PARASITOIDS

Atanycolus cappaerti used for this study were originally collected in the fall of 2012 and 2013 from central Michigan as diapausing mature larvae (in cocoons) from parasitized A. planipennis larvae infesting green ash (Fraxinus pennsylvanica Marshall) or white ash (F. americana L.) trees. These specimens were identified as A. cappaerti by using the Atanycolus key provided by Paul Marsh (North Newton, Kansas, formerly with United States Department of Agriculture, Agricultural Research Service, Systematic Entomology Laboratory) and confirmed by comparing the A. cappaerti voucher specimens collected from the same locations and identified by Paul Marsh.

Throughout the investigation, we utilized only naïve wasps of F0 to F5 generations reared on A. planipennis larvae infesting green ash or tropical ash (Fraxinus uhdei [Wenzig] Lingels) bolts (1.5–5 cm in diameter, 10–15 cm long). Green ash and greenhouse grown tropical ash were used in previous studies as an effective rearing medium depending on the season when the experiment was carried out (Duan et al. 2011). Before experiments, adult parasitoids were housed in ventilated clear-polystyrene crisper boxes (each 17.6 × 12.6 × 10 cm, State Plastics, Latonia, Kentucky) and maintained in environmental chambers (AR-66L2, Percival Scientific, Perry, Iowa) at 25 ± 1.5 °C, with 65 ± 10% RH and a 16:8 h L:D photoperiod. A water source was provided inside each crisper box (containing approximately 10 female and 10 male parasitoids) via a 10 dram clear plastic vial (US Plastics, Lima, Ohio) fitted with a 10 cm braided cotton dental wick (Richmond Dental, Charlotte, North Carolina) and replaced weekly; pure clover honey was streaked on the ventilation screen of rearing containers as a food source for adult parasitoids. Newly emerged adult parasitoids (<1 wk old), from solitary cocoons dissected from ash, were used for adult longevity, oviposition, lifetime fecundity, and progeny diapause experiments. Adult parasitoids of up to 4 wk of age after emergence were used in the primary parasitoid exposure trials for the larval development study.

HOST LARVAE

All host larvae used in the study were reared in freshly cut green ash or tropical ash bolts according to the method described in Duan et al. (2011, 2013c). Briefly, freshly cut ash bolts (2–3 cm in diameter, 10–25 cm long) were infested with A. planipennis eggs laid on coffee filter paper. Egg density per bolt varied with the diameter and length of the stick: 5 eggs for a small bolt (∼2 cm in diameter, 10–15 cm long) and 12 eggs for a large bolt (∼3 cm in diameter, 20–25 cm long). After infestation with A. planipennis eggs, ash bolts were placed in watersoaked floral foam bricks (OASIS®, Smithers-Oasis Company, Hent, Ohio) inside large plastic containers (58.4 × 41.3 × 31.4 cm WLH, Sterilite, Sterilite Corporation, Townsend, Massachusetts), and incubated in an environmental chamber at 27 ± 2 °C, 65 ± 10% RH, and a 16:8 h L:D photoperiod for 4.5 to 5 wk to produce 3rd to 4th instars of A. planipennis larvae or for more than 10 wk to produce mature J-shaped (4th instar) larvae for use in different experiments.

ADULT LONGEVITY, OVIPOSITION RATE, FECUNDITY, AND PROGENY DIAPAUSE

All adult parasitoids used for this experiment originated from diapaused larvae in cocoons that had been chilled at 2 to 4 °C for at least 3 mo. Newly emerging A. cappaerti adults (<1 wk old) were reared in single pairs (1 female and 1 male per container) on late (3rd to 4th) instars of A. planipennis infesting tropical ash sticks. On a weekly basis, pairs of newly emerging female and male parasitoids (n = 16) were exposed to ash bolts hosted in clear acrylic cylinders (5 cm in diameter × 15 cm in height; Consolidated Plastics, Stow, Ohio), each with 4 screened cuts on the side or lid of the container for ventilation. Pure clover honey was streaked on the screen of containers and maintained throughout the study as a food source for parasitoids. Each parasitoid pair was given 1 or 2 ash bolts containing 4 or more late instars of A. planipennis larvae twice a week until the death of the female parasitoid. On a weekly basis, mortality or survivorship of adult parasitoids was recorded.

All parasitoid-exposed ash bolts were immediately placed in fresh containers and incubated at 25 ± 2 °C, 65 ± 10% RH, and a 16:8 h L:D photoperiod for adult wasp emergence. Adult parasitoids normally emerged from parasitized host larvae within 3 to 4 wk after the primary exposure to parental parasitoids. One wk after the last parasitoid emerged, all exposed ash sticks were dissected to determine the fate and stage of each viable host larva (parasitized or not parasitized) as well as parasitoid cocoons and/or larval cadavers. The number of progeny produced weekly for each pair was calculated as the sum of parasitoid adults emerged and diapausing parasitoid larvae for each week, and used as a measurement of weekly oviposition rate by the pair. Non-emerged parasitoid cocoons (with no exit holes) containing mature live larvae were classified as diapausing parasitoid progeny. These diapausing parasitoid larvae could resume development to adults only after were they chilled at 2 to 4 °C for 2 to 4 mo. The number of diapausing (non-emerged) progeny produced by each pair was recorded upon dissection of each exposed ash stick. Throughout the study, we observed a few dead parasitoid cadavers as larvae (n = 12) and excluded these dead parasitoid progeny (∼3% of the total progeny) from data analysis.

DEVELOPMENT STAGE AND TIME OF IMMATURE LARVAE

We observed immature stages (eggs, larvae, and cocoons) of A. cappaerti and their development time using a similar procedure described for the braconid parasitoid S. galinae in Duan et al. (2014). This procedure involved using mature (J-shaped) host larvae artificially inserted into green ash sticks (2 larvae per stick), which were then exposed to gravid female parasitoids to obtain parasitism. The procedure for insertion of host larvae into ash sticks was described in detail by Duan et al. (2014). Briefly, grooves (0.3 mm deep × 5 mm wide × 30 mm long) with bark flaps were first fashioned on each stick with a utility knife and a palm-handled straight groover (Style # 11, 3 mm in size, Woodcraft Supply LLC, Parkersburg, West Virginia). A single host larva was then placed in the groove and covered with the bark flap. All host larvae inside the grooves of sticks were secured with bands of Parafilm at the top and bottom of the flap and exposed to adult parasitoids at a host-to-parasitoid (female) ratio of 1:2 in a crisper box as described previously.

Approximately 24 h after parasitoid exposure, exposed bolts were removed and immediately examined under a dissection microscope for parasitism of host larvae by A. cappaerti (i.e., presence of parasitoid eggs). Exposures continued until 20 parasitized host larvae, each with an A. cappaerti egg, were obtained from 24 h of the primary parasitoid exposure. After observation of the parasitism of each host larva, ash sticks with parasitized host larvae were placed into 200 mL plastic cups with a moist paper towel and a screened lid and returned to the rearing environmental chamber for daily observations of immature stages by just opening the flap and observing the parasitoid stage under a dissection microscope. To assist in the determination of instars of A. cappaerti, we made a small dot on each parasitoid larva before each molt by using a fine-point blue permanent marker (Sharpie, Newell Rubbermaid Office Supplies, Oak Brook, Illinois). When each larva molted to the next stage, the dot was seen on the shed exuvia, or was absent from integument, and the next-stage larva was then marked.

Because of the diapause behavior in mature A. cappaerti larvae, our observation of larval development stopped once mature larvae completed formation of cocoons. However, we set up additional parasitoid exposure trials as described previously and determined the development time from eggs to adult emergence of non-diapaused A. cappaerti. For diapausing A. cappaerti, we chilled the mature larvae in cocoons at 2 to 4 °C for approximately 2 to 4 mo. This period of chilling was effective in terminating the diapause of A. cappaerti larvae (J. J. D., unpublished data). Following diapause termination, we then placed the cocoons under the normal rearing conditions described previously and determined their development time from diapaused mature (6th instar) larvae to adult emergence.

DATA ANALYSES

The median survival time (wk from eclosion) and 95% confidence interval (CI) as well as the mean survival time (± SE) of both sexes were estimated with survival analysis based on the Kaplan-Meier survival platform. The realized lifetime fecundity was estimated by the total number of progeny produced by each female parasitoid over her lifetime, and oviposition rates were estimated based on the mean numbers of progeny (± SE) produced per wk by each female parasitoid. The relationship between the probability of progeny entering diapause and the age of their parental parasitoids was analyzed with nominal logistic regression analysis. Median development times (in d) and 95% CI as well as the mean survival time (in d ± SE) for recognized immature stages including eggs, 1st to 5th instars, cocoons (6th instars), and adults were estimated by the same survival analysis procedure described previously. All statistical analyses and calculations were carried out with JMP Pro Version 12.1.0 (SAS Institute 2015).

ADULT LONGEVITY, OVIPOSITION RATE, FECUNDITY, AND PROGENY DIAPAUSE

The median survival time of A. cappaerti adults was 9 wk (95% CI = 3-9 wk) for males and 11 wk (95% CI = 8-12 wk) for females (Fig. 1A). The mean (± SE) survival times varied slightly from the medians, with the males living an average of 7.4 wk (± 0.89) and females living an average of 10.5 wk (± 1.09). The median survival time differed between male and female parasitoids (log rank test, χ² = 5.42, df = 1, P = 0.0199). The oviposition rate peaked at week 3 with 5.4 (± 1.7) progeny produced per parental female (Fig. 1B). Over the entire 19 wk period, 3 female parasitoids failed to produce any progeny, and the rest of the females (n = 13) produced a mean of 28 (± 3.1) progeny over their lifetime with a minimum of 8 and maximum of 42 progeny. The host utilization (parasitism) rate peaked at week 8 with 22% (± 7.1%) and then declined to 0% in week 13 (with spikes in weeks 14 and 17 and a large spike to 57% in week 19 with 1 remaining female parasitoid).

Throughout the entire period (19 wk) of progeny production (n = 352), approximately 43.2% of progeny developed to adult parasitoids, whereas the remaining 56.8% of the progeny diapaused as mature larvae in cocoons. The proportion of diapausing progeny varied with parental age (log likelihood ratio test: χ² = 5.56; df = 1; P = 0.0184). As they aged, parental parasitoids produced a higher proportion of diapausing progeny (Fig. 2).

Fig. 1.

(A) Longevity and (B) realized fecundity and host utilization (parasitism) rate of adults of Atanycolus cappaerti when reared in single mating pairs (n = 16 for both sexes) and continually provided with emerald ash borer larvae on a weekly basis for their lifespan.

DEVELOPMENT TIME OF IMMATURE LARVAE

We observed 6 instars after egg eclosion (Fig. 3A-G) of A. cappaerti, with the last (6th) instars forming solitary silk cocoons (Fig. 3H). Different instars of larvae had similar morphology and differed mainly in size as measured with a micrometer; however, the 1st instar had noticeable mandibles and antennal appendages that disappeared from its successive stages. Median and mean development times for each instar or stage can be found in Table 1. Eggs developed fairly quickly with a 50% hatch rate by 1.6 d after the host had been parasitized. The 1st instar molted 5 times into the 6th instar in approximately 4.8 d, and 50% of 6th instars formed solitary cocoons in another 1.9 d (8.3 d after 1st instars had hatched).

Fig. 2.

Diapause behavior of Atanycolus cappaerti progeny when reared in normal rearing conditions (25 ± 2 °C, 65 ± 10% RH, and a photoperiod of 16:8 h L:D).

Once cocoons were formed, a proportion of the mature larvae continued development to adult parasitoids and the rest diapaused. The median development time from egg to adult for non-diapaused parasitoids was 20 d and differed between female and male parasitoids (log rank test, χ² = 0.45, df = 1, P = 0.5037; Table 2). Diapausing larvae from separate experiments resumed development to adult parasitoids in 28 d (± 2 d 95% CI) after being chilled for 2 to 4 mo at 2 to 4 °C. There was no difference in the development time of the diapaused larvae to adults between female and male parasitoids (log rank test, χ² = 1.76, df = 1, P = 0.1847; Table 2).