Appl. Entomol. Zool. 37 (3): 469–475 (2002)

Ecological significance of adult summer diapause after nymphal winter diapause in Poecilocoris lewisi (Distant) (Heteroptera: )

Shinichi I. Tanaka,*,1,2 Chobei Imai3 and Hideharu Numata1 1 Department of Bio- and Geosciences, Graduate School of Science, Osaka City University, Osaka 558–8585, Japan 2 Osaka Environmental Technology and Research Co., Ltd., Osaka 558–0013, Japan 3 Osaka City Institute of Public Health and Environmental Sciences, Osaka 543–0026, Japan (Received 8 February 2002; Accepted 17 May 2002)

Abstract Seasonal adaptations were studied in a partially bivoltine population of Poecilocoris lewisi that overwinters as nymphs. Under short-day conditions, the final (5th) instar nymphs entered diapause, whereas under long-day condi- tions they emerged as adults within 2 weeks. The critical daylength for induction of the nymphal diapause was be- tween 14 and 15 h at 25°C in the Osaka population. P. lewisi entered diapause in the adult stage also, under long-day conditions. Adults emerging in late spring started oviposition only after July. transferred from long-day to short-day conditions at adult emergence began to lay eggs earlier and more synchronously than those kept continu- ously under long-day conditions due to prevention of the adult diapause. The critical daylength for induction of the adult diapause was also between 14 and 15 h at 25°C, as in the induction of nymphal diapause. The dogwood Cornus controversa, a principal host plant of P. lewisi, did not develop seeds until early July. Therefore, this suggests the ab- sence of host plant seeds in late spring is the ultimate factor for the evolution of adult diapause in P. lewisi.

Key words: Nymphal diapause, adult diapause, photoperiodic response, voltinism, host plant

regarded as having such an adaptation mechanism. INTRODUCTION Carbula humerigera Uhler has a winter nymphal At higher latitudes, univoltinism can be realized diapause and summer adult diapause (Kiritani, as a passive consequence of the relative paucity 1985a, 1987). Picromerus bidens L. has a winter of effective temperatures for growth. Two het- egg diapause and summer adult diapause (Musolin eropteran species, Pyrrhocoris apterus and Ly- and Saulich, 2000). gaeus equestris, believed to be strictly univoltine, Poecilocoris lewisi is distributed in Central and have been shown to possess the potential to pro- Southern Japan, China and Taiwan, and overwin- duce a second generation as a response to long-day ters as final (5th) instar nymphs (Tomokuni et al., conditions in unusually warm years (Honeˇk and 1993). This species has been considered to be uni- Sˇrámková, 1976; Solbreck and Sillén-Tullberg, voltine (Kobayashi, 1967; Moriya et al., 1987). 1981; Saulich et al., 1994). In warmer climates, When P. lewisi was reared under long-day condi- however, univoltine insects often have specific con- tions in the laboratory, only a small proportion of trol mechanisms to avoid producing a second gen- females laid eggs with a long preoviposition period eration (Masaki, 1980; Saulich and Musolin, (Moriya et al., 1987). Moreover, our preliminary 1996). observations showed that adults emerging in spring One peculiar mechanism has been found in sev- after overwintering soon dispersed from overwin- eral temperate insects (Masaki, 1980), in which tering sites and did not start reproduction until late two diapauses occur at different developmental June. Therefore, P. lewisi seems to have an adult stages within the same generation and enforce uni- diapause induced by long-day conditions. If this is voltinism, i.e. the insects actively suppress devel- the case, the adult diapause could be regarded as an opment at the first diapause stage and ensure enter- adaptation to ensure univoltinism in combination ing the second diapause at the following proper with a winter diapause at the final instar nymphal seasonal time. In Heteroptera, two pentatomids are stage. However, P. lewisi may have a partially bi-

* To whom correspondence should be addressed: E-mail: [email protected]

469 470 S. I. Tanaka et al. voltine life cycle in Osaka, because we observed placed in the outdoor cage. Nymphs hatching from some adults emerging in early August. In the pres- these eggs entered diapause at the final instar. ent study, we attempt to clarify the relationship be- These insects were kept under natural photoperiods tween the two diapauses in different developmental and temperatures through autumn, winter and stages and the partially bivoltine life cycle in P. spring. Adult emergence, mortality and oviposition lewisi. of female adults were recorded from 26 April every second day in both 1995 and 1996. Emerging adults were kept with peanuts as single male-fe- MATERIALS AND METHODS male pairs. Insects. Adults and final (5th) instar nymphs of Seasonal development of host plant seeds. De- P. lewisi were collected in the Botanical Garden of velopment of seeds of the dogwood Cornus contro- Osaka City University, Katano City, Japan (34.8°N, versa Hemsley, a principal host plant of P. lewisi, 135.7°E) in 1993–1998. These insects and their was monitored in 1996. Thirty to 50 fruits of C. progeny were used in these experiments. Collected controversa were collected from the Botanical Gar- adults were kept as single male-female pairs in den of Osaka City University every week from 1 plastic cups (9 cm diameter, 4.5 cm depth) with a June to 27 July. Only one seed was formed in each piece of folded filter paper as a substratum for fruit in C. controversa. The seed was weighed after oviposition. Eggs laid by the adults were collected removing the fleshy tissue. and kept in plastic petri dishes (5.5 cm diameter, Photoperiodic response in female adults. Final 1 cm depth) as clutches of about 14 eggs under LD instar nymphs after overwintering were collected in 16 : 8 (16 h light and 8 h darkness) at 25Ϯ1°C. the field on 12 and 19 May in 1994 and reared Hatching nymphs were transferred to various pho- under LD 16 : 8 at 25°C. Emerging adults were toperiodic conditions within 24 h with a piece of kept as single male-female pairs in plastic cups water-soaked cotton. After the first ecdysis, they under the same photoperiod, or transferred to LD were provided with food and moved into the plastic 13 : 11. In addition, adults reared from eggs under cups. Final instar nymphs were transferred to plas- LD 16 : 8 at 25°C were kept under the same pho- tic containers (15 cm diameter, 6.5 cm depth) and toperiod, or transferred to LD 13 : 11, LD 14 : 10 or provided folded filter paper as a foothold for adult LD 15 : 9. Oviposition and mortality were recorded emergence. Emerging adults were kept in the same daily. way as those collected in the field. Moriya et al. Nutritional conditions on oviposition. Nymphs (1987) successively reared this species on raw were reared on raw peanuts under LD 16 : 8 at peanuts, soybean and water. In the present study, 25°C and emerging adults kept under the same however, only raw peanuts were provided as food photoperiod, or transferred to LD 13 : 11. The and water was made available in vials with cotton adults were provided only water or 10% sucrose plugs for both nymphs and adults. solution. Oviposition and mortality were recorded Photoperiodic response in nymphs. Nymphs daily. were kept under various stationary photoperiods Statistical analysis. The statistical significance between LD 10 : 14 and LD 16 : 8 at 25°C. Their of differences in the preoviposition period and ecdysis and mortality were recorded daily. When a adult longevity was examined by Mann-Whitney U nymph did not emerge as an adult within 30 days test between groups with two samples. For groups after the final nymphal ecdysis, it was regarded as with three or more samples, the Bonferroni-type having entered diapause. multiple comparison was applied among all sample Oviposition under quasi-natural conditions. groups after Kruskal-Wallis test. For all tests, sig- An outdoor cage with no artificial illumination or nificance was set at a level of pϭ0.05. heating was placed on the Campus of Osaka City University, Osaka City (34.6°N, 135.5°E). In 1994, RESULTS diapause nymphs collected in the field on 17 Sep- tember or 11 October were kept in plastic contain- Photoperiodic response in nymphs ers in the outdoor cage. In August 1995, eggs laid Figure 1 shows the nymphal development under by adults raised under LD 13 : 11 at 25°C were LD 15 : 9 and LD 11 : 13 at 25°C. Under LD 15 : 9, Seasonal Adaptations in a Scutellerid 471

Fig. 1. Nymphal development of Poecilocoris lewisi under long-day (LD 15 : 9) and short-day (LD 11 : 13) pho- toperiods at 25°C. Roman numerals indicate instars of nymphs.

Fig. 3. Relationship between oviposition in Poecilocoris lewisi (A) and growth of seeds of its host plant Cornus contro- versa (B). Nymphs of P. lewisi after overwintering were kept under quasi-natural conditions in 1995 and 1996. Each line represents a female; closed circles, adult emergence; open cir- cles, onset of oviposition; crosses, death. Seeds of C. contro- versa were collected in the field in 1996, and their dry weights (meansϮSD) are shown (nϭ29 to 48).

29 July. In 1996, female adults emerged between 25 May and 3 June. Five of 11 females laid eggs with the onset of oviposition between 4 and 20 Fig. 2. Photoperiodic induction of nymphal diapause in Poecilocoris lewisi at 25°C (nϭ23 to 181). July. Although a notable proportion of adults died without oviposition, oviposition started almost at the same seasonal timing in the two years. Daily most nymphs emerged as adults within 50 days mean temperatures in Osaka showed no obvious after hatching and within 30 days after the final differences between the years from April to July nymphal ecdysis. Under LD 11 : 13, nymphs grew (see Fig. 6). as under LD 15 : 9 until the final instar but none emerged as adults within 30 days after the final Seasonal development of host plants nymphal ecdysis. Thus, P. lewisi entered a faculta- Figure 3B shows the seasonal development of C. tive diapause at the final instar in response to short- controversa seeds in June and July 1996. Although day conditions. Figure 2 shows the photoperiodic C. controversa flowered in mid-April and produced response curve for the induction of nymphal dia- fruits in early May, seeds were still very small in pause at 25°C. The critical daylength for the induc- May and it was impossible to measure their weight tion of diapause was about 14.5 h, which corre- individually. The weight of collected seeds in- sponds to the natural daylength in early August, in- creased with time until 6 July at which point they cluding a 1.0 h twilight period, in Osaka. reached a maximum weight.

Oviposition under quasi-natural conditions Photoperiodic response in female adults Figure 3A shows the emergence and oviposition The preoviposition period was significantly of female adults under quasi-natural conditions in longer in insects collected from the field as nymphs 1995 and 1996. In 1995, female adults emerged be- and kept under LD 16 : 8 than those transferred to tween 17 May and 6 June. Nine of 36 females laid LD 13 : 11 at adult emergence (Fig. 4). When in- eggs with the onset of oviposition between 2 and sects were reared from eggs under LD 16 : 8 in the 472 S. I. Tanaka et al.

Fig. 4. Preoviposition periods (meansϮSD) in Poecilo- coris lewisi under various photoperiodic conditions at 25°C. Open circles, females reared from eggs in the laboratory (nϭ9 to 15); closed circles, females collected in the field as final in- star nymphs after overwintering in 1995 (nϭ4 and 5). laboratory, the preoviposition period was signifi- cantly longer in insects kept continuously under LD 16 : 8 or transferred to LD 15 : 9 at adult emer- gence than those transferred to LD 13 : 11 or LD Fig. 5. Survival and oviposition under various conditions 14 : 10 (Fig. 4). Thus, female adults that had been of nutrition in Poecilocoris lewisi at 25°C. Nymphs were raised under LD 16 : 8 showed a photoperiodic re- reared on raw peanuts under LD 16 : 8 at 25°C, and emerging adults were kept under the same photoperiod, or transferred to sponse in the preoviposition period and the critical LD 13 : 11. Adults were provided with only water or 10% su- daylength was between 14 and 15 h. Furthermore, crose solution. Shaded areas, surviving females; columns, cu- laboratory-reared females started oviposition sig- mulative ovipositing females. nificantly earlier than those collected in the field under both LD 13 : 11 and LD 16 : 8. As a result, difference was observed between the two photope- field-collected insects transferred to LD 13 : 11 riods when sucrose solution was provided (Fig. 5). showed a similar preoviposition period to that of insects reared in the laboratory under LD 15 : 9 or DISCUSSION LD 16 : 8. Although P. lewisi has been recognized as a uni- Nutritional conditions on oviposition voltine species with nymphal diapause (Kobayashi, When sufficient food was supplied as raw 1967; Moriya et al., 1987), nymphs emerged as peanuts, 15 of 18 and 14 of 22 females laid eggs adults without an intervening of diapause under under LD 13 : 11 and LD 16 : 8, respectively. Even long-day conditions. Nymphal diapause for over- when only sucrose solution was supplied, 9 of 15 wintering is scarce in Heteroptera, and has been of females laid eggs and 2 of 15 of females did studied by laboratory experiments only in the fol- with only water under LD 13 : 11. However, each lowing three species, Carbula humerigera, Copto- female under these unfavorable conditions pro- soma scutellatum and P. lewisi (Kiritani, 1985a, b, duced only one egg mass, although they produced 1987; Saulich and Musolin, 1996; see Musolin and 5.5Ϯ4.5 (meanϮSD) egg masses on the average Saulich, 1999 for review). with sufficient food. In contrast under LD 16 : 8 no The critical daylength for the induction of females laid eggs with only water or sucrose solu- nymphal diapause in P. lewisi in Osaka was about tion. Longevity of female adults was significantly 14.5 h, which was longer than that of other het- longer under LD 16 : 8 than under LD 13 : 11, when eropterans with adult diapause for overwintering in only water was provided. However, no significant the same region (e.g., Kobayashi and Numata, Seasonal Adaptations in a Scutellerid 473

Fig. 6. Schematic representation of the life cycle of Poecilocoris lewisi. The upper panel shows the daylength including a 1.0 h twilight period and daily mean temperatures recorded at an outdoor cage on the Campus of Osaka City University in 1995 and 1996. The shaded bar shows the critical daylength for the induction of nymphal and adult diapause. The lower panel shows the pos- sible pathways of life cycle in Osaka. OW, overwintering generation composed of the first and the second generations that overwin- tered; I and II, the first and the second generations, respectively.

1993; Numata and Kobayashi, 1994). However, Some of the first generation nymphs develop to the Aelia fieberi has a critical daylength for the induc- final instar in early August when the daylength is tion of adult diapause similar to that for the induc- still longer than the critical value for the induction tion of nymphal diapause in P. lewisi. This bug of nymphal diapause, and therefore they emerge as shows a bivoltine life cycle imposed by the lack of adults in mid-August. Then, the daylength will a favorable food supply after late summer (Naka- soon decrease to less than the critical value for the mura and Numata, 1997). The photoperiodic re- induction of adult diapause and adults will avert di- sponse in nymphs of P. lewisi indicates that it is apause. Consequently, adults of the first generation possible to produce two generations per year in this never enter diapause. This prevention of adult dia- region. pause allows nymphs of the next generation to Not only nymphs but also adults of P. lewisi re- reach the diapause stage at an appropriate time of sponded to photoperiod. Both laboratory-reared year (Fig. 6). In fact, some nymphs hatching in and field-collected females began to lay eggs sig- early October successfully grow to the final instar nificantly earlier under short-day conditions than by early November under quasi-natural conditions under long-day conditions. From the results in lab- on seeds of C. controversa, which are available oratory-reared adults, the critical daylength for until early November in Osaka (unpublished). adult diapause was estimated to be about 14.5 h. These observations raise a question as to why This value corresponded to that for the induction of adults of the overwintering generation enter dia- nymphal diapause and these two response curves pause in early summer. Without this diapause, all are mirror images. The daylength in mid- or late adults of the overwintering generation would repro- May when adults of P. lewisi emerge in the field is duce in June and more individuals could achieve about 15.0 h including a 1.0 h twilight period, bivoltine life cycles. The food supply is considered which is longer than the critical daylength for the the most critical factor for the evolution of summer induction of adult diapause. Therefore, all adults of diapause (Masaki, 1980). Seeds of C. controversa the overwintering generation enter diapause. In do not reach the maximum weight until early July contrast, adults of the first generation that emerge in Osaka. This suggests the availability of the host around mid-August lay eggs soon after emergence. plant in spring is related to the evolution of the 474 S. I. Tanaka et al. adult diapause in P. lewisi. pause as shown in some other insects (Danks, Summer diapause sometimes has synchronizing 1987). and stabilizing effects in life cycles (Masaki, In the present study, the insects were reared on a 1980). In univoltine bugs C. humerigera and P. substitute diet, raw peanuts only. To determine the bidens, adult summer diapause is maintained by seasonal adaptive mechanisms of this species in long-day photoperiod and stabilizes the life cycle more detail, it is necessary to examine develop- as strictly univoltine (Kiritani, 1985a, b; Musolin ment and reproduction when feeding on natural and Saulich, 2000). In contrast, adult summer dia- food. pause in P. lewisi is terminated near the summer ACKNOWLEDGEMENTS solstice and adults begin to lay eggs in early July. Thus, a proportion of nymphs in the first genera- We would like to thank Dr. Yosihiro Natuhara, Osaka Pre- tion emerged as adults and produced an extra gen- fecture University, for valuable suggestions in the early stage of this study, Dr. Keiji Nakamura, Okayama University of Sci- eration in August (Fig. 6). Therefore, adult dia- ence, and Dr. Dmitry L. Musolin, National Agricultural Re- pause in P. lewisi has not evolved to maintain uni- search Center for Hokkaido Region, for helpful comments. We voltinism but produce partial bivoltinism. also thank Professor Hiroshi Okada for allowing us to use the The evolution of spring adult diapause in P. Botanical Garden of Osaka City University as a study site. lewisi is still unclear, because in insects with long This research was partly supported by an OCU Grant for adult longevity, such as some dipterans, adults with Graduate Course Students to S. I. T. and a collaboration proj- ect between Osaka Environmental Technology and Research a poor protein diet show suppression of ovarian de- Co., Ltd. and Osaka City Institute of Public Health and Envi- velopment without diapause (e.g., Harlow, 1956; ronmental Sciences in 1993–1994. Spielman and Wong, 1974; see Engelmann, 1970 for review). Among phytophagous heteropterans, REFERENCES suppression of ovarian development with starvation Danks, H. V. (1987) Insect Dormancy: An Ecological Perspec- has been observed in Oncopeltus fasciatus, tive. Biological Survey of Canada (Terrestrial Arthro- Pyrrhocoris apterus and Riptortus clavatus (Jo- pods), Ottawa. 439 pp. Engelmann, F. (1970) The Physiology of Insect Reproduction. hansson, 1954; Hodková, 1982; Numata and Hi- Pergamon Press, Oxford. 307 pp. daka, 1984). In P. lewisi, however, some females Harlow, P. M. 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