Apis Cerana Japonica): a Lowland Case in Southwestern Honshu

Original paper Seasonal change and diversity of pollen-source plants used by the Japanese honeybee (Apis cerana japonica): a lowland case in southwestern Honshu

Ai INOUE1 and Hiroyuki TAKASAKI1*

岡山理科大学周辺におけるニホンミツバチ(Apis cerana japonica)の花粉源植物の季節変移と多様性

井上愛1・高崎浩幸1*

Abstract: A handmade trap for collecting pollen pellets was set on a hive of Japanese honeybees (Apis cerana japonica) on the campus of Okayama University of Science, southwestern Honshu, to study the seasonal change and diversity of their pollen-source plants. Pollen pellets gathered by the bees were trapped for an hour once every week, 29 times in total, from June to December 2014. The hourly yield count of trapped pellets peaked in September and October, while the counts in mid August and December were low. In total, 61 types of color and morphology patterns were recognized in the pollen grains in 1493 trapped pellets. The hourly sample comprised 2-9 types of pollen pellets differing in grain color and morphology. Throughout the study, 12 of the 61 pollen types were identified to the species, genus, or family, despite efforts in collecting pollen samples from 74 flowering species in total, both cultivated and wild in the neighborhood. In the autumn, the abundance of the wild asteraceous plants growing in patches around the campus was likely advantageous for the bee colony in their pollen gathering.

I. Introduction (e.g. Miyamoto 1958, Kubota 1984, Sakai and Ono 1991, Suryanarayana et al. 1992, Nagamitsu There are two apiculturally important bee and Inoue 1999) have been made with regard to species, Apis mellifera and A. cerana. The latter the pollen-source plants for Asian honeybees (A. species is widespread in Asia, from Afghanistan cerana subspp.). in the west to Japan in the east, and from Indo- The present study aimed to clarify the seasonal nesia in the south to China and Russian Far East change and diversity of the pollen-source plants in the north; and it has been introduced to Papua used by the Japanese honeybee. The study area New Guinea (Bradbear 2009, Radloff et al. 2010). was in Okayama City, southwestern Honshu. The It is a natural gift to local sustainable beekeepers preceding studies of A. c. japonica on this topic in regions where apiculture with A. mellifera is (Miyamoto 1958, Kubota 1984, Sakai and Ono impractical (Bradbear 2009, Hisashi 2010). 1991, Nagamitsu and Inoue 1999) have been Honeybees visit various flowers to gather made only further north in Japan. We sampled nectar and pollen. The diversity of flowering pollen pellets for analysis from the returning plants visited by them has been studied through worker bees at the hive entrance with a handmade observation of bees visiting flowers and analyses trap. The results suggested us to view the inter- of gathered pollen pellets (e.g. Synge 1947, Levin dependence between the regional plants and this and Bohart 1955, Keller et al. 2005a, b). The native honeybee. studied bees have been mainly some breeds of the domesticated European species (A. mellifera). By contrast, only a small number of scientific studies

*To whom correspondence should be addressed. E-mail: [email protected] １．岡山理科大学動物学科 700-0005 岡山市北区理大町1-1 Department of Zoology, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama-shi, Okayama-ken 700-0005, Japan.

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Fig. 1. Handmade pollen trap in use for the Japanese honeybee (Apis cerana japonica) (Photo by A. Inoue, 24 June 2014).

Table 1. List of the 74 flower species, either wild (w) or cultivated (c), for which reference pollen samples were collected.

- 48 - Seasonal change and diversity of pollen-source plants used by the Japanese honeybee (Apis cerana japonica): a lowland case in southwestern Honshu Table 2. Pollen pellets sorted by color and grain morphology, number of pollen pellets, and monthly diversity index. (Diversity index was calculated using Shannon-Weaver function [Shannon and Weaver 1949].) Weaver [Shannon and function index was calculated using Shannon-Weaver diversity index. (Diversity and monthly of pollen pellets, number and grain morphology, 2. Pollen pellets sorted by color Table

- 49 - Ai INOUE and Hiroyuki TAKASAKI II. Materials and Methods hive, pollen samples were collected from flowers in bloom in the neighborhood. We observed the 1. Study bee colony and its habitat pollen grains under the microscope, and photo- We used a newly hived colony in the experi- graphed them. The photos recording the pollen ment in the 2014 undergraduate fieldwork class grains collected from flowering plants were at the Department of Zoology, Okayama Uni- compared for identifying the source species of versity of Science (OUS). On 17 April 2014 in the trapped pellets. the swarming season (April-June), a Japanese honeybee swarm settled in one of the “jubako” III. Results and Discussion bait hives placed on the OUS campus, Okayama City, southwestern Honshu, Japan. (For the bait 1. Pollen trap passage hole adjusted hive design, see Takasaki et al. [2014].) This hive Trial and error (data not shown), starting from was the only inhabited one on the campus in 2014. the passage hole standard used for A. mellifera, The OUS campus is located on the northen led us to adoption of the diameter of the hole edge of the urban area of the city. To the west of being about 4.5mm, usable for sampling pollen the campus lies the peak of Daimisen (160m a.s.l., pellets (Fig. 1). This measurement almost equals 34°41′44″N, 133°55′12″). Its slopes are mostly 95% of the diameter of the worker larval cell (re- covered with a mixed deciduous and evergreen portedly 4.65−4.68mm [mean 4.65mm], Tokuda secondary broadleaf forest dominated by Quer- 1924; 4.7mm, Yoshida 2000 ) in A. c. japonica. cus serrata, Q. variabilis, Q. glauca, and Ilex Using the pollen trap, we sampled pollen pellets chinensis. On the southeastern side, the campus 29 times from 10 June to 23 December. borders with Handayama Botanical Garden. In the north, woody areas and grassy fields spread 2. Bees' seasonal activity patterns in Sangenya Garrison of the Japan Self-Defense For identification of the pollen species, we col- Force. Within 1km of the hive, secondary forest lected reference pollen samples directly from 74 patches mostly covered with Q. serrata and Q. plant species, either wild or cultivated, flowering variabilis spread in the Kake Botanical Garden during the study period in the vicinity (Table 1). (Morita et al. 1998) beyond the garrison from the As the cultivated flowers were conspicuous and north to the northeast. The riverbed of the Asahi easily collected, we assume almost all flowering River lies within 1km east of the hive, providing in the neighborhood were sampled. bees with various flowers on it and its banks. A total of 1493 pellets were trapped, and sorted into 61 types by pollen grain color and 2. Study methods morphology (Tables 2 and 3). In particular, Table (1) No commercial pollen trap designed for the 2 gives the monthly pellet count of each pollen Japanese honeybee was available. We handmade type, and the diversity index (calculated using a trap by trial and error, starting from an imitation Shannon-Weaver function [Shannon and Weaver of those used for the European honeybee, for 1949]) of the collected pollen pellet types for each sampling pollen pellets at the hive. month. Table 3 shows the duration of each pollen (2) Once every week during the study period pellet type, picturing the seasonal shift in the use (June-December) the trap was set to sample the of pollen species. pollen pellets gathered and brought into the hive Fig. 2 shows the change in the count of pollen by worker bees. The trap was kept set at the hive pellets trapped per hour on each weekly examina- entrance for an hour somewhere between 10:00 tion day. The count showed a progressive increase and 14:00. (When the number of samples was until October. This likely indicates the increase too small, the duration of trapping was somehow of brood and workers in the colony, as a positive extended to avoid the statistical error due to the small sample number, and the count per hour was calculated for comparison.) The trapped pollen pellets were observed using a transmitted-light microscope (Leica DM500 at 400× magnification, 10× ocular and 40× ob- jective), and prepared for identifying the pollen species using their morphology and color patterns in comparison with pollen samples obtained in (3). The number, color, and likely pollen-source plants concurrently in flower of trapped pellets were recorded. Fig. 2. Change in the count of pollen pellets trapped per hour on each (3) Along with trapping pollen pellets at the weekly examination day.

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Table 3. The duration of each pollen pellet type trapped. (● indicates the pellet type which recorded the maximum number trapped on the day.)

correlation between the brood and the amount Nansen and Holst 2002, Baum et al. 2011, Dalio of pollen pellets brought into the hive has been 2012). Therefore, the reality may somehow differ reported for the European honeybee (e.g. Allen in detail from the rough sketch obtained in this and Jeffree 1957, Free and Williams 1974, Keller study. et al. 2005b). The worker bees' activity is likely affected 3. Diversity of pollen-source plants by the conditions of the surrounding environ- Fig. 3, drawn from Table 2, shows the change ment (Keller et al. 2005a, b). The decrease of of the number of pollen pellet types, i.e. the as- pollen-source plants due to the summer growth sumed number of pollen-source plant species, depression in August and the low temperature as the season progressed. The results indicate in December inactivated them greatly, as often the bees visited a plural number of species of observed for A. mellifera (Keller et al. 2005a, b). pollen-source plants each month (range 5–17, Throughout the study, the pollen pellets were lowest [5] in December and highest [17] in Sep- trapped in the same time zone, although it has tember and October). Fig. 4, also drawn from been reported for A. mellifera that particular pol- Table 2, shows the monthly change in the diver- len species tend to be gathered more frequently sity index, indicating a similar seasonal pattern. either in the morning or in the afternoon (e.g. Close examination of Table 3 reveals the plural - 51 - Ai INOUE and Hiroyuki TAKASAKI 4. Identification of pollen-source plant species The present study attempted to identify each pollen-source plant species by color and morphology of pollen grains under the microscope. However, it was difficult to identify them specifically. We only succeeded in inference of 12 species (including those identified to the genus or family) despite there were 61 sorted types (Tables 1 and 2). Fig. 3. Seasonal change in the number of pollen pellet types. Nagamitsu and Inoue (1999) stated that the Japanese honeybee tends to prefer dioecious and andromonoecious flowers, often bearing inconspicuous green petals. In the present study, the pollen specimens collected for comparison in the identification procedure were mostly from flowers of herbs and shrubs relatively low in height, being easily found and collected. Probably for this reason in retrospect, the wild flowers visited by the bees in reality and those sampled for reference were not in good matching. Furthermore, even if morphological traits look the same, it does not necessarily mean the sample is of the same species as the reference Fig. 4. Seasonal change in the pollen diversity. specimen. Some species resemble one another in pollen grain color and morphology. The pollen grain size and shape may change depending on species were visited also each day (range 2–9). the condition of the sample such as its moisture The plurality even on the daily basis in the season content. Use of photos in plates (e.g. Nakamura with abundant preferred pollen-source flowers 1980) and color charts in literature (e.g. Sasaki may indicate that the bee balance the required 2010) led us to no conclusive identification of nutrients (e.g. Keller et al. [2005a]), or it may also the pollen grains which we had once failed to reduce detoxication loads due to excessive intake “identify” through comparison with the pollen of particular foods as discussed for mammalian samples collected directly from flowers. Neither herbivores (e.g. Wiggins et al. 2006). the color charts of pellets nor the numerous photos The number of pollen-source species (Fig. 3) of exines prepared for palynological studies were gives a seasonal change pattern similar to that helfpul. Instead of the term “identified,” therefore, in Fig. 2. Close comparison of Figs. 2, 3, and 4 we use “inferred” to signify the case in which the reveals the following. In September and October, trapped pollen pellet was likely of the species when both the count of trapped pellets (Fig. 2) concurrently in bloom with pollen grains very and the number of species (Fig. 3) peaked, the similar in color and morphology. Application of diversity (Fig. 4) was unexpectedly low. To re- more rigorous standard methods (e.g. acetolysis, phrase, despite the numerosity of pollen-source scanning electron microscopy, etc. as briefly species available then, the bee showed a promi- described in Delaplane et al. 2013) might solve nent preference of particular plants. By contrast the taxonomic identification of the trapped pollen in July and November, although the number of specimens (stored at OUS Zoology Department) species was small, all were rather evenly used, in the future. resulting in the high diversities. In August and December, the data were insufficient for any good 5. Foul smell around the hive in the autumn comparison owing to the inactivity of the worker The asteraceous pollen pellets trapped in Oc- bees outside the hive. tober and November (No. 38) emitted a peculiar Up to this step in the procedure, the same would foul smell. The same peculiar odor many bee- be applicable at research sites of which flora have keepers in Japan smell around hives in the autumn not been clarified yet, e.g. tropical forest in remote is likely ascribable to this pollen. We inferred places. Even with little taxonomic knowledge of pollen No. 38, from its color and morphology as the pollen-source plants, similar analyses would well as its season, to be of the Canada goldenrod be feasible. (Solidago canadensis var. scabra) (Figs. 5, 6). However, neither the pollen collected directly from its flowers nor the pollen mixture with

- 52 - Seasonal change and diversity of pollen-source plants used by the Japanese honeybee (Apis cerana japonica): a lowland case in southwestern Honshu 6. Annual cycle During the present study, the bee colony, after moving in the hive in April, increased its population size and intensified its pollen gathering activity, changing their seasonal pollen-source plants one after another (Table 3). In mid Au- gust, it dropped its activity level markedly, likely owing to the heat and the decreased number of pollen-source plants. Then it reached its activity peak in October. Bimodal increase of pollen harvest before the colony's growth in spring and before wintering has been pointed out for the European honeybee (e.g. Synge 1947, Moriya 1960, 1961, Fukuda 1983, Keller et al. 2005b). The Japanese honeybee, living in the temperate climate zone, is expected to have a similar annual cycle. At the time of drafting this paper in March 2015, we observed the colony showed an intensive daily transportation of pollen pellets Fig. 5. Pollen grains in a trapped pellet likely of Canada goldenrod into its hive. Kubota (1984) and Sakai and Ono (Solidago canadensis var. scabra) (Photo by A. Inoue, 26 October (1991) suggested the same cycle pattern in their 2014). studies on this species at Tamagawa University in Machida City, Tokyo. 7. Intensive use of Asteraceae in autumn Notably, when the harvest of pollen peaked in October, 60% of the harvest was of the Asterace- ae. This high percentage of the asteraceous pollen pellets is likely due to the location of the hive. Within 500m of the study hive, there are many asteraceous plant patches mainly consisting of the Canada goldenrod. The Canada goldenrod in particular is regarded as an important pollen-source plant in the autumn, when other plant flowers decline in number. As Sasaki (2010) pointed, three main factors are involved in the choice of flowers by the honeybee to visit, i.e. (1) quantity, (2) quality, and (3) distance. The asteraceous plant patches in the present study Fig. 6. Canada goldenrod (Solidago canadensis var. scabra) visited by site favorably satisfied at least (1) and (3). The a Japanese honeybee (Photo by R. Murakami, 15 October 2014). Canada goldenrod and Bidens spp., both of the Asteraceae, have their peak flowering season from late September through October, coinciding honey kept at 37°C overnight emitted the same with the pollen harvest peak in the present study. repugnant odor. For the reason above, we could Among the pollen-source plants in the autumn not confirm our identification, although it is very other than the asteraceous, only a limited number likely. As Sugahara (2005) and Watanabe (2015) of species were inferred. As Nagamitsu and Inoue state, the non-native Canada goldenrod is now an (1999) suggested, inconspicuous tree and shrub important source of honey and pollen for bees in species are likely used. the autumn, when other flowers to visit decline in The data set obtained in the present study alone number. Similarly, some particular pollen-source is not enough for evaluating the environment of plant species or groups have been reported to be the study area in terms of food availability for the intensively collected by both A. mellifera and A. bee. Apparently, however, the asteraceous plant cerana (e.g. Suryanarayana et al. 1992, Keller et patches, or more specifically of the Canada gold- al. 2005a, Dalio 2012, Girard et al. 2012). enrod, provide bees with favorable conditions for securing pollen harvest in the autumn. In some studies in Machida City (Kubota 1984, Sakai and Ono 1991) the pollen harvest peaked in August,

- 53 - Ai INOUE and Hiroyuki TAKASAKI plants. Nagamitsu and Inoue (1999) concluded in their comparative study of A. cerana japonica and A. mellifera in a primary beech forest in Ashiu, Kyoto, central Japan, that both honeybee species, being flexible and adaptive to the given environment, are not too much different in their use of pollen sources. They also noted the former's particular preference of some inconspicuous tree flowers. Their study did not mean, therefore, that the native former species is replaceable with the introduced latter in the cool temperate beech forest ecosystem. The present study also has Fig. 7. Cayratia japonica, none of which pollen pellet was sampled suggested the Japanese honeybee's dependence in the pollen trap, visited by a Japanese honeybee (Photo by A. on the local flora in the wild for pollen sources in Inoue, 23 June 2014). and around the OUS campus, largely consisting of the warm temperate secondary vegetation. while the harvest peaked in October in the present study. Besides the climatic difference, the growth Acknowledgments condition of the Canada goldenrod may explain this difference in the peak month. Inoue did the present study as part of prepara- tion for her graduation thesis. Takasaki supple- 8. Dependence of the Japanese honeybee on mented her discussion with additional references the local flora and drafted the final manuscript. All students in The present study trapped 61 distinct types of the 2014 OUS Zoology Department (OUSDZ) pollen pellets. To rephrase, if all were insect-pol- fieldwork class and Prof. S. Kobayashi contrib- linated species, the studied colony was likely uted to the setup of the experiment on which the involved in the pollination of 61 species at least. present study was based. Our mates in OUSDZ Miyamoto (1958) recorded 42 species in Sanda, Animal Socioecology Laboratory helped us in Hyogo Pref., including additions from Kagoshima many ways. R. Murakami offered the photograph Pref., Kyushu, while Okada (1991) listed 104 used as Fig. 6. We are grateful to these people. species in Machida, Tokyo, including additions from all over Japan. Given our coverage of only References less than 7 months (June-December) in the year, the record of 61 taxa of pollen-source plants from Allen, M.D., and Jeffree, E.P. (1957). The annual cycle of a single locality is significant. pollen storage by honeybees. J. Econ. Entomol., 50: In field observation, we found this bee species 211–212. visiting many other wild flowering plants (e.g. Bradbear, N. (2009). “Bees and their role in forest liveli- Castanea crenata, Swida macrophylla, Partheno- hoods―a guide to the services provided by bees and cissus tricuspidata, Ampelopsis glandulosa var. the sustainable harvesting, processing and marketing heterophylla, Cayratia japonica [Fig. 7], Aralia of their product” 204pp. Food and Agriculture Orga- elata, Eriobotrya japonica, in the order of their nization of the United Nations, Rome. http://www. flowering seasons) which were not sampled in the fao.org/docrep/012/i0842e/i0842e00.htm (accessed pollen trap though checked with respective pollen October 2013) specimens from flowers. The bee may visit some Baum, K. A., Rubink, W., L., Coulson, R. N., and Bryant, flowers only for nectar, and likely pollinate them V. M., Jr. (2011). Diurnal patterns of pollen collection too. Nectar-source plants and pollen-source plants by feral honey bee colonies in southern Texas, USA. must be studied separately in the future. Palynology 35(1):85–93. 2011. In the trapped pellets, we found no pollen Dalio, J. S. (2012). Cannabis sativa―an important subsis- from the cultivated, horticultural, or agricultural tence pollen source for Apis mellifera. IOSR Journal of species in the neighborhood (except Triadica Pharmacy and Biological Sciences 1 (4): 1–3 sebifera and Camellia sasanqua), although their Delaplane, K.S., Dag, A., Danka, R.G., Freitas, B.M., reference specimens were thoroughly searched Garibaldi, L.A., Goodwin, R.M., Hormaza, J.I. (2013) (Table 1). The pollen-source species inferred were Standard methods for pollination research with Apis mostly wild-growing (Table 2). Many of those mellifera. In Dietemann, V., J D Ellis, J.D., Neumann, “unidentified” pollen pellets are also likely to P. (eds.) The COLOSS BEEBOOK, Volume I: stan- be of wild trees and shrubs, possibly including a dard methods for Apis mellifera research. 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Jpn. J. Ecol. 11: 82–86. (in Japanese) 井上愛・高崎浩幸：岡山理科大学周辺におけるニ Nagamitsu, T., Inoue, T. (1999). Differences in pollen sources of Apis mellifera at a primary beech forest in central ホンミツバチ(Apis cerana japonica)の花粉源植物の Japan. Journal of Apicultural Research 38: 71–78. 季節変移と多様性 Nakamura, J. (1980). “Diagonistic characters of pollen grains of Japan, part II” 157pp. Special Publications from the Osaka Museum of Natural History, vol. 12. 要約 Friends Association for Osaka Museum of Natural History. (in Japanese) ニホンミツバチの花粉源植物の多様性と季節変 Nansen, C., Holst, N. (2002). Dynamics of short-term 移を明らかにするために，岡山理科大学構内で花粉 variation in pollen foraging by honey bees. Portugaliae Acta Biol. 20: 249–264. 採取器を用いた調査を行った．花粉ダンゴの採取は Okada, I. (1991). Memorandum on Japanese honeybees 週に一度一時間程度行い，６月から12月の間に計29 (I, II). Honeybee Science 12(1, 2) : 13-26. 61-76. (in Japanese) 回採取した．採取された花粉は，色や形状によって Radloff, S. E., Hepburn, C, Hepburn, H.R., Fuchs, S, Had- 61種類に分類できた．どの調査日においても，複数 isoesilo, S., Tan, K., Engel, M.S., and Kuznetsov, V. (2010). Population structure and classification ofApis 種の花粉ダンゴが検出された．一時間当たりの花粉

- 55 - Ai INOUE and Hiroyuki TAKASAKI ダンゴの収量は９月・10月に著しく上昇した．一む 12種の推定にとどまった．秋季はセイタカアワ方，８月と12月は気温と花粉源植物減少の影響で，ダチソウやセンダングサ等のキク科の花粉が多くみ花粉収集はほとんど行われなかった．巣箱から採取られ，岡山理科大学周辺のキク科植物の群生地が秋した総数1493個の花粉ダンゴと，野生および栽培の季の花粉収集活動に有利に働いていることが明らか 74種の植物から採取した花粉を比較して花粉源植物になった．の同定を試みたが，属あるいは科までの同定を含 (Accepted 28 December 2015)

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