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Kabocha squash, Cucurbita maxima Duchesne, in delayed-start culture is one of the major cultivation systems in southwestern Japan. Honeybee introduction or hand-pollination has been practiced in this crop system, but the species composition, abun- dance, and pollination efficiency of major pollinators remain unclear. In the current study, we investigated which insect(s) accomplish pollination of kabocha squash. Among the total visitations to female flowers, honeybees, mostly Apis mellifera L. (Hymenoptera: Apidae), dominated at 94.4% and 85.3% at the experimental field in 2019 and 2020, respectively, followed by the bumblebee Bombus diversus diversus Smith (Hymenoptera: Apidae) and the hummingbird hawkmoth Macroglossum pyrrhosticta Butler (Lepidoptera: Sphingidae). The number of pollen grains on the body surface of B. diversus diversus individuals was significantly greater than that on honeybees, such that a single visit by B. diversus diversus deposited 5–6 times more pollen grains on a stigma than a honeybee visit, meaning that the bumblebee is a more efficient pollinator at the individual level. However, visitations by numerous honeybees compensated for their lower individual efficiency and resulted in fruit set. In addition, we report for the first time that a lepidopteran can pollinate squash: our data indicate that pollen grains deposited by several visits of M. pyrrhosticta were likely to exceed the estimated minimum number of pollen grains required for fruit set. Our survey at the experimental field and at three private farms revealed that far more pollen than is required for fruit set was deposited on the stigmas by wild pollinators and wintering honeybees. These findings indicate that kabocha squash production in delayed-start culture in this region is practicable without pollination enhancement effort, such as honeybee introduction or hand-pollination. Keywords Apis mellifera · Bombus diversus diversus · Cucurbita maxima · Macroglossum pyrrhosticta · Pollinator efficiency * Tsunashi Kamo Research Center for Agricultural Information Technology, firstname.lastname@example.org National Agriculture and Food Research Organization, Kowa Nishishimbashi Building B, Nishishimbashi 2-14-1, Institute for Agro-Environmental Sciences, National Minato-ku, Tokyo 105-0003, Japan Agriculture and Food Research Organization, 3-1-3 Present Address: Forestry and Forest Products Research Kannondai, Tsukuba, Ibaraki 305-8604, Japan Institute, Forest Research and Management Organization, Kagoshima Prefectural Institute for Agricultural 1833-81 Todori, Hachioji 193-0843, Japan Development, 2200 Ohno, Kinpo-cho, Minamisatsuma, Kagoshima 899-3401, Japan Forestry and Forest Products Research Institute, Forest Research and Management Organization, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan Vol.:(0123456789) 1 3 120 Applied Entomology and Zoology (2022) 57:119–129 systems have contrasting requirements for pollination in Introduction response to the commercial price. While farmers are willing to pay costs for hand-pollination to ship ahead of the rest of Both wild insects and the domesticated European honeybee, the producing areas in Japan to sell fruits at higher prices in Apis mellifera L. (Hymenoptera: Apidae) significantly con- semi-forcing culture, there is an increasing demand for sav- tribute to the pollination of many crops (Breeze et al. 2011; ing labor and costs for pollination in delayed-start culture to Potts et al. 2010). Though recent studies have emphasized survive price competition with other producing areas. How- the importance of wild pollinators in the production of many ever, farmers have continued to hand-pollinate the crop or to crops (e.g., Garibaldi et al. 2013; Mallinger and Gratton introduce honeybee colonies in their farms also in delayed- 2015), the domesticated honeybee is still a reliable partner start culture, concerned about the yield reduction and quality considering the fluctuating visitations and variable foraging deterioration of the crop. There is a possibility that pollina- traits of wild pollinators all over the world (Breeze et al. tion service provided by wild and domesticated pollinators 2014; Decourtye et al. 2010). Thus, which bees, wild ones is enough in that culture, but no studies have evaluated the or domesticated honeybees, are better pollinators should availability of pollination service and the efficiency and con- be carefully evaluated, depending on the crops, localities, tribution of pollinator species. seasons, and species composition of wild pollinators (Potts Our preliminary survey at our experimental field in et al. 2010). Kagoshima demonstrated that the amount of kabocha squash Squash, Cucurbita spp., is a monoecious entomophilous production per area in delayed-start culture was satisfactory plant that have hundreds of ovules in a female flower. Basi- without pollination enhancement effort. Pollinators would cally, the larger the number of pollinator visits to a female be more active in delayed-start culture than in semi-forcing flower, the greater the probability of the fruiting becomes, culture, because higher temperature enhances pollinators’ and more seeds the fruit has (Artz and Nault 2011; McGrady activity (Hoehn et al. 2008). In addition, abundant winter- et al. 2020; Vidal et al. 2010; Winsor et al. 2000). The fruit ing honeybees would be available in autumn, because many weight is positively correlated to the number of mature seeds migratory beekeepers spend the autumn and winter months (McGrady et al. 2020; Petersen et al. 2013). Aside from the with their A. mellifera hives in Kagoshima (Sakai and Mat- squash bees, which are major pollinators of squash plants in suka 1982). origin, bumblebees and honeybees contribute to the produc- To explore the possibility of producing kabocha squash tion worldwide (Bomfim et al. 2016; Knapp and Osborne without pollination enhancement efforts in delayed-start 2019). culture in warm Kagoshima prefecture, we first aimed to Whereas pollination of squash crops is well studied in identify pollinator species and evaluate their pollination effi- cool regions, few studies have reported the efficiency of ciency in the region in autumn. We monitored flower visi- pollinators or even lists of pollinating species in warm pro- tors and fruiting success for several locations and years, and ducing areas. According to the studies conducted in rela- counted the number of pollen grains on their body surfaces tively cool locations, i.e., northeastern United States (Artz and those deposited on a stigma by a single visit. Then we and Nault 2011; Vidal et al. 2010), southwestern Germany estimated the minimum number of pollen grains required for (Pfister et al. 2017 ), and highland in southwestern China fruit set by analyzing the relationship between the number of (Xie and An 2014), bumblebees are found to be the most pollen grains deposited on a stigma and fruit set. Integrating efficient pollinator of the plant per visit, that is, they deposit those results, we discussed the contribution of visiting spe- more pollen grains on a stigma per visit than other sympat- cies and necessity for pollination enhancements. ric visitor insects. Similar results have been reported in the northern areas in Japan (Matsumoto and Yamazaki 2013; Nagamitsu et al. 2012; Nakamura et al. 2021). Method Meanwhile, kabocha squash has been widely cultivated in Japan, from the southern to the northern part of the archi- Study sites pelago. Kagoshima prefecture, located in the southwestern part of Japan, has the second greatest amount of kabocha Field observations and experiments were performed in the squash production following Hokkaido, located in northeast experimental el fi d of the Kagoshima Prefectural Institute for (Statistics Bureau, Ministry of Internal Aa ff irs and Commu - Agricultural Development (31.48° N, 130.34° E, 44 m asl, nications 2019). In contrast to the shorter growing season hereafter “the experimental field”) and at three private farms in Hokkaido, farmers in Kagoshima use the advantage of (Farms A–C; Table S1) in Kagoshima. The surrounding area warm climate to produce kabocha squash in two cropping of the experimental field was largely agricultural crops with systems: semi-forcing culture (flowering in March–April) relatively small secondary forests (Fig. S1). The surrounding and delayed-start culture (September–October). These two areas of Farm A (31.44° N, 130.33° E, 20 m asl) and Farm 1 3 Applied Entomology and Zoology (2022) 57:119–129 121 B (31.51° N, 130.35° E, 10 m asl) were similar to that of clothianidin (I) on 25 August, copper sulfate (P) on 3 and 8 the experimental field. Farm C (31.54° N, 130.35° E, 20 m September, chlorothalonil (P) on 18 September, flutianil (P) asl) located near a mountainous area. The growing area for on 25 September, and copper sulfate (P) and spinetoram (I) squash varied, from 180 m in the experimental field to 3000 on 9 October, following the standard practices. m at Field A (Table S1). Cucurbita maxima Duchesne ‘Ebisu’ was grown in the Identification of flower‑visiting species experimental field and Farm A, and farmers of Farms B and C grew ‘Kuriyutaka’. Anthesis started in the middle to To identify flower-visiting species, we captured at least a late September. Flowers bloomed before 05:00 h prior to few individuals from each taxonomic group based on the dawn and withered by approximately 11:30 h on the same visual identification in the field. Individuals were captured day. Okra, Abelmoschus esculentus (L.) Moench (Malvales: during late September to early October in 2019 and 2020 in Malvaceae), was the sole crop that bloomed simultaneously the experimental field, using a method in which a researcher with kabocha squash in the experimental field. No domes- moved the aperture of a 5- or 50-mL vial (depending on the ticated pollinators, such as A. mellifera, were placed in any size of insects) toward an insect that had entered a flower. On of the study fields. At Farms A and B, farmers performed 26 and 29 September 2020, we specifically captured flower- hand-pollination, while at Farm C, farmers relied on insects visiting Macroglossum pyrrhosticta Butler (Lepidoptera: for pollination. Sphingidae), which was not observed in the preceding year. The 10-year mean monthly temperature ranged from Among the taxonomic groups, honeybees were captured 7.8 °C in January to 28.3 °C in August (25.2 °C, 20.5 °C, exceptionally in large numbers (> 100 individuals per year), and 15.2 °C in September, October, and November, respec- to identify species composition (European or Asian honey- tively), and the mean annual precipitation was 2544 mm, bees). Captured insects were morphologically identified in recorded at the nearest automated meteorological data acqui- our laboratory. sition point of the Japan Meteorological Agency (Minami- satsuma, Kagoshima; 31.42° N, 130.33° E, 9 m asl) from Monitoring frequency of flower visitations 2009 to 2018. and fruiting success in open pollination Cultivation and management at the experimental In the experimental field (in 2019 and 2020) and Farms field A–C (in 2020), the numbers of visiting insects, pollen grains deposited on stigmas, and fruiting success were We grew squash in a plot (30 m × 6 m) located in the experi- monitored for 12‒15 flowers in each year and location to mental field. We seeded kabocha squash, C. maxima ‘Ebisu’, understand the available amount of pollination service. in pots on 19 August in 2019 and on 11 August in 2020, and For 4 to 6 consecutive days in each research year in every transplanted 74 or 75 seedlings in a row at intervals of 0.4 m location, we selected two or three female flowers every day on 28 August 2019 and 25 August 2020 (Fig. S2). The plant in each field and placed a video camera (Handycam HDR- tops were pinched on 9 October 2019 and 5 October 2020. PJ675, Sony Corp., Tokyo, Japan) fixed on a tripod about We applied fertilizers and controlled insect herbivores 30 cm away from each flower, and videotaped the flowers and pathogens following the standard practice described from daybreak (approximately 06:00 h) until withering of below, while weeds were controlled by hand. In 2019, 0.9 kg the flower (approximately 11:30 h). From these video files, each of nitrogen, phosphorus, and potassium was applied we classified every flower visitor into the following taxo- to the ridge (30 m × 0.5 m) formed in the experimental plot nomic groups: honeybee, Bombus diversus diversus Smith (180 m ) before planting. The plot was side-dressed with (Hymenoptera: Apidae), Campsomeriella annulata annu- 0.4 kg each of nitrogen and potassium after planting. We lata (Fabricius) (Hymenoptera: Scoliidae), M. pyrrhosticta, applied clothianidin (insecticide [I]) on 28 August, fluben- small bee, or other, and calculated taxonomic composition. diamide (I) and penthiopyrad (pesticide [P]) on 9 September, To count the number of pollen grains deposited, the stig- Bacillus thuringiensis formulations (I), oxolinic acid (P), mas of these flowers were collected at approximately 24 h and thiophanate-methyl (P) on 19 September, azoxystrobin (in 2019) or 48 h (in 2020) after anthesis, which did not (P), flonicamid (I), and flubendiamide (I) on 7 October, and affect ovary growth. Suzuki (1969) reported that the time quinoxaline (P) on 25 October. required for pollen-tube growth and fertilization in Cucur- In 2020, 1.35 kg each of nitrogen, phosphorus, and potas- bita sp. is 3–5 and 9–11 h after pollination, respectively. sium was applied to the ridge (30 m × 0.5 m) before plant- To deal with the large stigmas under a microscope (SZ61; ing, which was equivalent to 150% of the preceding year Olympus, Tokyo, Japan), we cut the stigmas into three por- (based on the result of soil analysis). The plot was side- tions without dyeing, and counted the pollen grains of C. dressed in the same manner as the previous year. We applied 1 3 122 Applied Entomology and Zoology (2022) 57:119–129 maxima while continuously shifting the focusing distance. Pollen grains on the stigmas were counted in the same Fruit set of these flowers was checked 14 days after anthesis. manner as described above by collecting stigmas 48 h after flowering, and fruiting success was assessed on 15 October Counting pollen grains on insects’ body surfaces 2020. We excluded flowers on plants that already had grow - ing fruits from the assessment of fruiting success, because We counted the numbers of C. maxima pollen grains on the C. maxima plants often abort fruits when another fruit is body surfaces (excluding pollen loads) of flower visitors cap- growing (Stephenson 1981). In case the plant already had tured on male and female flowers, following the experimental growing fruit(s), we counted the pollen grains deposited on protocol of Nikkeshi et al. (2019). We captured 23 and 14 the stigma, but then removed the ovary of those flowers. The honeybees, and 9 and 4 B. diversus diversus from female and numbers of flowers used for checking fruit set were 11, 13, male flowers, respectively, at Farm A on 20 September 2020. 17, 9, 15, and 10 for the NP, 1-Mp, 1-HB, 1-Bd, OP, and Each tube containing a captured insect was immediately put HP treatments. on ice in a cooler bag to quell its activity and prevent decom- position of the pollen loads. The insects were then taken to Relationship between the number of pollen grains our laboratory and stored in the tubes at − 30 °C in the freezer on a stigma and yields until pollen-counting analysis. After cutting away the insect’s hind legs by using scissors to exclude pollen loads, each body To clarify the relationship between the number of pollen sample was put into 0.4 M sucrose solution and vortexed. We grains deposited on a stigma and resulting seed numbers then counted the number of pollen grains in small drops of and fruit weight, we used fruits that had developed from the the solution under a microscope (SZ61), and calculated the flowers from which the stigmas were collected for counting total number of pollen grains attached on the individual’s body deposited pollen grains. We harvested and weighed fruits on surface. 13 November 2019 or 19 November 2020, and the numbers of mature seeds were counted afterwards. We limited the Evaluating pollination efficiency by a single visit fruits to those grown in the experimental field because of the difference of growing conditions and cultivar used. Thus, To evaluate pollination efficiency of visitor species, we per - 33 and 32 flowers were used for this experiment in 2019 and formed a field experiment to limit the number of visits at the 2020, respectively. experimental field in 2020. The number of pollen grains on a stigma and the probability of fruit set were compared among Statistical analysis the following 6 pollination treatments: (1) no pollination (NP; 11 flowers), bagged with white polypropylene cloth as a nega - To test whether the number of C. maxima pollen grains on tive control; (2) single visitation by M. pyrrhosticta (1-Mp; 22 the insect body surface differed significantly between major flowers); (3) single visitation by a honeybee (1-HB; 22 flow - visitors (honeybees vs. B. diversus diversus) and between ers); (4) single visitation by B. diversus diversus (1-Bd; 19 the individuals collected on female versus male flowers, we flowers); (5) open-pollination (OP; 24 flowers); and (6) hand- fitted generalized linear models (GLMs) assuming a Poisson pollination (HP; 10 flowers). For hand-pollination, an operator distribution in which the response variable was the number picked up a male flower, removed the petal off, and touched of pollen grains and the explanatory variables were visitor a stigma with the stripped anther. A male flower was used for species, flower sex, and the interaction effect. Tukey’s HSD one to three female flowers. This procedure was finished by test was carried out for post hoc multiple comparisons. 08:00 h. To compare the number of pollen grains on a stigma All of the flower buds except for the OP and HP treatments among the six pollination treatments (i.e., NP, 1-Mp, were covered with polypropylene cloth bags the previous day 1-HB, 1-Bd, OP, and HP), we performed a non-parametric of the experiment to prevent unexpected visitations by insects Mann–Whitney U test for pairwise comparisons. Follow- before experiment. For the 1-Mp, 1-HB, and 1-Bd treatments, ing Bonferroni correction, we set the threshold value to flowers were left opened on the morning of anthesis until we p = 0.05/15 = 0.0033. confirmed a single visit by one of the targeted species, and re- To compare the probability of fruiting success among bagged immediately after that. This experiment was performed the six treatments, we calculated the probability distribution during 06:00–08:00 h to avoid depletion of pollen in male of the fruiting success for each treatment using a Bayes- flowers. Some of the flowers randomly selected from the OP ian model. In the model, we assumed that the result of treatment were concurrently used as targets for the monitoring each treatment (i.e., the number of fruit set out of the total of pollinators and fruiting success (see subsection “Monitoring replicates) was caused by Bernoulli trials with the certain frequency of flower visitations and fruiting success in open success probability. To fit a Baysian model, we ran 2000 pollination” in Methods). iterations for each of four chains in Markov chain Monte 1 3 Applied Entomology and Zoology (2022) 57:119–129 123 Carlo sampling. We calculated the mean value and the 95% confidence interval (CI) of the estimated probability distri- bution of each treatment and compared them among the six treatments. To test whether the fruiting success increased with the number of pollen grains on a stigma, we fitted a GLM to the data in which the response variable was fruiting success, assuming a binomial distribution, the link function was the logit, and the explanatory variable was the number of pollen grains on a stigma. The number of pollen grains at the inflec- tion point on a fitted logistic curve, where the probability of fruiting success was 0.5, was calculated from the estimated values of the best-fitting model. We examined whether the number of mature seeds in a fruit increased with the number of pollen grains on a stigma. We fitted a generalized linear mixed model (GLMM) to the dataset in which the response variable was the number of mature seeds in a fruit, with Poisson distribution, the link function was the logarithm, the explanatory variable was the number of pollen grains on a stigma, and year was a random effect. To test whether the fresh weight of a fruit increased with the number of pollen grains on a stigma, we fitted a linear regression mixed model to the dataset, in which the response variable was the fresh weight of a fruit, the explan- atory variable was the number of pollen grains on a stigma, and year was a random effect. All statistical analyses were performed in R statistical software, version 4.0.4 (R Development Core Team 2021). We used the rstan package for data sampling when using the Markov chain Monte Carlo method in a Bayesian model (Stan Development Team 2020). Results Flower‑visitor species Morphological identification indicated that the flower visitors captured in the experimental field in the 2 years consisted of at least 16 species. The honeybees captured in 2019 (126 individuals) were identified as A. mellifera (107 individuals; 84.9%) and Apis cerana japonica Rado- szkowski (Hymenoptera: Apidae) (19 individuals; 15.1%), and those captured in 2020 (113 individuals) were identi- fied as A. mellifera (108 individuals; 95.6%) and A. cerana japonica (5 individuals; 4.4%). The other flower visitors were identified as Aulacophora nigripennis Motchulsky (Coleoptera: Chrysomelidae), Nitidulidae sp. (Coleoptera), Stomorhina obsoleta (Wiedemann) (Diptera: Calliphoridae), B. diversus diversus (Hymenoptera: Apidae), Lasioglossum mutilum (Vachal), Lasioglossum occidens (Smith) (Hyme- noptera: Halictidae), C. annulata annulata, Megacampsom- eris schulthessi (Betrem), Scolia decorata ventralis Smith 1 3 Table 1 All flower visitors to a single female flower of Cucurbita maxima during anthesis Site (year) No. of flow - No. of flower visitors (mean ± standard deviation) No. of pollen Fruiting success (%) ers vide- grains deposited Honeybees Bombus Small bees Campsomeriella Macroglossum Others Total otaped diversus annulata annulata pyrrhosticta diversus Experimental field (2019) 12 81.5 ± 27.0 2.8 ± 2.8 0.4 ± 1.2 0.3 ± 0.7 0.0 ± 0.0 1.3 ± 1.8 86.3 ± 25.2 555.8 ± 229.3 10/12 (83.3) Experimental field (2020) 15 128.1 ± 23.6 7.1 ± 7.1 4.1 ± 2.5 2.0 ± 4.1 7.9 ± 5.7 1.1 ± 2.3 150.2 ± 21.3 1061.3 ± 247.8 13/13 (100) Farm A (2020) 12 147.3 ± 49.7 17.2 ± 18.8 2.9 ± 4.2 1.7 ± 4.2 1.7 ± 1.8 1.8 ± 2.0 172.4 ± 47.1 1033.6 ± 373.7 10/11 (90.9) Farm B (2020) 12 123.2 ± 47.2 23.4 ± 21.8 3.9 ± 3.4 0.3 ± 0.6 11.1 ± 6.3 1.0 ± 1.2 162.8 ± 52.5 677.2 ± 235.9 10/12 (83.3) Farm C (2020) 15 121.6 ± 30.1 0.1 ± 0.5 0.5 ± 1.3 0.5 ± 1.2 8.2 ± 5.9 0.3 ± 0.8 131.3 ± 33.2 735.5 ± 168.4 13/15 (86.7) 124 Applied Entomology and Zoology (2022) 57:119–129 (Hymenoptera: Scoliidae), Vespa analis Fabricius (Hyme- pyrrhosticta set fruit (flower ID: 20F041, 20F043, 20F048, noptera: Vespidae), Diaphania indica (Saunders) (Lepidop- 20F050, 20F053, and 20F054 in Table S2). tera: Crambidae), Parnara guttata guttata (Bremer et Grey), Pelopidas mathias oberthuri Evans (Lepidoptera: Hesperii- Number of pollen grains on insect body surfaces dae), and M. pyrrhosticta (Lepidoptera: Sphingidae). The number of C. maxima pollen grains on the body surfaces Frequency of flower visitations of insect visitors significantly differed among major species (GLM: z = − 42.51, p < 0.0001; Fig. 1). Individuals captured Although numbers varied among locations, video record- on male flowers carried more pollen grains compared to ing of all the flower-visiting insects showed that the mean those on female flowers (z = 53.18, p < 0.0001). An interac- number of total visits to a single female flower by any insect tion effect of the two factors was also detected (z = − 9.20, was more than 80 (Table 1). Most of the visits were made by p < 0.0001). Thus, the number of pollen grains on B. diver- honeybees, accounting for 94.4% and 85.3% at the experi- sus diversus individuals was significantly greater than those mental field in 2019 and 2020, respectively, and 85.4%, on honeybee individuals, and the number of pollen grains 75.7%, and 92.6% at Farms A, B, and C, respectively. In on an individual collected on a male flower was significantly contrast, the percentages of flower visitations by the other greater than that on a female flower. Furthermore, the dif- insects, such as B. diversus diversus, small bees, C. annu- ference between the number of pollen grains on individuals lata annulata, and M. pyrrhosticta, were inconsistent among captured on female and male flowers was large in B. diver - years and locations. sus diversus compared to that of honeybees. While 26.9% (74 out of 275 individuals) of the captured honeybees pos- Fruiting success by open pollination sessed pollen loads, most of which consisted of C. maxima pollen grains, on their hind legs, none of the captured 18 For each stigma examined, more than 500 pollen grains B. diversus diversus possessed pollen loads (Kamo et al., on average were observed at any location (Table 1). Fruit unpublished data). sets ranged between 83.3% and 100%. Six female flow - ers that received visitations only from honeybees and M. Pollination efficiency of each species by single visits The numbers of pollen grains (mean ± SD) on a stigma in the six treatments were 0 ± 0 (NP), 15.1 ± 16.8 (1-Mp), 29.3 ± 48.6 (1-HB), 170.1 ± 114.7 (1-Bd), 1058.0 ± 305.5 (OP), and 1507.2 ± 173.0 (HP). Between any pair of the six treatments, the number of pollen grains on a stigma was significantly different (OP–HP: W = 25, p < 0.0001, OP–NP: W = 2 7 5 , p < 0 . 0 0 0 1 , O P – 1 - H B : W = 5 5 0 , p < 0.0001, OP–1-Bd: W = 475, p < 0.0001, OP–1-Mp: W = 550, p < 0.0001, HP–NP: W = 110, p < 0.0001, HP–1-HB: W = 220, p < 0.0001, HP–1-Bd: W = 190, p < 0.0001, HP–1-Mp: W = 220, p < 0.0001, NP–1-HB: W = 22, p < 0.0001, NP–1-Bd: W = 0, p < 0.0001, NP–1-Mp: W = 27.5, p < 0.001, 1-HB–1-Bd: W = 46, p < 0.0001, 1-Bd–1-Mp: W = 388.5, p < 0.0001), except for the pair of 1-HB and 1-Mp (W = 284.5, p = 0.316) (Fig. 2). Because all of the R-hat convergence diagnostic values were within the range of 0.999 and 1.000, we considered that all Bayesian models converged well. The estimated mean value of the fruiting success probability was 0.077 (95% CI, 0.0019–0.270) in NP, 0.067 (0.0021–0.0229) in 1-Mp, Fig. 1 The numbers of Cucurbita maxima pollen grains on the body 0.105 (0.012–0.282) in 1-HB, 0.725 (0.444–0.934) in 1-Bd, surface of major insect visitors, Bombus diversus diversus (Bd) and honeybees (HB), captured on female and male flowers. The top, mid- 0.938 (0.782–0.998) in OP, and 0.917 (0.703–0.998) in HP dle, and bottom lines of each box indicate the third, second (median), (Fig. 3). Thus, the 95% CIs of 1-Bd, OP, and HP widely and first quartiles, respectively. The top and bottom end of whiskers overlapped, but were distinct from those of NP, 1-Mp, and indicate the maximum and minimum values between the third quar- 1-HB, which overlapped. tile + 1.5 interquartile range (IQR) and the first − 1.5 IQR 1 3 Applied Entomology and Zoology (2022) 57:119–129 125 Fig. 2 The number of pollen grains deposited on a stigma of Cucur- bita maxima ‘Ebisu’ in six pollination treatments: no pollination (NP); single visitation by Macroglossum pyrrhosticta (1-Mp); single visitation by honeybees (1-HB); single visitation by Bombus diversus diversus (1-Bd); open pollination (OP); and hand-pollination (HP). The top, middle, and bottom lines of each box indicate the third, sec- ond (median), and first quartiles, respectively. The top and bottom end of whiskers indicate the maximum and minimum values between the third quartile + 1.5 interquartile range (IQR) and the first − 1.5 Fig. 3 Distribution of the probability density of fruit setting estimated IQR. Closed circles are outliers. A significant difference was detected by Baysian models of Cucurbita maxima ‘Ebisu’ in six pollination between any pair of the six treatments (see text for details) except for treatments: no pollination (NP); single visitation by Macroglossum the pair of 1-HB and 1-Mp pyrrhosticta (1-Mp); single visitation by honeybees (1-HB); single visitation by Bombus diversus diversus (1-Bd); open pollination (OP); and hand-pollination (HP) Relationship between the number of pollen grains deposited on stigmas and yields Behaviors of flower visitors The success of fruit set increased with the increase in Common behaviors of honeybees and a bumblebee in a the number of the pollen grains on a stigma (z = 3.46, female flower are presented in Supplementary Video 1. As p = 0.00055; Fig. 4). The estimated values of the intercept shown in these images, in which two honeybee individuals and coefficient in the estimated logistic curve were − 4.063 (A. mellifera and A. cerana japonica, in order of appear- and 0.024, such that the number of pollen grains at the ance) were sucking nectar, the honeybees’ bodies made inflection point of the curve was 167.7. inconsistent contact with the pistil. In contrast, B. diversus GLMM detected a significant effect of the number of pol- diversus, which visited the same flower later, moved clum- len grains on the number of mature seeds in a fruit (z = 4.16, sily inside the corolla, resulting in more contacts with the p < 0.0001, Fig. 5a). The coefficient value of the explanatory reproductive organs. In addition, a large amount of pollen variable was 0.000114, meaning that the number of mature was visible on the fuzzy body of the bumblebee. seeds increases exp(0.000114) = 1.000114 times when the Two different types of contact with a female flower by number of pollen grains deposited on a stigma increases by M. pyrrhosticta are presented in Supplementary Videos 2 one. However, the fresh weight of a fruit did not significantly and 3. When this species sucks nectar with its extended increase with the number of pollen grains deposited on a proboscis, it makes few contacts with the flowers’ stigma (t = − 1.60, p = 0.115, Fig. 5b). reproductive organs (Supplementary Video 2). We also observed, however, that M. pyrrhosticta often held onto a stigma when sucking nectar, and the behavior is likely 1 3 126 Applied Entomology and Zoology (2022) 57:119–129 Fig. 4 Probability of fruit set of Cucurbita maxima ‘Ebisu’ when the given number of pollen grains is deposited on a stigma. Open circles indicate observed data and the dashed line indicates a fitted logistic curve to involve depositing pollen on a stigma (Supplementary of pollen grains deposited during a single visit by a honey- Video 3). bee (29.3 ± 48.6), only six visitations by honeybees might meet the threshold number. Therefore, we conclude that hon- eybees were also effective pollinators for the kabocha squash Discussion at our experimental field. Artz and Nault (2011) reported a positive relationship between the number of visitations The current study revealed that B. diversus diversus was the and the subsequent fruiting success of Cucurbita pepo L., most efficient pollinator per visit in delayed-start culture of for both European honeybee and the bumblebee Bombus kabocha squash in Kagoshima, Japan. The number of pol- impatiens Cresson, but under a low visitation rate the ratio len grains on individuals of this species was significantly of fruiting success was lower for flowers pollinated by hon- greater than that on honeybee individuals. The average eybees compared to those pollinated by bumblebees. Thus, number of pollen grains deposited on a stigma by a single honeybees’ low efficiency of pollination is compensated for B. diversus diversus visit was also larger than that from a by their large numbers, whereas the highly efficient pollina- single honeybee visit or a single M. pyrrhosticta visit. In tion of bumblebees requires fewer visits to achieve fruit set. the pollination experiment, even a single visit by B. diver- Furthermore, our study is the first to indicate that a lepi- sus diversus nearly achieved fruit set (success probability: dopteran species, the hummingbird hawkmoth, M. pyrrho- 0.725), whereas visitation by a single honeybee or a single sticta, is likely to provide significant pollination service to M. pyrrhosticta hardly achieved set fruit (success probabil- Cucurbita spp. Although Bomfim et al. (2016) noted flower ity: 0.105 and 0.067, respectively). Thus, this bumblebee visitation to cucurbit crops by lepidopterans, their deposi- pollinates far more efficiently than honeybees and the hawk - tion of pollen on stigmas was thought to be little or none. moth at the individual level. The bumblebee’s excellent per- In a study using Cucurbita andreana Naudin, the behavior visit pollination efficiency is consistent with previous studies of flower visitors was monitored and pollen grains attached performed in other countries (Artz and Nault 2011; Pfister on their body surfaces were counted (Ashworth and Galetto et al. 2017; Vidal et al. 2010; Xie and An 2014). Our field 2001); the authors concluded that Eudiopthis hyalinata Stoll. observations suggest that their large and hairy body and the (Lepidoptera: Pyraustidae) only took nectar, without touch- clumsy movements in the flowers likely contribute to the ing fertile whorls of the flower. In contrast, M. pyrrhosticta pollination efficiency of bumblebees. often landed on the reproducing organs of kabocha squash We confirmed that female flowers that were exclusively flowers instead of hovering while sucking nectar. This visited by honeybees or M. pyrrhosticta, less efficient pol- behavior might be due to the mismatch between the length of linators than B. diversus diversus, could set fruit (Table S2). proboscis and the depth of flower (Cucurbita maxima have Taking account of the average minimum number of pollen quite large flower compared to C. andreana; see Nakamura grains required for fruit set (167.7) and the average number et al. 2021; Teppner 2004). Our results demonstrated that M. 1 3 Applied Entomology and Zoology (2022) 57:119–129 127 relatively limited due to its lower per-visit efficiency and its smaller and fluctuating population (no individual was recorded in the experimental field in 2019). Further detailed studies may reveal other overlooked relationships between lepidopterans and cucurbit crops. Although there should be clear species differences in pollination ability among pollinators, the number of pol- len grains deposited during a single visit could depend on multiple factors. The numbers of pollen grains deposited during a single bumblebee or honeybee visit in our study are comparable with those reported by Xie and An (2014) (bumblebees, 14–604; honeybees, 0–12), but much smaller than those reported by Pfister et al. (2017) (Bombus ter - restris L., mean 3369; A. mellifera, 582). These large differences might be partially due to the spatial arrange- ments of flowers in the field. At our intensively managed study site, like other small-scale kabocha squash fields in Japan, vines are arranged to grow in one direction. This results in spatial separation of flower sexes in two lines, because female flowers tend to bloom near the tip of a vine and male flowers clump at the base. In addition to field management, differences in pollinator density, sex ratio of flowers, and environmental conditions also could have affected the results (McGrady et al. 2020). The estimated threshold number of pollen grains needed for fruit set, 167.7, is comparable with those reported for C. pepo, with reported estimates of 70 and 174 grains (Artz and Nault 2011; Vidal et al. 2010). However, our estimate is much less than the approximately 500 grains estimated for C. maxima ‘Hokkaido’ (Pfister et al. 2017). Several factors, such as differences in species and temperature, might explain this difference (Pfister et al. 2017). We investigated whether additional visitations by polli- nators over the minimum threshold for fruit set are neces- sary for plants to produce kabocha squash fruit with higher economic value. As the number of pollen grains deposited on a stigma increased, that of mature seeds also increased. Fig. 5 The number of mature seeds developed in a Cucurbita maxima No correlation was detected between the number of pollen ‘Ebisu’ fruit (a) and the fruit weight (b) when the given number of grains and the fresh weight of the fruit, however, as oppo- pollen grains was deposited on the stigma. Open and closed circles indicate observed data in 2019 and 2020, respectively. Dashed lines site trends were noted in the two research years. In a previ- show a linear regression line for each research year ous study, Nicodemo et al. (2009) reported an inconstant relationship between visitation number by A. mellifera to a female flower of C. maxima and fruit weight, but the number pyrrhosticta could be a significant pollinator of squash at our of visits to a female flower is not always proportional to the study sites. On average, this species deposited 15.1 pollen number of pollen grains deposited on the stigma. It might be grains per visit on a stigma. In 2020, this species visited each necessary, therefore, to obtain more data in multiple years female flower 7.9–11.1 times in three of the four study sites. for more accurate analysis of the relationship between the Therefore, the minimum number of pollen grains required number of pollen grains and the fresh weight of fruit. How- for fruiting success, 167.7, was potentially exceeded by M. ever, farmers in this region aim to produce kabocha squash pyrrhosticta visitations. The genus Macroglossum is diurnal fruit of more than 0.8 kg fresh weight (minimum market- or crepuscular (Kendrick 2010), which suits the flowering able size) and ideally 2.5 kg fresh weight (suitable size for a hour of Cucurbita spp. However, the contribution of M. pyr- standard box for six fruits). Given that the fresh weight of all rhosticta to total squash pollination in this region would be the fruit produced in our pollination experiment far exceeded 1 3 128 Applied Entomology and Zoology (2022) 57:119–129 need to obtain permission directly from the copyright holder. To view a 0.8 kg, pollen deposition above the level of the minimum copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . requirement for fruit set would not necessarily be required for kabocha squash production. In addition to the experimental field, we confirmed that References the female flowers of C. maxima received pollen sufficient for fruit set in three private farms. Nevertheless, hand-polli- Albrecht M, Kleijn D, Williams NM, Tschumi M, Blaauw BR, Bom- nation is in practice in two of these farms. 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Applied Entomology and Zoology – Springer Journals
Published: May 1, 2022
Keywords: Apis mellifera; Bombus diversus diversus; Cucurbita maxima; Macroglossum pyrrhosticta; Pollinator efficiency
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