STUDY AREA AND SAMPLING

Green ash trees (Fraxinus pennsylvanica Marshall; Oleaceae) infested with emerald ash borer were selected and felled to quantify emerald ash borer overwintering stages in North Carolina. Live trees with signs of decline, such as bark splits, epicormic shoots, or emerald ash borer exit holes were chosen from a 3-ha area of bottomland hardwood forest; selected trees were at least 3 m apart. Each tree was cut near the soil line and the bole sawed into manageable sections labelled sequentially from the base to the top; log sections ranged from 7 to 28 cm in diameter. These were transported to a North Carolina Department of Agriculture & Consumer Services property, Raleigh, North Carolina, USA, where the logs were debarked and inspected for life stages of emerald ash borer.

In 2017, we worked in 2 sites in Granville County, near the northern border of North Carolina with Virginia: a residential property north of Stovall (36.463046°N, 78.566499°W), and a state-owned forestland in Butner (36.15290°N, 78.767530°W). The 2 sites are approximately 39 km apart. Two ash trees were felled in Stovall and 3 in Butner on 28 Feb 2017; 4 trees were felled in Butner on 9 Feb 2018 and 5 Feb 2019. Because our 2017 results indicated that the emerald ash borer infestation in Stovall had already progressed to near total mortality of the trees, our studies in 2018 to 2020 were conducted exclusively at the site in Butner.

Debarking took place 1 to 14 Mar 2017, 12 to 21 Feb 2018, and 6 to 13 Feb 2019. In 2017, the results from the Stovall and Butner sites were combined; just 15% (9 of 59) of the insects collected were from Stovall. On 4 Feb 2020, we travelled to the Butner site with the intention of again felling trees for debarking in the laboratory, but the ash had reached near complete mortality. Instead, trees were debarked in the field until we located emerald ash borer life stages. Eventually these were found in 3 trees, where n = 13, 1, and 1 live emerald ash borer life stages were found, respectively.

The number of cumulative developmental degree d on the date of tree harvest were taken from the Duarte 2013 OSU IPPC model, base 50 ( http://uspest.org/cgi-bin/ddmodel.us; last accessed 8 Apr 2021). The weather station used was at Creedmoor, located 8 km southeast of the Butner collection site.

DETERMINING DEVELOPMENTAL STAGES

The developmental stage of each emerald ash borer (larva, J-larva, pupa) was recorded, and those that were undamaged during collection were preserved in alcohol. The instar of each alcohol-preserved larva was determined by measuring, from the dorsal aspect, the length of sclerotized terminal processes on the abdomen using an ocular micrometer (0.05 mm) on a dissecting microscope (Nikon SMZ1500, Melville, New York, USA). The length of these terminal processes, also called urogomphi (see Chamorro et al. 2012), are a consistent character for separating larval instars of emerald ash borer (Wang et al. 2005; Orlova-Bienkowskaja & Bienkowski 2016). The formula we used for categorizing instars was a combination of the ranges published in Wang et al. (2005) and Orlova-Bienkowskaja and Bienkowski (2016) as follows: < 0.2 mm (first instar), 0.21 to 0.37 mm (second instar), 0.38 to 0.72 mm (third instar), and > 0.73 (fourth instar).

COLLECTION AND IDENTIFICATION OF POSSIBLE PARASITOIDS

From 2017 to 2020, cocoons of possible parasitoids encountered during debarking were collected from emerald ash borer overwintering chambers and reared to maturity. Adult wasps that successfully emerged were identified and photographed by M. Bertone. We note that our technique did not allow for identification of larval endoparasitoids and those failing to emerge, disallowing estimates of parasitism rates. Three emerald ash borer-infested trees additional to the ones used for debarking were harvested at the Butner site on 13 Mar 2019 to better screen for emergence of overwintering parasitoids. These trees were sectioned into logs of a length that would fit into 61 cm L × 61 cm W × 49 cm H laboratory built Plexiglas emergence cages (n = 42 logs in 30 cages), held at 24 to 25 °C, and checked every 1 to 3 d for emerged Hymenoptera. The insects have been deposited in the North Carolina State University Insect Museum, Raleigh, North Carolina, USA.

EMERALD ASH BORER DEVELOPMENT

In 2017, 252 degree d already had accumulated near the field sites by 28 Feb; the majority (85%) of emerald ash borer collected (n = 59) had developed to the pupal stage. In 2018 and 2019, a combination of earlier log harvesting and a winter season more typical for the area led to a better reflection of emerald ash borer development prior to the onset of spring warming (harvested at 78.3 and 76.6 degree d, respectively). In both yr, more than 92% of emerald ash borer overwintered as larvae, with just a small percentage classified as J-larvae (Table 1).

Urogomphi length indicated that all collected emerald ash borer larvae fell into the range reported for the fourth instar in 2018; in 2019 all but 1 larva (n = 466) measured as fourth instar (Fig. 1). The lone exception measured 0.70 mm; however, 2 additional larvae measured at 0.75 mm, which might be interpreted as third instar according to the criteria presented by Wang et al. (2005).

PARASITOIDS ASSOCIATED WITH EMERALD ASH BORER GALLERIES

Three specimens identified as Xorides (Exomus) humeralis (Say) (Hymenoptera: Ichneumonidae) (Fig. 2E) were dissected from cocoons collected from emerald ash borer galleries in 2017 (reported in Bertone et al. 2017). In 2018, 7 adult specimens of Atanycolus cf. cappaerti Marsh and Strazanac (Hymenoptera: Braconidae) (Fig. 2D) were reared from cocoons. One specimen of Eurytoma sp. (Hymenoptera: Eurytomidae) also emerged from the collected material (Fig. 2B), but because Eurytoma is a known hyperparasitoid of Atanycolus (Cappaert & McCullough 2008), it is uncertain whether this wasp was parasitizing emerald ash borer or Atanycolus. In 2019, 6 adult specimens of Atanycolus cf. cappaerti and 1 Spathius sp. (Fig. 2C) were reared from cocoons collected from the emerald ash borer galleries. The single Spathius specimen did not match any species known (in litt. Robert Kula, USDA-SEL), including species introduced for control of emerald ash borer (S. agrili and S. galinae). Currently, it appears to be an undescribed species, though more specimens are needed to assess that hypothesis and formally describe the species. Three possible parasitoids of emerald ash borer collected from emergence cages await further study: a species of Atanycolus, a species of Wroughtonia, and the species of Spathius mentioned above. Morphologically, the Atanycolus identifies as Atanycolus cf. cappaerti, but a BLAST analysis (Basic Local Alignment Search Tool) places it closer to Atanycolus hicoriae Shenefelt (Hymenoptera: Braconidae); these 2 species are difficult to distinguish (Marsh et al. 2009).

Fig. 1.

Length of the terminal process of emerald ash borer (Agrilus planipennis) larvae collected under bark in the winters of 2017 to 2020 (n = 547 total).

Table 1.

Developmental stages of overwintering emerald ash borer (Agrilus planipennis) during winter in Granville County, North Carolina, ranked in order of increasing accumulated degree d on the date of tree harvest. Degree d were based on model of Duarte 2013 OSU IPPC, base 50, Version 5.52.

EMERALD ASH BORER DEVELOPMENT IN NORTH CAROLINA

Overall, our results indicate that emerald ash borer development is synchronous in north-central North Carolina, with the majority overwintering as fourth instar larvae that advance to the J-larva stage as degree d accumulate in the new yr. Although 15% of collected emerald ash borer in 2017 were classified as fourth instar larvae by measuring their urogomphi, these likely were prepupae, but not noted as such at the time of collection. Prepupae are the stage between J-larva and pupa, when the larva contracts prior to pupation; urogomphi length, however, remains the same in fourth instars, J-larvae, and prepupae. The distinction between fourth instars and prepupae is not crucial, because both are likely to develop to the emergent adult stage in early spring. On the other hand, a recent laboratory study (Jian et al. 2021) suggests that only those emerald ash borers overwintering as J-larvae emerge as adults when the growing season begins, because it is the only stage that undergoes an obligatory diapause. All other larval stages seem to require another growing season to reach adulthood. Only field sampling that continues throughout the yr will determine if this is the case in North Carolina.

A key result is that no early instars overwintered, thus supporting the idea that the different life cycle in the South may influence the timing of release of parasitoids for classical biological control and the subsequent establishment of these wasps in the southern US. It has been noted that as one moves from north to south in the US, the proportion of emerald ash borer overwintering as fourth instar larvae versus prepupae decreases until in the Deep South it reaches 0% (Jones et al. 2020). Although we acknowledge the limitations of primarily using a single sampling site in our study, proportions of the different developmental stages overwintering would depend not only on the location but the yr, because winter temperatures can vary widely in North Carolina. In 2019, for example, there were 77.5 accumulated degree d on 5 Feb at our experimental site, but 131.7 degree d on 4 Feb in 2020 (Table 1).

Fig. 2.

(A) Balcha indica (Eupelmidae); (B) Eurytoma sp. (Eurytomidae); (C) Spathius sp. (Braconidae: Doryctinae); (D) Atanycolus cf. cappaerti (Braconidae: Braconinae); (E) Xorides humeralis (Ichneumonidae: Xoridinae). Not to scale. Photographs by Matt Bertone.

EMERGED PARASITOIDS

Recorded hosts of X. humeralis in the US include Dicerca Eschscholtz (Coleoptera: Buprestidae) (Porter 2005), but other species of Xorides are reported to parasitize Agrilus in Michigan and Europe (Kenis & Hilszczanski 2007; Petrice & Haack 2014). Related species in the genus use emerald ash borer as hosts in China (Wei et al. 2004; Wang et al. 2016). Wroughtonia species are known parasitoids of Coleoptera, but may attack both Cerambycidae and Buprestidae (Taylor et al. 2012). We collected several adults of the cerambycid Obrium rufulum Gahan (Coleoptera: Cerambycidae) from our emergence cages, and therefore cannot be certain if Wroughtonia was attacking O. rufulum, emerald ash borer, or possibly both.

Atanycolus is a cosmopolitan genus of solitary ectoparasitoids that attack the larvae of various wood-boring beetles (Wang et al 2009). There are at least 7 species throughout the Holarctic that use emerald ash borer as host (Taylor et al. 2012), but exact determination of Atanycolus species is extremely difficult (Abell et al. 2012), and individual Atanycolus species often attack both buprestid and cerambycid larvae (P. Marsh, personal communication). Atanycolus cappaerti is native to North America and appears to be the native species with most potential to have a role in managing emerald ash borer (Cappaert & McCullough 2008). It has been considered a candidate for potential mass rearing for augmentative biocontrol (Taylor et al. 2012; Duan & Schmude 2016).

In Michigan, A. cappaerti emerges from overwintering cocoons and attacks emerald ash borer larvae that overwinter as early instars (Cappaert & McCullough 2008). It is thought that in a univoltine host, emerging wasps have a minimal window of opportunity and may not encounter suitable hosts (second, third, and fourth instar larvae) for most of their life span (Cappaert & McCullough 2008). It will be interesting then to see how host usage or the life history of this native parasitoid may have shifted to accommodate emerald ash borer in North Carolina, where emerald ash borer in the site we studied overwinters as late stage fourth instars, pupates in late winter or early spring, and emerges as adults shortly after. This and ongoing work will inform release decisions on future biocontrol efforts in the southern US.