in four growth chambers. Two chambers will be set to 17°C and two set to 25°C. One of each temperature chamber groups will then be set to a photoperiod of 16:8 L:D and the other two for each temperature chamber set to 8:16 L:D. This will be done to test the effect of photoperiod when S. agrili larvae are exposed to a diapause inducing temperature (17°C) and non-diapause inducing temperature (25°C). Ten female and five male wasps will be placed on a single bolt for one week in the walk-in chamber described in previous sections. After a one-week parasitization period, wasps will be removed, and 5 bolts will be maintained inside each of the four treatment combinations of temperature and photoperiod. Following the initial 1-wk parasitoid exposure, we will monitor adult parasitoid emergence from each exposed bolt (replicate) on a weekly basis for 16 weeks and then dissect all exposed bolts to determine the fate of parasitoid progeny – exit as adults, live or dead larvae, pupae or pharate adults. Liveparasitoid (mature) larvae inside the cocoon are considered to be in diapause.