Geographic variation in embryonic diapause ... - Wiley Online Library

Entomological Science (2008) 11, 327–339

doi:10.1111/j.1479-8298.2008.00284.x

ORIGINAL ARTICLE

Geographic variation in embryonic diapause, cold-hardiness and life cycles in the migratory locust Locusta migratoria (Orthoptera: Acrididae) in China Seiji TANAKA1 and Dao-Hong ZHU2 1 Laboratory of Insect Life Cycles and Physiology, National Institute of Agrobiological Sciences at Ohwashi, Tsukuba, Ibaraki, Japan; and 2Laboratory of Entomology, College of Resources and Environment, Central South Forestry University, Changsha, Hunan, China

Abstract To investigate geographic adaptation of the migratory locust Locusta migratoria in China, locusts were collected from six localities, ranging from 47.4°N to 19.2°N. Using offspring from the various populations, we compared embryonic diapause, reproductive traits, cold-hardiness and adult body size. The incidence of embryonic diapause was influenced by the genetic makeup, parental photoperiod, and incubation temperature of the eggs. The northern strain (47.4°N) produced diapause eggs under all photoperiodic conditions, whereas the other strains produced a higher proportion of diapause eggs when exposed to a short photoperiod. The incubation temperature greatly influenced diapause induction. At a low temperature, all eggs entered diapause, even some of those from a tropical strain (19.2°N) in which no diapause was induced at high temperatures. Photoperiodic changes during the parental generation affected the incidence of embryonic diapause. Diapause intensity decreased with decreasing original latitude. Cold hardiness was compared by exposing eggs in diapause to either -10 or -20°C for various periods; the northern strain was more cold-hardy than the southern strain, although some eggs in the tropical strain were probably not in a state of diapause. Adult body size and head width showed a complicated pattern of variation along the latitudinal gradient, whereas egg pod size (egg pod width and egg number) and hatchling weight tended to decrease with decreasing latitude. These results reveal that L. migratoria has adapted to local environments and that the latitudinal gradient appears to play an important role in shaping L. migratoria life cycle and development. Key words: Acrididae, body size, cold-hardiness, embryonic diapause, phase polyphenism, photoperiodism, progeny size.

INTRODUCTION The migratory locust Locusta migratoria L. is distributed throughout Old World grasslands. Specimens have been found in sub-Saharan Africa, Madagascar, southern Arabian Peninsula, Pakistan, India, South-East Asia,

Correspondence: Seiji Tanaka, Laboratory of Insect Life Cycles and Physiology, National Institute of Agrobiological Sciences at Ohwashi (NIASO), Tsukuba, Ibaraki 305-8634, Japan. Email: [email protected] Received 19 March 2008; accepted 14 May 2008.

© 2008 The Entomological Society of Japan

China, Japan, Australia and New Zealand, and on many islands of the Indian and Pacific Oceans (Uvarov 1966, 1977; Farrow & Colless 1980; Chen 1999). Like the desert locust Schistocerca gregaria Forskål, L. migratoria often forms large swarms and undergoes longdistance migration. However, the extent to which migration occurs appears to be limited compared with S. gregaria, which covers a wide area over successive generations in sub-Saharan Africa, the Scandinavian Peninsula, the Middle East and India. This difference could be one of the reasons why L. migratoria has various distinct populations or subspecies living in different

S. Tanaka and D.-H. Zhu

climatic regions (Uvarov 1966; Farrow & Colless 1980). Another difference between the two species is that L. migratoria has evolved a special adaptation, diapause, which is probably one of the most important physiological traits involved in its northward progression. In contrast, S. gregaria has no diapause at any stage. The role of diapause in controlling life cycle in L. migratoria has been described for Japanese populations (Tanaka 1992, 1994; Ando 1993; Tanaka 1994a,b). Diapause is often accompanied by a variety of other physiological adaptations, known as diapause syndromes (Tauber et al. 1986; Danks 1987). One such syndrome is coldhardiness, which is associated with diapause in many insects; acquisition of this trait plays an important role in the expansion of their distribution to high latitudes (Danilevsky 1966; Danks 1987; Lee & Denlinger 1991). Locusta migratoria ranges from the equator to cool temperate regions in the northern hemisphere (Uvarov 1966; Farrow & Colless 1980; Tanaka 1994a; Chen 1999). In Europe and Japan, various geographic strains of this locust have been studied, and embryonic diapause is suggested to play an important role in their adaptation to local climate (Verdier 1967, 1971, 1972; Hakomori & Tanaka 1992; Tanaka 1992; Ando 1993; Tanaka 1994a,b). Locusta migratoria is also widely distributed in China (Chen 1999), and numerous studies have documented its biology and ecology there (Ma 1958; Guo et al. 1991; Chen 1999; Stige et al. 2007). Despite much attention and effort to control the locust, frequent outbreaks continue to pose a serious problem to agriculture (e.g. Tanaka & Zhu 2005). Information about the physiological adaptations of this species to local climates is still fragmental, although recent studies on embryonic diapause and cold-hardiness have produced some important results (Li et al. 1998, 2001; Jing & Kang 2003a, 2004; Wang & Kang 2003, 2005; Wang et al. 2006; Qi et al. 2007). The present study was designed to compare embryonic diapause, reproductive performance and cold-hardiness among L. migratoria populations from different regions, covering the whole latitudinal range of this species in China. For Japanese populations of this locust (Tanaka 1994), embryonic diapause is determined not only by maternal influence but also by environmental conditions, particularly temperature, during embryonic development. Therefore, to understand the ecological significance of diapause, it is important to observe the response of diapause at different temperatures.

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Figure 1 Map showing the collection sites for Locusta migratoria in China.

Table 1 Collection sites in China Collection site

Latitude, longitude

Jiminay, Xinjiang Huludao, Liaoning Dagang, Tianjin Jining, Shandong Fengqiu, Henan Baisha, Hainan

47.4°N, 85.8°E 40.8°N, 120.0°E 38.8°N, 117.4°E 35.3°N, 116.6°E 35.0°N, 114.0°E 19.2°N, 109.4°E

MATERIALS AND METHODS Insects Locusts were collected in Xinjiang Uyghur Autonomous Region, Liaoning, Shandong, Tianjin, Henan and Hainan, as shown in Figure 1 and Table 1. Adults and nymphs collected were reared at 30°C and under conditions of 12 h light : 12 h dark. Eggs were laid into moist sand held in 340 mL plastic cups. To avoid the possible influence of maternal conditions such as crowding and temperature, approximately 100 individuals obtained from each local strain were reared in a wood-framed cage (42 cm ¥ 22 cm ¥ 42 cm) under crowded conditions as above, and their progeny were used for experiments. Eggs of some populations entered diapause. These eggs were kept at 20°C for 2 months and then chilled at 10°C for 2 months before being returned to 30°C for hatching according to the method of Tanaka (1992). Most experiments were conducted with the third generations, but some were conducted with the fourth generations. The minimum, mean and maximum temperatures for each locality are given in Figure 2.

Entomological Science (2008) 11, 327–339 © 2008 The Entomological Society of Japan

Locusta in China

Effects of maternal photoperiod

Figure 2 (A) Minimum, (B) mean and (C) maximum temperatures at collection sites. Data are based on means calculated from data collected by the China Meteorological Administration from 1961 to 1990. The name of each locality is abbreviated using the first three letters (see Fig. 1).


Approximately 100 newly hatched nymphs derived from each strain were kept in a cage at 30°C at each of three different photoperiods: 12 h light : 12 h dark (12 h photoperiod), 14 h light : 10 h dark (14 h photoperiod) and 16 h light : 8 h dark (16 h photoperiod). Upon ecdysis to the second nymphal stadium, four groups of ten males and ten females taken from each cage were reared as described above. Approximately a week after adult emergence, maximum head width was measured by using digital calipers (Mitsutoyo Co., Tokyo, Japan). Because locusts were reared in groups, it was not possible to determine the pre-ovipositional period for each individual. Thus, the period between the time of adult emergence of the first female individual and that of first oviposition was determined for each cage, and this period was regarded as the pre-ovipositional period in this study, in accordance with the methods of Tanaka (1994a) and Li et al. (1998). After mating began, a plastic cup (diameter, 10 cm; height, 5 cm) filled with moist sand was placed in each cage. Egg pods deposited in the sand were collected every day and incubated at 30, 25 or 20°C. Two days after deposition, egg pods were washed with tap water and individually kept in small plastic cups (bottom diameter, 3 cm; height, 4 cm) with moist sand. The maximum width of egg pods was measured for some egg pods for each strain within 2 days of deposition. All egg pods were then returned to the same temperature and observed for hatching. Eggs hatching within 16, 25 and 60 days of incubation at 30, 25 and 20°C, respectively, were regarded as non-diapause individuals (Tanaka 1992, 1994). Those that remained unhatched were usually chilled at 10°C for 2–3 months to terminate diapause and then incubated at 30°C for hatching (Tanaka 1992). In this case, the individuals that hatched after chilling were regarded as having emerged from diapause eggs (Tanaka 1992). The number of dead eggs was also recorded for each egg pod. In one strain, no egg pods were obtained during the longer photoperiods. A similar phenomenon has been observed for certain strains of this locust when reared under crowded conditions with a long photoperiod (Tanaka et al. 1993; Tanaka 1994a; Hasegawa & Tanaka 1996). Thus, adults of this strain were reared individually at each photoperiod both as nymphs and adults, except for a short period for mating. In the present analysis, only egg pods with a >50% survival rate were used. The incidence of diapause was subjected to arcsine trans-

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formation before being analyzed by anova or Fisher’s protected least significant difference (PSLD) test.

Effects of changes in photoperiod on embryonic diapause Locusts reared under different photoperiods as described above were exposed to either an increase or a decrease in photoperiod at adult emergence, and their eggs were incubated at either 30 or 25°C to determine the incidence of embryonic diapause as described above. The incidences of diapause were thus compared with those obtained under constant photoperiods. Locusts from Liaoning were used for this experiment.

Comparison of diapause intensity among strains Locusts of four strains, from Xinjiang, Liaoning, Henan and Hainan, were reared in groups of approximately 100 individuals at 30°C and a 12 h photoperiod, and their eggs were incubated at 20°C for 45, 60 and 75 days before being transferred to a 25°C temperature regime. Some nymphs hatched before the shift in temperature. Those eggs that hatched within 60 days of incubation at 20°C were regarded as non-diapause eggs, whereas those that hatched later than this time were regarded as diapause eggs. In those eggs transferred from 20 to 25°C on day 45 after deposition, a bimodal pattern was observed in hatching frequency. The group that hatched earlier were regarded as non-diapause eggs, and those that hatched later were regarded as diapause eggs. The details of these groups are provided in the text.

Cold hardiness of eggs To assess the cold-hardiness of eggs, eggs obtained from the above-mentioned four strains under the same conditions as described above were exposed to -10 or -20°C for 0, 5, 10 or 20 days and their survival rates were compared by determining the numbers of eggs that gave rise to hatchlings at 30°C. In this experiment, newly deposited egg pods were first incubated at 25°C for 16–18 days to allow them to reach the diapause stage (Tanaka 1992). Preliminary observations using ten eggs from each strain confirmed that embryonic diapause occurs at the end of anatrepsis in Chinese strains (S. Tanaka & D.-H. Zhu, unpublished data). Five to ten egg pods from each strain were then broken, and the eggs were separated, mixed, and dried on tissue paper for several minutes at room temperature. Groups of 30 randomly chosen eggs were kept in 1.5 mL airtight tubes

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and acclimated at 5°C for 5 days and then at 0°C for another 5 days in incubators before being exposed to temperatures of -10 ⫾ 0.5°C or -20 ⫾ 0.5°C in a lowtemperature tank (Eyela NCB-1200; Tokyo Rika Co., Tokyo, Japan), in which the sealed tubes containing eggs were immersed in 70% alcohol. The temperature inside the tank was monitored every hour using a thermo recorder (RT-12; Espec Co., Tokyo, Japan). Our preliminary observations indicated that all eggs from Xinjiang, Liaoning and Henan entered diapause at 20°C, whereas only some eggs from Hainan did so (S. Tanaka & D.-H. Zhu, unpublished data). Therefore, after exposure to subzero temperatures, eggs of the former strains were chilled at 10°C for 2 months before being incubated at 30°C for hatching. Those from Hainan were directly incubated at 30°C for hatching after exposure to subzero temperatures.

RESULTS Pre-ovipositional period Adults started ovipositing beginning 2 weeks after the last ecdysis. Figure 3 shows the mean pre-ovipositional periods from the day of female adult emergence to the day of first oviposition per cage for four strains. In the Xinjiang strain, females laid eggs only at a 12 h photoperiod during the 75-day observation period. Adults at the longer photoperiods died without laying any eggs. In the other strains, adults started laying eggs relatively rapidly, as also observed in the Xinjiang strain at a 12 h photoperiod. When all data were analyzed by anova (excluding the results for the Xinjiang strain at the long photoperiods), no significant differences were found in mean pre-ovipositional period either among populations or among photoperiods (P > 0.05).

Incidence of embryonic diapause Egg pods produced by females of different strains were incubated at either 25 or 30°C to determine the incidence of embryonic diapause. Because none of the Xinjiang strain females reared at a 14 or 16 h photoperiod produced eggs, nymphs of this strain were reared individually and kept under isolated conditions during the adult stage, except for a few days for mating. Eggs produced by these adults were used to assess embryonic diapause. Different strains of locusts displayed different patterns of response to photoperiod and temperature. The inci-


Locusta in China

Figure 4 Effects of parental photoperiod on induction of embryonic diapause in different strains of Locusta migratoria when eggs were incubated at either 25 or 30°C. (A) Xinjiang (47.4°N); (B) Liaoning (40.8°N); (C) Tianjin (38.8°N); (D) Shandong (35.3°N); (E) Henan (35.0°N); (F) Hainan (19.2°N). Different letters in each panel indicate significant differences between different photoperiods (Fisher’s protected least significant difference test after anova; P < 0.05). Vertical bars indicate either +SD or -SD. n = 7–20 pods. Figure 3 Pre-ovipositional periods at 30°C with different photoperiods in different strains of Locusta migratoria. (A) Xinjiang (47.4°N); (B) Liaoning (40.8°N); (C) Henan (35.0°N); (F) Hainan (19.2°N). In the Xinjiang strain, oviposition did not begin for photoperiods of 14 and 16 h within 2 months of adult emergence (arrows). Vertical bars indicate either +SD or -SD. No significant differences were found between different photoperiods for any strains except for the Xinjiang strain (anova; P > 0.05). n = 4 for each.

dence of diapause was not influenced either by photoperiod or temperature in the Xinjiang and Hainan strains, which were derived from the northernmost (100% incidence) and southernmost (no incidence) populations sampled, respectively (Fig. 4A,F). The strains derived from intermediate latitudes showed similar patterns of response to photoperiod and temperature; that is, the incidence of diapause was higher at a shorter parental photoperiod and at a lower egg temperature (Fig. 4B–D).


Some eggs from the strains from Liaoning, Henan and Hainan were incubated at 20°C, and the incidence of diapause was determined. Virtually all eggs entered diapause at this temperature in the Liaoning and Henan strains (Fig. 5). The eggs hatched at 30°C after being chilled at 10°C for 2 months. On the other hand, many eggs from Hainan hatched within 60 days after deposition. However, approximately two-thirds of the eggs obtained at a 12 h photoperiod entered diapause and remained unhatched until they were transferred to 30°C at day 60 without chilling. Preliminary observations suggested that chilling was not required to terminate diapause in this strain (S. Tanaka & D.-H. Zhu, unpublished data). The incidence of diapause was significantly lower at a 14 or 16 h photoperiod than at a 12 h photoperiod (Fisher’s PLSD test after anova; P < 0.05 each; Fig. 5).

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Figure 5 Effects of parental photoperiod on induction of embryonic diapause in different strains of Locusta migratoria when eggs were incubated at 20°C. Different letters in each panel indicate significant differences (Fisher’s protected least significant difference test; P < 0.05). Vertical bars indicate either +SD or -SD. n = 20–30.

Effect of a shift in photoperiod on diapause induction Nymphs from Liaoning were reared at a 12, 14 or 16 h photoperiod and transferred to a different photoperiod at adult emergence. Egg pods obtained were incubated at 25 or 30°C to determine the incidence of diapause per pod. Adults transferred from a 12 h photoperiod to either a 14 or 16 h photoperiod deposited only a few diapause eggs when incubated at 30°C (Fig. 6A). However, these incidences did not differ significantly from the values obtained for the constant 14 or 16 h photoperiod (P > 0.05). Adults exposed to a decreased photoperiod (from 16 h to 12 or 14 h) produced significantly greater proportions of diapause eggs than those kept at constant photoperiods (P < 0.05). At an incubation temperature of 25°C, however, a significant effect on the induction of diapause was obtained for adults exposed to an increase in photoperiod from 12 h to 14 h: they had a lower incidence of diapause eggs (70%) compared with those kept at either of the constant photoperiods (98–100%; Fig. 6B). An increase in photoperiod from 12 to 16 h was also associated with a low incidence of diapause eggs (48%), which was significantly lower than that for the 12 h photoperiod (100%; P < 0.05) or the constant 16 h photoperiod (63%; P < 0.05). A shift from a 16 h to a 12 or 14 h photoperiod produced a high incidence of diapause, which was similar to the value at a constant 12 or 14 h photoperiod (P > 0.05). These results indicate that locusts respond to a shift in photoperiod by modifying the incidence of diapause in their progeny, but the effects were manifested only at a particular temperature.

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Figure 6 Effects of changes in photoperiod on induction of diapause in the Liaoning strain (40.8°N) of Locusta migratoria when eggs were incubated at (A) 30°C or (B) 25°C. Different letters in each panel indicate significant differences between photoperiods (Fisher’s protected least significant difference test; P < 0.05). Vertical bars indicate either +SD or -SD. Mean n = 5–20.

Variation in diapause intensity The intensity of diapause was compared among four geographic strains by exposing eggs to a shift in temperature from 20 to 25°C at 45, 60 or 75 days after deposition. Here, the intensity of diapause was quantified as the proportion of diapause eggs that terminated diapause and hatched in response to an increase in incubation temperature. An example showing their responses is given for the Hainan strain in Figure 7, where the frequencies of hatched eggs are plotted against the time after deposition. Eggs transferred from 20 to 25°C at day 45 showed a bimodal pattern of hatching. The group that hatched first probably consisted of non-diapause eggs and some eggs with a weak diapause, whereas the late-hatching group comprised diapause eggs that terminated diapause in response to an increase in temperature (Fig. 7A). In the present study, the first hatchling group was regarded as nondiapause individuals, and the latter as diapause


Locusta in China

Figure 7 Patterns of egg hatching for the Hainan strain (19.2°N) of Locusta migratoria that were first incubated at 20°C and then transferred to 25°C on day (A) 45, (B) 60 or (C) 75. Arrows indicate the time of transfer. Closed horizontal bars indicate the range of hatching for non-diapause eggs, and open bars indicate the range for diapause eggs.

individuals. Eggs exposed to a shift in temperature at day 60 showed a tri-modal pattern (Fig. 7B). Here, eggs of the first group were apparently non-diapause, whereas those in the second-hatching group consisted of eggs with a weak diapause and had already started post-diapause embryogenesis before the shift to 25°C. In the latter, an increase in temperature only accelerated post-diapause morphogenesis, because it normally takes approximately 15 days for eggs at the diapause stage (late anatrepsis) to hatch at this temperature (S. Tanaka & D.-H. Zhu, unpublished data). The eggs in the last hatchling group were diapause eggs that terminated diapause in response to an increase in temperature and hatched simultaneously. The first group was regarded as non-diapause, and the last two groups as diapause. Eggs exposed to a shift in temperature at day 75 also exhibited a tri-modal hatching pattern (Fig. 7C), although the second hatching group was not as obvious as in the eggs treated at day 60. The distinction between non-diapause and diapause eggs was the same in the last two treatments.


Figure 8 (A) Incidence of diapause eggs for different strains of Locusta migratoria and (B) proportions of diapause eggs that hatched spontaneously or after a transfer from 20 to 25°C on day 45, 60 or 75. See Figure 7 for the ranges of hatching time for diapause and non-diapause eggs. Different letters in each panel indicate significant differences between the photoperiods (Fisher’s protected least significant difference test; P < 0.05). Vertical bars indicate either +SD or -SD. n = 152–238.

Figure 8A summarizes the incidences of diapause eggs among four strains that were first incubated at 20°C and then transferred to 25°C at day 45, 60 or 75 after deposition. Almost all eggs of the northern strains from Henan, Liaoning and Xinjiang entered diapause, whereas approximately two-thirds of the tropical strain from Hainan entered diapause (Fig. 8A), the results being comparable to those in Figure 3. Upon transfer to 25°C, almost all diapause eggs of this tropical strain hatched in a short time (Fig. 7B). The proportions of such eggs tended to decrease as the original latitude increased. In the three northern strains, more eggs terminated diapause when the time of transfer to 25°C was delayed. These results indicate that diapause is terminated more easily in eggs from southern strains than in those from northern strains.

Variation in cold-hardiness Eggs in which the embryo was at the diapause stage were exposed to subzero temperatures. Significant differences were observed in mean egg hatching (survival) among the four strains studied after exposure to either -10 or -20°C temperatures (Fig. 9). Egg survival was

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Figure 9 Effects of exposing Locusta migratoria eggs to (A) -10°C or (B) -20°C for various lengths of time. Eggs were incubated at 25°C for 16–18 days to reach the end of anatrepsis and then acclimated at 5°C and then 0°C for 5 days each before being exposed to a subzero temperature. After subzero exposure, the eggs were then transferred to 30°C for hatching either directly or after being chilled at 10°C for 2 months to terminate diapause. Asterisks indicate a significant difference among the four strains at P < 0.05 as determined using anova). Vertical bars indicate +SD. n = 7–10.

generally lower when the eggs were exposed to the lower temperature and when the exposure time was longer. In the Xinjiang strain, however, egg survival was relatively high, even after exposure to -20°C for 20 days (Fig. 9B). For the Hainan strain, on the other hand, eggs were highly susceptible to subzero temperatures, and most eggs did not survive even a 5-day exposure to -10°C (Fig. 9A).

Variation in adult morphology and progeny characteristics To examine how adult body size varied with latitude, head widths were measured. The patterns of latitudinal variation in head width with different photoperiods were similar, and thus only the results for locusts reared at the 12 h photoperiod are given in Figure 10A. Head widths were largest in the Xinjiang strain for both sexes. The mean values decreased as the latitude decreased to 41°N (Liaoning), but increased again as the latitude decreased further. In both sexes, mean head width was significantly smaller at 41°N than at 35°N (Fisher’s PLSD test; P < 0.05 for both). As the latitude further decreased to 19°N (Hainan), head widths decreased significantly (P < 0.05). These results show that there is no simple relationship between latitude and adult body size. Egg pod sizes were compared among strains by measuring the maximum widths of egg pods and the number of eggs per pod. A significant positive correlation was found between egg pod width and original latitude (y = 0.020 x + 6.369 where y is egg pod width

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(mm) and x the latitude (°N); R = 0.351; d.f. = 1, 229; F = 32.26; P < 0.0001; Fig. 10B). The number of eggs per pod varied among different strains. The mean number of eggs per pod ranged from 50.6 (n = 68) in the Xinjiang strain to 31.7 (n = 52) in the Hainan strain, the values being comparable to those (29–62) reported by Chen (1999). There was a weak positive correlation between the number of eggs per pod and the latitude (y = 0.456 x + 27.645 where y is the number of eggs per pod and x the latitude (°N); R = 0.226; d.f. = 1, 456; F = 24.46; P < 0.001; Fig. 10C). Both mean egg pod width and egg number correlated positively with mean head width of female adults (R = 0.833; n = 6; P < 0.05 for egg pod width; R = 0.830; n = 6; P < 0.05 for egg number). A statistically significant correlation was detected between hatchling weight and latitude, but the regression line (y = 0.072x + 12.224 where y is hatchling weight (mg) and x the latitude (°N); R = 0.325; n = 291; P < 0.001) was situated below most of the data points for the Xinjiang strain (not shown). The correlation coefficient increased to 0.510 (n = 224; P < 0.0001) when the data for the Hainan strain were not included (y = 0.249x + 5.076; Fig. 10D).

DISCUSSION Overall patterns The latitudinal adaptation of L. migratoria has been studied in Europe (Verdier 1967, 1971, 1972) and Japan (Hakomori & Tanaka 1992; Tanaka 1994; Tanaka


Locusta in China

Figure 10 Comparisons of (A) adult head widths, (B) egg pod widths, (C) egg number per pod, and (D) hatchling weights for different strains of Locusta migratoria. Adults and eggs were obtained at a 12 h photoperiod and 30°C. Different letters in each panel indicate significant differences between photoperiods (Fisher’s protected least significant difference test; P < 0.05). In part A, vertical bars indicate SD, and statistical comparisons for males and females was performed separately. Mean n = 26, 39, 76 and 26 in A, B, C and D, respectively. For explanation of the regression lines, see text.

1994a,b). In these two geographic regions, this locust shows similar patterns of variation in life cycle and developmental traits, including maternal control of embryonic diapause over the latitudinal range of its distribution. That is, the northern populations have a univoltine life cycle with an embryonic diapause that occurs in every generation. Diapause in these populations is determined genetically and induced without being influenced by seasonal changes in day length or temperature. The life cycles of southern populations are multivoltine, and there is little or no tendency to enter diapause in the egg stage. Populations inhabiting intermediate latitudes have a bivoltine life cycle, with their embryonic diapause controlled by photoperiod and temperature. The present study demonstrated that the way in which embryonic diapause is controlled in the Chinese populations of this locust along the latitudinal gradient is similar to what has been shown for the European and Japanese populations, except that in China diapause potential is retained even in the tropical population.

Diapause and life cycles Much information about the life cycle in different geographic regions is available for Chinese populations (Guo et al. 1991; Chen 1999). Cool-temperate populations from high latitudes have a univoltine life cycle, whereas temperate populations at latitudes ranging from 28°N to 39°N produce two generations a year


and tropical populations four generations. The results of the present study are consistent with this pattern of life cycle variation. The cool-temperate population (47.4°N) of Xinjiang underwent embryonic diapause that was induced under any photoperiodic conditions. The diapause was so intense that it persisted even at a high incubation temperature. This result, together with information from local entomologists, suggests that this locust is univoltine in this region. Li et al. (1998) reported that populations from Hami, Xinjiang, (43.0°N) and Beidagang, Tianjin (38.9°N), are bivoltine and show a long-day type of response in the control of embryonic diapause. In the present study, diapause was induced in all or most eggs when their parental generations experienced a short photoperiod in populations from Liaoning (40.8°N), Tianjin (38.8°N), Shandong (35.3°N) and Henan (35°N). Some eggs failed to enter diapause when their parents were exposed to a long photoperiod. The incidence of diapause eggs was further reduced when the eggs were incubated at a high temperature. These results confirm that the induction of diapause depends on the original latitude (or genetic composition), the parental photoperiod, and the incubation temperature of the egg in L. migratoria (Tanaka 1994). In temperate populations, eggs produced by the first (summer) generation would hatch without diapause and emerge as adults in the autumn as a second generation. However, some eggs produced by the first generation may enter diapause

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without producing a second generation if they are deposited late in the season and encounter relatively cool conditions. The proportion of such univoltine individuals may depend on the local temperature to which the eggs are exposed, and it is also likely to be influenced by annual fluctuations in temperature. The present results suggest that locusts may respond to seasonal changes in day length by modifying the incidence of diapause in their progeny. This seems particularly likely in the northern part of the temperate region, where the day length decreases rapidly in the summer (e.g. Liaoning). Therefore, it may be important to consider seasonal changes in day length as well as seasonal and annual changes in temperature when analyzing the life cycle of this locust. There is a difference between China and Japan in the latitudinal range of bivoltine life cycles in L. migratoria. Bivoltine life cycles with a facultative embryonic diapause occur at 43°N (Li et al. 1998) or 40.8°N (the present study) in China, whereas a univoltine life cycle with an obligatory embryonic diapause is predominant at 43.1°N (Sapporo) or at 38.8°N (Amarume) in Japan (Tanaka 1994a). Bivoltine life cycles become common in areas south of 37°N (Nasu) in Japan (Tanaka 1994). In the Nasu population (37°N), only half of all eggs can develop without diapause when their parents experience long days and the eggs are kept at a high temperature (Tanaka 1994), suggesting that the life cycle is mainly univoltine but partially bivoltine. The latitudinal differences in the life cycles between the two countries are likely to be related to differences in temperature, but further analysis considering longitudinal variations should be carried out.

Diapause status in winter It is often mentioned that Chinese populations of L. migratoria do not enter embryonic diapause for overwintering because different embryonic stages are sometimes found during the winter (Chin et al. 1954; Chen 1999). In the present study, diapause was found in all populations examined, and diapause occurs at the end of anatrepsis, which is reached approximately 7, 16 and 24 days after deposition at 30, 25 and 20°C, respectively (Tanaka 1992). In central Japan, locusts start laying diapause eggs in August and continue to do so until early December. However, the diapause stage is achieved by only those eggs that are deposited by mid-October (Tanaka 1992, 1994). Those deposited after midOctober encounter winter at various pre-diapause embryonic stages, yet they are apparently cold-hardy even during the early embryonic stage, and some over-

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winter successfully (Tanaka 1992). It is likely that the long ovipositing period of this locust is responsible for the occurrence of eggs overwintering at different embryonic stages in China. Tropical populations of L. migratoria may produce four generations per year (Chen 1999). Little information is available about the winter biology of the tropical populations except that they overwinter as eggs (Chen 1999). In the present study, no attempt was made to investigate how many generations were produced each year in tropical populations. However, we conducted a preliminary survey on 1 November in 2005 to study the overwintering biology of this locust in sugarcane fields on Hainan Island (S. Tanaka & D.-H. Zhu, unpublished data). Most individuals collected were adults (60 females, 116 males), but a few late-stadium nymphs were also collected (three last-stadium nymphs, and one penultimate nymph). When these adults were kept at 30°C with a 12 h photoperiod in the laboratory, some started laying eggs within a week, whereas most took much longer to do so. Therefore, at least some of them were reproductively active in November, suggesting that eggs are laid in the autumn and overwinter on Hainan, although many adults also overwinter there (Z.-Q. Peng, personal communication). Whether tropical populations enter embryonic diapause has not been investigated. Jing and Kang (2003a) used tropical strains to study the cold-hardiness of eggs, but diapause was not reported, probably because they incubated the eggs at a high temperature (28°C). The results of the present study confirmed the absence of diapause at high incubation temperatures such as 30 or 25°C, but also demonstrated the presence of diapause in some eggs by incubating eggs at a low temperature (20°C). The incidence of diapause eggs was also influenced by the parental photoperiod. In a separate study, we confirmed the presence of embryonic diapause in two strains from Hainan Island when the parents were reared at a 12 h photoperiod and 30°C and their eggs were incubated at 20°C (Zhu et al., unpublished data). This is the first report of the presence of embryonic diapause in tropical populations of L. migratoria.

Variation in diapause intensity and sexual maturation Diapause intensity varies with latitude in some insects (for reviews, see Masaki 1978, 2002). For example, Masaki (1965) demonstrated that the eggs of the Emma field cricket, Teleogryllus emma, undergo a more intense diapause in the south than in the north of Japan. He ascribed this cline to the latitudinal variation in the risk


Locusta in China

of untimely hatching before winter. Interestingly, an opposite cline was found for L. migratoria. This difference in diapause intensity may be related to the life cycle differences between the two species. Teleogryllus emma has a univoltine life cycle, and adults appear in late summer throughout the distribution range, whereas L. migratoria has variable life cycles, as mentioned above. For T. emma, eggs laid in the autumn are likely to be exposed to higher temperatures in the south than in the north in Japan. Therefore, to avoid untimely hatching before winter, eggs with a more intense diapause would be selectively favored in the southern populations compared with the northern populations. In L. migratoria, adults start laying diapause eggs early in the summer in the northern region (Chen 1999). On the other hand, diapause eggs are laid only by autumn generations in the southern areas, where two or more generations are produced each year. In those areas, diapause eggs are laid shortly before winter (Chen 1999). Therefore, the variation in diapause intensity may be related to the time of onset of diapause, as suggested by Masaki (1978) for the beach ground cricket, Pteronemobius csikii. Another factor contributing to the prevention of untimely hatching before winter is known for a Japanese population of L. migratoria. That is, adult locusts control the timing of sexual maturation in response to photoperiod and temperature (Tanaka et al. 1993; Hasegawa & Tanaka 1996). In this case, the photoperiodic effects on the rate of sexual maturation depend on the population density: oviposition is delayed most at a long photoperiod under gregarious (crowded) conditions, whereas it starts relatively rapidly either at a short or long photoperiod, but tends to be delayed at an intermediate photoperiod under solitarious (isolated) conditions. Strong suppression of sexual maturation by a long photoperiod was observed in the Xinjiang population when the locusts were reared in a group, but no data are available about their response under isolated conditions. Li et al. (1998) detected a delay of sexual maturation under long-day conditions in group-reared locusts from Xinjiang (43.0°N) and Tianjin (38.8°N). Whether adults of these populations use photoperiod to control the timing of sexual maturation under outdoor solitarious conditions is yet to be investigated. In the other populations tested in the present study, there was no significant delay in sexual maturation at any particular photoperiod.

18 to 41°N) in eastern China and found that the northern population was more cold-hardy than the southern population. However, the eggs in their study were obtained with a long photoperiod (14 h) and incubated at a high temperature of 25 or 28°C, and eggs appeared to have hatched without diapause even in the controls that had not been exposed to subzero temperatures. In another study, Jing and Kang (2003b) compared the cold-hardiness of eggs produced by locusts reared at a long (14 h) and a short (10 h) photoperiod and found that long-day eggs were significantly less cold-hardy than short-day eggs. This fact may suggest that comparisons of cold-hardiness using non-diapause eggs may be of limited value in terms of ecological significance. In the present study, eggs were obtained at a short photoperiod (12 h). Before being exposed to subzero temperatures, they were allowed to develop at 25°C for 16–18 days, during which the embryo reached stage XIV (Shulov & Pener 1959), that is, the end of anatrepsis, at which diapause occurs in this locust (Umeya 1946). These results confirmed that northern strain eggs are more cold-hardy than southern strain eggs. However, significant numbers of eggs derived from Liaoning (41°N) and Henan (35°N) populations survived at -10°C for 10 days in the present study, whereas most eggs derived from similar localities died after a 10-day exposure to -10°C in a previous study (Jing & Kang 2003a). Furthermore, the results of the present study demonstrated extraordinary tolerance to subzero temperatures in a strain from Xinjiang: approximately half of the eggs exposed to -20°C survived for 20 days. Because cold-hardiness is influenced by various factors, as mentioned above, it is possible to argue that the present results with eggs tested under dry conditions alone may not be sufficient to infer how cold-hardy eggs would be in the cool-temperate region. However, winter air temperatures in Jiminay, Xinjiang, go down to below -20°C (Fig. 2A). Furthermore, preliminary measurements of soil temperature at a depth of 3 cm at an exposed site in this locality indicated that eggs deposited into the soil would experience temperatures below -20°C for as long as 2 weeks during the winter (S. Tanaka & D.-H. Zhu, unpublished data). The eggs of this locust appear to require a considerable degree of cold-hardiness to survive the winter in cool-temperate regions where the winter is dry and eggs are not protected from extreme temperatures by thick snow cover.

Cold-hardiness

Body-size variation

Jing and Kang (2003a) studied the cold-hardiness of L. migratoria eggs originating from various regions (from

Adult head width showed a rather complicated pattern of variation over the latitudinal gradient. Adult head


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width was largest in the northern population and smallest in the southern population. However, in the range from 35°N to 41°N, mean head width was significantly smaller in the northern strain than in the southern strain (Fig. 10A). A similar pattern appears for solitarious individuals when the head widths reported by Kang et al. (1989) are plotted against the original latitude (data not shown). It is possible that the variation in adult body size is related to life cycles or voltinism, as observed in crickets (Masaki 1978), but the small sample size of the present study did not allow this possibility to be examined in detail. Both egg pod width and egg number per pod exhibited a simple tendency over the latitudinal range: both values decreased with a decrease in latitude. Because the locusts were reared under the same environmental conditions, the differences observed are likely to have a genetic origin. Such a geographic tendency apparently suggests that these traits have been exposed to selection by the climatic gradient. One candidate might be the severity of winter as inferred from the monthly fluctuations in temperature (Fig. 2). However, we cannot rule out the possibility that the two traits are products of different selection pressures. It is possible that the larger egg pod size is simply associated with the larger adult body size of the female parent. In fact, there is a positive correlation between the head widths of female adults and these traits, as described above. However, because the variation in head width did not show a simple relationship with latitude, adult body size alone does not fully account for the variation in egg pod size in this locust. Another trait displaying a tendency to decrease with latitude is hatchling weight (Fig. 10D). In this case, however, a minimum weight appears to be reached at approximately 35°N, and no further reduction was observed in a more southern strain (19.2°N). At present, it is difficult to determine the evolutionary factor(s) responsible for the development of these variations in this locust. To address this issue, a larger study must be undertaken that includes locusts from more diverse geographic locations, including various latitudes, longitudes and elevations, in China.

ACKNOWLEDGMENTS We thank N. Kemmochi, S. Ogawa, H. Ikeda, C. Ito and K. Maeno (National Institute of Agrobiological Sciences at Ohwashi) for assistance with rearing insects. We are also grateful to many friends and scientists, including Xiao-Gang Wu, Jing-Fei Yang, Feng-Sen Liu, Xi-Jing Hou, Zheng-Qiang Peng, Jia-Jun Zhu, Lian-Sheng Zhao

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and Shi-Da Liu, who helped us with locust collection in China. We are also indebted to the Yokohama Plant Protection Station for permission (no. 8-1115) to import live locusts from China. The present study was partly supported by Kakenhi funds (Japan) to S.T. and by a PhD Program Grant (China; no. 20050538003) to D.H.Z. The grass used was raised by the Field Management Section of the National Institute of Agrobiological Sciences at Ohwashi.

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