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Quantitative short-day photoperiodic response in larval development and its adaptive significance in an adult-overwintering cerambycid beetle, Phytoecia rufiventris
Journal of Insect Physiology 57 (2011) 1053–1059
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Quantitative short-day photoperiodic response in larval development and its adaptive significance in an adult-overwintering cerambycid beetle, Phytoecia rufiventris Yoshinori Shintani * Laboratory of Entomology, Department of Environmental and Horticultural Sciences, Minami Kyushu University, Tateno 3764-1, Miyakonojo, Miyazaki 885-0035, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 October 2010 Received in revised form 1 May 2011 Accepted 9 May 2011
The chrysanthemum longicorn beetle, Phytoecia rufiventris, overwinters in the adult stage and reproduces in spring. Larvae of this beetle develop during summer inside a host stem or root. In the present study, photoperiodic control of larval development and its adaptive significance were examined in this beetle using an artificial diet. Larvae showed a short-day photoperiodic response at 25 8C with a critical day length of around 14 h; larvae reared under short-day conditions pupated, whereas those reared under long-day conditions entered summer diapause with some supernumerary molts and did not pupate. A similar response was found at 30 8C, but with a shorter critical day length. Below the critical day length, a shorter day length corresponded to a shorter larval period. Larvae transferred from long-day conditions to various photoperiods showed a similar quantitative response. Field rearing of larvae starting at various times of year showed that pupation occurs within a relatively short period in early autumn. Field rearing of pupae and adults at various times indicated that only pupation in early autumn results in a high survival rate until winter. Earlier or later pupation led to a low survival rate due to death before overwintering in the adult and pupal stages, respectively. Thus, in P. rufiventris, timing of pupation regulated by the quantitative short-day photoperiodic response is vital for survival. Relatively lower developmental threshold in the pupal stage supports this hypothesis. ß 2011 Published by Elsevier Ltd.
Key words: Quantitative short-day photoperiodic response Larval summer diapause Pupation timing Chrysanthemum longicorn beetle Phytoecia rufiventris
1. Introduction As an adaptation to seasonally changing environments, many insects that inhabit temperate zones divide their life cycle into an active phase and a diapause phase. These insects often use the photoperiod as a seasonal cue for switching between these phases, although temperature and other environmental factors can modify the photoperiodic response. Diapause has a pivotal role in regulating seasonal life cycles of insects, and the developmental stage and season in which diapause is induced are diverse due to the variety of species-specific seasonal strategies (Tauber et al., 1986; Danks, 1987; Kosˇta´l, 2006). Cerambycids are among the most diverse insects, with more than 35,000 species in about 4000 genera (Lawrence, 1982). They are phytophagous in both the larval and adult stages: the larvae usually burrow in the tissues of woody plants, but comparatively few species feed within the stems or roots of living herbaceous plants. Many species are important pests in forests, plantations, and street trees (e.g., Linsley, 1959). Despite wealth of general
* Tel.: +81 986 46 1057; fax: +81 986 21 2113. E-mail address: [email protected]. 0022-1910/$ – see front matter ß 2011 Published by Elsevier Ltd. doi:10.1016/j.jinsphys.2011.05.005
biological information, the seasonal adaptation of cerambycids has received relatively little attention, primarily because they live inside plants over their whole life span, except for the adult stage, and cause negligible direct damage (Hanks, 1999). Depending on the species, cerambycids have different seasonal life cycles with respect to the overwintering stage; larval- or adultoverwintering. Although the larvae may live inside thick trunks, they are able to perceive the photoperiod, since it has been shown that larvae enter diapause by responding to the photoperiod. For example, larvae of the grape borer, Xylotrechus pyrrhoderus (Ashihara, 1982), and the yellow-spotted longicorn beetle, Psacothea hilaris (Emori, 1976; Shintani et al., 1996), enter winter diapause under short-day conditions. In the udo longicorn beetle, Acalolepta luxuriosa, the photoperiod influences the maintenance and termination of larval diapause (Honda et al., 1981). In the Japanese pine sawyer, Monochamus alternatus, post-chilling development is controlled by the photoperiod (Ueda and Enda, 1995). However, the photoperiodic response has only been examined for cerambycids that overwinter in a larval diapause state. Photoperiodic control of the seasonal life cycle of adultoverwintering cerambycids has not been examined. The chrysanthemum longicorn beetle, Phytoecia rufiventris, is a very small cerambycid (adult body length < 1 cm) that is
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distributed in eastern Asia (Ohbayashi et al., 1992). P. rufiventris infests herbaceous plants in the Asteraceae family and may cause serious damage to commercial chrysanthemums (Uezumi, 2003). The beetle has an adult-overwintering univoltine life cycle, in which adults emerge from host roots in the soil in early spring and reproduce during spring. These adults bite leaf veins or stems and females lay eggs inside the stems. After hatching, larvae burrow inside the stem and move to the root in summer. The larvae pupate in autumn and the pupae eclose to adults before overwintering (Uezumi, 2003). Therefore, the mechanisms controlling this seasonal life cycle are likely to differ from those in larvaloverwintering cerambycids. In the present study, to examine the mechanisms controlling the seasonal life cycle in P. rufiventris, the effects of temperature and photoperiod on larval development were examined for a local population in Japan. Furthermore, the adaptive significance of the photoperiodic response was examined by observing outdoor development and survival of larvae, pupae and adults at various times of year.
When the larvae were fully grown and had ceased feeding, they were transferred to another Petri dish in which a piece of wet filter paper (15 cm2) had been placed. These post-feeding larvae were kept until pupation, with the filter paper moistened with a sprayer once every few days. Pupae and resultant adults were kept in the same way. Preliminary experiments indicated that larvae did not grow well at 20 8C or lower temperatures (unpublished data). Therefore, laboratory rearing of larvae was performed at 25 8C, unless otherwise stated. P. rufiventris cannot be sexed until adult eclosion and the developmental period, and body weights did not differ significantly between the sexes. Thus, data for both sexes were pooled in all experiments. Data for individuals that died in the larval stage were excluded from analyses. Body weights of some newly ecdysed larvae that were mass-reared under 16L–8D were determined on the day of molting, using an electronic balance (UX620H, Shimadzu Co., Kyoto).
2. Materials and methods
To examine the effects of the photoperiod on larval development, larvae were first reared from hatching under five stationary photoperiodic conditions (12L–12D, 13L–11D, 14L– 10D, 15L–9D and 16L–8D) at 25 8C and three stationary photoperiodic conditions (12L–12D, 14L–10D, and 16L–8D) at 30 8C. Larvae reared under long-day conditions did not pupate (see Section 3.1). Thus, second, to examine the effects of photoperiodic change on larval development, larvae that had been reared from hatching under 16L–8D at 25 8C (designated as ‘long-day larvae’) for 40 days were transferred to 12L–12D, 13L– 11D, 14L–10D or 15L–9D conditions. Third, to compare the responses of larvae of various ages to photoperiodic change, long-day larvae were transferred to 12L–12D on day 20, 30, 40, 50, 60 or 70 after hatching.
2.1. Insects Adults of P. rufiventris were collected from Asteraceae herbs, including the mugwort, Artemisia indica; the Canada goldenrod, Solidago canadensis; the annual fleabane, Erigeron annuus; and the yomena, Aster yomena; in Takanabe (1318310 E, 328060 N), Miyazaki Prefecture, Japan, from early April to early June, 2006–2009. Collected adults were kept in acrylamide insect cages (34 cm 25 cm 34 cm) at 25 8C under a photoregime of 16L– 8D (16 h light and 8 h dark), with cut host plants arranged in glass cups containing water. Adults laid eggs inside the stems, and these eggs were collected and placed on wet filter paper in a glass Petri dish (9-cm diameter). The eggs were kept at 25 8C under 16L–8D until hatching, unless otherwise stated. Although adults had disappeared in early June in the field, the adults in the laboratory continued to lay eggs until late July. To obtain newly ecdysed pupae in autumn as experimental samples (see Section 2.7), some eggs were kept at 15–20 8C to elongate the egg period. This manipulation did not have any effect on larval development. 2.2. Rearing larvae with an artificial diet Since P. rufiventris larvae live inside the stems or roots of their natural hosts, it was not feasible to examine their development by rearing them on plants. Thus, an artificial diet for P. rufiventris larvae was prepared from a commercial diet (Insecta F-II; a diet for phytophagous insects, dry formula, Nihon Nosan Co., Yokohama) and powdered leaves of a host plant. Host plant powder was prepared in the laboratory; young leaves of A. indica collected in early spring were dried at room temperature (ca. 25 8C) and relative humidity (ca. 50%) for two weeks and powdered with an electric blender (SKP-C700, Tiger Co., Kadoma). A mixture of Insecta F-II and the host plant powder (3:1 in weight) was placed in a stainless steel container. After adding water at a weight of 2.6 times that of the mixture, the diet was steamed for 20 min. After cooling, the diet was kept at 5 8C in a refrigerator until use. All rearing of larvae in the study was performed using this diet. Newly hatched larvae were transferred separately to a plastic Petri dish (6-cm diameter) with a piece of artificial diet. To maintain the humidity, the Petri dishes were placed in transparent plastic containers (3 L) with loose lids for ventilation. Larval development was observed every day and the artificial diet was changed or replenished every two or three days. In each experiment, dates of molts, death and pupation were recorded.
2.3. Effects of photoperiod on larval development
2.4. Developmental parameters To determine developmental parameters in the egg, larval and pupal stages, newly laid eggs, newly hatched larvae, and newly ecdysed pupae were kept or reared at 17.5, 20, 22,5, 25, 27.5 or 30 8C under 16L–8D (egg and larva) or 12L–12D (pupa). Since larvae might not complete their development under all of these conditions, the duration from the 1st to 3rd instars was determined. Based on data for the developmental period, the developmental threshold (T0) and thermal constant (K) were estimated, as described by Ikemoto and Takai (2000). 2.5. Quasi-natural conditions and meteorological data To examine the significance of seasonal timing of pupation, timing-manipulation experiments were performed with insects reared under outdoor conditions in 2009 (see Sections 2.6 and 2.7). For outdoor rearing, a soil surface (50 cm 50 cm) was dug at 10 cm depth in a half-shed area near forests on the campus of Minami Kyushu University, Takanabe. A stainless shelf for horticulture (180 cm 90 cm 75 cm) was put on the hole, and a transparent nylon sheet (3-mm thickness) was placed on the top of the shelf as a cover from rain. Semi-transparent plastic containers (3 L) including insect Petri dishes were placed in the hole and leaf litter was placed on the containers. Data for soil temperatures (5-cm depth) were obtained from the Meteorological Observation System of Minami Kyushu University. In 2009, the seasonal change in air temperature was almost typical of a normal year, except that late winter was relatively warmer. Day length included civil twilight; i.e., sunrise to sunset plus 1 h (Beck, 1980).
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2.6. Effects of hatching timing on larval development in the field To examine the effects of the timing of hatching on larval development and survival in the field, larvae that had been reared in the laboratory until the first larval molt (7–10 days after hatching) were transferred to outdoor conditions at four times from spring to summer in 2009: 10 May (Cohort L1), 31 May (Cohort L2), 20 June (Cohort L3), and 10 July (Cohort L4). Larvae were reared on the artificial diet and their development was observed every day until they pupated or died. 2.7. Effects of pupation timing on survival in the pupal and adult stages in the field To examine the effects of the timing of pupation on pupal development and survival in the field, laboratory-raised pupae were transferred to outdoor conditions on the day of pupation at five times from summer to autumn: 4–7 August (Cohort P1), 21–26 August (Cohort P2), 10–15 September (Cohort P3), 1–6 October (Cohort P4), and 21–29 October (Cohort P5). To obtain pupae, longday larvae were transferred to 12L–12D at 25 8C on day 40 after hatching (see Section 3.2). Survival of the pupae and resultant adults was monitored daily until 31 December. 2.8. Survival of adults at various temperatures Pupae obtained by transferring long-day larvae to 12L–12D at 25 8C on day 40 after hatching were kept under the same conditions (12L–12D, 25 8C). Eclosed adults were assigned to groups kept at 20, 22.5, 25 or 27.5 8C under 12L–12D on the day of eclosion, and survival was monitored every other day for 80 days. The surviving adults were inactive and did not feed for more than 80 days, suggesting that they were in diapause. 3. Results 3.1. Larval development under various stationary photoperiodic conditions Larvae of P. rufiventris showed a clear short-day photoperiodic response at 25 8C (Fig. 1). Regardless of the photoperiod, larvae developed similarly from the 1st to 3rd instars. However, development in later instars differed among the photoperiodic conditions. Larvae reared under 12L–12D pupated from day 39 to 72 from the 4th to 6th instars, and those reared under 13L–11D pupated from day 41 to 107 from the 4th to 10th instars. Under 14L–10D, 60% of the larvae pupated from day 63 to 110 from the 4th to 8th instars, but the remainder ceased feeding in the 4th to 8th instars and did not pupate within the 120-day experimental period. The larval durations for those that pupated (mean SD) were 50.0 7.7, 83.5 14.7, and 95.2 15.5 days under 12L–12D, 13L–11D and 14L–10D, respectively, and differed significantly between photoperiods (Steel–Dwass test, P < 0.05). This difference was due to variation in the number of molts and/or the duration of the final instar (Fig. 1). Under 15L–9D and 16L–8D, all larvae underwent five or more instars and ceased feeding, and no larvae pupated. The body weight of these larvae increased with development, but the effect of an additional molt tended to be smaller as larvae grew (Table 1). After day 120, larvae neither pupated nor molted into the next instar, and thus these larvae were considered to be in diapause. At 30 8C, larvae showed a similar response to photoperiods: all larvae reared under 14L–10D and 16L–8D, and more than half of those reared under 12L–12D entered diapause with some supernumerary molts (Fig. 2). Thus, the critical day lengths for the photoperiodic response in P. rufiventris larvae were about 14 h and 12 h at 25 8C and 30 8C, respectively.
Fig. 1. Photoperiodic response in Phytoecia rufiventris larvae at 25 8C. Larvae were reared from hatching under five photoperiodic conditions at 25 8C. Each bar indicates development of an individual larva and the right end of the bar indicates pupation. For larvae that did not pupate within the experimental period (120 days), the bars end on day 120. Triangles indicate the mean larval period for individuals that pupated. The initial sample size was 40 for each experimental group.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059
Table 1 Body weight of Phytoecia rufiventris larvae in various instars.a
4th 5th 6th 7th 8th 9th
n 24 23 22 15 11 10
20.8 5.2 36.2 10.9 51.5 15.0 59.3 9.0 66.3 8.3 72.9 9.8
60
c b
b
Body weight (mg, mean SD)
50
75
% pupation
Instar
100
40
a
30
50
20 25
a Newly ecdysed larvae were sampled randomly from larvae mass-reared under 16L–8D at 25 8C.
10
Duration until pupation (days)
1056
0
0 12
13
13.5
14
15
16
Photophase (h/day) Fig. 3. Responses of Phytoecia rufiventris larvae to photoperiodic change. Larvae reared from hatching under 16L–8D at 25 8C were transferred on day 40 to various photoperiodic conditions. The percentage pupation (*) and the duration until pupation (column) are shown. Error bars indicate SD. n = 27–30. Values with different letters are significantly different at P = 0.05 by Tukey–Kramer multiple comparison test.
3.3. Developmental parameters The egg, larval and pupal periods at various temperatures in P. rufiventris are shown in Table 2. Based on these data, the developmental threshold and thermal constant were determined to be 13.1 8C and 66.6 degree-days, respectively, in the egg stage; 13.5 8C and 269.1 degree-days, respectively, in the larval stage (hatching to the 3rd larval molt); and 8.7 8C and 240.9 degree-days, respectively, in the pupal stage. 3.4. Effects of hatching timing on larval development in the field Development of P. rufiventris larvae transferred to outdoor conditions at various times from spring to summer is shown in Fig. 5. Most larvae died in summer, independently of the timing of transfer. In Cohort L4, no larvae survived to pupation. This high mortality might have been due to fungi that rapidly spread on the artificial diet. Only 7 larvae pupated from 4 to 17 September. This result suggests that larvae pupate within a very short period in early autumn, regardless of the time of hatching.
Fig. 2. Photoperiodic response in Phytoecia rufiventris larvae at 30 8C. Larvae were reared from hatching under three photoperiodic conditions at 30 8C. The initial sample size was 40 for each experimental group. For details, see legend for Fig. 1.
The responses of long-day larvae of P. rufiventris to photoperiodic change are shown in Fig. 3. Without photoperiodic transfer, no larvae pupated. When the larvae were transferred to 12L–12D, 13L–11D or 13.5L–10.5D, 87–93% pupated, whereas only about half pupated under14L–10D and none pupated under 15L–9D. The duration from transfer to pupation (mean SD) varied from 33.5 3.3 days (12L–12D) to 49.1 5.9 days (14L–10D). There was a significant difference among later photoperiods, with a shorter photoperiod corresponding to a shorter duration until pupation (Tukey–Kramer multiple comparison test, P < 0.05). The responses of long-day larvae of various ages to photoperiodic change to 12L–12D are shown in Fig. 4. Most larvae of all ages pupated after the photoperiodic change, but the duration until pupation differed among ages and was significantly longer in younger larvae. However, for larvae transferred on day 50 or later, the duration was about 27 days, irrespective of the age.
Duration until pupation (days)
3.2. Responses to photoperiodic change
50
a b
40
b
30
c
c
c
50
60
70
20
10
0
20
30
40
Larval age (days after hatching) Fig. 4. Responses to photoperiodic change from long-day to short-day conditions in Phytoecia rufiventris larvae of various ages. Larvae reared from hatching under 16L– 8D at 25 8C were transferred to 12L–12D on day 20, 30, 40, 50, 60 or 70. The duration until pupation is shown. Error bars indicates SD. n = 23–28. Values with different letters are significantly different at P = 0.05 by Tukey–Kramer multiple comparison test.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059 Table 2 Developmental periods at various temperatures and developmental parameters in the egg, larval and pupal stages in Phytoecia rufiventris.a Developmental period (days, mean SD)
Temperature (8C) 30 27.5 25 22.5 20 17.5 T0 (8C) K (degree-days) r2
Egg
Larvab
Pupa
4.0 0.6 4.6 0.7 5.4 0.7 7.1 0.4 10.1 0.7 14.7 1.1 13.1 66.6 0.999
15.6 1.0 20.0 2.1 23.9 1.4 29.1 1.3 41.1 3.8 13.5 269.1 0.996
11.2 0.5 12.5 0.6 14.9 0.6 17.8 0.6 21.8 0.8 26.5 1.0 8.7 240.9 0.988
n = 25–34 for eggs, 14–21 for larvae, and 17–27 for pupae. a Under 16L–8D for the egg and larval stages and under 12L–12D for the pupal stage. b For the first three instars.
20
Cohort L3
No. surviving larvae
Cohort L4 15
Cohort L2
1057
3.5. Effects of pupation timing on survival in the pupal and adult stages Development and survival of P. rufiventris pupae transferred to outdoor conditions at various times from summer to autumn is shown in Fig. 6. In Cohorts P1, P2, P3 and P4, all pupae eclosed to adults. In contrast, in Cohort P5, 60% of the pupae died before eclosion and 5 out of 8 survivors eclosed with lethally deformed wings. There was a significant difference in the eclosion rate between Cohort P5 and the other cohorts (Tukey-type multiple comparisons for proportions, P < 0.05 [Zar, 1999]). The survival rate of eclosed adults clearly differed among the four cohorts with higher eclosion rates: in Cohorts P1 and P2, most adults died in 20– 40 days and only 3 adults in Cohort P2 survived until winter. In Cohort P4, nearly half of the eclosed adults died within 10 days, but 50% survived until winter. In contrast, 95% of adults in Cohort P3 survived until winter. In Cohort P5, 3 of 8 adults survived until winter. There was a significant difference in the rate of survival from pupation until winter between cohorts; the survival rates in Cohort P3 and P4 were significantly higher than that in other cohorts, and that in Cohort P3 was significantly higher than that in Cohort P4 (Tukey-type multiple comparisons for proportions, P < 0.05 [Zar, 1999]). 3.6. Effects of temperature on survival of adults
10
Cohort L1 Pupation
5
Survival of P. rufiventris adults at various temperatures is shown in Fig. 7. There was a clear tendency for reduced longevity at higher temperatures: all the adults died within 32 days at 27.5 8C and 95% died within 80 days at 25 8C. In contrast, most adults remained alive at 22.5 and 20 8C.
0 May
Jun
Jul
Aug
Sept
4. Discussion
Fig. 5. Outdoor development and survival of Phytoecia rufiventris larvae with different hatching times. Newly ecdysed 2nd-instar larvae were transferred to outdoor conditions on 10 May (Cohort L1), 31 May (Cohort L2), 20 June (Cohort L3), and 10 July (Cohort L4) in 2009. n = 19 or 20.
The results of the study show that P. rufiventris has a short-day photoperiodic response in larval development. Under short-day conditions larvae pupate, whereas under long-day conditions they enter diapause with some supernumerary molts and do not
20 15
Cohort P2
10 5 0 20 15
Cohort P3
10 5 0 20 15 Pupa
Cohort P4
10
Adult
5
No. surviving pupae or adults
0 20 15
20 Cohort P1
15
10
10
5
5
0
0 Aug
Sept
Oct
Cohort P5
Nov
Dec
Fig. 6. Outdoor development and survival of Phytoecia rufiventris pupae and adults with different pupation times. Newly ecdysed pupae were transferred to outdoor conditions on 4–7 August (Cohort P1), 21–26 Aug (Cohort P2), 10–15 September (Cohort P3), 1–6 October (Cohort P4), and 21–29 October (Cohort P5) in 2009. n = 20.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059
22.5ºC 20ºC
Survival rate (%)
80
60
27.5ºC
25º C
40
20
0
0
10
20
30
40
50
60
70
80
Days after eclosion Fig. 7. Survival of Phytoecia rufiventris adults at various temperatures. Larvae reared under 16L–8D at 25 8C were transferred to 12L–12D on day 40 and the resultant adults were kept at various temperatures under 12L–12D. n = 20 at each temperature.
pupate. This diapause is induced under long-day conditions and terminated after transfer to short-day conditions; therefore, it is the typical summer diapause defined by Masaki (1980). The shift to a shorter critical day length at higher temperature found in this photoperiodic response is a common feature of summer diapause (Masaki, 1980; Danks, 1987). Several other insects, such as the southwestern corn borer, Diatraea grandiosella (Chippendale and Reddy, 1973), and the cornstalk borer, Sesamia nonagrioides (Gadenne et al., 1997), undergo supernumerary molts during photoperiodically induced diapause. Among such insects, the yellow-spotted longicorn beetle, Psacothea hilaris, provides a good contrast to P. rufiventris. P. hilaris has a long-day photoperiodic response in larval development: under long-day conditions larvae pupate after the 4th or 5th instar, whereas under short-day conditions they enter diapause with some supernumerary molts and do not pupate unless transferred to long-day conditions or exposed to low temperature (Shintani et al., 1996; Shintani and Ishikawa, 1997, 1998a, 1998b). Larvae of P. rufiventris reared under long-day conditions pupate after transfer to short-day conditions, but the response to photoperiodic change varies with age. For larvae transferred on day 20, the whole larval duration is 64 (20 + 44) days, which is much longer than the larval duration under stationary short-day conditions (50 days, Fig. 1). This indicates that the larvae have already become sensitive to the photoperiod in the 3rd instar. For larvae transferred in the 5th or later instars, the duration until pupation was almost the same indicating that they are in a similar physiological state with respect to diapause. The short-day photoperiodic response found in P. rufiventris larvae had a quantitative feature, since a shorter day length corresponded to a shorter larval duration for day lengths shorter than the critical day length. This difference is due to variation in the number of molts and/or the duration of the final instar. Short-day photoperiodic responses in insects clearly play a role in termination of summer diapause and regulation of the timing of developmental events in autumn (Masaki, 1980; Tauber et al., 1986; Danks, 1987). For example, termination of reproductive diapause is photoperiodically regulated in the carabid beetle, Leptocarabus kumagaii (Sota, 1987), and the viburnum leaf beetle, Pyrrhalta humeralis (Nakai and Takeda, 1995a). Summer diapause in prepupae of the tailed zygaenid moth, Elcysma westwoodii (Gomi and Takeda, 1992; Nakai and Takeda, 1995b), and pupae in the Japanese giant silkmoth, Dictyoploca japonica (Nagase and Masaki, 1991), is also terminated under short-day conditions. In P. rufiventris, the short-day photoperiodic response of larvae clearly regulates termination of summer diapause. Seasonal
changes in temperature and day length at the research site are shown in Fig. 8. Larvae hatch in spring and grow under long days in summer at temperatures fluctuating around 25 8C. Under these conditions larvae enter and maintain summer diapause. As the day length becomes shorter than the critical day length in late summer, the larvae terminate diapause and then pupate within a relatively short period in early autumn (Fig. 5). This synchronization of pupation may be due to the quantitative feature of the short-day photoperiodic response. The temperature-dependent shift in the critical day length may compensate for yearly differences in temperature. However, it was somewhat surprising that the day length became shorter than the critical day length (14 h at 25 8C) as late as late-August, since at least 27 days are necessary before pupation (Fig. 4), and the actual time of pupation (early autumn) cannot be explained based on these results. This contradiction may arise from the assumption of the natural day length including 1-h twilight; the actual duration perceived as the photophase may be shorter, since diapause larvae inhabit roots underground. The results also showed that only individuals that pupated in early autumn could survive as adults until the end of December, indicating that pupation timing has a crucial effect on survival. If larvae pupated earlier (in mid-summer), the pupae would eclose as adults within two weeks, but the resultant adults may not survive until mid-autumn (Fig. 6). High mortality in these adults is probably caused by high temperatures during mid- to late summer, because the adults are intolerant of temperatures of 27.5–30 8C for a long period (Fig. 7). In contrast, if larvae pupated later (in midautumn), high mortality would occur in the pupal or adult stage, probably due to low temperatures during late autumn to early winter. The pupa of most coleopterans is the exarate type (i.e., without puparium), which is generally susceptible to physical stresses such as cold and drought, and no coleopterans use this stage as the diapause stage (Masaki, 1980). In P. rufiventris pupae, the thermal threshold is much lower than in other stages (Table 2) and this ensures that the pupal period is shortened at moderate autumnal temperatures. Such a trend in thermal threshold is not found in other cerambycids, such as P. hilaris (Shimane and Kawakami, 1991; Sakakibara, 1995; Watari et al., 2002) and X. pyrrhoderus (Ashihara, 1982). Thus, this low thermal threshold in P. rufiventris pupae is an adaptation to reduce the risk of injury due to cold. The early autumn pupation in P. rufiventris seems to be evolutionarily stable for the following reasons. First, larvae are
(A)
30
Soil temperature
25 20
Air temperature
15 16
(B)
15
10
14
5
Temperature (ºC)
100
Day length (h)
1058
13 12 11 10
Apr
May
Jul
Jun
Aug Sept
Oct
Nov
Dec
Fig. 8. Seasonal changes in temperature and day length at the research site in Takanabe (1318310 E, 328060 N), Miyazaki, Japan in 2009. (A) Air temperature and soil temperature (5 cm beneath the surface). (B) Natural day length. Temperature is shown as a 10-day average and natural day length is shown with an additional 1 h of twilight.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059
clearly tolerant of high temperature in summer, and if eclosed adults also acquired high-temperature tolerance, earlier pupation might not lead to high mortality. In this case, however, P. rufiventris would have high temperature tolerance in both the larval and adult stages, which may lead to more cost. Second, if pupae had lowtemperature tolerance, later pupation might not lead to high mortality. However, this situation is unlikely because pupae of P. rufiventris may be susceptible to winter physical stresses due to the physiological restrictions in coleopterans discussed above. In summary, pupation timing in P. rufiventris is restricted by high temperature in summer and low temperature in autumn. The quantitative short-day photoperiodic response in P. rufiventris larvae provides precise regulation of timing for pupation and might have evolved as an adaptation to seasonally changing temperature. Acknowledgments I am very grateful to Dr. S. Kawanobu (Minami Kyushu University) for providing meteorological data. This study was supported in part by a grant from Minami Kyushu University from 2006 to 2009. References Ashihara, W., 1982. Effects of temperature and photoperiod on the development of the grape borer. Xylotrechus pyrrhoderus Bates (Coleoptera: Cerambycidae). Japanese Journal of Applied Entomogy and Zoology 26, 15–22 (in Japanese with English summary). Beck, S.D., 1980. Insect Photoperiodism, 2nd ed. Academic Press, New York. Chippendale, G.M., Reddy, A.S., 1973. Temperature and photoperiodic regulation of diapause of the southwestern corn borer, Diatraea grandiosella. Journal of Insect Physiology 19, 1397–1408. Danks, H.V., 1987. Insect Dormancy: An Ecological Perspective. Biological Survey of Canada, Ottawa. Emori, T., 1976. Ecological study on the occurrence of the yellow-spotted longihorn beetle (Psacothea hilaris Pascoe). I. Effects of temperature and photoperiodic conditions on their development. Japanese Journal of Applied Entomology and Zoology 20, 129–132 (in Japanese with English summary). Gadenne, C., Dufour, M.C., Rossignol, F., Be´card, J.M., Couillaud, F., 1997. Occurrence of non-stationary larval moults during diapause in the corn-stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). Journal of Insect Physiology 43, 425–431. Gomi, T., Takeda, M., 1992. A quantitative photoperiodic response terminates summer diapause in the tailed zygaenid moth, Elcysma westwoodii. Journal of Insect Physiology 38, 665–670. Masaki, S., 1980. Summer diapause. Annual Review of Entomology 25, 1–25. Nagase, A., Masaki, S., 1991. Thermal and photoperiodic responses in aestivasting pupae of Dictyoploca japonica (Lepidoptera: Saturniidae). Applied Entomology and Zoology 26, 387–396. Nakai, T., Takeda, M., 1995a. Temperature and photoperiodic regulation of summer diapause and reproduction in Pyrrhalta humeralis (Coleoptera: Chrysomelidae). Applied Entomology and Zoology 30, 295–301.
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Nakai, T., Takeda, M., 1995b. Effect of temperature on the estivation of Elcysma westwoodii (Lepidoptera, Zygaenidae). Applied Entomology and Zoology 30, 594–596. Hanks, L.M., 1999. Influence of the larval host plant on reproductive strategies of cerambycid beetles. Annual Review of Entomology 44, 483–505. Honda, K., Akutsu, K., Arai, S., 1981. Photoperiodic response of Acalolepta luxuriosa Bates (Coleoptera: Cerambycidae): effect of photoperiodic change for induction of larval diapause. Japanese Journal of Applied Entomology and Zoology 25, 108–112 (in Japanese with English summary). Ikemoto, T., Takai, K., 2000. A new linearized formula for the law of total effective temperature and the evaluation of line-fitting methods with both variables subject to error. Environmental Entomology 29, 671–682. Kosˇta´l, V., 2006. Eco-physiological phases of insect diapause. Journal of Insect Physiology 52, 113–127. Lawrence, J.F., 1982. Coleoptera. In: Parker, S.P. (Ed.), Synopsis and Classification of Living Organisms, vol. 2. McGraw-Hill, New York, pp. 482–553. Linsley, E.G., 1959. Ecology of Cerambycidae. Annual Review of Entomology 4, 99–138. Ohbayashi, N., Sato, M., Kojima, K., 1992. An Illustrated Guide to Identification of Longicorn Beetles of Japan. Tokai University Press, Hadano (in Japanese). Sakakibara, M., 1995. Egg periods in several populations of yellow-spotted longicorn beetle. Psacothea hilaris Pascoe (Coleoptera: Cerambycidae). Japanese Journal of Applied Entomology and Zoology 39, 59–64 (in Japanese with English summary). Shimane, T., Kawakami, K., 1991. Method for successive mass-rearing of the yellowspotted longicorn beetle, Psacothea hilaris Pascoe (Coleoptera: Cerambycidae). Bulletin of the National Institute of Sericultural and Entomological Science 2, 65–112 (in Japanese with English summary). Shintani, Y., Ishikawa, Y., 1997. Effects of photoperiod and low temperature on diapause termination in the yellow-spotted longicorn beetle (Psacothea hilaris). Physiological Entomology 22, 170–174. Shintani, Y., Ishikawa, Y., 1998a. Diapause avoidance induced by low temperature in the yellow-spotted longicorn beetle, Psacothea hilaris. Entomologia Experimentalis et Applicata 85, 11–15. Shintani, Y., Ishikawa, Y., 1998b. Photoperiodic control of larval diapause in the yellow-spotted longicorn beetle. Psacothea hilaris: analysis by photoperiod manipulation. Entomologia Experimentalis et Applicata 86, 41–48. Shintani, Y., Ishikawa, Y., Tatsuki, S., 1996. Larval diapause in the yellow-spotted longicorn beetle. Psacothea hilaris (Pascoe) (Coleoptera: Cerambycidae). Applied Entomology and Zoology 31, 489–494. Sota, T., 1987. Effects of temperature and photoperiod on the larval hibernation and adult aestivation of Leptocarabus kumagaii (Coleoptera: Carabidae). Applied Entomology and Zoology 22, 617–623. Tauber, M.J., Tauber, C.A., Masaki, S., 1986. Seasonal Adaptations of Insects. Oxford University Press, New York. Ueda, A., Enda, N., 1995. Photoperiodic and thermal control on diapause of the Japanese pine sawyer. Monochamus alternates Hope (Coleoptera: Cerambycidae). Transactions of Kansai Branch of the Japanese Forestry Society 4, 163–166 (in Japanese). Uezumi, Y., 2003. Phytoecia rufiventris Gautier. In: Umeya, K., Okada, T. (Eds.), Agricultural Insect Pests in Japan. Zenkoku Noson Kyoiku Kyokai, Tokyo, Japan, (in Japanese), pp. 738–739. Watari, Y., Yamanaka, T., Asano, W., Ishikawa, Y., 2002. Prediction of the life cycle of the west Japan type yellow-spotted longicorn beetle. Psacothea hilaris (Coleoptera: Cerambycidae) by numerical simulations. Applied Entomology and Zoology 37, 559–569. Zar, J.H., 1999. Biostatistical Analysis, 4th ed. Prentice Hall Inc., Upper Saddle River.
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Quantitative short-day photoperiodic response in larval development and its adaptive significance in an adult-overwintering cerambycid beetle, Phytoecia rufiventris Yoshinori Shintani * Laboratory of Entomology, Department of Environmental and Horticultural Sciences, Minami Kyushu University, Tateno 3764-1, Miyakonojo, Miyazaki 885-0035, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 October 2010 Received in revised form 1 May 2011 Accepted 9 May 2011
The chrysanthemum longicorn beetle, Phytoecia rufiventris, overwinters in the adult stage and reproduces in spring. Larvae of this beetle develop during summer inside a host stem or root. In the present study, photoperiodic control of larval development and its adaptive significance were examined in this beetle using an artificial diet. Larvae showed a short-day photoperiodic response at 25 8C with a critical day length of around 14 h; larvae reared under short-day conditions pupated, whereas those reared under long-day conditions entered summer diapause with some supernumerary molts and did not pupate. A similar response was found at 30 8C, but with a shorter critical day length. Below the critical day length, a shorter day length corresponded to a shorter larval period. Larvae transferred from long-day conditions to various photoperiods showed a similar quantitative response. Field rearing of larvae starting at various times of year showed that pupation occurs within a relatively short period in early autumn. Field rearing of pupae and adults at various times indicated that only pupation in early autumn results in a high survival rate until winter. Earlier or later pupation led to a low survival rate due to death before overwintering in the adult and pupal stages, respectively. Thus, in P. rufiventris, timing of pupation regulated by the quantitative short-day photoperiodic response is vital for survival. Relatively lower developmental threshold in the pupal stage supports this hypothesis. ß 2011 Published by Elsevier Ltd.
Key words: Quantitative short-day photoperiodic response Larval summer diapause Pupation timing Chrysanthemum longicorn beetle Phytoecia rufiventris
1. Introduction As an adaptation to seasonally changing environments, many insects that inhabit temperate zones divide their life cycle into an active phase and a diapause phase. These insects often use the photoperiod as a seasonal cue for switching between these phases, although temperature and other environmental factors can modify the photoperiodic response. Diapause has a pivotal role in regulating seasonal life cycles of insects, and the developmental stage and season in which diapause is induced are diverse due to the variety of species-specific seasonal strategies (Tauber et al., 1986; Danks, 1987; Kosˇta´l, 2006). Cerambycids are among the most diverse insects, with more than 35,000 species in about 4000 genera (Lawrence, 1982). They are phytophagous in both the larval and adult stages: the larvae usually burrow in the tissues of woody plants, but comparatively few species feed within the stems or roots of living herbaceous plants. Many species are important pests in forests, plantations, and street trees (e.g., Linsley, 1959). Despite wealth of general
* Tel.: +81 986 46 1057; fax: +81 986 21 2113. E-mail address: [email protected]. 0022-1910/$ – see front matter ß 2011 Published by Elsevier Ltd. doi:10.1016/j.jinsphys.2011.05.005
biological information, the seasonal adaptation of cerambycids has received relatively little attention, primarily because they live inside plants over their whole life span, except for the adult stage, and cause negligible direct damage (Hanks, 1999). Depending on the species, cerambycids have different seasonal life cycles with respect to the overwintering stage; larval- or adultoverwintering. Although the larvae may live inside thick trunks, they are able to perceive the photoperiod, since it has been shown that larvae enter diapause by responding to the photoperiod. For example, larvae of the grape borer, Xylotrechus pyrrhoderus (Ashihara, 1982), and the yellow-spotted longicorn beetle, Psacothea hilaris (Emori, 1976; Shintani et al., 1996), enter winter diapause under short-day conditions. In the udo longicorn beetle, Acalolepta luxuriosa, the photoperiod influences the maintenance and termination of larval diapause (Honda et al., 1981). In the Japanese pine sawyer, Monochamus alternatus, post-chilling development is controlled by the photoperiod (Ueda and Enda, 1995). However, the photoperiodic response has only been examined for cerambycids that overwinter in a larval diapause state. Photoperiodic control of the seasonal life cycle of adultoverwintering cerambycids has not been examined. The chrysanthemum longicorn beetle, Phytoecia rufiventris, is a very small cerambycid (adult body length < 1 cm) that is
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distributed in eastern Asia (Ohbayashi et al., 1992). P. rufiventris infests herbaceous plants in the Asteraceae family and may cause serious damage to commercial chrysanthemums (Uezumi, 2003). The beetle has an adult-overwintering univoltine life cycle, in which adults emerge from host roots in the soil in early spring and reproduce during spring. These adults bite leaf veins or stems and females lay eggs inside the stems. After hatching, larvae burrow inside the stem and move to the root in summer. The larvae pupate in autumn and the pupae eclose to adults before overwintering (Uezumi, 2003). Therefore, the mechanisms controlling this seasonal life cycle are likely to differ from those in larvaloverwintering cerambycids. In the present study, to examine the mechanisms controlling the seasonal life cycle in P. rufiventris, the effects of temperature and photoperiod on larval development were examined for a local population in Japan. Furthermore, the adaptive significance of the photoperiodic response was examined by observing outdoor development and survival of larvae, pupae and adults at various times of year.
When the larvae were fully grown and had ceased feeding, they were transferred to another Petri dish in which a piece of wet filter paper (15 cm2) had been placed. These post-feeding larvae were kept until pupation, with the filter paper moistened with a sprayer once every few days. Pupae and resultant adults were kept in the same way. Preliminary experiments indicated that larvae did not grow well at 20 8C or lower temperatures (unpublished data). Therefore, laboratory rearing of larvae was performed at 25 8C, unless otherwise stated. P. rufiventris cannot be sexed until adult eclosion and the developmental period, and body weights did not differ significantly between the sexes. Thus, data for both sexes were pooled in all experiments. Data for individuals that died in the larval stage were excluded from analyses. Body weights of some newly ecdysed larvae that were mass-reared under 16L–8D were determined on the day of molting, using an electronic balance (UX620H, Shimadzu Co., Kyoto).
2. Materials and methods
To examine the effects of the photoperiod on larval development, larvae were first reared from hatching under five stationary photoperiodic conditions (12L–12D, 13L–11D, 14L– 10D, 15L–9D and 16L–8D) at 25 8C and three stationary photoperiodic conditions (12L–12D, 14L–10D, and 16L–8D) at 30 8C. Larvae reared under long-day conditions did not pupate (see Section 3.1). Thus, second, to examine the effects of photoperiodic change on larval development, larvae that had been reared from hatching under 16L–8D at 25 8C (designated as ‘long-day larvae’) for 40 days were transferred to 12L–12D, 13L– 11D, 14L–10D or 15L–9D conditions. Third, to compare the responses of larvae of various ages to photoperiodic change, long-day larvae were transferred to 12L–12D on day 20, 30, 40, 50, 60 or 70 after hatching.
2.1. Insects Adults of P. rufiventris were collected from Asteraceae herbs, including the mugwort, Artemisia indica; the Canada goldenrod, Solidago canadensis; the annual fleabane, Erigeron annuus; and the yomena, Aster yomena; in Takanabe (1318310 E, 328060 N), Miyazaki Prefecture, Japan, from early April to early June, 2006–2009. Collected adults were kept in acrylamide insect cages (34 cm 25 cm 34 cm) at 25 8C under a photoregime of 16L– 8D (16 h light and 8 h dark), with cut host plants arranged in glass cups containing water. Adults laid eggs inside the stems, and these eggs were collected and placed on wet filter paper in a glass Petri dish (9-cm diameter). The eggs were kept at 25 8C under 16L–8D until hatching, unless otherwise stated. Although adults had disappeared in early June in the field, the adults in the laboratory continued to lay eggs until late July. To obtain newly ecdysed pupae in autumn as experimental samples (see Section 2.7), some eggs were kept at 15–20 8C to elongate the egg period. This manipulation did not have any effect on larval development. 2.2. Rearing larvae with an artificial diet Since P. rufiventris larvae live inside the stems or roots of their natural hosts, it was not feasible to examine their development by rearing them on plants. Thus, an artificial diet for P. rufiventris larvae was prepared from a commercial diet (Insecta F-II; a diet for phytophagous insects, dry formula, Nihon Nosan Co., Yokohama) and powdered leaves of a host plant. Host plant powder was prepared in the laboratory; young leaves of A. indica collected in early spring were dried at room temperature (ca. 25 8C) and relative humidity (ca. 50%) for two weeks and powdered with an electric blender (SKP-C700, Tiger Co., Kadoma). A mixture of Insecta F-II and the host plant powder (3:1 in weight) was placed in a stainless steel container. After adding water at a weight of 2.6 times that of the mixture, the diet was steamed for 20 min. After cooling, the diet was kept at 5 8C in a refrigerator until use. All rearing of larvae in the study was performed using this diet. Newly hatched larvae were transferred separately to a plastic Petri dish (6-cm diameter) with a piece of artificial diet. To maintain the humidity, the Petri dishes were placed in transparent plastic containers (3 L) with loose lids for ventilation. Larval development was observed every day and the artificial diet was changed or replenished every two or three days. In each experiment, dates of molts, death and pupation were recorded.
2.3. Effects of photoperiod on larval development
2.4. Developmental parameters To determine developmental parameters in the egg, larval and pupal stages, newly laid eggs, newly hatched larvae, and newly ecdysed pupae were kept or reared at 17.5, 20, 22,5, 25, 27.5 or 30 8C under 16L–8D (egg and larva) or 12L–12D (pupa). Since larvae might not complete their development under all of these conditions, the duration from the 1st to 3rd instars was determined. Based on data for the developmental period, the developmental threshold (T0) and thermal constant (K) were estimated, as described by Ikemoto and Takai (2000). 2.5. Quasi-natural conditions and meteorological data To examine the significance of seasonal timing of pupation, timing-manipulation experiments were performed with insects reared under outdoor conditions in 2009 (see Sections 2.6 and 2.7). For outdoor rearing, a soil surface (50 cm 50 cm) was dug at 10 cm depth in a half-shed area near forests on the campus of Minami Kyushu University, Takanabe. A stainless shelf for horticulture (180 cm 90 cm 75 cm) was put on the hole, and a transparent nylon sheet (3-mm thickness) was placed on the top of the shelf as a cover from rain. Semi-transparent plastic containers (3 L) including insect Petri dishes were placed in the hole and leaf litter was placed on the containers. Data for soil temperatures (5-cm depth) were obtained from the Meteorological Observation System of Minami Kyushu University. In 2009, the seasonal change in air temperature was almost typical of a normal year, except that late winter was relatively warmer. Day length included civil twilight; i.e., sunrise to sunset plus 1 h (Beck, 1980).
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2.6. Effects of hatching timing on larval development in the field To examine the effects of the timing of hatching on larval development and survival in the field, larvae that had been reared in the laboratory until the first larval molt (7–10 days after hatching) were transferred to outdoor conditions at four times from spring to summer in 2009: 10 May (Cohort L1), 31 May (Cohort L2), 20 June (Cohort L3), and 10 July (Cohort L4). Larvae were reared on the artificial diet and their development was observed every day until they pupated or died. 2.7. Effects of pupation timing on survival in the pupal and adult stages in the field To examine the effects of the timing of pupation on pupal development and survival in the field, laboratory-raised pupae were transferred to outdoor conditions on the day of pupation at five times from summer to autumn: 4–7 August (Cohort P1), 21–26 August (Cohort P2), 10–15 September (Cohort P3), 1–6 October (Cohort P4), and 21–29 October (Cohort P5). To obtain pupae, longday larvae were transferred to 12L–12D at 25 8C on day 40 after hatching (see Section 3.2). Survival of the pupae and resultant adults was monitored daily until 31 December. 2.8. Survival of adults at various temperatures Pupae obtained by transferring long-day larvae to 12L–12D at 25 8C on day 40 after hatching were kept under the same conditions (12L–12D, 25 8C). Eclosed adults were assigned to groups kept at 20, 22.5, 25 or 27.5 8C under 12L–12D on the day of eclosion, and survival was monitored every other day for 80 days. The surviving adults were inactive and did not feed for more than 80 days, suggesting that they were in diapause. 3. Results 3.1. Larval development under various stationary photoperiodic conditions Larvae of P. rufiventris showed a clear short-day photoperiodic response at 25 8C (Fig. 1). Regardless of the photoperiod, larvae developed similarly from the 1st to 3rd instars. However, development in later instars differed among the photoperiodic conditions. Larvae reared under 12L–12D pupated from day 39 to 72 from the 4th to 6th instars, and those reared under 13L–11D pupated from day 41 to 107 from the 4th to 10th instars. Under 14L–10D, 60% of the larvae pupated from day 63 to 110 from the 4th to 8th instars, but the remainder ceased feeding in the 4th to 8th instars and did not pupate within the 120-day experimental period. The larval durations for those that pupated (mean SD) were 50.0 7.7, 83.5 14.7, and 95.2 15.5 days under 12L–12D, 13L–11D and 14L–10D, respectively, and differed significantly between photoperiods (Steel–Dwass test, P < 0.05). This difference was due to variation in the number of molts and/or the duration of the final instar (Fig. 1). Under 15L–9D and 16L–8D, all larvae underwent five or more instars and ceased feeding, and no larvae pupated. The body weight of these larvae increased with development, but the effect of an additional molt tended to be smaller as larvae grew (Table 1). After day 120, larvae neither pupated nor molted into the next instar, and thus these larvae were considered to be in diapause. At 30 8C, larvae showed a similar response to photoperiods: all larvae reared under 14L–10D and 16L–8D, and more than half of those reared under 12L–12D entered diapause with some supernumerary molts (Fig. 2). Thus, the critical day lengths for the photoperiodic response in P. rufiventris larvae were about 14 h and 12 h at 25 8C and 30 8C, respectively.
Fig. 1. Photoperiodic response in Phytoecia rufiventris larvae at 25 8C. Larvae were reared from hatching under five photoperiodic conditions at 25 8C. Each bar indicates development of an individual larva and the right end of the bar indicates pupation. For larvae that did not pupate within the experimental period (120 days), the bars end on day 120. Triangles indicate the mean larval period for individuals that pupated. The initial sample size was 40 for each experimental group.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059
Table 1 Body weight of Phytoecia rufiventris larvae in various instars.a
4th 5th 6th 7th 8th 9th
n 24 23 22 15 11 10
20.8 5.2 36.2 10.9 51.5 15.0 59.3 9.0 66.3 8.3 72.9 9.8
60
c b
b
Body weight (mg, mean SD)
50
75
% pupation
Instar
100
40
a
30
50
20 25
a Newly ecdysed larvae were sampled randomly from larvae mass-reared under 16L–8D at 25 8C.
10
Duration until pupation (days)
1056
0
0 12
13
13.5
14
15
16
Photophase (h/day) Fig. 3. Responses of Phytoecia rufiventris larvae to photoperiodic change. Larvae reared from hatching under 16L–8D at 25 8C were transferred on day 40 to various photoperiodic conditions. The percentage pupation (*) and the duration until pupation (column) are shown. Error bars indicate SD. n = 27–30. Values with different letters are significantly different at P = 0.05 by Tukey–Kramer multiple comparison test.
3.3. Developmental parameters The egg, larval and pupal periods at various temperatures in P. rufiventris are shown in Table 2. Based on these data, the developmental threshold and thermal constant were determined to be 13.1 8C and 66.6 degree-days, respectively, in the egg stage; 13.5 8C and 269.1 degree-days, respectively, in the larval stage (hatching to the 3rd larval molt); and 8.7 8C and 240.9 degree-days, respectively, in the pupal stage. 3.4. Effects of hatching timing on larval development in the field Development of P. rufiventris larvae transferred to outdoor conditions at various times from spring to summer is shown in Fig. 5. Most larvae died in summer, independently of the timing of transfer. In Cohort L4, no larvae survived to pupation. This high mortality might have been due to fungi that rapidly spread on the artificial diet. Only 7 larvae pupated from 4 to 17 September. This result suggests that larvae pupate within a very short period in early autumn, regardless of the time of hatching.
Fig. 2. Photoperiodic response in Phytoecia rufiventris larvae at 30 8C. Larvae were reared from hatching under three photoperiodic conditions at 30 8C. The initial sample size was 40 for each experimental group. For details, see legend for Fig. 1.
The responses of long-day larvae of P. rufiventris to photoperiodic change are shown in Fig. 3. Without photoperiodic transfer, no larvae pupated. When the larvae were transferred to 12L–12D, 13L–11D or 13.5L–10.5D, 87–93% pupated, whereas only about half pupated under14L–10D and none pupated under 15L–9D. The duration from transfer to pupation (mean SD) varied from 33.5 3.3 days (12L–12D) to 49.1 5.9 days (14L–10D). There was a significant difference among later photoperiods, with a shorter photoperiod corresponding to a shorter duration until pupation (Tukey–Kramer multiple comparison test, P < 0.05). The responses of long-day larvae of various ages to photoperiodic change to 12L–12D are shown in Fig. 4. Most larvae of all ages pupated after the photoperiodic change, but the duration until pupation differed among ages and was significantly longer in younger larvae. However, for larvae transferred on day 50 or later, the duration was about 27 days, irrespective of the age.
Duration until pupation (days)
3.2. Responses to photoperiodic change
50
a b
40
b
30
c
c
c
50
60
70
20
10
0
20
30
40
Larval age (days after hatching) Fig. 4. Responses to photoperiodic change from long-day to short-day conditions in Phytoecia rufiventris larvae of various ages. Larvae reared from hatching under 16L– 8D at 25 8C were transferred to 12L–12D on day 20, 30, 40, 50, 60 or 70. The duration until pupation is shown. Error bars indicates SD. n = 23–28. Values with different letters are significantly different at P = 0.05 by Tukey–Kramer multiple comparison test.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059 Table 2 Developmental periods at various temperatures and developmental parameters in the egg, larval and pupal stages in Phytoecia rufiventris.a Developmental period (days, mean SD)
Temperature (8C) 30 27.5 25 22.5 20 17.5 T0 (8C) K (degree-days) r2
Egg
Larvab
Pupa
4.0 0.6 4.6 0.7 5.4 0.7 7.1 0.4 10.1 0.7 14.7 1.1 13.1 66.6 0.999
15.6 1.0 20.0 2.1 23.9 1.4 29.1 1.3 41.1 3.8 13.5 269.1 0.996
11.2 0.5 12.5 0.6 14.9 0.6 17.8 0.6 21.8 0.8 26.5 1.0 8.7 240.9 0.988
n = 25–34 for eggs, 14–21 for larvae, and 17–27 for pupae. a Under 16L–8D for the egg and larval stages and under 12L–12D for the pupal stage. b For the first three instars.
20
Cohort L3
No. surviving larvae
Cohort L4 15
Cohort L2
1057
3.5. Effects of pupation timing on survival in the pupal and adult stages Development and survival of P. rufiventris pupae transferred to outdoor conditions at various times from summer to autumn is shown in Fig. 6. In Cohorts P1, P2, P3 and P4, all pupae eclosed to adults. In contrast, in Cohort P5, 60% of the pupae died before eclosion and 5 out of 8 survivors eclosed with lethally deformed wings. There was a significant difference in the eclosion rate between Cohort P5 and the other cohorts (Tukey-type multiple comparisons for proportions, P < 0.05 [Zar, 1999]). The survival rate of eclosed adults clearly differed among the four cohorts with higher eclosion rates: in Cohorts P1 and P2, most adults died in 20– 40 days and only 3 adults in Cohort P2 survived until winter. In Cohort P4, nearly half of the eclosed adults died within 10 days, but 50% survived until winter. In contrast, 95% of adults in Cohort P3 survived until winter. In Cohort P5, 3 of 8 adults survived until winter. There was a significant difference in the rate of survival from pupation until winter between cohorts; the survival rates in Cohort P3 and P4 were significantly higher than that in other cohorts, and that in Cohort P3 was significantly higher than that in Cohort P4 (Tukey-type multiple comparisons for proportions, P < 0.05 [Zar, 1999]). 3.6. Effects of temperature on survival of adults
10
Cohort L1 Pupation
5
Survival of P. rufiventris adults at various temperatures is shown in Fig. 7. There was a clear tendency for reduced longevity at higher temperatures: all the adults died within 32 days at 27.5 8C and 95% died within 80 days at 25 8C. In contrast, most adults remained alive at 22.5 and 20 8C.
0 May
Jun
Jul
Aug
Sept
4. Discussion
Fig. 5. Outdoor development and survival of Phytoecia rufiventris larvae with different hatching times. Newly ecdysed 2nd-instar larvae were transferred to outdoor conditions on 10 May (Cohort L1), 31 May (Cohort L2), 20 June (Cohort L3), and 10 July (Cohort L4) in 2009. n = 19 or 20.
The results of the study show that P. rufiventris has a short-day photoperiodic response in larval development. Under short-day conditions larvae pupate, whereas under long-day conditions they enter diapause with some supernumerary molts and do not
20 15
Cohort P2
10 5 0 20 15
Cohort P3
10 5 0 20 15 Pupa
Cohort P4
10
Adult
5
No. surviving pupae or adults
0 20 15
20 Cohort P1
15
10
10
5
5
0
0 Aug
Sept
Oct
Cohort P5
Nov
Dec
Fig. 6. Outdoor development and survival of Phytoecia rufiventris pupae and adults with different pupation times. Newly ecdysed pupae were transferred to outdoor conditions on 4–7 August (Cohort P1), 21–26 Aug (Cohort P2), 10–15 September (Cohort P3), 1–6 October (Cohort P4), and 21–29 October (Cohort P5) in 2009. n = 20.
Y. Shintani / Journal of Insect Physiology 57 (2011) 1053–1059
22.5ºC 20ºC
Survival rate (%)
80
60
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0
0
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20
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50
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Days after eclosion Fig. 7. Survival of Phytoecia rufiventris adults at various temperatures. Larvae reared under 16L–8D at 25 8C were transferred to 12L–12D on day 40 and the resultant adults were kept at various temperatures under 12L–12D. n = 20 at each temperature.
pupate. This diapause is induced under long-day conditions and terminated after transfer to short-day conditions; therefore, it is the typical summer diapause defined by Masaki (1980). The shift to a shorter critical day length at higher temperature found in this photoperiodic response is a common feature of summer diapause (Masaki, 1980; Danks, 1987). Several other insects, such as the southwestern corn borer, Diatraea grandiosella (Chippendale and Reddy, 1973), and the cornstalk borer, Sesamia nonagrioides (Gadenne et al., 1997), undergo supernumerary molts during photoperiodically induced diapause. Among such insects, the yellow-spotted longicorn beetle, Psacothea hilaris, provides a good contrast to P. rufiventris. P. hilaris has a long-day photoperiodic response in larval development: under long-day conditions larvae pupate after the 4th or 5th instar, whereas under short-day conditions they enter diapause with some supernumerary molts and do not pupate unless transferred to long-day conditions or exposed to low temperature (Shintani et al., 1996; Shintani and Ishikawa, 1997, 1998a, 1998b). Larvae of P. rufiventris reared under long-day conditions pupate after transfer to short-day conditions, but the response to photoperiodic change varies with age. For larvae transferred on day 20, the whole larval duration is 64 (20 + 44) days, which is much longer than the larval duration under stationary short-day conditions (50 days, Fig. 1). This indicates that the larvae have already become sensitive to the photoperiod in the 3rd instar. For larvae transferred in the 5th or later instars, the duration until pupation was almost the same indicating that they are in a similar physiological state with respect to diapause. The short-day photoperiodic response found in P. rufiventris larvae had a quantitative feature, since a shorter day length corresponded to a shorter larval duration for day lengths shorter than the critical day length. This difference is due to variation in the number of molts and/or the duration of the final instar. Short-day photoperiodic responses in insects clearly play a role in termination of summer diapause and regulation of the timing of developmental events in autumn (Masaki, 1980; Tauber et al., 1986; Danks, 1987). For example, termination of reproductive diapause is photoperiodically regulated in the carabid beetle, Leptocarabus kumagaii (Sota, 1987), and the viburnum leaf beetle, Pyrrhalta humeralis (Nakai and Takeda, 1995a). Summer diapause in prepupae of the tailed zygaenid moth, Elcysma westwoodii (Gomi and Takeda, 1992; Nakai and Takeda, 1995b), and pupae in the Japanese giant silkmoth, Dictyoploca japonica (Nagase and Masaki, 1991), is also terminated under short-day conditions. In P. rufiventris, the short-day photoperiodic response of larvae clearly regulates termination of summer diapause. Seasonal
changes in temperature and day length at the research site are shown in Fig. 8. Larvae hatch in spring and grow under long days in summer at temperatures fluctuating around 25 8C. Under these conditions larvae enter and maintain summer diapause. As the day length becomes shorter than the critical day length in late summer, the larvae terminate diapause and then pupate within a relatively short period in early autumn (Fig. 5). This synchronization of pupation may be due to the quantitative feature of the short-day photoperiodic response. The temperature-dependent shift in the critical day length may compensate for yearly differences in temperature. However, it was somewhat surprising that the day length became shorter than the critical day length (14 h at 25 8C) as late as late-August, since at least 27 days are necessary before pupation (Fig. 4), and the actual time of pupation (early autumn) cannot be explained based on these results. This contradiction may arise from the assumption of the natural day length including 1-h twilight; the actual duration perceived as the photophase may be shorter, since diapause larvae inhabit roots underground. The results also showed that only individuals that pupated in early autumn could survive as adults until the end of December, indicating that pupation timing has a crucial effect on survival. If larvae pupated earlier (in mid-summer), the pupae would eclose as adults within two weeks, but the resultant adults may not survive until mid-autumn (Fig. 6). High mortality in these adults is probably caused by high temperatures during mid- to late summer, because the adults are intolerant of temperatures of 27.5–30 8C for a long period (Fig. 7). In contrast, if larvae pupated later (in midautumn), high mortality would occur in the pupal or adult stage, probably due to low temperatures during late autumn to early winter. The pupa of most coleopterans is the exarate type (i.e., without puparium), which is generally susceptible to physical stresses such as cold and drought, and no coleopterans use this stage as the diapause stage (Masaki, 1980). In P. rufiventris pupae, the thermal threshold is much lower than in other stages (Table 2) and this ensures that the pupal period is shortened at moderate autumnal temperatures. Such a trend in thermal threshold is not found in other cerambycids, such as P. hilaris (Shimane and Kawakami, 1991; Sakakibara, 1995; Watari et al., 2002) and X. pyrrhoderus (Ashihara, 1982). Thus, this low thermal threshold in P. rufiventris pupae is an adaptation to reduce the risk of injury due to cold. The early autumn pupation in P. rufiventris seems to be evolutionarily stable for the following reasons. First, larvae are
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Fig. 8. Seasonal changes in temperature and day length at the research site in Takanabe (1318310 E, 328060 N), Miyazaki, Japan in 2009. (A) Air temperature and soil temperature (5 cm beneath the surface). (B) Natural day length. Temperature is shown as a 10-day average and natural day length is shown with an additional 1 h of twilight.
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clearly tolerant of high temperature in summer, and if eclosed adults also acquired high-temperature tolerance, earlier pupation might not lead to high mortality. In this case, however, P. rufiventris would have high temperature tolerance in both the larval and adult stages, which may lead to more cost. Second, if pupae had lowtemperature tolerance, later pupation might not lead to high mortality. However, this situation is unlikely because pupae of P. rufiventris may be susceptible to winter physical stresses due to the physiological restrictions in coleopterans discussed above. In summary, pupation timing in P. rufiventris is restricted by high temperature in summer and low temperature in autumn. The quantitative short-day photoperiodic response in P. rufiventris larvae provides precise regulation of timing for pupation and might have evolved as an adaptation to seasonally changing temperature. Acknowledgments I am very grateful to Dr. S. Kawanobu (Minami Kyushu University) for providing meteorological data. This study was supported in part by a grant from Minami Kyushu University from 2006 to 2009. References Ashihara, W., 1982. Effects of temperature and photoperiod on the development of the grape borer. Xylotrechus pyrrhoderus Bates (Coleoptera: Cerambycidae). Japanese Journal of Applied Entomogy and Zoology 26, 15–22 (in Japanese with English summary). Beck, S.D., 1980. Insect Photoperiodism, 2nd ed. Academic Press, New York. Chippendale, G.M., Reddy, A.S., 1973. Temperature and photoperiodic regulation of diapause of the southwestern corn borer, Diatraea grandiosella. Journal of Insect Physiology 19, 1397–1408. Danks, H.V., 1987. Insect Dormancy: An Ecological Perspective. Biological Survey of Canada, Ottawa. Emori, T., 1976. Ecological study on the occurrence of the yellow-spotted longihorn beetle (Psacothea hilaris Pascoe). I. Effects of temperature and photoperiodic conditions on their development. Japanese Journal of Applied Entomology and Zoology 20, 129–132 (in Japanese with English summary). Gadenne, C., Dufour, M.C., Rossignol, F., Be´card, J.M., Couillaud, F., 1997. Occurrence of non-stationary larval moults during diapause in the corn-stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). Journal of Insect Physiology 43, 425–431. Gomi, T., Takeda, M., 1992. A quantitative photoperiodic response terminates summer diapause in the tailed zygaenid moth, Elcysma westwoodii. Journal of Insect Physiology 38, 665–670. Masaki, S., 1980. Summer diapause. Annual Review of Entomology 25, 1–25. Nagase, A., Masaki, S., 1991. Thermal and photoperiodic responses in aestivasting pupae of Dictyoploca japonica (Lepidoptera: Saturniidae). Applied Entomology and Zoology 26, 387–396. Nakai, T., Takeda, M., 1995a. Temperature and photoperiodic regulation of summer diapause and reproduction in Pyrrhalta humeralis (Coleoptera: Chrysomelidae). Applied Entomology and Zoology 30, 295–301.
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