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Effects of egg temperature and moisture on phase characteristics of the hatchlings in Locusta migratoria
Journal of Arid Environments 134 (2016) 56e61
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Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv
Effects of egg temperature and moisture on phase characteristics of the hatchlings in Locusta migratoria Amel Ben Hamouda*, Seiji Tanaka Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi (NIASO), Tsukuba, Ibaraki 305-8634, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2015 Received in revised form 24 November 2015 Accepted 1 July 2016 Available online 9 July 2016
The effects of temperature and moisture during the egg stage on hatchling phase characteristics of Locusta migratoria were investigated. The incubation temperature of eggs laid by crowd-reared females affected embryonic development and some phase characteristics of the hatchlings. Incubation at 20 C caused eggs to develop more slowly and to produce more black hatchlings than those incubated at 30 C. A correlation was found between body size and cuticular melanism of hatchlings at these temperatures; however, the correlation was lower at the lower temperature. When large eggs expected to produce large and black hatchlings were exposed to dry conditions, they produced small, whitish hatchlings, typical of the solitary phase. However, unlike typical solitary individuals, these nymphs did not show extra moulting and had only 5 stadia before reaching the adult stage. These results suggested that deprivation of water from eggs may produce progeny looking like solitarious forms, but developmental traits such as numbers of nymphal stadia were unaffected. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Locusts Phase polyphenism Body size Body color Offspring Abiotic factors
1. Introduction Studies on many organisms have demonstrated significant influences of the hatchling’s phenotype and its plasticity, on organism fitness (Newman, 1994; Via et al., 1995). The most profound cases of phenotypic plasticity in color patterns are those associated with polyphenism. Polyphenism consists of discrete or continuous phenotypes that develop in response to specific signals from the environment. Among the best known of these is phasepolyphenism of migratory locusts, Locusta migratoria, which show continuous variation in various traits (Nijhout, 2010). During changes from solitary to gregarious phases, morphological, physiological, biochemical and behavioral traits change continuously in response to the population density (Uvarov, 1977; Ferenz, 1990; Loher, 1990; Byers, 1991; Hassanali et al., 2005; Simpson et al., 2005; Song, 2005; Pener and Simpson, 2009; Verlinden et al., 2009). It is well known that solitary migratory locust hatchlings maintained under isolated conditions are typically whitish and small, while gregarious hatchlings maintained at crowded conditions are typically black and large (Pener, 1991; Tanaka and Maeno, 2006, 2008; Ben Hamouda et al., 2009). This variation in body
* Corresponding author. E-mail address: [email protected] (A. Ben Hamouda). http://dx.doi.org/10.1016/j.jaridenv.2016.07.002 0140-1963/© 2016 Elsevier Ltd. All rights reserved.
coloration and size is caused by the different population densities to which adult female parents are exposed (Hunter-Jones, 1958; Albrecht et al., 1959a,b). In addition to the maternal role and adult density (Hunter-Jones, 1958; Uvarov, 1966; Injeyan and Tobe, 1981; Pener, 1991) in the control of hatchling body size and color, the environment can alter the coloration in a variety of ways. The most important environmental factors influencing embryonic development are temperature and moisture (Hamilton, 1950). They affect various vital phenomena at all levels of biological organization (Stillman and Somero, 2000). Temperature influences morphological characteristics of locusts. Hunter-Jones (1970) reported that desert locust, Schistocerca gregaria, hatchling’s body color depends on temperature. Bernays (1994) showed that hatchling’s body size increased at higher temperature in S. gregaria. Maeno and Tanaka (2009) also found that dehydration during embryogenesis in S. gregaria led to green and small hatchlings, instead of the expected black, large hatchlings. It has been reported the influence of additional environmental factors, particularly crowding, also influence body color (Pener, 1991; Pener and Simpson, 2009). We infer that body coloration is a very complex aspect of locust biology, influenced by genetic, endocrine and environmental factors. Temperature and moisture influences morphological characteristics of locusts. Hunter-Jones (1970) reported that desert locust, S. gregaria, hatchling’s body color depends on temperature. Bernays
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(1994) showed that hatchling’s body size increased at higher temperature in S. gregaria. Maeno and Tanaka (2009) also found that dehydration during embryogenesis in S. gregaria led to green and small hatchlings, instead of the expected black, large hatchlings. In locusts, gregarious nymphs have usually only five nymphal stadia, whereas extra molting is often observed in solitarious phase (Uvarov, 1966). Hatchling body size and rearing density are important factors which influence the number of molts (HunterJones, 1958; Maeno and Tanaka, 2008). Studies on the incubation environment for L. migratoria hatchlings are scarce with no information regarding the number of nymphal stadia. For this reason, the current study investigated the effect of thermal and hydric environment on phase characteristics of hatchlings of L. migratoria in relation with the number of nymphal stadia. We applied this study to answer two questions. Are body phase characteristics of hatchlings sensitive to the environment conditions of incubation in L. migratoria? Is the moisture and temperature at incubation period induced variation in the number of nymphal stadia? Investigating the effect of temperature and moisture in producing different qualities of hatchlings should help in understanding the ecological role of phase-related characteristics of hatchlings in L. migratoria. 2. Material and methods 2.1. Locusts Experiments were carried out with a gregarious colony of Locusta migratoria. This strain was maintained for more than 6 years at the High Institute of Agronomy of Chott-Mariem, Tunisia. The colony was brought to the National Institute of Agrobiological Sciences at Ohwashi by permission from the Yokohama Plant Protection Station. Preliminary analysis of mitochondria DNA suggests that this strain belongs to the southern clade of this species that includes strains from Africa, France, Australia, Timor, Southern China and Southern Islands of Japan, and is distinctly different from the northern clade that includes strains of most areas of Japan and China (Tokuda et al., 2010). The crowd-reared colony was mainC tained at 30 in large wood-framed cages (42 cm 22 cm 42 cm; 0.038 m3). Each cage was covered with nylon screen mesh except for the wood floor and the front sliding door, which was composed of a transparent acrylic plate. Locusts were fed cut grass inserted in water jars and wheat bran. Grass was changed every 1 or 2 days. Bromus grass was grown in crop fields by the Field Management Department of the National Institute of Agro-biological Sciences at Ohwashi. Locusts laid eggs in moist sand held in plastic cups (vol. 380 ml) which were incubated at 30 C.
57
chosen for experiments, because in the case of S. gregaria, large eggs are known to produce black hatchlings characteristic of gregarious forms (Tanaka and Maeno, 2008). The eggs were held on wet filter paper in Petri dishes (9 cm in diameter; 1.5 cm in height) and incubated until used. Healthy looking eggs were sampled every day from day 2 to day 10 after oviposition. Sixty eggs derived from the prepared groups of large eggs were placed on dry filter paper (9 cm in diameter) in a Petri dish. The eggs in Petri dishes were then held in an air-tight plastic container in which relative moisture was kept close to 100% by putting moist tissue paper at a corner of the container. Eggs of S. gregaria lose some water under such conditions, because they cannot absorb water from air and can imbibe water only as liquid and not as vapor (Shulov and Pener, 1963). Hatchlings obtained from dried and moistened eggs were transferred to a plastic bag in which they were kept for more than 6 h before being weighed and checked for body coloration. 2.4. Measurements of hatchling body coloration, size and head luminance To quantify the moisture effect on head luminance, locusts were photographed using a scanner (Epson GT-X770, Japan) connected to a computer using commercial software, Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA). Locusts were chilled on ice for 15 min and placed with one side down on the glass table of the scanner for photographing. The image type used was 48-bit color at a resolution of 1200 d.p.i. Using the histogram function, luminance of body color (head) was measured and the mean luminance was recorded for each individual. The body color of hatchlings was observed 6e12 h after hatching. Hatchling body color was divided into three hatchling color groups based on the method of Ben Hamouda et al. (2009): whitish (W), grey (G) and black (B). After body color was scored, hatchlings were weighed individually using an electronic balance (METTLER AT201, Japan). 2.5. Measurements of the number of nymphal stadia To know whether incubation of eggs under dry conditions induces the solitarization of hatchlings, treated and control (untreated) nymphs were reared in isolation and checked for ecdysis every day. 2.6. Statistical analysis
Forty new egg pods laid by gregarious crowd-reared females were incubated separately in a small plastic cup containing moist sand at two temperatures: twenty egg pods at 30 C and another twenty at 20 C.
Statistical analysis was performed using IBM SPSS 20 software. The normal distribution was performed using the Shapiro-WilkTest. For normally distributed data or after transformation if necessary, differences between groups were calculated using analysis of variance (ANOVA). The Student-Newman-Keuls posthoc multiple comparison test was used if ANOVA indicated a significant effect. Categorical data were compared by Chi-square test and correlations were determined by Pearson’s correlation coefficient. The null hypothesis was rejected at the 0.05 level. For nonnormally distributed data, the Kruskal-Wallis test was used to investigate differences between groups. Subsequently, the Mann Whitney test was applied to detect the pairwise differences. Asterisks indicate levels of significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).
2.3. Incubation of eggs under moist and dry conditions
3. Results
In this experiment, we adopted the method described for S. gregaria by Maeno and Tanaka (2009). Eggs deposited by gregarious females were incubated at 30 C. The day of oviposition was designated as day 0. On day 2, eggs longer than 6.0 mm were
3.1. Effect of different egg temperature on hatchling body coloration and size
2.2. Incubation of eggs at high and low temperatures
At a high temperature (30 ± 2 C), embryogenesis proceeded
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3.2. Effect of drying eggs on hatchling body coloration and size No hatchling was obtained from eggs that had been preincubated on wet paper up to 4 days and then transferred to dry paper. Hatching was observed among eggs that had been kept on wet paper for at least 5 days. 36.66%, 43.33% and 56.66% were the hatching rates of hatchlings produced from eggs transferred to dry paper on day 5, 6 and 7, respectively. On day 8, 9 and 10, 66.66% of hatchlings were obtained. The maximum of hatching rate (83.33%) was observed under continuous moist conditions (Fig. 4). Whitish hatchlings were predominant when the eggs were transferred from wet to dry conditions, whereas such individuals appeared only in a small proportion under continuous moist conditions (Fig. 5). These results indicated that a reduction of water reserve of egg by drying reduced the probability to develop black hatchlings (Fig. 6). The lowest (darkest) value of mean luminance was observed only when eggs were incubated under wet conditions (Fig. 7).
(n=171)
100
Percentage of hatchlings (%)
rapidly for the majority of egg pods except for the last three egg pods that probably contained infertile eggs. Hatching started 12 days after oviposition and was completed in 4 days. The highest proportion of hatched nymphs was observed at day 1 of the hatching period (Fig. 1). At 20 C, hatching started after 39 days of incubation. It required 12 days to complete (Fig. 1). The highest proportion of hatched nymphs was observed at day 3 of the hatching period. The eggs incubated at 30 C showed significantly higher survival rates than those incubated at 20 C. Indeed, the hatchability at 30 C was 1.27 times higher than that at 20 C. The proportions of black, grey and whitish hatchlings were significantly different at 30 C and 20 C (Fig. 2; X2 ¼ 36.96; df ¼ 2; P < 0.001). Gregarious eggs incubated at 30 C produced a mixture of whitish (28.07%), grey (24.56%) and black (47.37%) hatchlings. At a low temperature (20 C), most hatchlings exhibited dark body coloration. We noted the disappearance of whitish coloration at this low temperature. Fig. 3 shows that hatchlings derived from eggs that were incubated at 30 C were larger than those derived from eggs that were incubated at 20 C. The largest grey hatchlings derived from eggs incubated at 20 C were characterized by the same weight of whitish hatchlings from 30 C. Moreover, the cuticular melanism in hatchlings exhibits a correlation with body size at 30 C (r ¼ 0.600; n ¼ 171; P < 0.01) than at 20 C (r ¼ 0.345; n ¼ 181; P < 0.01).
90
(n=181) 19.89%; 36
28.07%; 48
80
W
70 60
24.56%; 42
G
50 40
B
80.11%; 145
30 47.37%; 81
20 10 0
30°C
20°C
Incubation temperatures
Fig. 2. The effect of egg temperature on hatchling body color. Numbers in parentheses (n) indicate the number of individuals. Percentage of black, grey and whitish hatchlings was compared by the chi-square test for the two temperatures of eggs incubation (X2 ¼ 36.96; df ¼ 2; P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
18
(a)
16
Hatchling body weight (mg)
58
(b) (c)
14
(d)
(d)
12 10 8 6 4 2 0 30°C B
20°C B
1
30°C G
20°C G
30°C W
Hatchling body color at different temperatures Fig. 3. Relationship between hatchling body weight and color at different temperatures of eggs incubation. Letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by StudenteNewmaneKeuls test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
90 80
Hatching rate (%)
70
Percentage of hatchlings (%)
100 90
551
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20°C
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30°C
60 50
60 50 40 30 20 10
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20 10 20
37
0
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40
41
42
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48
10
49
5
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5 6 7 8 9 Days of transfer to dry filter paper
10
Moist
Fig. 4. Relationship between the hatching rate and the incubation of eggs at dry and moist conditions.
50
Days after oviposition Fig. 1. The pattern of hatching at 20 C and 30 C. Numbers above each histogram indicate the number of individuals.
Drying caused the eggs to produce lighter hatchlings (KruskaleWallis test: X2 ¼ 108.82, df ¼ 6, P < 0.01). This effect was more pronounced if the transfer from wet to dry conditions took place between the 5th and 7th days. Deprivation of water from eggs had a
A. Ben Hamouda, S. Tanaka / Journal of Arid Environments 134 (2016) 56e61
(0)
(0)
(0)
(22)
(26)
(34)
(40)
(40)
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Percentage of hatchlings (%)
100 1 0,9 90 0,8 80 0,7 70
B
60 0,6
G
50 0,5 40 0,4
W
30 0,3 20 0,2 10 0,1 00
2
3
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5
6
7
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9
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Moist
Days of transfer to dry filter paper Fig. 5. Effect of drying of eggs on hatchling body color. B, G and W: Black, Grey and Whitish body color. Numbers in parentheses (n) indicate the number of individuals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
20
(a)
Hatchlings weight (mg)
18
***
16
14 12
***
(b)
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***
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8
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***
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10 8 6
4 2 0
0 Moist
Days of transfer to dry conditions Fig. 6. Photographs showing the effect of eggs water loss on hatchling body size and color. (A) Large, black hatchling, obtained from moist conditions and (B) small, whitish hatchling, obtained from dry conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Head luminance means
120
100
(50)
(22) *** (d)
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(26) (34) (40) (40) *** *** *** *** (c) (d) (b) (b)
(40) *** (b)
n
80
60
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20
0
0 Moist 1
2
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4
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Fig. 8. Effect of drying of eggs on hatchlings weight. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by nonparametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions.
11
Days of transfer to dry conditions Fig. 7. Mean luminance values for the head of hatchlings derived from moistened and dried eggs. Open circles indicate individual datum points and closed ones indicate means. Different letters in parentheses indicate a significant difference among head luminance at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions. Numbers in parentheses (n) indicate the number of individuals.
significant effect on body size of the hatchlings. Fig. 8 indicates a significant difference in weight between hatchlings from wet and dry conditions (KruskaleWallis test: X2 ¼ 71.27, df ¼ 6, P < 0.01). It
was considered that hatchling size was closely related to that of eggs, and the loss of some water from eggs due to desiccation resulted in the appearance of small hatchlings. At wet conditions, black (B) hatchlings had the highest mean body weight, followed by grey (G) and whitish (W) ones. At dry conditions, black (B) and grey (G) hatchlings had the highest mean body weight, whitish ones (W) the lowest (Fig. 9). Thus, when the weight increases, the body coloration becomes darker and vice versa. The correlation between cuticular melanism and body size of hatchlings was apparent for both grey and whitish hatchlings in wet conditions and for whitish hatchlings that experienced dry conditions. The duration of embryonic development increases with the early transfer to dry conditions (KruskaleWallis test: X2 ¼ 132.01, df ¼ 6, P < 0.01) (Fig. 10). However, no change in the duration was observed at the 9th (ManneWhitney U ¼ 940, P ¼ 0.611) and 10th days (ManneWhitney U ¼ 913.5, P ¼ 0.460) of dry conditions.
3.3. Effect of drying eggs on the number of molts in the hatched nymphs To determine if drying eggs influenced the number of molts in the hatched nymphs, nymphs obtained in the above experiment were reared in isolation and the number of nymphal molts was
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Hatchling body weight (mg)
30
*
25 20
*
**
**
(a)
(b)
(ab)
(b)
15
(ab)
(c)
10 5
0 B
G
W
B
G
W
Dry conditions
Moist conditions
Hatchling body color under moist and dry conditions Fig. 9. Relationship between body weight of hatchlings and their body color. B, G and W: Black, Grey and Whitish hatchlings. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Differences in the hatchlings body weight in each body color for moist and dry conditions were compared with the ManneWhitney U test: *, P < 0.05 and **, P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Development duration (day)
20
18 16
*** (a)
*** (b)
***
***
(c)
(d)
14
(e)
(e)
(e)
9
10
Moist
12 10
8 6 4 2
0 5
6
7
8
Days of transfer to dry filter paper Fig. 10. Effect of drying of eggs on embryonic development duration. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions.
determined. All hatchlings derived from the eggs that were exposed to dry conditions became adults after five nymphal stadia (n ¼ 193) as observed in control nymphs derived from eggs constantly kept under wet conditions (n ¼ 48). 4. Discussion and conclusions Progeny body size and color and head luminance in L. migratoria were significantly influenced by the temperature of eggs incubation. Hatchlings displayed a distinct response to different temperatures during the egg stage; the percentage of hatching was significantly higher at a high temperature (30 C) than at a low temperature (20 C). Moreover, the period of incubation was shorter if eggs were incubated at the higher temperature. These results suggest that temperature is an important factor affecting their embryonic development and can thus be an indicator of predicting the state of locust populations in subsequent generations. The concept that the body color of solitary L. migratoria hatchlings was whitish and that of gregarious ones was black has been known in some strains for a long time (Uvarov, 1966). This
difference in body coloration was correlated with the parental phase state and population density (Hunter-Jones, 1958; Albrecht et al., 1959a,b). Tanaka and Maeno (2008) have demonstrated that the hatchling body color in S. gregaria is closely related to egg size that is pre-determined in the ovaries of the female parent. Small eggs, typical of solitary phase, tend to give rise to green hatchlings and those of large eggs, typical of gregarious phase were black. In L. migratoria, we found the same assumption, namely that in addition to the contribution of some abiotic factors that can modify the maternal effect on progeny characteristics, the temperature during embryogenesis also has a pronounced effect on the body size and color of hatchlings. Uvarov (1966) and Hunter-Jones (1970) have shown that the body color is influenced by the temperature during the egg incubation. The offspring of crowd-reared gregarious females emerged with a predisposition toward gregarious or solitarious body coloration and size depending upon the incubation temperature (Fig. 2). The correlation between body weight and cuticular melanism persists also at a low temperature but it is less obvious. It seems that in L. migratoria, the incubation temperature has an important effect on the activity of certain factors responsible for the control of hatchling body color. Body size and color at hatching are closely correlated: hatchlings with darker body coloration were heavier. Recent studies with S. gregaria (Tanaka and Maeno, 2006, 2008) and L. migratoria (Ben Hamouda et al., 2009) have confirmed this relationship. The important role of abiotic factors on hatchlings phase characteristics has been shown in our experiments: the present study tested the hypothesis that eggs thought to produce black hatchlings give rise to whitish hatchlings if they are incubated at dry conditions. Shulov and Pener (1963) studied the effect of moisture on egg weight and hatching rate in S. gregaria but did not observe the body color and size of hatchlings. Hunter-Jones (1964, 1970) showed that the weight and body color of hatchlings were influenced by the soil moisture and the temperature of egg incubation. In the present study, eggs were transferred from wet to dry filter paper at various timings after oviposition and were kept at nearly saturated conditions. It was known that eggs can imbibe water only as liquid and not as vapor from the air in S. gregaria (Shulov and Pener, 1963). Likewise, L. migratoria eggs also lost weight probably on dry paper under nearly saturated conditions, indicating that the eggs cannot take up water from the air. The present results raise two important questions: first, how are hatchling phase characteristics affected so that the body size and color of the offspring vary according to the time of transfer to dry conditions? In a previous study by Maeno and Tanaka (2009) it has been demonstrated that S. gregaria eggs transferred from wet to dry filter paper at various timings after deposition produced black hatchlings but some of those transferred on day 4 or 5 produced green hatchlings. Our results and those of Maeno and Tanaka (2009) may indicate the importance of water reserve in the egg in the regulation of hatchlings body coloration in L. migratoria and S. gregaria. The first change observed following drying was a loss of egg weight, which causes hatchling weight and darkening to be reduced. It has been known that the weight difference between hatchlings of different body colors is largely attributable to higher water content of the heavier ones (Blackith, 1961; Albrecht, 1962). Maeno and Tanaka (2009) suggested in similar experiments with S. gregaria that green hatchlings retained a relatively large amount of egg yolk in the gut compared to black hatchlings, suggesting that the availability of yolk material for embryogenesis is modified by water loss. Albrecht et al. (1959a) showed that the quantitative reduction of egg yolk led to changes in hatchlings body size and color. The second question raised is whether deprivation of egg water will be a source of hatchling solitarization? It would also be
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interesting to determine if small, whitish hatchlings produced by egg drying show other solitarious characteristics in their development. The number of nymphal stadia might be involved: Extra molting is typically observed in the solitarious phase (Uvarov, 1966), although some exception has been reported recently for a laboratory strain of S. gregaria in which phase did not affect the number of nymphal instars (Lester et al., 2005). It has been suggested that body weight at hatching is critical for the determination of extra molting (Hunter-Jones, 1958) and hatchlings that have a heavy weight at hatching do not go through supplementary nymphal stadia (Albrecht et al., 1959a). Because solitarious locusts often exhibit extra molting, the effects of drying eggs on extra molting in nymphs were examined in the present study. The results indicated that all gregarious nymphs derived from wet conditions undergo only five nymphal stadia under isolated conditions. The same number of nymphal stadia was obtained for nymphs derived from dry conditions and reared in isolation. Nymphs used in our experiments came from eggs that were laid by crowd-reared gregarious females. Therefore, rearing these nymphs in isolation during the nymphal stage does not affect the number of nymphal stadia even when they are characterized by small size and light body color at hatching. The present study is consistent with the observations of Albrecht et al. (1959b) who found that the number of nymphal stadia was not influenced in small hatchlings obtained by ligating the egg with a thread. Acknowledgements We thank Ms. H. Ikeda, Ms. N. Totsuka, Ms. Y. Yokota and Ms. M. Higuchi for laboratory assistance. We are grateful to Dr. M. Koutaro and Dr. K. Harano for kind advice and encouragement at the Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi, Tsukuba. The grass used was raised by Field Management Section of NIASO. This study was supported by the “Japanese Association of University Woman” Research Fellowships. References Albrecht, F.O., 1962. Some physiological and ecological aspects of locust phases. Trans. Entomol. Soc. Lond. 114 (11), 335e375. termination de la fertilite par Albrecht, F.O., Verdier, M., Blackith, R.E., 1959a. De l’effet de groupe chez le criquet migrateur (Locusta migratoria migratorioides R. & F.). Bull. Biol. Fr. Belg. 92, 349e427. Albrecht, F.O., Verdier, M., Blackith, R.E., 1959b. Maternal control of ovariole number in the progeny of the migratory locust. Nature 184, 103e104. Ben Hamouda, A., Ammar, M., Ben Hamouda, M.H., Bouain, A., 2009. The role of egg pod foam and rearing conditions on the phase state of the Asian migratory locust Locusta migratoria migratoria (Orthoptera, Acrididae). J. Insect Physiol. 55, 617e623. BernaysE.A.,Some factors affecting size in first-instar larvae of Schistocerca gregaria (Forskal), Acrida 1, 1972, 189-195, in: Atkinson, D. (Eds.), Temperature and Organism Size-a Biological Law for Ectotherms? Begon, M. and Fitter A.H. Adv. Ecol. Res. 25, 1994, pp. 133. Blackith, R.E., 1961. The water reserves of hatchling locusts. Comp. Biochem. Physiol. 3, 99e107. Byers, J.A., 1991. Pheromones and chemical ecology of locusts. Biol. Rev. 66, 347e378.
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Ferenz, H.J., 1990. Locust pheromones - basic and applied aspects. Bol. San. Veg. Plagas (Fuera de serie) 20, 29e37. Hamilton, A.G., 1950. Further studies on the relation of humidity and temperature to the development of two species of African locusts, Locusta migratoria migratorioides (R. et F.) and Schistocerca gregaria (Forsk.). Trans. Entomol. Soc. Lond. 101, 1e58. Hassanali, A., Njagi, P.G.N., Bashir, M.O., 2005. Chemical ecology of locusts and related acridids. Annu. Rev. Entomol. 50, 223e245. Hunter-Jones, P., 1958. Laboratory studies on the inheritance of phase characters in locusts. Anti-Locust Bull. 29, 1e32. Hunter-Jones, P., 1964. Egg development in the desert locust (Schistocerca gregaria Forsk.) in relation to the availability of water. Proc. R. Entomol. Soc. Lond. (A) 39, 25e33. Hunter-Jones, F., 1970. The effect of constant temperature on egg development in the desert locust Schistocerca gregaria (Forsk.). Bull. Entomol. Res. 59, 707e718. Injeyan, H.S., Tobe, S.S., 1981. Phase polymorphism in Schistocerca gregaria: reproductive parameters. J. Insect Physiol. 27, 97e102. Lester, R.L., Grach, C., Pener, M.P., Simpson, S.J., 2005. Stimuli inducing gregarious colouration and behaviour in nymphs of Schistocerca gregaria. J. Insect Physiol. 51, 737e747. Loher, W., 1990. Pheromones and phase transformation in locusts. In: Chapman, R.F., Joern, A. (Eds.), Biology of Grasshoppers. Wiley, New York, pp. 337e355. Maeno, K., Tanaka, S., 2008. Maternal effects on progeny size, number and body color in the desert locust, Schistocerca gregaria: density- and reproductive cycledependent variation. J. Insect Physiol. 54, 1072e1080. Maeno, K., Tanaka, S., 2009. Artificial miniaturization causes eggs laid by crowdreared (gregarious) desert locust to produce green (solitarious) offspring in the desert locust, Schistocerca gregaria. J. Insect Physiol. 55, 849e854. Newman, R.A., 1994. Genetic variation for phenotypic plasticity in the larval life history of spadefoot toads (Scaphiophus couchii). Evolution 48 (6), 1773e1785. Nijhout, F.H., 2010. Molecular and physiological basis of colour pattern formation. Adv. Insect Physiol. 38, 219e265. Pener, M.P., 1991. Locust phase polymorphism and its endocrine relations. Adv. Insect Physiol. 23, 1e79. Pener, M.P., Simpson, S.J., 2009. Locust phase polyphenism: an update. Adv. Insect Physiol. 36, 1e272. Shulov, A., Pener, M.P., 1963. Studies on the development of eggs of the desert locust (Schistocerca gregaria Forskål) and its interruption under particular conditions of humidity. Anti-Locust Bull. 41, 1e59. Simpson, S.J., Sword, G.A., De Loof, A., 2005. Advances, controversies and consensus in locust phase polyphenism research. J. Orthoptera Res. 14, 213e222. Song, H., 2005. Phylogenetic perspectives on the evolution of locust phase polyphenism. J. Orthoptera Res. 14, 235e245. Stillman, J.H., Somero, G.N., 2000. A comparative analysis of the upper thermal tolerance limits of eastern Pacific porcelain crabs, Genus Petrolisthes: influences of latitude, vertical zonation, acclimation, and phylogeny. Physiol. Biochem. Zool. 73, 200e208. Tanaka, S., Maeno, K., 2006. Phase-related body-color polyphenism in hatchlings of the desert locust, Schistocerca gregaria: re-examination of the maternal and crowding effects. J. Insect Physiol. 52, 1054e1061. Tanaka, S., Maeno, K., 2008. Maternal effects on progeny body size and color in the desert locust, Schistocerca gregaria: examination of a current view. J. Insect Physiol. 54, 612e618. Tokuda, M., Tanaka, S., Zhu, D.H., 2010. Multiple origins of Locusta migratoria (Orthoptera: Acrididae) in the Japanese Archipelago and the presence of two major clades in the world: evidence from a molecular approach. Biol. J. Linn. Soc. 99, 570e581. Uvarov, B.P., 1966. Grasshoppers and Locusts-a Handbook of General Acridology, vol. 1. Cambridge University Press, Cambridge, pp. 332e386. Uvarov, B.P., 1977. Grasshoppers and Locusts, vol. 2. Centre for Overseas Pest Research, London. Verlinden, H., Badisco, L., Marchal, E., van Wielendaele, P., Vanden Broeck, J., 2009. Endocrinology of reproduction and phase transition in locusts. General Comp. Endocrinol. 162, 72e92. Via, S., Gomulkiewicz, R., De Jong, G., Scheiner, S.M., Schlichting, C.D., Van Tienderen, P.H., 1995. Adaptive phenotypic plasticity. Trends Ecol. Evol. 10 (5), 212e217.
Contents lists available at ScienceDirect
Journal of Arid Environments journal homepage: www.elsevier.com/locate/jaridenv
Effects of egg temperature and moisture on phase characteristics of the hatchlings in Locusta migratoria Amel Ben Hamouda*, Seiji Tanaka Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi (NIASO), Tsukuba, Ibaraki 305-8634, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2015 Received in revised form 24 November 2015 Accepted 1 July 2016 Available online 9 July 2016
The effects of temperature and moisture during the egg stage on hatchling phase characteristics of Locusta migratoria were investigated. The incubation temperature of eggs laid by crowd-reared females affected embryonic development and some phase characteristics of the hatchlings. Incubation at 20 C caused eggs to develop more slowly and to produce more black hatchlings than those incubated at 30 C. A correlation was found between body size and cuticular melanism of hatchlings at these temperatures; however, the correlation was lower at the lower temperature. When large eggs expected to produce large and black hatchlings were exposed to dry conditions, they produced small, whitish hatchlings, typical of the solitary phase. However, unlike typical solitary individuals, these nymphs did not show extra moulting and had only 5 stadia before reaching the adult stage. These results suggested that deprivation of water from eggs may produce progeny looking like solitarious forms, but developmental traits such as numbers of nymphal stadia were unaffected. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Locusts Phase polyphenism Body size Body color Offspring Abiotic factors
1. Introduction Studies on many organisms have demonstrated significant influences of the hatchling’s phenotype and its plasticity, on organism fitness (Newman, 1994; Via et al., 1995). The most profound cases of phenotypic plasticity in color patterns are those associated with polyphenism. Polyphenism consists of discrete or continuous phenotypes that develop in response to specific signals from the environment. Among the best known of these is phasepolyphenism of migratory locusts, Locusta migratoria, which show continuous variation in various traits (Nijhout, 2010). During changes from solitary to gregarious phases, morphological, physiological, biochemical and behavioral traits change continuously in response to the population density (Uvarov, 1977; Ferenz, 1990; Loher, 1990; Byers, 1991; Hassanali et al., 2005; Simpson et al., 2005; Song, 2005; Pener and Simpson, 2009; Verlinden et al., 2009). It is well known that solitary migratory locust hatchlings maintained under isolated conditions are typically whitish and small, while gregarious hatchlings maintained at crowded conditions are typically black and large (Pener, 1991; Tanaka and Maeno, 2006, 2008; Ben Hamouda et al., 2009). This variation in body
* Corresponding author. E-mail address: [email protected] (A. Ben Hamouda). http://dx.doi.org/10.1016/j.jaridenv.2016.07.002 0140-1963/© 2016 Elsevier Ltd. All rights reserved.
coloration and size is caused by the different population densities to which adult female parents are exposed (Hunter-Jones, 1958; Albrecht et al., 1959a,b). In addition to the maternal role and adult density (Hunter-Jones, 1958; Uvarov, 1966; Injeyan and Tobe, 1981; Pener, 1991) in the control of hatchling body size and color, the environment can alter the coloration in a variety of ways. The most important environmental factors influencing embryonic development are temperature and moisture (Hamilton, 1950). They affect various vital phenomena at all levels of biological organization (Stillman and Somero, 2000). Temperature influences morphological characteristics of locusts. Hunter-Jones (1970) reported that desert locust, Schistocerca gregaria, hatchling’s body color depends on temperature. Bernays (1994) showed that hatchling’s body size increased at higher temperature in S. gregaria. Maeno and Tanaka (2009) also found that dehydration during embryogenesis in S. gregaria led to green and small hatchlings, instead of the expected black, large hatchlings. It has been reported the influence of additional environmental factors, particularly crowding, also influence body color (Pener, 1991; Pener and Simpson, 2009). We infer that body coloration is a very complex aspect of locust biology, influenced by genetic, endocrine and environmental factors. Temperature and moisture influences morphological characteristics of locusts. Hunter-Jones (1970) reported that desert locust, S. gregaria, hatchling’s body color depends on temperature. Bernays
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(1994) showed that hatchling’s body size increased at higher temperature in S. gregaria. Maeno and Tanaka (2009) also found that dehydration during embryogenesis in S. gregaria led to green and small hatchlings, instead of the expected black, large hatchlings. In locusts, gregarious nymphs have usually only five nymphal stadia, whereas extra molting is often observed in solitarious phase (Uvarov, 1966). Hatchling body size and rearing density are important factors which influence the number of molts (HunterJones, 1958; Maeno and Tanaka, 2008). Studies on the incubation environment for L. migratoria hatchlings are scarce with no information regarding the number of nymphal stadia. For this reason, the current study investigated the effect of thermal and hydric environment on phase characteristics of hatchlings of L. migratoria in relation with the number of nymphal stadia. We applied this study to answer two questions. Are body phase characteristics of hatchlings sensitive to the environment conditions of incubation in L. migratoria? Is the moisture and temperature at incubation period induced variation in the number of nymphal stadia? Investigating the effect of temperature and moisture in producing different qualities of hatchlings should help in understanding the ecological role of phase-related characteristics of hatchlings in L. migratoria. 2. Material and methods 2.1. Locusts Experiments were carried out with a gregarious colony of Locusta migratoria. This strain was maintained for more than 6 years at the High Institute of Agronomy of Chott-Mariem, Tunisia. The colony was brought to the National Institute of Agrobiological Sciences at Ohwashi by permission from the Yokohama Plant Protection Station. Preliminary analysis of mitochondria DNA suggests that this strain belongs to the southern clade of this species that includes strains from Africa, France, Australia, Timor, Southern China and Southern Islands of Japan, and is distinctly different from the northern clade that includes strains of most areas of Japan and China (Tokuda et al., 2010). The crowd-reared colony was mainC tained at 30 in large wood-framed cages (42 cm 22 cm 42 cm; 0.038 m3). Each cage was covered with nylon screen mesh except for the wood floor and the front sliding door, which was composed of a transparent acrylic plate. Locusts were fed cut grass inserted in water jars and wheat bran. Grass was changed every 1 or 2 days. Bromus grass was grown in crop fields by the Field Management Department of the National Institute of Agro-biological Sciences at Ohwashi. Locusts laid eggs in moist sand held in plastic cups (vol. 380 ml) which were incubated at 30 C.
57
chosen for experiments, because in the case of S. gregaria, large eggs are known to produce black hatchlings characteristic of gregarious forms (Tanaka and Maeno, 2008). The eggs were held on wet filter paper in Petri dishes (9 cm in diameter; 1.5 cm in height) and incubated until used. Healthy looking eggs were sampled every day from day 2 to day 10 after oviposition. Sixty eggs derived from the prepared groups of large eggs were placed on dry filter paper (9 cm in diameter) in a Petri dish. The eggs in Petri dishes were then held in an air-tight plastic container in which relative moisture was kept close to 100% by putting moist tissue paper at a corner of the container. Eggs of S. gregaria lose some water under such conditions, because they cannot absorb water from air and can imbibe water only as liquid and not as vapor (Shulov and Pener, 1963). Hatchlings obtained from dried and moistened eggs were transferred to a plastic bag in which they were kept for more than 6 h before being weighed and checked for body coloration. 2.4. Measurements of hatchling body coloration, size and head luminance To quantify the moisture effect on head luminance, locusts were photographed using a scanner (Epson GT-X770, Japan) connected to a computer using commercial software, Photoshop 7.0 (Adobe Systems Incorporated, San Jose, CA). Locusts were chilled on ice for 15 min and placed with one side down on the glass table of the scanner for photographing. The image type used was 48-bit color at a resolution of 1200 d.p.i. Using the histogram function, luminance of body color (head) was measured and the mean luminance was recorded for each individual. The body color of hatchlings was observed 6e12 h after hatching. Hatchling body color was divided into three hatchling color groups based on the method of Ben Hamouda et al. (2009): whitish (W), grey (G) and black (B). After body color was scored, hatchlings were weighed individually using an electronic balance (METTLER AT201, Japan). 2.5. Measurements of the number of nymphal stadia To know whether incubation of eggs under dry conditions induces the solitarization of hatchlings, treated and control (untreated) nymphs were reared in isolation and checked for ecdysis every day. 2.6. Statistical analysis
Forty new egg pods laid by gregarious crowd-reared females were incubated separately in a small plastic cup containing moist sand at two temperatures: twenty egg pods at 30 C and another twenty at 20 C.
Statistical analysis was performed using IBM SPSS 20 software. The normal distribution was performed using the Shapiro-WilkTest. For normally distributed data or after transformation if necessary, differences between groups were calculated using analysis of variance (ANOVA). The Student-Newman-Keuls posthoc multiple comparison test was used if ANOVA indicated a significant effect. Categorical data were compared by Chi-square test and correlations were determined by Pearson’s correlation coefficient. The null hypothesis was rejected at the 0.05 level. For nonnormally distributed data, the Kruskal-Wallis test was used to investigate differences between groups. Subsequently, the Mann Whitney test was applied to detect the pairwise differences. Asterisks indicate levels of significant differences (*p < 0.05; **p < 0.01; ***p < 0.001).
2.3. Incubation of eggs under moist and dry conditions
3. Results
In this experiment, we adopted the method described for S. gregaria by Maeno and Tanaka (2009). Eggs deposited by gregarious females were incubated at 30 C. The day of oviposition was designated as day 0. On day 2, eggs longer than 6.0 mm were
3.1. Effect of different egg temperature on hatchling body coloration and size
2.2. Incubation of eggs at high and low temperatures
At a high temperature (30 ± 2 C), embryogenesis proceeded
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3.2. Effect of drying eggs on hatchling body coloration and size No hatchling was obtained from eggs that had been preincubated on wet paper up to 4 days and then transferred to dry paper. Hatching was observed among eggs that had been kept on wet paper for at least 5 days. 36.66%, 43.33% and 56.66% were the hatching rates of hatchlings produced from eggs transferred to dry paper on day 5, 6 and 7, respectively. On day 8, 9 and 10, 66.66% of hatchlings were obtained. The maximum of hatching rate (83.33%) was observed under continuous moist conditions (Fig. 4). Whitish hatchlings were predominant when the eggs were transferred from wet to dry conditions, whereas such individuals appeared only in a small proportion under continuous moist conditions (Fig. 5). These results indicated that a reduction of water reserve of egg by drying reduced the probability to develop black hatchlings (Fig. 6). The lowest (darkest) value of mean luminance was observed only when eggs were incubated under wet conditions (Fig. 7).
(n=171)
100
Percentage of hatchlings (%)
rapidly for the majority of egg pods except for the last three egg pods that probably contained infertile eggs. Hatching started 12 days after oviposition and was completed in 4 days. The highest proportion of hatched nymphs was observed at day 1 of the hatching period (Fig. 1). At 20 C, hatching started after 39 days of incubation. It required 12 days to complete (Fig. 1). The highest proportion of hatched nymphs was observed at day 3 of the hatching period. The eggs incubated at 30 C showed significantly higher survival rates than those incubated at 20 C. Indeed, the hatchability at 30 C was 1.27 times higher than that at 20 C. The proportions of black, grey and whitish hatchlings were significantly different at 30 C and 20 C (Fig. 2; X2 ¼ 36.96; df ¼ 2; P < 0.001). Gregarious eggs incubated at 30 C produced a mixture of whitish (28.07%), grey (24.56%) and black (47.37%) hatchlings. At a low temperature (20 C), most hatchlings exhibited dark body coloration. We noted the disappearance of whitish coloration at this low temperature. Fig. 3 shows that hatchlings derived from eggs that were incubated at 30 C were larger than those derived from eggs that were incubated at 20 C. The largest grey hatchlings derived from eggs incubated at 20 C were characterized by the same weight of whitish hatchlings from 30 C. Moreover, the cuticular melanism in hatchlings exhibits a correlation with body size at 30 C (r ¼ 0.600; n ¼ 171; P < 0.01) than at 20 C (r ¼ 0.345; n ¼ 181; P < 0.01).
90
(n=181) 19.89%; 36
28.07%; 48
80
W
70 60
24.56%; 42
G
50 40
B
80.11%; 145
30 47.37%; 81
20 10 0
30°C
20°C
Incubation temperatures
Fig. 2. The effect of egg temperature on hatchling body color. Numbers in parentheses (n) indicate the number of individuals. Percentage of black, grey and whitish hatchlings was compared by the chi-square test for the two temperatures of eggs incubation (X2 ¼ 36.96; df ¼ 2; P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
18
(a)
16
Hatchling body weight (mg)
58
(b) (c)
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12 10 8 6 4 2 0 30°C B
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30°C W
Hatchling body color at different temperatures Fig. 3. Relationship between hatchling body weight and color at different temperatures of eggs incubation. Letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by StudenteNewmaneKeuls test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
90 80
Hatching rate (%)
70
Percentage of hatchlings (%)
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551
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5 6 7 8 9 Days of transfer to dry filter paper
10
Moist
Fig. 4. Relationship between the hatching rate and the incubation of eggs at dry and moist conditions.
50
Days after oviposition Fig. 1. The pattern of hatching at 20 C and 30 C. Numbers above each histogram indicate the number of individuals.
Drying caused the eggs to produce lighter hatchlings (KruskaleWallis test: X2 ¼ 108.82, df ¼ 6, P < 0.01). This effect was more pronounced if the transfer from wet to dry conditions took place between the 5th and 7th days. Deprivation of water from eggs had a
A. Ben Hamouda, S. Tanaka / Journal of Arid Environments 134 (2016) 56e61
(0)
(0)
(0)
(22)
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(34)
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100 1 0,9 90 0,8 80 0,7 70
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2
3
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Moist
Days of transfer to dry filter paper Fig. 5. Effect of drying of eggs on hatchling body color. B, G and W: Black, Grey and Whitish body color. Numbers in parentheses (n) indicate the number of individuals. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
20
(a)
Hatchlings weight (mg)
18
***
16
14 12
***
(b)
(b)
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***
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(b)
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8
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10 8 6
4 2 0
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Days of transfer to dry conditions Fig. 6. Photographs showing the effect of eggs water loss on hatchling body size and color. (A) Large, black hatchling, obtained from moist conditions and (B) small, whitish hatchling, obtained from dry conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Head luminance means
120
100
(50)
(22) *** (d)
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(26) (34) (40) (40) *** *** *** *** (c) (d) (b) (b)
(40) *** (b)
n
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0 Moist 1
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Fig. 8. Effect of drying of eggs on hatchlings weight. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by nonparametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions.
11
Days of transfer to dry conditions Fig. 7. Mean luminance values for the head of hatchlings derived from moistened and dried eggs. Open circles indicate individual datum points and closed ones indicate means. Different letters in parentheses indicate a significant difference among head luminance at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions. Numbers in parentheses (n) indicate the number of individuals.
significant effect on body size of the hatchlings. Fig. 8 indicates a significant difference in weight between hatchlings from wet and dry conditions (KruskaleWallis test: X2 ¼ 71.27, df ¼ 6, P < 0.01). It
was considered that hatchling size was closely related to that of eggs, and the loss of some water from eggs due to desiccation resulted in the appearance of small hatchlings. At wet conditions, black (B) hatchlings had the highest mean body weight, followed by grey (G) and whitish (W) ones. At dry conditions, black (B) and grey (G) hatchlings had the highest mean body weight, whitish ones (W) the lowest (Fig. 9). Thus, when the weight increases, the body coloration becomes darker and vice versa. The correlation between cuticular melanism and body size of hatchlings was apparent for both grey and whitish hatchlings in wet conditions and for whitish hatchlings that experienced dry conditions. The duration of embryonic development increases with the early transfer to dry conditions (KruskaleWallis test: X2 ¼ 132.01, df ¼ 6, P < 0.01) (Fig. 10). However, no change in the duration was observed at the 9th (ManneWhitney U ¼ 940, P ¼ 0.611) and 10th days (ManneWhitney U ¼ 913.5, P ¼ 0.460) of dry conditions.
3.3. Effect of drying eggs on the number of molts in the hatched nymphs To determine if drying eggs influenced the number of molts in the hatched nymphs, nymphs obtained in the above experiment were reared in isolation and the number of nymphal molts was
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Hatchling body weight (mg)
30
*
25 20
*
**
**
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15
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10 5
0 B
G
W
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G
W
Dry conditions
Moist conditions
Hatchling body color under moist and dry conditions Fig. 9. Relationship between body weight of hatchlings and their body color. B, G and W: Black, Grey and Whitish hatchlings. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Differences in the hatchlings body weight in each body color for moist and dry conditions were compared with the ManneWhitney U test: *, P < 0.05 and **, P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Development duration (day)
20
18 16
*** (a)
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***
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(d)
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(e)
9
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Moist
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8 6 4 2
0 5
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Days of transfer to dry filter paper Fig. 10. Effect of drying of eggs on embryonic development duration. Different letters in parentheses indicate a significant difference among hatchlings weight at P < 0.05 by non-parametric test KruskaleWallis followed by ManneWhitney test. Asterisks (***) indicate statistical significances after the paired ManneWhitney test (P < 0.001) between moist and dry conditions.
determined. All hatchlings derived from the eggs that were exposed to dry conditions became adults after five nymphal stadia (n ¼ 193) as observed in control nymphs derived from eggs constantly kept under wet conditions (n ¼ 48). 4. Discussion and conclusions Progeny body size and color and head luminance in L. migratoria were significantly influenced by the temperature of eggs incubation. Hatchlings displayed a distinct response to different temperatures during the egg stage; the percentage of hatching was significantly higher at a high temperature (30 C) than at a low temperature (20 C). Moreover, the period of incubation was shorter if eggs were incubated at the higher temperature. These results suggest that temperature is an important factor affecting their embryonic development and can thus be an indicator of predicting the state of locust populations in subsequent generations. The concept that the body color of solitary L. migratoria hatchlings was whitish and that of gregarious ones was black has been known in some strains for a long time (Uvarov, 1966). This
difference in body coloration was correlated with the parental phase state and population density (Hunter-Jones, 1958; Albrecht et al., 1959a,b). Tanaka and Maeno (2008) have demonstrated that the hatchling body color in S. gregaria is closely related to egg size that is pre-determined in the ovaries of the female parent. Small eggs, typical of solitary phase, tend to give rise to green hatchlings and those of large eggs, typical of gregarious phase were black. In L. migratoria, we found the same assumption, namely that in addition to the contribution of some abiotic factors that can modify the maternal effect on progeny characteristics, the temperature during embryogenesis also has a pronounced effect on the body size and color of hatchlings. Uvarov (1966) and Hunter-Jones (1970) have shown that the body color is influenced by the temperature during the egg incubation. The offspring of crowd-reared gregarious females emerged with a predisposition toward gregarious or solitarious body coloration and size depending upon the incubation temperature (Fig. 2). The correlation between body weight and cuticular melanism persists also at a low temperature but it is less obvious. It seems that in L. migratoria, the incubation temperature has an important effect on the activity of certain factors responsible for the control of hatchling body color. Body size and color at hatching are closely correlated: hatchlings with darker body coloration were heavier. Recent studies with S. gregaria (Tanaka and Maeno, 2006, 2008) and L. migratoria (Ben Hamouda et al., 2009) have confirmed this relationship. The important role of abiotic factors on hatchlings phase characteristics has been shown in our experiments: the present study tested the hypothesis that eggs thought to produce black hatchlings give rise to whitish hatchlings if they are incubated at dry conditions. Shulov and Pener (1963) studied the effect of moisture on egg weight and hatching rate in S. gregaria but did not observe the body color and size of hatchlings. Hunter-Jones (1964, 1970) showed that the weight and body color of hatchlings were influenced by the soil moisture and the temperature of egg incubation. In the present study, eggs were transferred from wet to dry filter paper at various timings after oviposition and were kept at nearly saturated conditions. It was known that eggs can imbibe water only as liquid and not as vapor from the air in S. gregaria (Shulov and Pener, 1963). Likewise, L. migratoria eggs also lost weight probably on dry paper under nearly saturated conditions, indicating that the eggs cannot take up water from the air. The present results raise two important questions: first, how are hatchling phase characteristics affected so that the body size and color of the offspring vary according to the time of transfer to dry conditions? In a previous study by Maeno and Tanaka (2009) it has been demonstrated that S. gregaria eggs transferred from wet to dry filter paper at various timings after deposition produced black hatchlings but some of those transferred on day 4 or 5 produced green hatchlings. Our results and those of Maeno and Tanaka (2009) may indicate the importance of water reserve in the egg in the regulation of hatchlings body coloration in L. migratoria and S. gregaria. The first change observed following drying was a loss of egg weight, which causes hatchling weight and darkening to be reduced. It has been known that the weight difference between hatchlings of different body colors is largely attributable to higher water content of the heavier ones (Blackith, 1961; Albrecht, 1962). Maeno and Tanaka (2009) suggested in similar experiments with S. gregaria that green hatchlings retained a relatively large amount of egg yolk in the gut compared to black hatchlings, suggesting that the availability of yolk material for embryogenesis is modified by water loss. Albrecht et al. (1959a) showed that the quantitative reduction of egg yolk led to changes in hatchlings body size and color. The second question raised is whether deprivation of egg water will be a source of hatchling solitarization? It would also be
A. Ben Hamouda, S. Tanaka / Journal of Arid Environments 134 (2016) 56e61
interesting to determine if small, whitish hatchlings produced by egg drying show other solitarious characteristics in their development. The number of nymphal stadia might be involved: Extra molting is typically observed in the solitarious phase (Uvarov, 1966), although some exception has been reported recently for a laboratory strain of S. gregaria in which phase did not affect the number of nymphal instars (Lester et al., 2005). It has been suggested that body weight at hatching is critical for the determination of extra molting (Hunter-Jones, 1958) and hatchlings that have a heavy weight at hatching do not go through supplementary nymphal stadia (Albrecht et al., 1959a). Because solitarious locusts often exhibit extra molting, the effects of drying eggs on extra molting in nymphs were examined in the present study. The results indicated that all gregarious nymphs derived from wet conditions undergo only five nymphal stadia under isolated conditions. The same number of nymphal stadia was obtained for nymphs derived from dry conditions and reared in isolation. Nymphs used in our experiments came from eggs that were laid by crowd-reared gregarious females. Therefore, rearing these nymphs in isolation during the nymphal stage does not affect the number of nymphal stadia even when they are characterized by small size and light body color at hatching. The present study is consistent with the observations of Albrecht et al. (1959b) who found that the number of nymphal stadia was not influenced in small hatchlings obtained by ligating the egg with a thread. Acknowledgements We thank Ms. H. Ikeda, Ms. N. Totsuka, Ms. Y. Yokota and Ms. M. Higuchi for laboratory assistance. We are grateful to Dr. M. Koutaro and Dr. K. Harano for kind advice and encouragement at the Locust Research Laboratory, National Institute of Agro-biological Sciences at Ohwashi, Tsukuba. The grass used was raised by Field Management Section of NIASO. This study was supported by the “Japanese Association of University Woman” Research Fellowships. References Albrecht, F.O., 1962. Some physiological and ecological aspects of locust phases. Trans. Entomol. Soc. Lond. 114 (11), 335e375. termination de la fertilite par Albrecht, F.O., Verdier, M., Blackith, R.E., 1959a. De l’effet de groupe chez le criquet migrateur (Locusta migratoria migratorioides R. & F.). Bull. Biol. Fr. Belg. 92, 349e427. Albrecht, F.O., Verdier, M., Blackith, R.E., 1959b. Maternal control of ovariole number in the progeny of the migratory locust. Nature 184, 103e104. Ben Hamouda, A., Ammar, M., Ben Hamouda, M.H., Bouain, A., 2009. The role of egg pod foam and rearing conditions on the phase state of the Asian migratory locust Locusta migratoria migratoria (Orthoptera, Acrididae). J. Insect Physiol. 55, 617e623. BernaysE.A.,Some factors affecting size in first-instar larvae of Schistocerca gregaria (Forskal), Acrida 1, 1972, 189-195, in: Atkinson, D. (Eds.), Temperature and Organism Size-a Biological Law for Ectotherms? Begon, M. and Fitter A.H. Adv. Ecol. Res. 25, 1994, pp. 133. Blackith, R.E., 1961. The water reserves of hatchling locusts. Comp. Biochem. Physiol. 3, 99e107. Byers, J.A., 1991. Pheromones and chemical ecology of locusts. Biol. Rev. 66, 347e378.
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