Geographic Variation in Diapause Induction and Termination of the Cotton Bollworm, Helicoverpa Armigera Hã¼bner (Lepidoptera

Journal of Insect Physiology 59 (2013) 855–862

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Geographic variation in diapause induction and termination of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) ⇑ Yuan-Sheng Chen a,b,c, Chao Chen a,b, Hai-Min He a,b, Qin-Wen Xia a,b, Fang-Sen Xue a,b, a Key Laboratory of Physiology, Ecology and Cultivation of Double Cropping Rice, Ministry of Agriculture, China b Institute of Entomology, Jiangxi Agriculture University, Nanchang 330045, China c Jiangxi Environmental Engineering Vocational College, Ganzhou 341000, China article info abstract

Article history: Overwintering diapause in Helicoverpa armigera, a multivoltine species, is controlled by response to pho- Received 19 March 2013 toperiod and temperature. Photoperiodic responses from 5 different geographical populations showed Received in revised form 5 June 2013 that the variation in critical photoperiod for diapause induction was positively related to the latitudinal Accepted 6 June 2013 origin of the populations at 20, 22 and 25 °C. Diapause response to photoperiod and temperature was Available online 18 June 2013 quite different between northern and southern populations, being highly sensitive to photoperiod in northern populations and temperature dependence in southern populations. Diapause pupae from south- Keywords: ern population showed a significantly shorter diapause duration than from northern-most populations Diapause when they were cultured at 20, 22, 25, 28 and 31 °C; by contrast, overwintering pupae from southern Photoperiod Temperature populations emerged significantly later than from northern populations when they were maintained in Chilling natural conditions, showing a clinal latitudinal variation in diapause termination. Diapause-inducing Geographical variation temperature had a significant effect on diapause duration, but with a significant difference between Helicoverpa armigera southern and northern populations. The higher rearing temperature of 22 °C evoked a more intense diapause than did 20 °C in northern populations; but a less intense diapause in southern population. Cold exposure (chilling) is not necessary to break the pupal diapause. The higher the temperature, the quicker the diapause terminated. Response of diapause termination to chilling showed that northern populations were more sensitive to chilling than southern population. All results demonstrate that H. armigera is not genetically homogeneous throughout its range, but rather is composed of distinct populations genetically adapted to local environmental conditions despite the potential for gene flow via seasonal migration of adults. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction for diapause induction being the most common (see Tauber et al., 1986; Danks, 1987). However, few have examined variation Diapause is a state of arrested development that allows insects in sets of seasonal responses (e.g. Bradshaw and Lounibos, 1977; to survive unfavorable conditions and synchronize their life histo- Istock, 1978; Masaki, 1978; Dingle et al., 1980; Holzapfel and Brad- ries with conditions conducive to growth and reproduction (e.g. shaw, 1981; Tauber et al., 1986; Nechols et al., 1987; Wang et al., Beck, 1980; Tauber et al., 1986; Danks, 1987). In insects with a 2012). Thus, to initiate a study of the relationship among season- widespread distribution, diapause characteristics may vary in ally important ecophysiological traits, it is important to investigate relation to latitude, altitude and local climatic conditions. This geo- patterns of geographical variability in a suite of responses that con- graphical variation may concern characteristics such as the critical trol dormancy from its onset through its termination in a geo- photoperiod for diapause induction as well as diapause intensity, graphically widespread insect species. and may be seen as an adaptive mechanism to different local envi- The cotton bollworm, Helicoverpa armigera (Hübner) is one of ronmental conditions (Danilevskii, 1965; Tauber et al., 1986; the most serious pests of cotton, maize, wheat, sorghum and many Danks, 1987). Numerous studies with insects have examined vari- other crops and widely distributes in various climatic zones of the ability in a single seasonal response pattern, critical photoperiod world (Fitt, 1989; Luttrell et al., 1994; Guo, 1997). It enters pupal diapause in the soil. Diapause induction and termination in H. armigera have been investigated in detail in its distribution regions, ⇑ Corresponding author at: Institute of Entomology, Jiangxi Agriculture Univer- especially in China (e.g. Wu and Guo, 1995, 1996, 1997a,b; Li and sity, Nanchang 330045, China. Tel./fax: +86 791 382 8081. E-mail address: [email protected] (F.-S. Xue). Li, 1998; Jiang et al., 1999; Chen et al., 2012), Japan (e.g. Qureshi

0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.06.002 856 Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862 et al., 1999, 2000; Shimizu and Fujisaki, 2006; Shimizu et al., 2006; emergence of overwintering pupae. The emerged adults in the Kurban et al., 2005, 2007), Greece (Mironidis et al., 2010; Mironidis three experiments were recorded every day until all adults and Savopoulou-Soultani, 2012), Australia (Wilson et al., 1979; emerged. Kay, 1982), Sudan (Hackett and Gatehouse, 1982) and Botswana (Roome, 1979). However, most of these studies examined the effect 2.4. Identification of diapause induction and termination of environmental conditions on diapause induction and termination in a single population; only a few studies compared differ- Diapause was determined by a lack of eye spot movement. Pu- ences in induction and termination of diapause among different pae that retained eyespots in the post genal region for more than geographical populations (Wu and Guo, 1997a,b; Shimizu and Fuji- 15 days were considered to be in diapause (Cullen and Browning, saki, 2006; Qureshi et al., 2000). For better understanding of insect 1978). For convenience, the criterion of diapause termination life cycles, information about geographical variation in the induc- was adult emergence in our experiment. Thus, our estimates of tion and termination of diapause is desirable. This study was de- diapause duration include the period of post-diapause signed therefore to determine and compare the photoperiodic development. response for diapause induction and diapause termination of populations of H. armigera from different geographic latitudes under 2.5. Statistical analyses wide temperature conditions. The experimental data were analyzed by data-processing sys- 2. Materials and methods tem SPSS 17.0. Probit regression analysis was used to estimate critical photoperiods for each group and linear regression was used to 2.1. Insects and rearing conditions analyze the relationship between latitude and critical photoperiod. Effects of population, diapause-inducing and -terminating temper- Helicoverpa armigera was obtained from five field populations of ature on diapause duration were analyzed using general linear Guangzhou (GZ: 23.08°N, 113.14°E), Yongxiu (YX: 29.04°N, models (GLM) and Tukey’s test was used on multiple comparisons. 116.82°E), Taian (TA: 36.15°N, 116.59°E), Langfang (LF: 39.31°N, We used an alpha level of 0.05 in all analyses. 116.42°E) and Kazuo (KZ: 41.34°N, 120.27°E) collected from late June to early August in 2010 and were reared in the laboratory 3. Results for three generations under non-diapausing conditions (25 °C, LD 16:8) until adult eclosion by previous methods (Chen et al., 3.1. Geographic variations in photoperiod response for diapause 2012). Adults were fed with 10% honey solution. Eggs were col- induction lected on days 3, 4, and 5 after eclosion. After hatching, 3–5 newly hatched larvae were together transferred to the plastic plates with The critical photoperiods of five geographical populations were 24 holes (for each hole: diameter: 1.5 cm; height: 2 cm) and reared highly affected by latitude and temperature. The critical photope- on an artificial diet (Wu and Gong, 1997) until the 3rd instar larvae. riod for diapause induction had a positive relationship with the lat- The 3rd instar larvae were individually transferred to new plastic itudinal origin of populations at 20, 22 and 25 °C(Fig. 1 and plates with 24 holes until the 5th larval instar, then the larvae were Table 1). A very high correlation was found at 25 °C. There were individually reared in plastic plates with 21 holes (length: 2.5 cm; significant differences in critical photoperiod among different geo- width: 2.5 cm; height: 2.5 cm) until pupation. graphical populations at 20 °C. The critical photoperiod also shortened with increasing temperature, as seen in the LF population, the 2.2. Induction of diapause critical photoperiod was 14.28, 13.70 and 13.30 h, respectively, when reared at 20, 22 and 25 °C. However, the southern population To examine variability in photoperiodic induction of diapause from GZ exhibited a lower incidence of diapause (26.53–21.05%) among the five geographical populations, we reared individuals even under short daylengths of 12–13 h at the lower temperature from each population under a range of constant photoperiods from of 20 °C, without showing the critical photoperiod. Similarly, the LD11:13 to LD 16:8 at constant temperatures of 20, 22, 25 and incidence of diapause for southern population from YX was less 28 °C. We recorded the incidence of pupal diapause under each than 50% under the short daylengths of 11–13 h at 22 °C. At condition and used these data to determine critical photoperiod 25 °C, all individuals from the southern populations (GZ and YX) (50% of the pupae entered diapause). At least 100 larvae were used developed without diapause regardless of photoperiods, whereas in each experimental condition. northern populations (TA, LF and KZ) still exhibited a higher incidence of diapause (>58%) under short daylengths of 11–12 h, espe- 2.3. Termination of diapause cially in the northern-most population of KZ (>85%). At the high temperature of 28 °C, more than 70% individuals from northern To determine the variability in the length of diapause among populations developed without diapause. These results reveal that different geographical populations, three experimental methods higher temperatures significantly weaken and even completely in- were adopted. In the first experiment, diapausing pupae from YX, hibit photoperiodic induction of diapause; this effect is even more TA, LF and KZ induced under short photoperiod of LD 12:12 at 20 obvious in the southern populations. and 22 °C were maintained under long photoperiod of LD 16:8 at 20, 22, 25, 28 and 31 °C to observe diapause termination. In the 3.2. Geographical variation in diapause termination at different second experiment, diapausing pupae from YX, TA and KZ induced constant temperatures under LD 12:12 at 20 °C were placed at 5 and 9 °C for 20, 40, 60, 80 and 100 days in continuous darkness (DD). After chilling, pupae The duration of diapause of the 4 geographical populations was were transferred to 25 and 31 °C combined with the long photope- highly affected by latitude and diapause-inducing and -terminat- riod of LD 16:8 to terminate diapause. In the third experiment, ing temperature (F3, 2817 = 57.15, P = 0.000 for latitude effect, diapausing pupae from GZ, YX, TA and KZ induced under LD F1, 2819 = 94.39, P = 0.000 for diapause-inducing temperature effect 12:12 at 20 °C were maintained in natural conditions in the suburb and F4, 2816 = 1175.24, P = 0.000 for diapause-terminating tempera- of Nanchang (28.77°N, 115.83°E), Jiangxi Province to observe adult ture effect on the duration of diapause). The duration of diapause Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862 857

Fig. 2. A comparison of diapause duration among different geographical populations of H. armigera. Diapausing pupae induced under the short photoperiod of LD 12:12 at 20 and 22 °C were terminated under LD 16:8 at 20, 22, 25, 28 and 31 °C.

N = 33–121 for each treatment (Tukey’s multiple comparisons: 31 °C: F7, 448 = 14.06,

P < 0.05; 28 °C: F7, 562 = 14.04, P < 0.05; 25 °C: F7, 600 = 16.48, P < 0.05; 22 °C:

F7, 589 = 25.40, P < 0.05; 22 °C: F7, 581 = 18.65, P < 0.05).

southern YX population was significantly lower than those from Fig. 1. Photoperiodic response curves for diapause induction in different geographical populations of H. armigera at 20, 22, 25 and 28 °C. N = 34–109 for each point. northern populations (TA, LF and KZ, P < 0.05) at all temperatures. Interestingly, the diapause-inducing temperature of 22 °C evoked a was significantly different among different populations at all tem- more intense diapause than did 20 °C at all diapause-terminating peratures (Fig. 2, P < 0.05). Except for diapausing pupae from temperatures in northern populations. The duration of diapause southern YX population which were induced at 20 °C and termi- in northern populations was significantly different at 20 and nated at 20 °C, the duration of diapause induced at 22 °C from 22 °C between the two inducing temperatures (P < 0.05). At 25

Table 1 Geographic variation in critical photoperiod of diapause induction of H. armigera at different temperatures.

Temperature (°C) Critical photoperiod (h) The relationship between critical photoperiod and latitude GZ YX TA LF KZ 20 – 11.78 13.43 14.28 13.89 Y = 0.19X + 6.3, R2 = 0.858, P = 0.049 22 – – 12.55 13.7 13.73 Y = 0.24X + 4.0, R2 = 0.730, P = 0.240 25 – – 12.38 13.24 13.53 Y = 0.23X + 4.3, R2 = 0.953, P = 0.098 858 Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862 and 28 °C, the northern-most KZ population also showed that the emergence for 50% individuals was much later in the northern- duration of diapause was significantly higher at the inducing tem- most KZ population than in the southern YX population at the perature of 22 °C than that at the inducing temperature of 20 °C chilling treatments of 20 and 40 days; they were significantly dif- (P < 0.05). In contrast, diapause in a southern population (YX) ferent in diapause durations when the chilling period was included was significantly more intense at the inducing temperature of (P < 0.05). Interestingly, when the chilling period exceeded 40 20 °C than at 22 °C when diapause was terminated at 20 °C days, the adult emergence for 50% individuals was earlier at the (P < 0.05). There were also no significant differences in diapause northern-most population (KZ) than at the YX population in most duration between the two inducing temperatures at 22, 25, 28 cases, although there was no significant difference among popula- and 31 °C(P > 0.05). tions (P > 0.05). This result suggests that the northern-most popu- When diapausing pupae were cultured at 20, 22, 25, 28 and lation is more sensitive to chilling than the southern population. 31 °C combined with a long photoperiod of LD 16:8, the duration of diapause of the 4 geographical populations was gradually short- 3.4. Geographical variation in diapause termination under natural ened with increasing temperature (Fig. 3). For example, at the dia- conditions pause-inducing temperature of 22 °C, duration of diapause was significantly shortened from 72.4 days at 20 °C to 16.4 days at Fig. 5 shows termination of pupal diapause in H. armigera 31 °C in YX population, from 102 days to 23.8 days in TA popula- among different geographical populations under natural condition, from 116.4 days to 24.2 days in LF population and from tions. Adult emergence for 50% individuals from overwintering pu- 115.2 days to 27.1 days in KZ population. This result showed that pae was earliest in the northern-most KZ population, followed by high temperature significantly enhanced the termination of dia- northern TA, southern YX and GZ populations, showing that diapause in H. armigera. pause duration was negatively correlated with latitude. Duration of diapause was significantly different between the northern pop- 3.3. Geographical variation in diapause termination after different ulations (KZ and TA) and southern populations (YX and GZ) chilling periods (P < 0.01).

Figs. 4 and A1 show cumulative eclosion of diapausing pupae 4. Discussion under LD 16:8 at 25 and 31 °C after chilling at 5 °C and 9 °C for different days in the three geographical populations. From the two The photoperiodic responses for diapause induction in this figures we can see three important features. First, eclosion time study revealed that diapause response to photoperiod and temper- for the three populations was significantly delayed with increase ature in H. armigera was quite different between northern and in chilling time when chilling period was regarded as part of the southern populations (Fig. 1). The northern populations were duration of diapause development (P < 0.05). However, chilling highly sensitive to photoperiod; most individuals entered diapause played an important role in synchronizing adult emergence. All under short photoperiods at the high temperature of 25 °C. How- adults emerged within 6–10 days at 25 °C and 4–5 days at 31 °C ever, the southern populations were highly sensitive to tempera- at the chilling treatment of 100 days. Second, adult emergence ture; all individuals developed without diapause at 22 °CinGZ was much earlier and more synchronized at 31 °C than at 25 °C population and at 25 °C in YX population regardless of photope- at all chilling treatments, further suggesting that high temperature riod. Diapause can be induced only under short photoperiods at significantly accelerated diapause development. Third, influence of lower temperatures in southern populations. This result suggests chilling on diapause termination varied with chilling duration and that photoperiod plays a decisive role in the determination of dia- the latitudinal origin of the populations. For example, adult pause in northern populations under natural conditions, whereas

Fig. 3. The duration of diapause of different geographical populations of H. armigera at different temperatures. N = 33–121 for each treatment. Ã showing signiﬁcantly different between the diapause terminating temperatures (Tukey’s multiple comparisons: F19, 1414 = 367.57, P < 0.05 for inducing temperature of 20 °C; F19, 1366 = 171.32, P < 0.05 for inducing temperature of 22 °C). Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862 859

Fig. 4. A comparison of cumulative eclosion from winter diapausing pupae among different population of H. armigera. The diapausing pupae were treated under LD16:8 at 25 and 31 °C after exposure to 5 °C and DD for different days. N = 30–48 for each treatment. (Tukey’s multiple comparisons: F14, 496 = 367.57, P < 0.05 for terminating at 25 °C;

F14, 482 = 1897.17, P < 0.05 for terminating at 31 °C.)

temperature is more important than photoperiod in diapause for every 5° increase in latitude (e.g. Danilevskii, 1965; Tauber determination in southern populations. Such a difference in dia- et al., 1986; Danks, 1987). Of the traits we studied, the critical pho- pause regulating mechanism between northern and southern pop- toperiods for diapause induction in H. armigera at 20, 22 and 25 °C ulations may be seen as an adaptive mechanism to different local are also positively related with the latitudinal origin of the popula- environmental conditions. In nature, the response pattern of pho- tion (Table 1). There is a strong north-south cline in photoperiodic toperiodic control of diapause induction in northern populations responses – with diapause induction in northern populations allows individuals which pupated in mid-autumn under warm (KZ, LF, and TA) occurring at longer daylengths (13.89 h for KZ conditions to enter diapause, thus avoiding adult emergence in population, 14.28 h for LF population and 13.43 h for TA popula- early winter and increasing the size of the overwintering population) than in a southern population (11.78 h for YX population) tion. The response pattern of temperature dependence of diapause at 20 °C. The critical daylength changed by approximately 1.2 h induction in southern populations enables the moth to utilize a lar- with every 5° of latitude between the KZ population (41.34°N) ger amount of available environmental resources and longer time and TA population (36.15°N) at 22 and 25 °C; the critical daylength for growth and breeding most effectively. of TA population (36.15°N) increased 1.7 h beyond that of the YX In many wide-spread insect and mite species with a long-day population (29.04°N) at 20 °C. The longer critical photoperiod in response, the critical photoperiod for diapause induction is pro- populations from higher latitudes supports an earlier report (Wu longed with the latitude towards the north or with temperature and Guo, 1997a,b). However, previous research from Japan in H. drop, but the degree of variation varies with different insect spe- armigera did not ﬁnd a geographic cline in critical photoperiod cies. In a number of such long-day insect species, the critical pho- (Qureshi et al., 2000). The southern population from Ishigaki toperiod for diapause induction has been found to change 1–1.5 h (24.3°N, 124.2°E) had a critical photoperiod 1 h longer than the 860 Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862

A similar result also was found in the rice-stem borer Chilo suppressalis, in which overwintering larvae from the southern Saigoku population had a more intense diapause than the northern Shonai population (Inouye and Kamano, 1957). One surprising result of this study is the observation that the diapause-inducing temperature had a significant effect on diapause duration, but with a significantly different between southern and northern populations (Fig. 2). The higher rearing temperature of 22 °C evoked a more intense diapause than did 20 °C in northern TA, LF and KZ populations at all diapause-terminating temperatures, especially at the lower diapause-terminating temperatures of 20 and 22 °C with significantly different. In contrast, the lower rearing temperature diapause-inducing temperature of 20 °C evoked a more intense diapause than did 22 °C in southern YX population at the diapause-terminating temperatures of 20 and 22 °C, showing a significant difference at 20 °C. Influence of pre-diapause temperature on diapause intensity has been reported for a number Fig. 5. Diapause termination of hibernating pupae (pupated on 30 October 2010) in of insects. In some insects, high temperatures during diapause four geographical populations of H. armigera at a mean natural temperature of induction induce more intense diapause than that of low temper- 11.3 °C for GZ population, 11.3 °C for YX population, 10.7 °C for LF population and 10.6 °C for KZ population. The mean temperature was calculated from the day of atures, such as in the cricket Teleogryllus emma (Masaki, 1962), pupation to the day on which 50% of individuals emerged. N = 33–326 for each codling moth Cydia pomonella (Sieber and Benz, 1980), tobacco treatment. Values followed by different letters are significantly different (Tukey’s hornworm Manduca sexta (Denlinger and Bradfield, 1981), corn multiple comparisons: F3, 492 = 214.711, P = 0.000 < 0.01). borer Ostrinia nubilalis (Beck, 1989), Hessian fly Mayetiola destruc- tor (Wellso, 1991), grape berry moth Lobesia botrana (Roditakis and northern population from Okayama (34.6°N, 134.1°E) at 20 °C. The Karandinos, 2001), yellow-spotted longicorn beetle, Psacothea authors thought that the lack of clinal latitudinal difference in hilaris (Asano et al., 2004). In some species, diapause was more in- diapause induction may be attributable to two reasons: (1) The tense after rearing at a low temperature, such as the Indian meal strong flight capacity in this species may cause an active gene flow moth Plodia interpunctella (Bell, 1976), lacewing fly Mallada flavi- (active gene migration) between areas of very different climatic frons (Brauer) (Principi et al., 1990), corn stalk borer Sesamia non- conditions. (2) Introduction of this species to the Japanese agrioides (Fantinou et al., 1998), the cabbage butterfly, Pieris Archipelago has not been sufficiently long to establish a clear melete (Xiao et al., 2008). Our results further reveal that the effect latitudinal cline. Of course, further investigation is needed among of pre-diapause temperature on diapause intensity is different various other populations in Japan to clarify this point. even in the same insect species depending on the latitudinal origin Diapause termination from three different experimental meth- of the populations. ods in this study revealed that the effect of temperature on dia- Increasing evidence now shows that chilling is not a prerequi- pause termination was significantly different among different site for the completion of hibernation diapause in many insect spe- geographic populations, especially between the southern and cies (Hodek, 2002, see Tables 1 and 3; Takeda, 2006; Damos and northern populations. When diapausing pupae from the southern Savopoulou-Soultani, 2010) including other populations of H. armi- YX and northern TA, LF and KZ populations were transferred to gera (Wu and Guo, 1996; Jiang and Zhang, 1997; Qureshi et al., the constant temperatures of 20, 22, 25, 28 and 31 °C to terminate 2000; Mironidis and Savopoulou-Soultani, 2012). Results in the diapause, diapause duration was significantly shorter in southern present study further reveal that diapause can be terminated with- population than in northern populations in most cases (Figs. 2 out exposure to chilling in H. armigera. Furthermore, the higher the and 3), especially at high temperatures of 25, 28 and 30 °C temperature, the quicker the diapause was terminated, i.e. the (Fig. 2). When diapausing pupae from the southern YX, northern duration of diapause was significantly shortened from 72.4 days TA and northern-most KZ populations placed at 5 and 9 °C for 20, at 20 °C to 16.4 days at 31 °C in the YX population, from 102 days 40, 60, 80 and 100 days in continuous darkness were transferred to 23.8 days in the TA population, from 116.4 days to 24.2 days in to 25 and 31 °C to terminate diapause, influence of chilling on dia- LF population and from 115.2 days to 27.1 days in the KZ popula- pause termination varied with the chilling duration and the latitu- tion at the diapause-inducing temperature of 22 °C(Fig. 3), show- dinal origin of the populations (Figs. 4 and A1). Adult emergence ing that high temperature significantly accelerates diapause for 50% individuals was much later in the northern-most KZ popu- development. There is ample evidence that pupal diapause is lation than at the YX and TA populations at the chilling treatments caused by lack of ecdysteroids and is terminated when the protho- of 20 and 40 days; however, when the chilling period exceeded 40 racic glands are activated to produce ecdysteroid (Meola, 1977; days, adult emergence for 50% individuals was earlier at the north- Browning, 1981; Loeb, 1982; Wang and Gong, 1997). Therefore, ern-most population (KZ) than at the YX populations at most cases. it is possible that the neurosecretory cell in the brain can be acti- When diapausing pupae from southern GZ, YX and northern TA vated quickly by exposing diapause puae to high temperature to and KZ populations were maintained in natural conditions in the produce prothoracicotropic hormone, which in turn acts on the suburb of Nanchang to terminate diapause, the adult emergence prothoracic gland to release ecdysone and results in the prompt for 50% individuals from overwintering pupae was earliest in the termination of diapause. More recently, diapause hormone (DH) northern-most KZ population, followed by northern TA, southern was shown to break diapause by activating the prothoracic glands YX and GZ populations (Fig. 5), showing that diapause duration to synthesize ecdysone in H. armigera and H. zea (Zhang et al., 2004, was negatively correlated with latitude. It is clear that different 2008). However, this effect was highly temperature-dependent. In experimental methods produce different results. However, only H. armigera, DH could completely break diapause at high tempera- the result from the third experiment adequately reflects diapause tures of 25 and 27 °C but had no effect at the low temperature of termination under natural conditions. Therefore, diapause dura- 20 °C(Zhang et al., 2004). In H. zea, DH terminated diapause in tion in H. armigera is negatively correlated with latitude rather nearly all individuals at 25 °C but did not terminate diapause at than positively correlated with latitude under natural conditions. 18 °C(Zhang et al., 2008). Therefore, it is also possible that the Y.-S. Chen et al. / Journal of Insect Physiology 59 (2013) 855–862 861 brain can control pupal development through the regulation of DH, Hackett, D.S., Gatehouse, A.G., 1982. Diapause in Heliothis armigera (Hübner) and H. and DH can terminate diapause and promote development at high fletcheri (Hardwick) (Lepidoptera: Noctuidae) in the Sudan Gezira. Bulletin of Entomological Research 72, 409–422. temperatures. Moreover, our data showed that chilling at 5° and 9° Hodek, I., 2002. Controversial aspects of diapause development. European Journal of did not shorten the duration of diapause, rather it lengthened dia- Entomology 99, 163–174. pause in all tested populations when chilling period was included. Hodek, I., Hodková, M., 1988. Multiple role of temperature during insect diapause: a review. Entomologia Experimentalis et Applicata 49, 153–166. Adult emergence was much earlier and more synchronized at 31 °C Holzapfel, C.M., Bradshaw, W.E., 1981. Geographic of larval dormancy in the tree-hole than at 25 °C at all chilling treatments (Figs. 4 and A1). However, mosquito, Aedes triseriatus (Say). 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