Size of emerald ash borer in North Carolina, USA: Preliminary evidence for a sawtooth cline?

We test the hypothesis of Marshall et al. (2013) that in the United States there is a converse Bergmann's cline in body size of Agrilus planipennis (emerald ash borer, EAB), with the largest females ( x¯  = 12.6 mm length) collected at the most southern latitudes tested (37° N). In 2020, we employed three techniques to collect EAB at a North Carolina (NC) site located at 35.64° N (purple prism traps [PPTs], log emergence, Cerceris fumipennis biosurveillance). The study was repeated in 2021, but with green funnel traps replacing log emergence. EAB collected by C. fumipennis in three altitudinally disparate regions of NC were also measured. Overall, EAB collected in 2020 averaged 12.02 mm, with those emerged from logs significantly smaller than those from PPTs. Length of females collected from C. fumipennis at three elevations was not significantly different and averaged 12.01 mm. In 2021, females collected from funnel traps, PPTs and C. fumipennis were not significantly different and larger ( x¯  = 12.41 mm) than in 2020; eliminating the smaller log‐emerged EAB from the 2020 data set did not change the outcome. Mean EAB size in NC never reached the reported 12.6 mm at 37° N, regardless of the technique or altitude tested. Our expansion of the latitudinal range in which EAB body size has been studied may shift the proposed converse Bergmann's cline to that of a sawtooth or other non‐linear model, likely associated with a transition in EAB voltinism at or near NC latitudes.


INTRODUCTION
In the United States, the invasive woodboring beetle emerald ash borer [(EAB) Agrilus planipennis Fairmaire (Coleoptera: Buprestidae)] was first observed in Michigan in 2002.In the 20+ years since then, it has expanded its distribution into the eastern, southern and midwestern parts of the country, and into the western state of Oregon.It is now confirmed in 36 states and the District of Columbia (Emerald Ash Borer Information Network, 2022).Because of the location of its initial invasion, most studies on the biology and life history of EAB have been implemented in the northern United States.Regional climatic differences, however, may affect the applicability of these results to other geographic regions of the country (e.g., Abell et al., 2019;Bohannon, 2022;Bohannon et al., 2022;Nalepa et al., 2021).Emerald ash borer was first detected in North Carolina (NC) in 2013 and is now reported from 63 counties in the state.
Consequently, we began studying the basic biology of EAB in the south-eastern part of its current range.
The present study focuses on adult body size of EAB, as it affects a variety of life history traits that have fitness consequences in organisms (Chown & Gaston, 2010;LaBarbara, 1989).In 2013, Marshall et al. demonstrated that EAB exhibited a converse Bergmann's cline.
They showed that adult female EAB body size was negatively correlated with latitude, with the largest females occurring in the more southern sites sampled within the United States.Overall, female EAB removed from purple and green prism traps in their study averaged 12.2 mm in length, but those sampled from the most southern site, just north of 37 N latitude in Missouri, measured 12.6 mm.These authors additionally demonstrated that larger females produced more eggs, leading to the prediction that more southerly populations of this pest may expand more quickly than populations established at higher latitudes.
This study was undertaken to test the hypothesis that female EAB are larger in their recently expanded range into southern regions of the United States.To do so, we used two approaches.
First, we compared the body length of female EAB captured at a site in central NC, using four sampling methods over 2 years.In 2020, EAB were collected biweekly from purple prism traps (PPTs); collected as they emerged from infested ash logs; and retrieved from Cerceris fumipennis Say (Hymenoptera: Crabronidae) as the wasps returned from hunting trips carrying their buprestid prey.In 2021, we repeated the collections from the PPTs and from C. fumipennis, but instead of log-emerged beetles, we collected EAB from green funnel traps.Analyses of these data allowed for comparison among the collection techniques, with the goal of detecting any bias in the methods employed.
In the second approach, we compared the body length of EAB females captured by C. fumipennis in three geographically and altitudinally separated regions within the state: one site each in the Appalachian Mountains, the Piedmont and the Coastal Plain.The goal was to determine whether site elevation or local conditions influence female EAB body size in NC.

EAB collection from purple prism traps 2020 and 2021
At the Garner, NC site adult EAB were collected every 2 weeks from glue-coated USDA-APHIS-PPQ standard-issue PPTs (Francese et al., 2008) baited with (Z)-3-hexenol.Baits were regularly refreshed within the 60-day expiration period.Three traps were hung in each of the three ash stands along a transect running from the edge into the interior of the stands.Traps were located approximately 45-60 m apart along each transect and, using a telescoping pole, were hung 4.5-5.0m from the ground on the branch of a living ash tree.

EAB collection from green multifunnel traps 2021
Two green, Lindgren multifunnel traps consisting of 12 funnel units were deployed in each of the three ash stands.Adult EAB specimens were captured in a removable collection cup containing between 150 and 200 mL of propylene glycol as a preservative (Francese et al., 2011;Tobin et al., 2021).The traps were hung 30-70 m apart in the stand interior and 2.5-3 m from the ground.

EAB collection by Cerceris fumipennis wasps 2020 and 2021
Cerceris fumipennis biosurveillance (see Careless et al., 2014;Marshall et al., 2005) was conducted at a newly detected nesting aggregation site of the wasps on a baseball diamond (see Nalepa et al., 2012) on the grounds of South Garner High School about 550 m north of the Wrenn Road Facility in Garner (Figure 1b).

Biosurveillance consists of collecting buprestid beetles from
C. fumipennis females as they return to their nest after a hunting trip.
The wasps use these prey beetles to provision their larvae in the brood chambers of their underground nest.
Biosurveillance was conducted at the nesting site on 14 days, as weather allowed, between 5 June and 1 July 2020.The peak number of nests in the aggregation was 27.In 2021, biosurveillance was conducted at the same site on 10 days between 6 June and 30 June, with 22 nests in the aggregation at peak wasp activity.All EAB collected by the wasps were frozen on the same day they were collected, then measured within 3 days.The beetles were alive when frozen, but the  The sex of each EAB was determined under a microscope by looking for sexually dimorphic external characteristics, including the abundance of sternal setae in males and abdominal shape: a narrow, tapered abdomen in males, compared with the larger, rounded abdomen in females (Rodriguez-Saona et al., 2007;Wang et al., 2010).If there was any uncertainty in determining sex, the abdomen of the insect was dissected and the genitalia examined (e.g., Jendek & Nakládal, 2019).Dissections were commonly done when sexing EAB collected from PPTs, as the adhesive from the traps and subsequent cleaning often made it difficult to determine setal patterns.
Counts were log-transformed to help meet the assumptions of normally distributed residuals and homogeneity of variances.Analyses were conducted using analysis of variance (ANOVA) in Systat 13 (Systat Software, Inc., 2009) with significance based on an alpha level of 0.05.

Beetles on purple prism traps
Combined over the 2 years of the study, female EAB collected from PPTs at the Garner site averaged 12.20 ± 0.90 mm in length (mean ± standard deviation; n = 319) with a non-significant difference (F 1,317 = 1.87, p = 0.17) between the measurements taken in 2020 (n = 293) and 2021 (n = 26).

Beetles in multifunnel traps
The length of female EAB collected from multifunnel traps in 2021 (n = 127) was not significantly different (F 1,151 = 0.03, p = 0. 85) from those collected from PPTs in 2021 and averaged 12.40 ± 0.90 mm.

Garner site
In 2020, a total of 173 beetles were collected from C. fumipennis at the Garner nesting aggregation, 91 of which were EAB (52.6%).The first EAB was taken from the wasps on 7 June, and the last on 1 July, at which point the wasps in this site were at the end of their life cycle.Female EAB measured 12.07 ± 0.97 mm in length (n = 53), significantly larger than males (F 1,87 = 18.05, p < 0.001; n = 38), which measured 11.37 ± 0.66 mm.
In 2021, at the same nesting aggregation, 37 EAB were brought back to the nest by C. fumipennis: 23 females and 14 males.The body length of males was not significantly different between 2020 and 2021 (F 1,50 = 0.32, p = 0.57); females were larger in 2021 than in 2020, but not at the 0.05 significance level (F 1,72 = 3.67, p = 0.06; Figure 2a).Females were again significantly larger than males in 2021 (F 1,124 = 31.14,p < 0.001).

Mountain and coastal plain sites
There was no significant difference (F 2,81 = 0.484, p = 0.63) in female body length of EAB collected by C. fumipennis in three geographically and altitudinally disparate nesting aggregations in NC (Table 1).

Emergence from logs
A total of 1298 EAB emerged between 29 March and 22 April 2020 from the ash logs placed in emergence cages.Females were haphazardly selected from those that emerged each day (total n = 261).These were measured and averaged 11.84 ± 0.79 mm in length.

Comparison of techniques
There were significant differences in the length of female EAB from the Garner population in 2020 depending on the collection/ preservation technique (Table 2).Those EAB collected from PPTs were significantly larger than those that emerged from cut logs   1).
Collection technique did have some influence on results.Emerald ash borers that emerged from harvested logs had not undergone the typical maturational feeding period on ash leaves that precedes mating and egg development (Cappaert et al., 2005;Poland & McCullough, 2006).
This may have influenced body size, accounting for their slight but significantly shorter length when compared with those collected from PPTs (Table 2).It is possible that intersegmental membranes in the abdomen stretch as females feed, the gut fills and eggs begin maturing.2) suggests that Cerceris females were not choosing the larger EAB available as prey.
We acknowledge that it was fortuitous that a nesting aggregation of C. fumipennis was in the vicinity of our research site.
Our results do not fit the converse Bergmann's cline reported by Marshall et al. (2013), but insect body size trends with latitude or altitude are highly idiosyncratic.Different insect species may exhibit Bergmann's clines, converse Bergmann's clines, non-linear clines or display no detectable clinal trend (Blankenhorn & Demont, 2004;Shelomi, 2012).Terrestrial insects exhibit these diverse geographical patterns because body size is the result of complex interactions among multiple drivers, including temperature, photoperiod, season length, intraspecific larval competition, and resource quality and availability (Atkinson, 1994;Classen et al., 2017;Forrest & Miller-Rushing, 2010;Tseng et al., 2018;Zeuss et al., 2017).
Although there are numerous explanations for patterns of latitude-related variation in insect body size, the mechanisms responsible for intraspecific geographic variation in body size are not well known.Patterns and mechanisms of body size variation are often conflated, leading to some degree of confusion (Blackburn et al., 1999).Here, we have described patterns of intraspecific size variation of EAB in NC; the associated drivers and mechanisms are yet to be determined.Two notable results from this study nevertheless deserve further discussion.
First, females were significantly larger in 2021 than in 2020 at the Garner research site.Male EAB collected by Cerceris in our study averaged 11.40 mm (about 93% of female length) and were not significantly different between the 2 years of the study.The shift in female body size between successive years at a single site in NC is suggestive of phenotypic plasticity, but further study is required to resolve the issue (e.g., Austin et al., 2022).Emerald ash borers are known to exhibit phenotypic plasticity, defined as the ability of a genotype to exhibit a range of phenotypes in response to variation in the environment (Fordyce, 2006;Snell-Rood, 2012) in other studied life history traits (e.g., cold tolerance: Duell et al., 2022).Because they have restricted mobility and are unable to determine their own nutritional situation, tree-boring larvae need flexibility to optimally exploit the T A B L E 2 Comparison of body length of female emerald ash borers using three collection techniques at one site in Garner, North Carolina in 2020 and 2021.habitat available to them (Andersen & Nilssen, 1983).Season length is notably variable at NC latitudes (Nalepa et al., 2021: Table 1), but additional growing time does not always serve to increase adult size (reviewed by Chown & Gaston, 2010).Diet quality and larval competition are also influential (Garcia-Robledo et al., 2020;Gardner et al., 2011).The marked decline in the ash trees at the Garner site (Figure 1a) accompanied by a steep drop in the number of EAB collected on the same number of PPTs between 2020 (n = 293) and 2021 (n = 26) are indicative that host quality had deteriorated.Ash health assessments conducted on 26 July 2021 estimated that these trees experienced approximately 90% mortality from EAB (Bohannon, 2022;Bohannon et al., 2022).The influence of host condition on larval success and adult size nonetheless depends on a number of interacting factors (Jennings et al., 2014;MacQuarrie, 2020).
For example, as host quality declines, EAB intraspecific competition may increase prior to the population crash of the invasion wave, resulting in larval cannibalism (Duan et al., 2013;Lyons, 2015) and consequent shifts in adult EAB size.
Second, the smaller EAB body size at NC latitudes relative to Missouri latitudes may be associated with a shift in voltinism.A major reason that ecographical patterns of body size are still a matter of debate is because the number of generations per year (voltinism) and its relationship with body size have been neglected; there are tradeoffs between voltinism and body size (Horne et al., 2018;Zeuss et al., 2017).In the northern United States, the life cycle of EAB is well characterized.It is semivoltine, completing its life cycle in 1 or 2 years.
Not every member of each generation reaches the pupation stage in a given year, resulting in an asynchronous pattern of development and overlapping life stages (Cappaert et al., 2005;Duan et al., 2010;Tluczek et al., 2011).The life cycle of EAB at the Missouri site, to our knowledge, has not been studied.Consequently, it is unknown what percentage of the EAB population at 37 N latitude may be semivoltine versus univoltine, although a model by Gould et al. (2020) predicts that this site is near the shift from semivoltinism to univoltinism.
North Carolina appears to be at or near a geographic life cycle transition point where nearly all EAB larvae develop to a prepupal stage in a single year, that is, EAB is univoltine (Bohannon, 2022;Bohannon et al., 2022;Nalepa et al., 2021).The current results indicating that EAB body size in NC is smaller than expected if it were to fit a converse Bergmann's cline may be associated with this life cycle shift.
Nonetheless, we cannot reject Marshall et al.'s (2013) proposal that EAB may spread more quickly at southern latitudes.A univoltine life cycle indicates that all EAB within a given population emerge as adults and lay eggs each year, whereas in semivoltine areas, adult emergence and oviposition of a given cohort is at least partially staggered over 2 years, which may have an impact on rates of population growth.A univoltine life cycle also has major implications for developing a successful biological control program for EAB at southern latitudes.This is because, in a given year, larval stages susceptible to parasitism (typically third and fourth instars) are not abundant prior to the reproduction of adult EAB emerging in a population (Bohannon et al., 2022).
This constrained seasonal availability of parasitoid-susceptible EAB larvae would reduce appropriate host material for spring-emerging parasitoids, potentially preventing the successful establishment of these parasitoid species (Bohannon, 2022;Bohannon et al., 2022;Gould et al., 2020;Nalepa et al., 2021).
Converse Bergmann's clines exist because development time and body size are predicted to increase along a gradient of additional season length (Kivelä et al., 2011).However, sharp decreases in both traits are expected at season lengths that facilitate the emergence of a new generation within the year, because time available per generation decreases at these transitions (Kivelä et al., 2011;Sukhodolskaya et al., 2020).The outcome is that clinal variation is reversed at specific latitudes.This results in a sawtooth clinal pattern (Figure 2b), because after this inflection point the trend towards a larger body size with decreasing latitude continues (Hu et al., 2012;Kivelä et al., 2011;Roff, 1980).Sawtooth clines typically occur in species with long developmental times in relation to season length, that is, where converse Bergmann's clines are expected (Blankenhorn & Demont, 2004;Chown & Gaston, 2010).In nearly every case where a sawtooth cline is found, shifts in voltinism occur around the latitudes or elevations where cline directions change (Shelomi, 2012).
North Carolina is geographically and climatically notable, because life cycle shifts in other insects have been documented in the state.
For example, a shift in voltinism of the striped ground cricket Allonemobious fasciatus occurs at NC latitudes and is associated with a drop in body size in the transition zone (Mousseau & Roff, 1989)

F
I G U R E 1 (a) Stand of green ash Fraxinus pennsylvanica Marshall (Oleaceae) at our research site in Garner, NC, photo taken 7 July 2021; note high tree mortality.(b) Map of the Garner research site indicating location of the Cerceris fumipennis aggregation in relation to the three stands of ash.(c) Plexiglas ® emergence cages used to collect emerald ash borer adults emerged from infested ash logs.
majority had been paralysed by the wasp.Body lengths of female EAB collected by C. fumipennis were also obtained from two additional nesting sites in NC and were included in this study with the goal of detecting any altitudinal variation.(1) A ball diamond on the grounds of Mountain Heritage High School, Burnsville, in the mountains of NC [35.91107N, 82.33273 W, elevation 861 m (2825 ft)].(2) A ball diamond on the grounds of Faith Christian Academy, Goldsboro, in the Coastal Plain of NC [35.39785N, 78.01230 W, elevation 33 m (108 ft)].Emergence from felled logs 2020 On 20 March 2020, six EAB-infested ash trees (diameter breast height 15.5-22.4cm) at the Garner research site were harvested near ground level and the bole sectioned into logs 55 cm in length.Two trees from each of the three stands (total 6 trees, 60 logs) were harvested.Logs were brought to the North Carolina Department of Agriculture & Consumer Services Beneficial Insects Laboratory in Cary, NC.The ends of the logs were covered with Parafilm ® to retain moisture, placed into 61 cm L Â 61 cm W Â 49 cm H Plexiglas ®emergence cages at room temperature and monitored daily for the appearance of adult EAB (Figure1c).Emerged adults were placed in a freezer until they were immobile and then measured.
Measuring and sexing the insectsAs in theMarshall et al. (2013) study, the length of adult EAB was measured from the dorsal aspect using a Mitutoyo Absolute Digimatic Caliper (0.01 mm) from the top of the head to the tip of the abdomen.Males of Cerceris-collected EAB were measured to determine sexual dimorphism in body length.

(
F 1,552 = 22.62, p < 0.001).The length of EAB collected by C. fumipennis was intermediate between these two extremes and not significantly different from either technique.Overall (all methods within a year combined), female EAB were significantly larger in 2021 than in 2020 at the Garner research site (F 1,781 = 0.32, p < 0.001).Eliminating the significantly smaller log-emerged EAB from the 2020 data set (Table 2) did not change the outcome (F 1,520 = 9.22, p < 0.01).Female body size of PPT + Cerceris-collected EAB combined averaged 12.16 ± 0.89 mm F I G U R E 2 (a) Body length of female and male Agrilus planipennis (EAB) collected by Cerceris fumipennis at the Garner research site in 2020 and 2021.The length of each box shows the range within which the central 50% of values fall.The central horizontal line marks the median, and bars indicate the complete range of values.Female EAB were larger in 2021, but not at the 0.05 significance level (F 1,72 = 3.67, p = 0.06).(b) Depiction of theoretical sawtooth cline of EAB body size in the United States.It is unknown what percentage of the EAB population may be semivoltine versus univoltine at the Missouri (MO) site.NC = North Carolina.T A B L E 1 Comparison of body length of female emerald ash borers collected by Cerceris fumipennis in three geographically disparate regions of North Carolina in 2020.
Emerald ash borers collected by C. fumipennis may give the most efficient estimate of EAB body length.Beetles are collected by the wasp during most stages of the adult EAB life cycle, and the insects were alive (and most paralysed) when frozen prior to measurement.This technique avoids the bother of specimen removal from sticky traps and eliminates subsequent cleaning of the samples prior to measurement.The non-significant difference between the length of PPT and C. fumipennis collected EAB in both years (Table (Abell et al., 2019) data from additional US states where EAB is established are essential before we can reject the hypothesis that EAB fits a converse Bergmann's cline.Of particular relevance would be studies of EAB body size in additional populations bracketing the 40th parallel, especially in those locations where EAB populations are reported to be univoltine: Washington, D.C (38.91 N) and Maryland (39.16 N)(Abell et al., 2019).Because EAB is currently established in the United States at latitudes lower than NC (e.g., South Carolina, Georgia, Alabama, Louisiana, Texas), body size data from these locations are also necessary before a definitive pattern can be established.