Pollination of Cherimoya (Annona Cherimola Mill.) by the Mass-Propagated Beetles Haptoncus Ocularis, Mimemodes Monstrosus, and Carpophilus Marginellus

Trop. Agr. Develop. 58（1）：18 － 25，2014

Pollination of Cherimoya (Annona cherimola Mill.) by the Mass-propagated Beetles Haptoncus ocularis, Mimemodes monstrosus, and Carpophilus marginellus

Hirokazu HIGUCHI1, *, Morio TSUKADA2, Tomoko NINOMIYA1, Takayuki FURUKAWA2, Akihiro YOSHIDA2, and Masafumi KURIYAMA2

1 Graduate School of Agriculture, Kyoto University, Kitashirakawa Sakyo-ku, Kyoto 606-8502, Japan 2 Faculty of Bioresources, Mie University, Kurimamachiya-cho, Tsu, Mie 514-8507, Japan

Abstract Insect pollination of cherimoya (Annona cherimola Mill.) was evaluated using artificially propagated Haptoncus ocularis, Mimemodes monstrosus, and Carpophilus marginellus beetles, which are some of the main insect visitors to cherimoya grown on the Japanese mainland. Among these species, both H. ocularis and C. marginellus proved to be good pollinators leading to more seed formation and better fruit set. The harvested fruits obtained following the release of either of these two species were similar in size to hand-pollinated fruits, although the release of C. marginellus led to a better fruit set than the release of H. ocularis. The release of these species at about five individuals per flower, or possibly less for C. marginellus, under ideal cool and humid environmental conditions may be sufficient for the effective pollination of cherimoya. Key words: Annonaceae, Cantharophyly, Fruit set, Nitidulid beetle, Pollinator density

from our previous insect pollination experiment using Introduction feral beetles collected from a Japanese cherimoya or- The natural pollinators of cherimoya (Annona chard (Higuchi et al., submitting) as having the greatest cherimola Mill.) are beetles, although cherimoya grow- potential to serve as effective pollinators for cherimoya. ers worldwide commonly practice hand-pollination to To establish a mass rearing beetle pollination system ensure fruit set. Various studies have been conducted as a practical technique, such as in bee culturing, further to improve hand-pollination and fruit set in terms of the studies including a rigid selection of insect species and environmental physiology of reproductive organs (e.g., a determination of the optimum insect population are Richardson and Anderson, 1996; Higuchi et al., 1998; required. Therefore, a large quantity of insects is re- Rosell et al., 1999; Matsuda and Higuchi, 2012), whereas quired for testing. Recently, mass propagation methods fewer studies (e.g., George et al., 1989; Lopez and for H. ocularis (Tsukada et al., 2005b), M. monstrosus Uquillas, 1997; Peña et al., 2002) have been conducted (Asai and Tsukada, data not published), and Carpophilus on natural or insect pollination. Studies on the practical marginellus (Tsukada et al., 2008) were established. In utilization of insect pollinators have only recently begun this study, we conducted pollination experiments using (e.g., Tsukada et al., 2005b; Higuchi et al., submitted these artificially propagated species. with this study). Although we reared without live insects as bait The insect community of cherimoya visitors varies (see Materials and Methods), M. monstrosus has been according to region. Tsukada et al. (2005a) surveyed considered to be insectivorous (Hisamatsu, 1985), the cherimoya insect community in Japan and identi- suggesting a low potential to become a pest (Tsukada fied a large difference from other areas, which mainly et al., 2005a), and it may be an advantageous rather consisted of nitidulid beetles (Peña et al., 2002). The than herbivorous or frugivorous species. The two other Japanese community included Phloeonomus sp. (Staph- species, H. ocularis and C. marginellus, are frugivorous ilinidae), Cortinicara gibbosa (Lathridiidae), Mimemodes and belong to the family Nitidulidae, which is the main monstrosus (Rhizophagidae), and Haptoncus ocularis group of annona pollinators in Queensland (George et (Nitidulidae) as its major constituents. Among these al., 1989), Florida (Nagel et al., 1989), and Israel (Gazit species, H. ocularis and M. monstrosus were identified et al., 1982). Although H. ocularis and C. marginellus in these regions are not the main annona visitors, the allied

Communicated by H. Gemma species H. luteolus and C. hemipterus are often reported Received Jul. 12, 2013 as pollinators (Gazit et al., 1982; Nadel and Peña, 1994; Accepted Dec. 13, 2013 Lopez and Uquillas, 1997). Thus, H. ocularis and C. mar- * Corresponding author [email protected] ginellus might also have the potential to be pollination Higuchi et al.: Cherimoya pollination by rearing beetles 19 agents of cherimoya. Experiment 1 We first verified whether artificially propagated The experiment was conducted in 2000 under plas- these beetles fed with a fruit diet were attracted to tic-house conditions in a private commercial cherimoya female-stage flowers and pollinated them, following the orchard in Shimotsu, Wakayama, using 38 trees of 7- method of Higuchi et al. (2014). Generally, whether year-old ‘Big Sister’ cultivars and the nitidulid beetle H. insects raised on a fruit diet during larvae are attracted ocularis. Together with the basal stem and some leaves, to flowers is questionable because fruit-philic individu- floral buds at several days before anthesis (i.e., the petals may be selected via genetic inheritance or learning als were closed tightly so that insects could not enter) during the larval stage. Furthermore, while studies have were carefully covered with a 0.1-mm mesh cylindrical established the effective density of insect visitors inside gauze net (φ 0.45 m × 0.9 m) to exclude arachnids or flowers for pollination (Gazit et al., 1982; Nadel and other insectivorous arthropods. In the evening of the Peña, 1994), little is known about how many insects are following day, flowers that had reached the female stage necessary for adequate pollination using mass-release and whose petals had begun to separate were selected techniques. Thus, we also attempted to determine the for the experiment, and 1, 5, or 15 beetles were released population of mass-released beetles required to effec- into the bags after pollen was dusted onto their bodies. tively pollinate cherimoya. The release of insects was conducted on June 22 and Next, to investigate whether artificially mass-propa- July 28. Each treatment group was compared to a control gated insects displayed pollination behavior pertinent to group of open-pollinated flowers. The open pollination cherimoya, we released groups of beetles into bagging was performed on June 22, July 13, and July 28. enclosures containing both male- and female-stage In the evening 1 or 2 days after treatment, the flow- flowers to observe their behavior. To set fruit, insect ers reached the male stage; the bags were removed and pollinators need to remain inside female-stage flowers the flowers were treated with gibberellic acid (GA3) and throughout the transition to the male stage. They then tagged with the flowering date and treatment method. leave dusted in pollen and enter another female flower, Cherimoya trees drop fruit in their early stages if seed thereby pollinating it. formation is not sufficient, which depends on pollination. However, when treated with GA , trees retain their Materials and Methods 3 fruit, even when pollination is insufficient. Therefore,

Insect materials we applied 1500 ppm GA3 to the calyx of every tagged Wild H. ocularis and M. monstrosus were collected male-stage flower (Yonemoto et al., 2000). Thereafter, from cherimoya flowers in commercial orchards in GA3 treatment was repeated every 2 weeks. All the fruits Wakayama Prefecture, Japan, and C. marginellus was were harvested until September 27 and sliced to count collected from flowers on an experimental farm belong- the number of seeds, which can be used as a more ing to Mie University using an insect aspirator. The reliable index of pollination than the fruit-set percentage beetles were propagated in cages following the method (Higuchi et al., 2014). of Tsukada et al. (2005b). They were given a slice of pineapple every week, and no other food was provided. Experiment 2 The pineapple began to decay after several days, becom- In 2001, a similar experiment was conducted under ing a suitable site for laying eggs and a good medium plastic-house conditions in a private commercial orchard for larvae. For M. monstrosus, some pieces of sliced in Kokawa, Wakayama, using 75 trees of 6-year-old ‘Big cherimoya were supplied in addition to pineapple. The Sister’, and both the nitidulid beetle H. ocularis and the beetles were incubated at 20–25oC. rhizophagid beetle M. monstrosus. As in Experiment 1, buds at a few days before Release density of beetles for pollination anthesis were covered with gauze bags, and flowers that To verify whether the artificially reared beetles had reached the female stage the following day were would visit cherimoya flowers and contribute to pollina- selected, in which 1, 5, or 15 beetles, dusted in pollen, tion, and to determine how many beetles are required were released into the bags. Flowers that did not reach for effective pollination, we conducted the following the female stage were left bagged and were regarded as three experiments in which we released a different num- insect-exclusive treatments. Each treatment group was ber of beetles, dusted in pollen, into a bag containing a compared to both the hand- and open-pollinated control cherimoya flower at the female stage. groups. This experiment was performed on June 12–14. 20 Trop. Agr. Develop. 58（1）2014

In the evening of the day flowers reached the male stage flowers to coexist together in the same bag when stage, the bags were removed and the flowers were the beetles were released. To create such conditions, we treated with GA3 and tagged. Thereafter, GA3 was ap- placed the potted plants next to each other, arranged so plied every 2 weeks. All the fruits were harvested until that male-stage flowers from the smaller 4-year-old trees October 19, and seeds were counted as described in in the 10-L pots could be housed with female flowers Experiment 1. from the larger 5-year-old trees. The experiment consisted of replications of the Experiment 3 4-day treatments. On the morning of the first day, floral In 2004, another similar experiment was conducted buds with tightly closed petals, including their derived under greenhouse conditions at Kyoto University using stems and some leaves from the smaller trees, were seven 5-year-old ‘Big Sister’ cherimoya trees grown in 40- covered with gauze bags to exclude other arthropods. In L pots and fifteen 4-year-old trees in 10-L pots, together the evening, flowers that had begun to open, indicating with the nitidulid beetles H. ocularis and C. marginellus. that they were 2 days before anthesis, were selected Bagging was similar to that described for Experi- as pollen suppliers. Flower buds from the larger trees ments 1 and 2, and 1, 5, or 15 H. ocularis or C. marginellus were selected as target flowers (i.e., timed to reach the individuals dusted in pollen were released into the bags receptive female stage when housed with the pollen between May 1 and June 25. A hand-pollinated control suppliers). Pollen suppliers and target flowers were then group was used for comparison. housed together in gauze bags by moving the smaller After flowers reached the male stage, the bags were trees. removed and flowers were treated with GA3. The fruits In the evening of the second day, 15 H. ocularis or C. were harvested while still young (at a diameter of about marginellus individuals were released into the bags. By 5 cm) to control the competition for a limited assimilate. the time of release, the bagged flowers were 1 or 2 days All fruits were harvested until September 14 and the before anthesis, when the tips of the petals were half- number of seeds was counted. way or slightly open, respectively. In the evening of the third day, most flowers of the smaller trees and those of The fruit set of cherimoya is largely affected by the larger trees had reached the male and female stage, temperature and humidity (Higuchi et al., 1998), as well respectively. In bags in which flowers did not show the as the behavior of pollinators; thus, we monitored the expected stages at this point, the experiment was ter- temperature during the 2001 experiment, and both tem- minated. On the fourth day, the bags and insects were perature and humidity throughout the 2004 flowering removed, and the flowers were tagged with the date and season. The percentage of pollen germination, which is method of pollination. These steps were repeated 11 also influenced by environmental changes, affects fruit times for H. ocularis and 29 times for C. marginellus. set, growth, and development (Saavedra, 1977). Thus, A group of 558 open-pollinated flowers, and five rep- the fresh pollen germination percentage on the day of lications of hand-pollination, were used for comparison. pollination throughout the 2004 flowering period was For the open-pollinated group, insects were released examined following incubation at 25oC for 12 hours on during May 1–July 2, 2004, roughly approximating the 2% agar media containing 15% sucrose (Yonemoto et al., 2004 flowering period (April 25–July 18). Every evening 1999). The pollen germination percentage was deter- throughout this period, the flowering date and number of mined from the averages of more than nine replications all cherimoya trees inside the greenhouse were recorded. of 200 grains by microscopic observation. Pollen with the The fruit set percentage was calculated for all flowers 2 tube extended farther than its diameter was considered weeks after anthesis, and the fruits were harvested after to have germinated (Higuchi and Utsunomiya, 1999). dropping naturally. The weight, height, diameter, and number of seeds of the harvested fruits were measured. Released nitidulid beetles in bags with both female- Daily changes in temperature, humidity, and the pollen and male-stage flowers germination percentage in the greenhouse were moni- Experiment 4 tored throughout the flowering period. To verify whether H. ocularis and C. marginellus The experiment was conducted under the same would pollinate and set fruit by visiting both male and greenhouse conditions at Kyoto University using flow- female flowers in the correct order, a cantharophilous ers from the same trees as in Experiment 3. experiment was designed to allow male- and female- Higuchi et al.: Cherimoya pollination by rearing beetles 21

Statistical analysis unusually hot, as shown later, which detrimentally In Experiment 1, data were analyzed using a affected fruit set. The release of H. ocularis resulted in generalized linear model (GLM) with Tukey’s multiple fewer seeds than the release of M. monstrosus (Table comparison test. In Experiments 2 and 3, a two-way 2), implying that the latter was more effective under analysis was conducted using a GLM with the beetle hot environmental conditions, which is consistent with species as a fixed factor and the density as a numeral previous reports (Tsukada et al., 2005a). The estimated factor, and when the interaction was significant, Tukey’s percentages of fruit set for M. monstrosus were 27.3% multiple comparison test was conducted. The analysis when 5 individuals were released and 33.3% when 15 was carried out using R 2.15.1 (R Development Core individuals were released, indicating that this species Team, 2012). is an effective pollinator of cherimoya under such hot conditions. Figure 1 shows the changes in maximum Results and Discussion and minimum air temperature in the plastic house over Availability of artificially propagated beetles for the experimental period. The maximum temperature pollination frequently exceeded 35ºC, implying severe conditions According to the Experiments 1, 2, and 3, all three for insect survival (Tsukada et al., 2005b), and which artificially propagated, fruit-fed beetle species (H. ocu- negatively affected pollination and probably gave an laris, M. monstrosus, and C. marginellus) were proved unexpected comparative advantage to M. monstrosus. to visit cherimoya flowers and substantially contributed to set fruit. Experiment 3 We did not use M. monstrosus in this experiment Density of beetles reared for pollination primarily because of its slower reproduction rate com- Experiment 1 pared to both H. ocularis and C. marginellus. Moreover, The number of seeds increased as the number of M. monstrosus is insectivorous and requires live food for released H. ocularis individuals increased (Table 1). effective reproduction (Hérard and Mercadier, 1996), Previous studies on insect pollination of atemoya (An- which is disadvantageous for practical use. Cherimoya nona squamosa × A. cherimola; George et al., 1989), pollination strategies should not be conducted under sugar apple (A. squamosa), and atemoya (Nadel and such high temperature conditions as we encountered in Peña, 1994) indicated that fruit set percentages were Experiment 2, when the potential effects of M. monstro- proportional to the number of insects inside the flower. sus could be large. The average seed number obtained by releasing 5 pol- Both H. ocularis and C. marginellus performed well, len-dusted H. ocularis was 10.2, while the average seed resulting in the production of many seeds (Table 3). The number obtained by releasing 15 individuals was 13.8. release of 15 H. ocularis and C. marginellus individuals Both of these values were higher than the average seed produced an average of 70.1 and 53.0 seeds, respec- number achieved by open pollination, indicating that tively. Each fruit contained more than 40 seeds when 15 either 5 or 15 released beetles attained a high percent- individuals were released (Fig. 2). Under natural condi- age of fruit set. tions, fruits with less than 15 seeds are likely to drop, whereas fruits with more than 40 seeds will develop to Experiment 2 be harvested, suggesting that the fruit set percentage The summer in the year of this experiment was would be 100% with a release of 15 individuals of either

Table 1 Numbers of seeds formed by bagging entomophily of cherimoya Number of Number Average number Pollination beetles per flower of flowers of seeds per fruit Haptoncus ocularis 1 11 2.4 c 5 6 10.2 b 15 5 13.8 a Open pollination - 40 3.4 c The effect of pollination was estimated by the number of seeds formed. The flowers were

treated with GA3 immediately after insect rearing to keep the fruit on the tree even if seed formation was not sufﬁcient. Means with the same letters are not signiﬁcantly different at P < 0.05 by Tukey’s multiple comparison test. 22 Trop. Agr. Develop. 58（1）2014

Table 2 Effect of insect species combined with rearing density on cherimoya fruit set as estimated by seed formation Number Number Average number Estimated fruit-set Pollination Beetle species of beetles of ﬂowers of seeds per fruit percentagez Entomophily Haptoncus ocularis 1 11 0.0 c 0.0 5 9 0.8 c 0.0 15 8 2.9 b 12.5 Mimemodes monstrosus 1 13 0.4 c 0.0 5 11 6.5 a 27.3 15 9 8.4 a 33.3 Open pollination - 18 0.1 c 0.0 Exclusion of insects 0 23 0.2 0.0 Hand pollination 0 34 59.3 91.2 Two-way analysis for the entomophilous experimenty Effect of beetle species [B] *** Effect of density [D] *** [B] × [D] interaction *

Fig. 1 Maximum and minimum temperatures during the ﬂowering season for Experiment 2.

Table 3 Effect of insect species combined with rearing density on cherimoya fruit set as estimated by seed formation Number of Number of Average number Estimated fruit-set Pollination Beetle species beetles ﬂowers of seeds per fruit percentagez Entomophily Carpophilus marginallus 1 13 44.4 bc 69.2 5 17 45.6 bc 70.6 15 11 70.1 a 100.0 Haptoncus ocularis 1 7 21.0 d 28.6 5 6 39.7 c 66.7 15 5 53.0 b 100.0 Hand pollination - 5 77.8 100.0 Two-way analysis for the entomophilous experimenty Effect of beetle species [B] ** Effect of density [D] ** [B] × [D] interaction **

The effect of pollination was estimated by the number of seeds formed. The flowers were treated with GA3 immediately after insect rearing to keep the fruit on the tree even if seed formation was not sufficient. z: Attainable percentage of fruit set was estimated for fruit containing more y than 15 seeds and able to keep growing without artificial GA3 addition. : Two-way analysis was conducted by GLM for the entomophilous experiment with the beetle species as a fixed factor and the density as a numeral factor. ** indicates significant differences at P < 0.01. Means with the same letters are not significantly different at P < 0.05 by Tukey’s multiple comparison test. Higuchi et al.: Cherimoya pollination by rearing beetles 23 of these species, which was comparable to hand-pollina- marginellus was estimated to be 69.2%. This percentage tion. These results indicate that no need exists to release is promising from a practical viewpoint. more than 15 insects per flower for H. ocularis and C. The release of five H. ocularis individuals resulted in marginellus. an average of 39.7 seeds per fruit, and the attainable per- The release of C. marginellus, in any density of 1, 5, centage of fruit set was 66.7%. These performances were or 15 individuals, led to a larger number of seeds than the similar to a single release of C. marginellus, whereas the release of H. ocularis at a corresponding density (Table release of one H. ocularis beetle formed only 21 seeds 3), especially at low insect density. This suggests that C. and the estimated percentage of fruit set remained at marginellus may be a more effective pollinator of cheri- 28.6%. These results suggest that the release of five H. moya. A single release of C. marginellus resulted in the ocularis individuals was recommendable to ensure fruit production of more than 40 seeds per fruit on average, set. suggesting that the release of just one C. marginellus Figure 3 shows the changes in temperature and beetle might be practicable under ideal conditions. The humidity during the 2004 flowering period in Kyoto. Al- attainable percentage of fruit set for a single release of C. though ideal conditions for cherimoya pollination (cool

Fig. 2 The distribution of the number of seeds developed in a cherimoya fruit pollinated by beetles released into a bagged cherimoya ﬂower. Average seed numbers per fruit are shown in Table 3.

Fig. 3 Maximum and minimum temperatures (A), and minimum relative humidity (B) during the ﬂowering season for Experiment 3. 24 Trop. Agr. Develop. 58（1）2014

Fig. 4 Percentage of pollen germinating and the number of ﬂowers at anthesis observed during the ﬂowering season for Experiments 3 and 4.

Table 4 Effect of entomophily by propagated beetles on fruit set and fruit size of cherimoya Harvested fruits Number Number Number Fruit Number Diameter Height Weight of of of set Symmetricityz of seeds (mm) (mm) (g) Pollination Species beetles replications fruit set percentage Beetle pollination Carpophilus marginallus 15 11 5 45.5 40.8 76.72 76.53 221.0 3.3 Haptoncus ocularis 15 29 9 31.0 34.3 77.78 75.65 202.9 3.8 Hand pollination - 5 5 100.0 54.0 73.08 77.92 195.8 4.0 Open pollination - 558 0 0.0 - - - - - z: Fruit symmetricity was estimated at 5 levels from 1: deformative to 5: symmetrical. temperatures and high humidity) were recorded during C. marginellus, released in bags housing both male the early flowering season in May, the temperature then and female flowers were effective pollination agents. As increased gradually to reach 35ºC by the end of June shown in Table 4, the fruit set percentage was higher and often exceed 35ºC in the middle of July. Figure 4 for C. marginellus (45.5%) than for H. ocularis (31.0%), shows the changes in number of flowering and pollen consistent with the tendency in Experiment 3 indicated germination percentage throughout the 2004 flowering in Table 3, but either level is high enough for practical season. Flowering and germination displayed similar application. patterns; both were low at the beginning and end of the The average number of seeds produced was 34.3 season, and high in the latter half of May, when the cool for H. ocularis and 40.8 for C. marginellus, which is fewer temperatures with humid air are ideal conditions for than that achieved via hand-pollination (47.3), despite the cherimoya pollination (Fig. 3). In Japan, the last half of similar fruit sizes. Other than seed number, harvested the flowering season occurs after the rainy season, when fruits showed no significant differences in diameter, temperatures increase and humidity decreases. Thus, height, weight, and symmetry among treatments. The the fruit set will be limited not only by a lower survival release of 15 H. ocularis or C. marginellus individuals rate of beetle pollinators but also a reduced performance per flower led to fruits similar in size as hand-pollinated of floral factors, such as pollen-tube growth and pistil fruit. Hand-pollinated fruit typically produce a larger receptivity as exhibited by Higuchi et al., 1998; Matsuda number of seeds than naturally pollinated fruit, which et al., 2011; and Matsuda and Higuchi 2012. Therefore, is not desirable for consumers and reduces the market we recommend beetle release during the cooler early value. Thus, attempts have been made to reduce seed half of the flowering season when pollen germination is numbers by diluting pollen with Lycopodium spores high. or other materials such as polyvinyl chloride (George and Campbell, 1991; Richardson and Anderson, 1996); Released nitidulid beetles in bags with both female- however, our results indicate that beetle pollination can and male-stage flowers reduce the seed number without affecting fruit size. Experiment 4 Some studies have addressed the relationship be- Both of the two nitidulid beetles, H. ocularis and tween the number of visiting insects and fruit set (e.g., Higuchi et al.: Cherimoya pollination by rearing beetles 25

Gazit et al., 1982; Nadel and Peña, 1994). These studies (Osaka) pp. 169-172. (in Japanese) considered the number of insects visiting the flowers, Lopez, E. and C. Uquillas 1997. Carophilus hemipterus (Coleoptera: Nitidulidae) as Cherimoya (Annona cherimola Mill.) pollina- while we considered the number of released beetles as tion agent under controlled conditions. Acta Entomologica a practical parameter. Although directly comparing the Chilena 21: 89-92. results of these studies with our findings is difficult, we Matsuda, H., H. Higuchi, N. Kozai, and T. Ogata 2011. Effect of temperature on the time requirement of pollen tubes to observed that about 75% of released individuals remained penetrate into the embryo sac after pollination in cherimoya outside of the flower, suggesting that when 15 insects (Annona cherimola Mill.). Trop. Agr. Develop. 55: 157-161. were released, only 4–5 were likely to remain inside the Matsuda, H. and H. Higuchi 2012. 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