Andrena semirugosa brassicae (Hymenoptera: Andrenidae) as a major small bee species in apple orchards in Japan

Abstract

Wild small bees are known to visit apple flowers along with honeybees and other bees. However, their species composition in Japan has not been well investigated for more than 50 years. Here, we identified 791 wild small bee individuals captured in seven apple orchards in four prefectures. Andrena semirugosa brassicae (Hymenoptera: Andrenidae) was the predominant species visiting apple flowers in five orchards, although it had not been listed as a major visitor in past studies. This species was also the major visitor to dandelion flowers in the orchards. We deposited the sequences of the cytochrome c oxidase subunit I gene (CO1) of 250 individuals (7 genera; 32 species) in DDBJ to support the genetic identification of small bees widely observed in apple orchards in Japan.

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Applied Entomology and Zoology
https://doi.org/10.1007/s13355-022-00787-5
BRIEF REPORT
Andrena semirugosa brassicae (Hymenoptera: Andrenidae) asamajor
small bee species inapple orchards inJapan
TsunashiKamo1· KenFunayama2· HidenariKishimoto3· KoukiYoshida4· ShigekiKishi5
Received: 1 February 2022 / Accepted: 26 May 2022
© The Author(s) under exclusive licence to The Japanese Society of Applied Entomology and Zoology 2022
Abstract
Wild small bees are known to visit apple flowers along with honeybees and other bees. However, their species composition
in Japan has not been well investigated for more than 50years. Here, we identified 791 wild small bee individuals captured
in seven apple orchards in four prefectures. Andrena semirugosa brassicae (Hymenoptera: Andrenidae) was the predominant
species visiting apple flowers in five orchards, although it had not been listed as a major visitor in past studies. This species
was also the major visitor to dandelion flowers in the orchards. We deposited the sequences of the cytochrome c oxidase
subunit I gene (CO1) of 250 individuals (7 genera; 32 species) in DDBJ to support the genetic identification of small bees
widely observed in apple orchards in Japan.
Keywords Andrena semirugosa brassicae· Apple· Pollinator
Introduction
The contribution of diverse pollinators, including wild
insects, to the provision of pollination services for fruit
production is now considered greater than once thought
(Garibaldi etal. 2013). In crop production, wild bees often
transfer and deposit pollen with an efficiency that is equal
to, or greater than, that of honeybees (Frier etal. 2016; Pfis-
ter etal. 2017). In addition, the presence of wild bees can
affect honeybee foraging behavior, resulting in increased
pollen deposition (Greenleaf and Kremen 2006). Therefore,
enhancing the pollination services performed by diverse
local wild pollinators is likely to be a highly sustainable and
low-cost method for producing various crops that require
pollination (Albrecht etal. 2012; Hoehn etal. 2008).
Apple, Malus domestica Borkh. (Rosales: Rosaceae), is a
representative crop that requires pollination because of self-
incompatibility (Ramírez and Davenport 2013). In Japan,
since the yield of apple is the second largest among fruit
crops (MIAC 2019), pollination is an important concern for
farmers to optimize and stabilize production. European hon-
eybees, Apis mellifera L. (Hymenoptera: Apidae), have been
used commercially as pollinators in apple orchards, although
they are not the most efficient pollinators of this crop (Blitzer
etal. 2016; Russo etal. 2017). In Japan, Osmia cornifrons
(Radoszkowski) (Hymenoptera: Megachilidae) has been
kept and used as a pollinator (Maeta and Kitamura 1965)
and has been proved to be more efficient than the honeybee
in apple pollination (Matsumoto etal. 2009).
Periodic monitoring of the wild bee community that can
provide pollination services for apple is necessary because
the community and the resultant pollination services can
easily change in response to orchard management (Roquer-
Beni etal. 2021) and surrounding land use (Holzschuh etal.
2012). However, since studies that reported flower visitors
in Japan from 1962 to 1970 (Kobayashi 1971; Kobayashi
etal. 1966; Tsugawa etal. 1967), few studies have closely
surveyed them in recent years. In particular, small bees that
visit apple flowers have not been well surveyed in Japan,
although they would make up a considerable ratio of the
* Tsunashi Kamo
tkamo@affrc.go.jp
1 Institute forAgro-Environmental Sciences, National
Agriculture andFood Research Organization (NARO),
Tsukuba, Ibaraki305-8604, Japan
2 Akita Fruit-Tree Experiment Station, Yokote,
Akita013-0102, Japan
3 Institute forPlant Protection, NARO, Morioka,
Iwate020-0123, Japan
4 Fruit Tree Research Center, Fukushima Agricultural
Technology Center, Fukushima, Fukushima960-0231, Japan
5 Research Center forAgricultural Information Technology,
NARO, Minato-ku, Tokyo105-0003, Japan

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total visitor species and individuals. For example, wild small
bee species comprised 25 of 53 wild bee species collected
on apple flowers in New York state in the USA (Blitzer etal.
2016). A possible reason for the lack of reports of small bee
assemblages at the species level in Japan is that identifica-
tion of small bees is often more difficult for non-specialists
than that of major recognizable honeybees, bumblebees, and
the carpenter bee. DNA barcoding could be a good solution
for species-level identification without taxonomic skills,
although DNA databases are not always satisfactory in reso-
lution, especially when small bees are the target.
As the first step in understanding pollination services pro-
vided by small bees, we surveyed wild small bees in seven
apple orchards over four prefectures in Japan. We identi-
fied them based on morphological traits to species level and
confirmed the results by DNA barcoding as necessary. We
then deposited the sequences of the cytochrome c oxidase
subunit I gene (CO1) of the individuals in the DNA Data
Bank of Japan (DDBJ) to facilitate studies on the genetic
identification of small bees. We excluded the other visitors
from our targeted species in this study, although they would
be also important for apple production.
Methods
Study sites
We captured wild small bees in seven apple orchards—two
in Akita prefecture, AKT-1 (39.24°N, 140.53°E), AKT-2
(39.24°N, 140.56°E); three in Fukushima prefecture, FKS-1
(37.81°N, 140.44°E), FKS-2 (37.90°N, 140.43°E), FKS-3
(37.80°N, 140.49°E); one in Iwate prefecture, IWT-1
(39.77°N, 141.13°E); and one in Nagano prefecture, NGN-1
(36.77°N, 138.30°E)—from 2018 to 2020. Environmentally
friendly farming was practiced in AKT-1, FKS-1, and IWT-
1, and conventional farming in the rest. ‘Fuji’, an apple vari-
ety, was planted in AKT-1, AKT-2, FKS-1, FKS-2, FKS-
3, and IWT-1. ‘Sansa’ was planted with ‘Fuji’ in IWT-1.
‘Tsugaru’ was planted in NGN-1. The orchards are described
in TableS1.
Capturing flower visitors inapple orchards
From pre-anthesis to post-anthesis of apple, we collected
small bees on apple and weed flowers by placing the aper-
ture of a 5-mL vial over each bee. In 2018, we added 3–5mL
of ethanol to each tube and stored the samples at room tem-
perature until identification. In 2019 and 2020, each vial was
immediately cooled on ice in a cooler bag and then stored
at −30°C in a freezer in the laboratory until identification.
Flying bees in AKT-1 were also captured in an insect net on
23 and 24 April 2019.
Species identification offlower visitors
We identified 790 of the 791 small bees captured to the
species level based on their morphology; if necessary,
identification was confirmed by CO1 DNA barcoding. The
remaining one was identified to genus level.
DNA barcoding was used for 250 individuals. The
primer combination used for PCR amplification and
sequencing of a segment of the mitochondrial CO1 was
BarbeeF (forward), 5′-CAA CAA ATC ATA AAA ATA
TTGG-3′ (Françoso and Arias 2013), and MtD9 (reverse),
5′-CCC GGT AAA ATT AAA ATA TAA ACT TC-3′ (Simon
et al. 1994). DNA from one of the middle legs was
extracted with a DNeasy Blood and Tissue Kit (Qiagen,
Valencia, CA, USA) in accordance with the manufacturer’s
protocol. The amplification reaction contained 0.08μL of
ExTaq polymerase (TaKaRa Bio, Shiga, Japan; 5 U/μL),
1.6 μL of Ex Taq buffer (Mg2+ free), 1.3 μL of MgCl2
(25mM), 1.3 μL of dNTP mixture (2.5mM each), 0.5
μL of DNA template, 0.5 μL each of forward and reverse
primer (10μM), and purified water up to 16.6μL. PCR
cycles consisted of an initial denaturation step for 5min at
94°C; 35 cycles of denaturation (1min at 94°C), anneal-
ing (1min 20s at 40°C), and extension (2min at 64°C);
and a final extension at 64°C for 10min. The PCR prod-
ucts were treated with ExoSAP-IT (USB Corp., Cleveland,
OH, USA), and directly sequenced in both directions in
an ABI 3130XL or SeqStudio genetic analyzer (Applied
Biosystems, Foster City, CA, USA) with a Big Dye Ter-
minator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems), using the same primers as for PCR. Similar
sequences were searched by using an identification engine
searching for “All Barcode Records on BOLD” (http://
www. bolds ystems. org/ index. php/ IDS_ OpenI dEngi ne) in
BOLD (the Barcode of Life Data Systems) or the Basic
Local Alignment Search Tool (BLAST; https:// blast. ncbi.
nlm. nih. gov/ Blast. cgi) in GenBank (National Centre for
Biotechnology Information). Species were identified on
the basis of highly similar best matches (> 98% identity).
The sequences were deposited in DDBJ (accession num-
bers LC652088–LC652337; TableS2).
Results anddiscussion
In total, we collected 358 individual small bees of 26 spe-
cies on apple flowers at the seven sites (Table1). Andrena
semirugosa brassicae Hirashima (Hymenoptera: Andre-
nidae) numbered 192, accounting for 53.6%. This species
was the most abundant at five of the seven sites (AKT-
1, FKS-1, FKS-2, FKS-3, and IWT-1). At AKT-1 and

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Table 1 Wild small bees visiting apple flowers
Bee species* Sex Number of individuals captured on apple flowers
Orchard AKT-1 AKT-1 AKT-1 AKT-2 FKS-1 FKS-2 FKS-3 IWT-1 IWT-1 NGN-1 Total
Sampling start
date
2 May 2018 8 May 2019 5 May 2020 7 May 2018 27 Apr 2018 26 Apr 2018 26 Apr 2018 5 May 2018 11 May 2020 4 May 2019
Sampling end
date
6 May 2018 13 May 2019 5 May 2020 11 May 2018 1 May 2018 1 May 2018 1 May 2018 6 May 2018 11 May 2020 5 May 2019
Andrena benefica
Hirashima
F 1 1
Andrena dentata
Smith
F 1 1
Andrena
foveopunctata
Alfken
F 1 1 1 3
Andrena haemor-
rhoa japonibia
Hirashima
F 9 9
Andrena japonica
(Smith)
F 3 1 4
Andrena kaguya
Hirashima
F 2 1 1 4 8
Andrena komachi
Hirashima
F 1 1
Andrena loni-
cerae Tadauchi
et Hirashima
F 1 1
Andrena luri-
diloma Strand
F 10 5 6 1 9 2 11 2 46
Andrena semiru-
gosa brassicae
Hirashima
F 25 13 48 6 24 13 15 27 21 192
Andrena sub-
opaca Nylander
F 1 1 2
Andrena watasei
Cockerell
M 1 1
Andrena yamato
Tadauchi et
Hirashima
F 1 4 5
Ceratina flavipes
Smith
F 1 1
Ceratina japon-
ica Cockerell
F 5 5

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Table 1 (continued)
Bee species* Sex Number of individuals captured on apple flowers
Orchard AKT-1 AKT-1 AKT-1 AKT-2 FKS-1 FKS-2 FKS-3 IWT-1 IWT-1 NGN-1 Total
Sampling start
date
2 May 2018 8 May 2019 5 May 2020 7 May 2018 27 Apr 2018 26 Apr 2018 26 Apr 2018 5 May 2018 11 May 2020 4 May 2019
Sampling end
date
6 May 2018 13 May 2019 5 May 2020 11 May 2018 1 May 2018 1 May 2018 1 May 2018 6 May 2018 11 May 2020 5 May 2019
Lasioglossum
hoffmanni
(Strand)
F 1 26 1 1 5 34
Lasioglossum
primavera
Sakagami et
Maeta
F 4 5 2 5 1 17
Lasioglossum
proximatum
(Smith)
F 5 5
Lasioglossum sci-
tulum (Smith)
F 1 1
Lasioglossum
sibiriacum
(Blüthgen)
F 1 2 3 4 1 11
Lasioglossum
spectrum
Murao
F 1 1 1 1 4
Lasioglossum
taeniolellum
(Vachal)
F 1 1 2
Lasioglossum
vulsum
(Vachal)
F 1 1
Nomada
harimensis
Cockerell
M 1 1
Panurginus craw-
fordi Cockerell
F 1 1
Sphecodes sp. F 1 1
Total 45 25 56 37 35 30 27 35 41 27 358
* Honeybees, bumblebees, carpenter bees, and Osmia cornifrons (Radoszkowski) (Hymenoptera: Megachilidae) were excluded from this pollinator survey

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IWT-1, where pollinators were surveyed in multiple years,
it remained the most abundant (Table1). By tracking an
individual flying near the ground in AKT-1, we found the
entrance to a nest of A. semirugosa brassicae (data not
shown). The reason why A. semirugosa brassicae was not
captured at NGN-1 is unclear. The altitude of this site
(550m a.s.l.) has seemingly affected the species assem-
blage, because all other sites lie between 60 and 200m
a.s.l. (TableS1). However, this species has been found
in many mountainous areas in Japan (Tadauchi 1985).
Andrena luridiloma Strand and Lasioglossum hoffmanni
(Strand) (Hymenoptera: Halictidae) were the next most
abundant. Since we focused on the species identification of
small bees found in apple orchards, further studies of the
frequency of flower visitation and pollination efficiency
are needed to evaluate the effectiveness of these species
in pollination of apple.
In two orchards (AKT-1 and IWT-1), we collected 433
individuals on weed flowers or in flight and identified all to
species level (TableS3). Andrena semirugosa brassicae was
the most abundant species captured on the flowers of dande-
lion, Taraxacum officinale (L.) Weber ex F.H.Wigg. (Aster-
ales: Asteraceae). Since dandelion blooms earlier and ends
later than apple, it could feed the overwintering generation
of this bee species. One supplementary but notable result
is that 13 males of this species were captured on the flow-
ers of Erigeron annuus (L.) Pers. (Asterales: Asteraceae)
in the groundcover of AKT-1 in late June 2020, after apple
bloom. As this bee is bivoltine (Tadauchi and Murao 2014),
these males would have been the first generation reared by
the overwintering generation that had exploited other flower
resources such as the apple and dandelion flowers. So it may
be important that the blooming period of undergrowth flow-
ers extend outside the period of apple anthesis. Norfolk etal.
(2016) showed that abundance and richness of ground flow-
ers increased the abundance of wild bees and thus their fre-
quency of visitation to flowers of almond, Amygdalus dulcis
(Mill.) D.A.Webb (Rosales: Rosaceae). It will be necessary
to compare the effects of these flowers on the abundance
of A. semirugosa brassicae and other wild small bees in
orchards.
We found for the first time that A. semirugosa brassicae
was the predominant small bee species visiting apple flow-
ers. In the past study by Kobayashi etal. (1966), the three
major species here—A. semirugosa brassicae, A. luridiloma,
and L. hoffmanni—were listed as minor visitors (TableS4).
We chose not to compare our species composition with those
reported by Kobayashi (1971) and Tsugawa etal. (1967),
who identified almost half of the small bees they found only
to the genus level. Nevertheless, the species composition of
small bees observed here is different from those in previous
studies. We do not know why A. semirugosa brassicae was
the predominant species here, although changes in orchard
management and surrounding land use may be involved.
Further periodic surveys at more sites could answer this
question.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s13355- 022- 00787-5.
Acknowledgements This work was supported financially by the Min-
istry of Agriculture, Forestry, and Fisheries, Japan, through a research
project titled “Monitoring and enhancement of pollinators for crop
production (JPJ006239).” We are grateful to the members of this
project for capturing the bees. We thank Dr. Ryuki Murao (Regional
Environmental Planning Co., Ltd.) for identifying 23 individuals and
for his useful comments on the taxonomy of small bees. We thank Dr.
Aoi Nikkeshi and Mr. Ryohei Hoshino (NARO) for his technical help.
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