JHR 96: I-20 (2023) ages -&,* JOURNAL OF. “Sees

doi: |0.3897/jhr.96.99307 RESEARCH ARTICLE ) Hymenopter a
g

https://jhr.pensoft.net ‘The International Society of Hymenopterisss RESEARCH

Detection and description of four Vespa mandarinia
(Hymenoptera, Vespidae) nests in
western North America

Chris Looney', Brant Carman', Jenni Cena', Cassie Cichorz', Vikram Iyer’,
Jessica Orr', Nathan Roueché', Karla Salp', Jacqueline M. Serrano?, Landon Udo'!,
Paul van Westendorp’, Telissa M. Wilson', Rian Wojahn', Sven-Erik Spichiger'

| Washington State Department of Agriculture, 1111 Washington St SE, Olympia WA, 98501, USA 2. Paul
G. Allen School of Computer Science and Engineering, University of Washington, Seattle WA, 98101, USA
3 USDA-ARS Temperate Tree Fruit and Vegetable Research Unit, 5230 Konnowac Pass Rd., Wapato, WA,
98951, USA 4 British Columbia Ministry of Agriculture and Food, Animal Health Center, Abbotsford BC,
V3G 2M3, Canada

Corresponding author: Chris Looney (clooney@agr.wa.gov)

Academic editor: V. Lohrmann | Received 27 December 2022 | Accepted 17 February 2023 | Published 2 March 2023
https:/zoobank. org/EBE8F5 D9-C387-4712-AF5F-A9C624212D46

Citation: Looney C, Carman B, Cena J, Cichorz C, Iyer V, Orr J, Roueché N, Salp K, Serrano JM, Udo L, van
Westendorp P, Wilson TM, Wojahn R, Spichiger S-E (2023) Detection and description of four Vespa mandarinia
(Hymenoptera, Vespidae) nests in western North America. Journal of Hymenoptera Research 96: 1-20. https://doi.
org/10.3897/jhr.96.99307

Abstract

Vespa mandarinia Smith 1852 is a semi-specialized predator of other social Hymenoptera and one of the
two largest species of Vespa. Several individuals of this predatory wasp were detected in Canada and the
United States in 2019, including an entire nest that was located and destroyed on Vancouver Island, Brit-
ish Columbia. The Washington State Department of Agriculture and the United States Department of
Agriculture's Animal and Plant Health Inspection Service have collaborated to survey Washington State
for V. mandarinia since 2020, using traps staffed by agency personnel, collaborators from local govern-
ments and nongovernmental organizations, and the general public. Trap data and public reports were
used to select sites for live-trapping or net surveys, and live hornets captured in these efforts were subse-
quently collected and fitted with radio tags to locate nests. The survey ultimately led to the discovery of a
V. mandarinia nest in October 2020, and three nests in August and September 2021. All of the nests were

located within in red alder trees (Alnus rubra), with one just above the ground in a standing dead tree,

Copyright Chris Looney et al. This is an open access article distributed under the terms of the CCO Public Domain Dedication.

2 Chris Looney et al. / Journal of Hymenoptera Research 96: 1—20 (2023)

and the other three in cavities ~2 to 5 meters above the ground in living trees. The number of combs in
each nest varied between four and ten, cells between 418 and 1,329, and total hornets per nest between
449 and 1,474 (including immature and mature stages). Together, the four nests indicate an incipient
population of V. mandarinia in the Cascadia region, and ongoing action by local, state, provincial, and
federal governments, and residents of both countries is required to avoid the establishment of this exotic

species in the region.

Keywords

community science, Eradication, radio-tracking, social Hymenoptera

Introduction

Vespa mandarinia Smith, 1855, is one of the 22 species of hornet (Vespidae: Vespa)
(Smith-Pardo et al. 2020). Like all hornets, V. mandarinia is a eusocial predaceous
wasp that feeds on many different types of insects. One aspect of its behavior (shared
by the closely related V. soror) is the ability to mount group attacks on other social
Hymenoptera, including honey bees (Apis spp.), resulting in rapid destruction of entire
colonies of their prey (Matsuura and Sakagami 1973). This behavior makes it a peren-
nial pest of apiaries throughout its native range, and it is a notable predator of Apis
mellifera, which lacks effective defensive behaviors against V. mandarinia (Matsuura
and Sakagami 1973; Arca et al. 2014; Mattila et al. 2020).

The first verified North American specimens of V. mandarinia outside of ports of
entry were collected in British Columbia in 2019. Several wasps were observed that
year on Vancouver Island, British Columbia, culminating in the location and eradica-
tion of an entire nest in the city of Nanaimo in September 2019 (Bérubé 2020). In De-
cember 2019, V. mandarinia workers were found approximately 90 km SW in Blaine,
WA. The first confirmed specimen in the United States was a single dead worker that
was collected on a porch (Wilson et al. 2020). Two additional specimens that had been
collected earlier in the year were subsequently provided to the Washington State De-
partment of Agriculture (WSDA), both of which were associated with beehive attacks
— one hornet that had been “hawking” at a hive, and two specimens which were col-
lected from a killed colony. Two colony losses that strongly resembled descriptions of
V. mandarinia attacks in Japan (see Matsuura and Sakagami 1973) were also recorded
in fall 2019 in the same region, although wasp specimens were not collected at either
event. Based on the available evidence, WSDA, the United States Department of Agri-
culture’s Animal and Plant Health Inspection Service (USDA APHIS), and the British
Columbia Ministry of Agriculture implemented a robust trapping program in 2020,
with the goal of early detection and eradication of any nascent V. mandarinia popula-
tions. The program included trapping by agency personnel and the general public
and continuous public outreach to locate hornet populations. Because trapping hornet
workers will not lead to eradication, WSDA also developed a protocol for capturing
live hornets and tracking them to their nest, which could then be eliminated.

North American Vespa mandarinia nests i:

Public reporting

Washington State used active outreach to inform the public about V. mandarinia and
encourage residents to report sightings. Potential hornet sightings were received via
email, social media, phone calls, and through a purpose-built web application. All
sightings were reviewed and positive/probable reports were used to inform trapping
activities or initiate a site visit.

Lethal trapping

The basic survey approach was to use lethal traps and public reports to locate centers
of hornet activity, and then shift to capturing live hornets that could be tracked back
to nests. A variety of trapping approaches were identified from the literature and the
experiences of beekeepers in the hornet’s native range (Tatsuta and Makino 2003; Choi
et al. 2012; Paschapur et al. 2022). Although there is considerable diversity in trapping
approaches reported in the literature, many utilize a blend of fruit juices and ethanol.
WSDA used bottle traps armed with orange juice and rice cooking wine (Makino and
Sayama 2005; Okuda et al. 2011) because both components were available at gro-
cery stores and promoted consistency across our collaborator and community science
trapping programs. In 2020, traps were constructed from 1.65L (56 oz) clear plastic
bottles with 2 cm square openings on 3 sides (Fig. 1). Each bottle contained a bait/
killing solution comprising approximately 120 ml (4 oz) of orange juice and 120 ml
(4 oz) of rice wine of at least 10% ABV. Captured hornets would subsequently drown
in the bait. This approach was repeated in 2021, with the exception that the square-
shaped openings were replaced by star-shaped openings due to concerns that the hor-
nets might escape from the traps. This concern was raised when late-season captures of
other insects in 2020 were so massive that they formed a solid surface inside the traps,
allowing hornets to avoid the killing solution.

Traps maintained by WSDA were mostly placed in Whatcom County in north-
western Washington State and maintained from June through November, covering
the entire presumed area of potential hornet occupancy. In 2020, three traps were
placed per square km, within a 2 km radius from any detection made through May
2020. Trap density was decreased to two traps/km’ at a 4 km radius, and to one
trap/km* at an 8 km radius. The maximum distance for the 2020 trapping areas
was based on the maximum foraging distance of 8 km reported by Matsuura and
Sakagami (1973). Traps were placed on branches in trees at ca. 2-2.2 meters high,
depending on site characteristics and trapper height. In 2021, the trap density was
reduced to 1/km* throughout the trapping area, but the geographic area of trap
coverage was expanded. A series of traps testing alternative lures (e.g., isobutanol,
2-methyl butanol, and acetic acid; Landolt and Zhang 2016) was also maintained
at eight sites in 2020 during the trapping season. Those results are not analyzed in
this paper, but hornets caught using experimental lures are included in the results
reported here.

4 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

Figure |. A bottle trap used to survey for V. mandarinia by the Washington State Department of Agri-

culture, collaborating agencies, and community scientists.

Traps maintained by WSDA were inspected every 7 to 10 days, and each
trap action (e.g., trap placement, service, specimen collected) was recorded using
a smartphone. A short trap check interval was selected in part because the traps
lacked a preservative, with longer trap return intervals potentially compromising
DNA and morphological analysis of trap contents, in addition to being unpleasant
to service. The short interval also allowed the agency to rapidly respond to captures
and begin attempts to collect live specimens to track to a nest. In 2020, all trap
contents were strained, sorted for field detection of V. mandarinia, and all bycatch
collected into a sample bottle for further analysis. In 2021, contents were sorted
in the field to detect V. mandarinia, with one trap randomly selected daily by each
trapper for analysis of bycatch. Because trap contents were not returned to the
lab in 2021, the agency initiated a quality control program by placing a preserved
hornet in a random trap in each trapper’s field area once a week to ensure suspect
hornets were reported.

North American Vespa mandarinia nests 5

WSDA also recruited other agencies, nongovernmental organizations, and private
residents (i.e., community scientists) to employ the same trapping protocol and expand
the program more broadly in the state. This approach was also used in British Columbia,
with community scientists staffing most traps. Instructions for constructing and servicing
traps were provided on the WSDA website, and trap locations were logged by partici-
pants via an ArcGIS web app. These traps were located opportunistically and at the con-
venience of the participants, who were asked to check traps weekly. In 2020 the agency
requested participants to send all trap contents to the WSDA Entomology Laboratory
for analysis, in part to ensure hornets were not missed, and in part to analyze bycatch for
impacts on non-hornet species. In 2021 the agency only requested trap contents from
captures of potential hornets. In all, 1,681 traps were deployed in 2020 (797 WSDA, 263
collaborating agencies, 621 community science), and 1,648 total traps were deployed in
2021 (868 WSDA, 370 collaborating agencies, 410 community science) (Fig. 2).

Live trapping

An array of live traps was deployed near any hornet detections. Two live trap designs
were used. One was based on a modified bottle trap incorporating a screen to separate
the hornets from the attractant/kill solution. We also used translucent unitraps, again
with a screen to isolate hornets from the killing solution. The unitraps were further
modified by adding additional screened holes to the upper section to reduce heat and
fumes inside of the trap, and potentially increase the chemical plume released from the
baits (Fig. 3). Once deployed, live traps were checked daily, six days a week.

Tracking

Live hornets captured in traps or by net were chilled on ice and affixed with a radio tag
for tracking back to a nest. Bluetooth tags constructed at the University of Washington,
modified from tags developed to monitor bumble bee activity, were used for the first two
tracking attempts. Subsequent tracking efforts employed VHF radio tags produced by
Lotek (in 2020) or Advanced Telemetry Systems (in 2021). The initial attempt to glue
a tag to a captive hornet, following the approach used by Iyer et al. (2019, 2020) was
not successful. Bluetooth and VHF tags were subsequently attached by gluing them to
dental floss or Teflon thread, and then looping this around the petiole of the insect and
tightening it against the body (Fig. 4). After afhxing the tag to a captive hornet, it was
provided with commercially-produced jam or jelly, allowed to feed, and then followed
with antenna-enhanced cell phones (for the Bluetooth tags) or commercially-produced
receivers and Yagi antennas (for VHF tags) once the hornet commenced flying (Fig. 4).

Nest removal

Upon locating a nest, an electric vacuum with an in-line collection chamber was used to
capture as many worker hornets as possible, followed by physical removal of the nests.
The vacuum approach was preferred to insecticides to avoid contaminating sites and to

6 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

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Figure 2. A map of bottle traps maintained by WSDA B map of bottle traps maintained by private resi-
dents and collaborating agencies. (Trap sites in British Columbia were not typically logged on the website
and are thus underrepresented in these maps.) Map tiles by Stamen Design, under CC BY 3.0. Data by
OpenStreetMap, under ODbL.

North American Vespa mandarinia nests is

Figure 3. Two styles of live traps used to collect V mandarinia specimens for subsequent tracking back

to an active nest.

Figure 4. A antenna for Bluetooth tracking tag after Iyer et al. (2019) B V. mandarinia with afhxed
Bluetooth tag © V. mandarinia with ATS T-15 tag afhxed D VHF receiver and Yagi antenna (Advanced
Telemetry Systems).

8 Chris Looney et al. / Journal of Hymenoptera Research 96: 1—20 (2023)

facilitate safe study of nests following extraction. A 1% cyfluthrin dust was on hand for
any situations where the vacuum was impractical, but was never required. Once located,
hornets were vacuumed from the nest opening early in the morning, typically just before
or at dawn. After the majority of workers were captured using the vacuum, carbon dioxide
was used to anesthetize any hornets remaining in the nest. Nest openings were sealed with
foam or plastic wrap and the entire structure removed from the site for study, or, if hornet
activity was low, partially studied in situ. Live adult hornets collected with the vacuum or
found within the nests were placed on ice and transported to the laboratory for study. Pro-
tective suits constructed from a thick mesh were worn during all nest extraction activities.

Nest analysis

Nest cavities were measured in the laboratory or on site. Combs were separated, and
the depth and width of each cell was measured in the laboratory using a digital caliper,
with the exception of two combs from nest 4 that were provided to landowners before
they could be measured. ‘The caste of each adult hornet collected was recorded (queens
and workers were distinguished by size), and immature stages were counted as egg,
larva, or capped cell. Capped cells were opened for nest 4 and the sex of all pupae was
recorded. We did not distinguish between pupae and prepupae for the other three. Two
nests had abundant litter beneath the combs, which was retained and placed in Berlese
funnels to collect other insects living within the nest cavity.

A subset of hornets was collected alive at each eradication event, chilled, weighed,
and body length and mesosomal width measured with digital calipers. Length was
measured by pressing the chilled hornet gently onto a flat surface, positioning the head
so that it was vertical, and measuring between the frons and the tip of the metasoma.
Care was taken to ensure that the metasoma was not artificially extended by pushing
on it nor shortened by compressing it with the caliper. Mesosomal width was measured
at the wing hinge. Most hornets were well-chilled throughout the measurements; any
that showed signs of activity were chilled again and remeasured.

Results

Four hornets were found and one photograph was submitted by the public in 2019.
In 2020, 15 hornets were captured in WSDA or community science traps, one was
collected in a net by a WSDA entomologist, one was collected in a net by a commu-
nity member, and 17 confirmed hornet reports were received through photographs
or dead specimens. In 2021, four hornets were collected in traps, three were captured
by WSDA entomologists, one was captured by a community member, and three were
reported or found dead by community members (Table 1, Fig. 5).

Only a small amount of these detections led to opportunities to find nests. A home-
owner report in late September 2020 was followed by a site visit by a WSDA entomolo-
gist, who was able to capture a foraging hornet with a net. The following day a Bluetooth
tag was glued to the mesosoma of the hornet, but it failed to fly. It was initially supposed

North American Vespa mandarinia nests 9

@ Vespa mandarinia detections

oO Vespa mandarinia nests

Figure 5. Map of confirmed Vespa mandarinia specimens and nests recorded from British Columbia and
Washington State in 2020-2021. Details on the nest collected in British Columbia (not shown) in 2019
can be found in Bérubé, 2020. A single male specimen found in 2021 further south in Washington State
was unrelated to these sightings and is also not included here. Map tiles by Stamen Design, under CC BY
3.0. Data by OpenStreetMap, under ODbL.

that the wings were glued together while afhxing the tag, but later examination showed
that the wings were intact. We suspect instead that the hornet may have been chilled for
too long. The homeowner captured another hornet the following week, which was suc-
cessfully affixed with a Bluetooth tag by gluing it to floss and tying it around the hornet’s
petiole. The hornet successfully recovered and flew, but left the range of the antennas with-
in an hour and was not detected again. Four hornets were collected alive in nearby traps
the following week, two of which were affixed with VHF tags (NanoPin, Lotek Inc.). One
of these was subsequently tracked for approximately 240 m from the release site to the first
V. mandarinia nest located in the United States, and the second in North America. The
nest was found in a cavity within a living red alder (Alnus rubra Bong.), approximately 2.4
m above the ground (Fig. 6a). A few more hornets were subsequently found in the vicinity
of that nest after the nest was removed, but no more hornet nests were detected in 2020.
In 2021, several reports of hornets were received from a site located near the US/Can-
ada border. Upon intensive trap placement and survey of the area, WSDA entomologists
captured two hornets, which were subsequently tagged with VHF tags (T15, Advanced
Telemetry Systems). One of these was successfully followed for about 625 m to the second
nest detected in the US. Nest 2 was found within a completely dead alder tree, with
the entrance at roughly ground level and combs extending both into the tree and below

10 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

Table |. Vespa mandarinia specimens and confirmed sightings in British Columbia and Washington
State, 2019-2021.

Date State/County (US)/ regional Caste Method Number Notes
province district (BC)

Aug-Sep 2019 BC Vancouver Island W,Q found many Nanaimo nest; see Bérubé 2020 for more details

19-Oct-2019 WA Whatcom W found 2 workers collected from killed A. mellifera colonies

22-Oct-2019 WA Whatcom W found 1 dead worker, hornets observed hawking at apiary

13-Nov-2019 BC Fraser Valley Unk photo only 1

8-Dec-2019 WA Whatcom W found I dead worker found on porch

10-May-2020 BC Fraser Valley W _ hand capt. 1

13-May-2020 BC Fraser Valley Q found 1 queen found dead in garden

27-May-2020 WA Whatcom Q found 1 queen found dead in driveway

6-Jun-2020 WA Whatcom Q found 1 queen dead on porch

14-Jul-2020 WA Whatcom Q trap 1 orange juice/rice wine trap, WSDA

29-Jul-2020 WA Whatcom M trap 1 orange juice/rice wine trap, WSDA

17-Aug-2020 WA Whatcom Unk photo only 1

19-Aug-2020 WA Whatcom W trap 1 orange juice/rice wine trap, citizen science survey

21-Sep-2020 WA Whatcom W hand capt. 1 sprayed with pesticide

25-Sep-2020 WA Whatcom W trap 1 orange juice/rice wine trap, citizen science survey

29-Sep-2020 WA Whatcom Unk photo only 1

29-Sep-2020 WA Whatcom W hand capt. 1 collected by WSDA entomologist

30-Sep-2020 WA Whatcom W found 1 dead worker found in porch light

2-Oct-2020 WA Whatcom W trap 1 orange juice/rice wine trap, WSDA

5-Oct-2020 WA Whatcom W hand capt. 1 collected by private citizen

9-Oct-2020 BC Fraser Valley W trap 1 orange juice/rice wine trap

9-Oct-2020 WA Whatcom Ww trap 1 orange juice/rice wine trap, WSDA

15-Oct-2020 WA Whatcom W trap 1 orange juice/rice wine/honey bee comb, WSDA

15-Oct-2020 WA Whatcom W trap 1 isobutanol-acetic acid, WSDA

20-Oct-2020 WA Whatcom W trap 1 orange juice/rice wine/honey bee comb, WSDA

20-Oct-2020 WA Whatcom W trap 2 isobutanol-acetic acid, WSDA

21-Oct-2020 WA Whatcom W trap 2 orange juice/rice wine/honey bee comb/
isobutanol, WSDA

24-Oct-2020 WA Whatcom W ~~ nesterad. many US nest 1

27-Oct-2020 BC Fraser Valley M _ hand capt. 1 feeding on pumpkin

29-Oct-2020 WA Whatcom M found 1 sticky trap

29-Oct-2020 WA Whatcom W found 1 found dead in water bowl

29-Oct-2020 WA Whatcom Q found 3 found dead in water bowl

30-Oct-2020 WA Whatcom M trap 1 orange juice/rice wine trap, WSDA

1-Nov-2020 WA Whatcom M _ hand capt. 1 crawling in garage

4-Noy-2020 WA Whatcom W trap 1 orange juice/rice wine trap, WSDA

7-Nov-2020 BC Fraser Valley Q _ hand capt. 1 crawling in house

12-Nov-2020 WA Whatcom M _ hand capt. 1 crawling in driveway

4-Jun-2021 WA Snohomish M found 1 found dead in yard

12-Aug-2021 WA Whatcom W photo only 1 internet report

12-Aug-2021 WA Whatcom W hand capt. 1 collected by WSDA entomologist

13-Aug-2021 WA Whatcom W hand capt. 1 collected by WSDA entomologist

17-Aug-2021 WA Whatcom W _ hand capt. 1 collected by private citizen

8-Sep-2021 WA Whatcom W hand capt. 1 collected by WSDA entomologist

8-Sep-2021 WA Whatcom W trap 1 orange juice/rice wine trap, WSDA

9-Sep-2021 WA Whatcom WwW trap 1 orange juice/rice wine trap, WSDA

25-Aug-2021 WA Whatcom W ~~ nesterad. many US nest 2

10-Sep-2021 WA Whatcom W trap 1 orange juice/rice wine trap, WSDA

21-Sep-2021 WA Whatcom W = nesterad. many US nest 3

12-Sep-2021 WA Whatcom W hand capt. 1 crawling in yard

23-Sep-2021 WA Whatcom W ~~ nesterad. many US nest 4

22-Oct-2021 BC Fraser Valley W trap 1 dead in Japanese beetle trap

North American Vespa mandarinia nests inl

Figure 6. A nest 1 (23 October 2020) B nest 2 (25 August 2021) C nest 3 (11 September 2021) D nest
4 (23 September 2021). Arrows indicate location of nest entrance.

ground (Fig. 6b). Soon after the detection and removal of the second nest, a homeowner
captured a hornet using a makeshift trap, which was subsequently tagged and tracked
490 m from the release point to the third nest in the US, and fourth in North America. A
public report also led to the capture of a fifth living hornet in 2021, which was tagged and

12 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

tracked for 650 m to the fourth nest. Nests 3 and 4 were, like the first nest in 2020, located
within red alder trees well above ground (nest 3 — ~2.2 m, and nest 4— ~ 5.5 m; Fig. 6c, d).

All nest measurements and life stages are presented in Tables 2—6. ‘The nests varied in
the number of combs and total cells, ranging from 4 to 10 combs (Figs 7—8; Tables 2-4),
and 418 to 1,329 cells (Tables 2-6). Nest 1, found late in 2020, contained 76 queens,
and another 25 emerged while the nest was being studied. It was not possible to iden-
tify the foundress, which may have already died. Nine males were also recovered from
nest 1. Only a single queen was found in each of the other nests. In each of these, the
queen was not found until the nest was extracted, and in each case was the last adult
hornet to be collected in the nest. The only other nest that contained adult males
was nest 3 (the smallest), of which approximately 37% of adult hornets were males
(Table 4). All capped cells from nest 4 were opened, and found to contain 23 male
pupae, 112 female pupae, and 126 pre-pupae.

Table 2. Characteristics of V. mandarinia nest 1, collected 23 Oct 2020.

Comb Total cells Mean cell depth Mean cell width Eggs Larvae!’ Capped cells
1 238 21.82 10.34 0 0 0
2 212 44.49 11.52 0 6 y)
3 177 34.74 12.53 0 8 4]
4 137. 32.8 12.08 0 30 58
5 98 29.20 12.81 0 12 0
6 31 18.91 11.22 6 vi 0

‘Only larvae still in the combs are represented here. Many larvae fell from the combs when CO2 was applied to the nest while it was
still in the tree.

Table 3. Characteristics of Vv. mandarinia nest 2, collected 25 Aug 2021.

Comb Total cells' Mean cell depth Mean cell width Eggs Larvae Capped cells
i 79 21.66 9.65 18 12 28
2 86 24.40 10.56 12 9 62
3 129 26.47 10.78 25 52 47
4* 168 24.16 10.81 48 17 97
5 200 25.71 11.23 0 45 150
6 207 26.29 11.26 16 59 128
i 236 23.66 11.38 53 104 52
8° 170 27.39 11.75 66 104 0
9 54 22.06 11.53 54 0 0
* One cell with 2 eggs.

* One cell with a larva and an egg.
*Two cells with 2 eggs.

Table 4. Characteristics of V. mandarinia nest 3, collected 11 Sep 2021.

Comb Total cells Mean cell depth Mean cell width Eggs Larvae Capped cells
1* 178 25.79 10.12 28 24 88
2 143 26.32 10.89 16 44 79
3 92 23.46 11.70 42 33 12
4 5 10.97 9.35 5 0 0

* One cell with 2 eggs.

North American Vespa mandarinia nests 13

Table 5. Characteristics of V. mandarinia nest 4, collected 23 Sep 2021.

Comb Total cells Mean cell depth Mean cell width Eggs Larvae Capped cells
1 93 22.75 9.80 6 20 31
91 23.89 10.28 2; 15 52
3 88 25.75 11.08 2 16 56
4* 77 unmeasured unmeasured 17 37 17
5 57 31.99 12.50 1 2 49
6 62 unmeasured unmeasured 4 17 40
7 59 30.47 12.35 6 44 16
8 59 27.40 11.75 15 40 0
9 53 24.43 12.04 40 11 0
10 35 24.52 11.76 35 0 0

* One cell with 2 larvae, both appeared to be 2” instar.

Table 6. Overview of Vespa mandarinia nests found in the United States, 2020—202021.

Nest Collection date Combs Total cells Eggs Larvae Capped cells Workers Males Queens

1 23 Oct 2020 6 893 6 190° 108 112 9 76
2 25 Aug 2021 9 1329 292 422 564 195 0 1
3 11 Sep 2021 4 418 91 101 179 49 28 1
4 23 Sep 2021 10 674 128 202 261* 185 0 1

*23 of the capped cells contained male pupae, 112 contained female pupae, and 126 were prepupae.

A subsample of 366 hornets comprising 15 males, 249 workers, and 102 queens
was measured from the four nests. Worker mass ranged from 0.36 g to 1.41 g, and
males from 0.82 g to 1.3 g. Queens ranged between 1.84 g and 2.88 g (Fig. 9). The
three foundress queens massed 2.7 g (nest 2), and 2.16 g (nests 3 and 4). Length of
males ranged from 27.8 to 35.19 mm, workers from 21.83 to 37.08 mm, and queens
from 36.93 to 44.15 mm. Mesosomal width of males was between 7.3 to 8.99 mm,
workers from 6.2 to 9.1 mm, and queens from 8.94 to 10.96 mm. While queen mass
was always appreciably larger than workers and males, there was slight overlap at the
extremes of length and mesosomal width for a few individuals.

Other insects located in the nest included species of Staphylinidae, Elateridae,
and Cantharidae commonly associated with decaying tree environments. Two species
of flies were common in two nests, and have been recorded in other Vespidae nests
(Table 7). Other invertebrates (e.g., Acari, Collembola, Annelida) were also collected
in the Berlese funnels but were not enumerated.

Discussion

The nests located in the Pacific Northwest were generally somewhat smaller than those
described by Matsuura and Sakagami (1973). The nest removed on Vancouver Island
in mid-September 2019 contained approximately 400 cells and 200 adult hornets and
was located in a subterranean burrow (Bérubé 2020). The largest nest reported here,
collected in late August 2021, contained 1,329 cells, which is close to the size of the

14 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

Figure 7. Top — combs from nest 1, 23 Oct 2020; Bottom — combs from nest 2, 25 August 2021. Num-

bers next to the combs indicate the position in the nest (smaller numbers are older and higher in the nest).

Comb characteristics are recorded in Tables 2—5.

North American Vespa mandarinia nests 1S

Figure 8. Top — combs from nest 3, 11 September 2021; Bottom — combs from nest 4, 23 September
2021. Numbers next to the combs indicate the position in the nest (smaller numbers are older and higher

in the nest). Comb characteristics are recorded in Tables 2—5.

16 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

Mass and width of V. mandarinia
collected in the United States

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oOo males
© queens
0.5 1.0 1.5 2.0 2.5
Mass (g)
Length and width of V. mandarinia
collected in the United States

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es
Ss
x fo)
5
=
ow
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NR

workers
© males
© queens

25 30 35 40 45
Length (mm)

Figure 9. Body measurements of V. mandarinia collected from four nests in North America. The three
foundress queens are represented with solid circles.

North American Vespa mandarinia nests LF

Table 7. Insects collected from litter below three V. mandarinia nests in Washington State, USA.

Order Family Species Nest
Diptera Scatopsidae Coboldia fuscipes (Meigen, 1830) 1,4

Scatopse notata (Linnaeus, 1758) 1
Phoridae Dohrniphora cornuta (Bigot, 1857) 1

Triphleba lugubris (Meigen, 1830) 1, 3,4
Megaselia sp. 4
Sphaeroceridae Minilimosina parva (Malloch, 1913) 1
Milichiidae Leptometopa latipes (Meigen, 1830) 1
Fanniidae Fannia incisurata (Zetterstedt, 1838) 1
Coleoptera Staphylinidae Quedius sp. 1
Hylota ochracea Casey, 1906 1
Phloeopora oregona Casey, 1906 1
Crataraea suturalis (Mannerheim, 1830) 3

Silusa californica (Bernhauer, 1905) 1,4

Scydmaenus ovipennis Casey, 1897 1
Euplectus confluens LeConte, 1849 4
Lobrathium subseriatum LeConte 1880 4
Medon pugetense Hatch, 1957 4
Elateridae Limoniscus sp. 1
Leiodidae Ptomaphagus nevadicus Horn 1880 1
Cantharidae Silis lutea LeConte, 1853 1
Corylophidae Sericoderus lateralis (Gyllenhal, 1827) 3
Histeridae Bacanius hatchi Wenzel, 1960 3

smallest nest (collected in December) reported by Matsuura and Sakagami (1973). It is
possible that the August nest would have continued to grow and may have approached
the more typical size reported for nests in Japan. However, nests described by Matsuura
and Sakagami (1973) were collected from south-central Japan, and more southerly
nests may be able to expand more quickly to large sizes. Although details about nests
from northern parts of the hornet’s range are sparse, two nests collected on Hokkaido
(Yamane and Makino 1977) each comprised five combs, with 675 cells (col. Aug 26,
1973) and 1141 cells (col. Sep 15, 1976). The nests removed in Washington State and
British Columbia seem to be in accord with these northern records, so it is also possible
that the nests we removed were typically sized for this latitude and climate.

One factor that may have impacted nest size and shape, particularly for nests 1,
3, and 4, was the constraining geometry of the tree cavity they were located within.
The shape of the combs mirrored the internal shape of the cavities, and it is possible
that workers could not use the space as efficiently as a nest in excavated soil. Indeed,
the nest with the greatest number of cells reported here was nest 2, collected in August
and the only one of the Washington nests not wholly confined to a tree cavity. Nest 3
seemed exceptionally small and contained a high proportion of males early in the sea-
son. [he high number of males so early in the season is suggestive of inbreeding effects
causing the production of diploid males (Van Wilgenburg et al. 2006; Darrouzet et al.
2015), which may have contributed to the very small nest size. Body measurements
of all castes indicate that the largest workers and smallest queens could be confused if
mesosomal width or body length is the only characteristic available for distinguishing
between them, a phenomenon observed in other Vespidae (O’Donnell 1998).

18 Chris Looney et al. / Journal of Hymenoptera Research 96: 1-20 (2023)

It is interesting that all of the nests we located were in tree cavities in alder trees,
with three of them high above the ground in still-living trees. Even though this is a
small sample size, it is unexpected based on the most comprehensive reports of other
nest sites (Matsuura and Sakagami 1973), where less than 16% of nests were reported
from any sort of tree cavity. However, other data suggests that nests in cavities are some-
what more frequent (Choi et al. unpublished), and two of the three V. mandarinia nests
reported by Yamane and Makino (1977) from Hokkaido were in tree hollows, although
no further description is provided. Even so, the proportion of tree cavity nests in our
results seems unusual. This could be a result of heavily saturated ground in the study
area at the time queens are establishing nests, with more than 650 mm of total pre-
cipitation typical during the winter and spring months. Alternatively, nests from 2021
could have been established by queens that successfully dispersed from the 2020 nest
after imprinting on the cues of their natal home, biasing nest selection towards alders.

No hornets were detected in British Columbia or Washington State in 2022. It is
too early to feel confident that the species has been prevented from establishing, and
several years of survey remain to be conducted. Some of the findings described in this
paper suggest that small population effects may be impeding establishment, i.e., the
unseasonably high number of males observed in nest 3. However, the characteristics
of the other three nests, and our observations of foraging behavior and analysis of the
local prey base (unpublished) concur with climate modeling (Alaniz et al. 2020; Zhu
et al. 2020; Nufez-Penichet et al. 2021) in suggesting that the region provides viable
habitat for this species. Those results, and the recent spread of other hornet species
outside of their range in multiple countries, are evidence that Vespidae require further
study to either prevent, or mitigate the effects of, future introductions.

Acknowledgements

This project was the culmination of collaborative work by multiple agencies and numer-
ous members of the public. We are grateful to the Day, Bovenkamp, Shelton, Beard,
DeJong, Polinder, Morin and Tompkins, Kreider, Revak, Jordan, Tjoelker, Rodenberg,
and Barrett families for their hospitality and property access. We are indebted to Philip
Bovencamp and Dean Tjoelker for their hornet-hunting prowess and observations of
hornet behavior. Ted McFall, Ruthie Danielsen, and the Mt. Baker Beekeepers Associa-
tion have been tireless partners in survey, field experiments, and public outreach. We are
deeply indebted to the thousands of residents of Washington, British Columbia, and
beyond, who have reported observations, facilitated property access, and staffed their own
traps throughout this project. Dr. Peter Kennedy, University of Exeter, Cornwall, provid-
ed invaluable advice about radio-tagging Vespa species. Dr. Miriam Cooperband shared
insights into radio-tagging insects and provided equipment and training leading to our
first successful tracking. Shawn Cleveland provided timely instruction on how to best use
radio-telemetry equipment to track wily, fast-moving animals. Zach Techner of Cascadia
Venom Collection graciously showed us how to vacuum swarming, stinging wasps. We
are grateful for the assistance of Jake Bodart, Jessica Rendon, and Austin Johnson (Oregon

North American Vespa mandarinia nests 19

Department of Agriculture) in tracking nest 2 in 2021. Camilo Acosta, Andrew Bigelow,
Ryan Gelwicks, Brian Henderson, Stacy Herron, Soraya Jessa, Diane MacLean, Kris-
ten Mason, Hadley Ocheltree, Romie Pugh, Olivia Schmit, Katie Simon, Luke Turner,
and Ciara Varnum-Lowry were tireless field trappers throughout this program. Quinlyn
Baine, Chanda Bartholomew, Warren Hellman, Brandy Kamakawiwo ole, Wade Peters-
en, and Angela Yoder sorted the contents of more than 18,000 hornet traps in the first
year of this project. Dr. Dina Roberts helped rear hornet queens and suggested approaches
for measuring hornet nests. We are grateful to Rod Rood and Dr. Brian Brown for provid-
ing identifications of beetles and flies (respectively) collected from the hornet nests. We
are very thankful for the guidance and support of Anne LeBrun, Dr. Todd Gilligan, Josie
Ryan, and Dr. Tim St. Germain (USDA-APHIS) throughout this project. This project
was funded via the USDA-APHIS Plant Protection Act (PPA) Section 7721 and by the
State of Washington. Mention of trade names or commercial products in this publication
is solely for the purpose of providing specific information and does not imply recom-
mendation or endorsement by the U.S. Department of Agriculture or the Washington
State Department of Agriculture. USDA is an equal opportunity provider and employer.

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