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Juvenile hormone titer and wing-morph differentiation in the vetch aphid Megoura crassicauda
Journal of Insect Physiology 59 (2013) 444–449
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Juvenile hormone titer and wing-morph differentiation in the vetch aphid Megoura crassicauda Asano Ishikawa 1, Hiroki Gotoh, Taisuke Abe, Toru Miura ⇑ Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan
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Article history: Received 15 October 2012 Received in revised form 8 February 2013 Accepted 11 February 2013 Available online 19 February 2013 Keywords: Wing polyphenism Phenotypic plasticity Flight-apparatus development
a b s t r a c t Most aphids exhibit wing polyphenism, in which winged and wingless females are produced depending on aphid densities. Although juvenile hormone (JH) has been implicated in the regulation of aphid wing polyphenism, relatively few studies examining the direct relationship between JH titer and resultant wing morphs have been undertaken. We therefore investigated the relationship between JH III titer and the development of wing morphs in the vetch aphid Megoura crassicauda during postembryonic development. JH III measurements by liquid chromatography–mass spectrometry (LC–MS) revealed that, at the third instar, presumptive wingless nymphs had significantly higher JH III titers than winged nymphs. In winged nymphs at the third instar, JH III application inhibited wing development resulting in the appearance of winged/wingless intermediates as well as juvenilized individuals with supernumerary molting. These results suggest that JH III plays an important role in wing-morph differentiation during postembryonic development. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Juvenile hormone (JH) is one of the most important hormones, which play the various pleiotropic physiological roles in insect life cycles such as metamorphosis, reproduction, diapause, and the regulation of polyphenism in which different phenotypes are produced depending on environmental stimuli (Hartfelder and Emlen, 2005; Nijhout, 1994, 2003). In crickets, JH and its degradative enzyme, juvenile hormone esterase (JHE), are responsible for morph determination in brachypterous (short-winged) and macropterous (long-winged) morphs (Zera and Denno, 1997; Zera et al., 1989). In several coleopterans, such as dung beetles and stag beetles, JH regulates the dimorphic growth of male horns and/or mandibles in response to nutritional conditions (Emlen and Nijhout, 1999; Gotoh et al., 2011). In addition, JH also plays important roles in the regulation of caste differentiation in social insects (Miura, 2005; Nijhout, 1994; Nijhout and Wheeler, 1982). The role of JH in insect wing development has been investigated in many taxa, including the Orthoptera (Zera and Denno, 1997; Zera et al., 1989), Lepidoptera (Miner et al., 2000), and some Hemiptera (Iwanaga and Tojo, 1986). The most widely accepted hypothesis of endocrine control of wing-morph determination is that high JH titer during a critical developmental period either
⇑ Corresponding author. Tel./fax: +81 11 706 4524. E-mail address: [email protected] (T. Miura). Present address: Ecological Genetics Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. 1
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completely or partially blocks the normal development of wings, flight muscles, and structures related to the flight apparatus, producing brachypterous or wingless phenotypes (Hardie and Lees, 1985a; Nijhout, 1994; Zera and Denno, 1997). Most aphid species (Family Aphididae) exhibit two types of polyphenism; wing polyphenism and reproductive polyphenism in their annual life cycles (Dixon, 1998; Miyazaki, 1987). From spring to summer, aphids produce vast numbers of clonal offspring through parthenogenetic reproduction. Wing polyphenism refers to the production of winged and wingless adult females in response to the environmental conditions, such as population density, host-plant quality, and presence of natural enemies, during the parthenogenetic generations. On the other hand, reproductive polyphenism refers to the change of reproductive modes between parthenogenesis and sexual reproduction. At the beginning of fall, the males and sexual oviparous females that are produced in response to short-day length mate and produce overwintering eggs (Müller et al., 2001; Tagu et al., 2005). Most aphids alter their reproductive modes from parthenogenesis to sexual reproduction depending on seasonal conditions, and JH III plays a crucial role in this reproductive polyphenism (Hardie et al., 1985; Ishikawa et al., 2012b). Although the involvement of JH in the wing polyphenism in aphids has been studied since the 1960s, relatively little definitive information exists on the nature of the endocrinological mechanisms involved (Braendle et al., 2006). Some aphid species, such as the vetch aphid Megoura crassicauda and the pea aphid Acyrthosiphon pisum change their wing types trans-generationally
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depending on aphid densities encountered by the mother aphids. In these species, the determination and differentiation of wing morphs occur at completely different times in aphid development (Dixon, 1998). Specifically, morph determination occurs during embryogenesis in the maternal ovarian cavity and morph differentiation (i.e. wing development/degeneration) occurs during postembryonic development. Numerous studies have investigated the effect of JH on wing-morph determination in aphids, and some of them showed that JH and JH analogs (JHAs) exhibited the potential to inhibit wing development in winged aphids (Lees, 1966, 1980). However, Schwartzberg et al. (2008) found no correlation between JH titer in maternal hemolymph and winged- and wingless-morph production. It would seem to exclude a role for JH in winged/wingless determination, but not differentiation. In contrast, relatively few studies have examined wing-morph differentiation. In the vetch aphid, the topical application of JH on winged nymphs induced winged/wingless intermediates with undeveloped thoracic structures and juvenilized individuals with supernumerary molting, with sensitivity to JH being most apparent in the third instar (Lees, 1966, 1980). However, the effects of changes in JH titer during postembryonic development as well as the extent to which JH is correlated with wing-morph differentiation have not yet been clarified. We therefore investigated the transition in JH III titer during postembryonic development in winged and wingless morphs of the vetch aphid M. crassicauda. In addition, to confirm whether the topical application of JH III would induce individuals that were intermediate between winged and wingless aphids, JH III was topically applied to winged nymphs and the resulting morphs were compared by examining under a scanning electron microscope. 2. Materials and methods 2.1. Insects Mcr1 strain of vetch aphid M. crassicauda was collected in Japan and used as the focal species in this study because, by manipulating density conditions, winged and wingless aphids can easily be induced in the parthenogenetic viviparous generations (Ishikawa et al., 2012a; Ishikawa and Miura, 2009; Lees, 1966). Stock aphid populations were maintained for several generations under longday conditions (16L:8D, 20 °C) in tubes (diameter: 2.5 cm, height 10 cm) containing vetch seedlings (Vicia faba L.) growing on wet vermiculite (Wilkinson and Ishikawa, 2000). The postembryonic stages of the focal aphid species comprise four nymphal instars and a fifth adult instar (Blackman, 1987). As in a previous study (Ishikawa and Miura, 2007), hind-tibia length was used to discriminate nymphal instars. 2.2. Induction of winged and wingless morphs To analyze the relationship between JH titer and morph differentiation, we induced winged and wingless aphids by manipulating density, and measured JH titers in both wing morphs during postembryonic development. Since the effect of rearing conditions lasts for over two or three generations in the pea aphid (MacKay and Wellington, 1977), the stock aphids used in this study were maintained under low-density conditions (one wingless aphid per seedling) over three generations before beginning the density treatments. Since winged and wingless nymphs exhibited very similar external morphologies at the first or second instars (Ishikawa and Miura, 2009), we measured JH titers of aphids produced by mothers that were exposed high- and low-density conditions as described previously (Ishikawa et al., 2012). In the high-density
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treatment, 15–20 adult females at day 1 after the imaginal molt were placed on a single, approximately 1 cm-high vetch seedling for 48 h. In the low-density treatment, one adult aphid at day 1 after the imaginal molt was kept on a 1 cm-high vetch seedling for 48 h. During the 48 h, mother aphids started to produce progeny that had reached the first or second instar. The mother aphids were then removed from the seedlings and the offspring were collected as ‘‘younger-instar nymphs’’. At that time, more than fifty aphid nymphs were reared to confirm the efficacy of wing induction based on the wing-bud morphology, which was recognizable from the third instar (Ishikawa et al., 2008). In addition to the younger-instar nymphs, winged and wingless morphs at the third to fifth instars (based on wing-bud morphology), were collected from the stock aphid population. 2.3. JH quantification To investigate the correlation between JH titer and wing-morph differentiation, we quantified the JH titers of winged and wingless aphids during postembryonic development; ‘‘younger-instar’’, third, and fourth-instar nymphs, and fifth-instar adults. The physiology of winged aphids changes markedly during adulthood; the well-developed flight muscles that are present immediately after the imaginal molt are degraded to provide energy for developing ovaries before larviposition (Ishikawa and Miura, 2009). Thus, fifth-instar aphids (adults) were subdivided into two groups, i.e. the stage immediately after the imaginal molt (5th E), and the stage after the onset of larviposition (5th L). After collection, individual aphids were frozen and stored at 80°C until the JH assay. Replicated samples (each replicate derived from 60 first or second instars, ten third and fourth instars, and five fifth-instar adults) were prepared for each of the ten categories (winged: N = 5, wingless: N = 3, at the younger instar; winged: N = 10, wingless: N = 10, at the third instar; winged: N = 10, wingless: N = 7, at the fourth instar; winged: N = 4, wingless: N = 5, at the fifth instar just after the imaginal molt; and winged: N = 5, wingless: N = 5, at the fifth instar after the onset of larviposition). JH III extraction and quantification was performed as described previously (Ishikawa et al., 2012b). To determine whether differences in JH secretion existed among categories, a Peritz test followed by a Kruskal–Wallis test was performed. 2.4. Topical application of JH III To clarify the role of low JH III titers in differentiation of the winged morph, JH III was topically applied on winged nymphs at the third and fourth instars following the method of (Hardie and Lees, 1985b). Since from the third instar onward, we were able to distinguish winged lines from wingless lines based on the external morphology of the wing buds (Ishikawa and Miura, 2009), JH III treatments on the winged nymphs were performed at the third and fourth instar stage of development. Third- and fourth-instar winged nymphs were collected from the mother aphids maintained under high-density conditions. For the third-instar aphids, 0.05, 0.25 or 0.5 lg of JH III (P93%; Sigma–Aldrich, St. Louis, MO) diluted in 0.5 ll of acetone (i.e. 0.1, 0.5 or 1.0 lg/ll) was placed on the abdomen of the aphid (Hardie and Lees, 1985b). A total of 15 individuals were used for each concentration. For the fourth instar aphids, 0.25 lg or 0.5 lg of JH III diluted in 0.5 ll of acetone (i.e. 0.5 or 1.0 lg/ll) was applied to the abdomen of each aphid. A total of 20 individuals were used for each concentration. As controls, 0.5 ll of acetone without any JH III was applied to the abdomens of third and fourth instars of winged aphids. All of the aphids were kept on new vetch seedlings for several days and their thoracic structures were observed. Any aphids that died before the imaginal molt were excluded from the analysis.
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2.5. Scanning electron microscopy To examine the effect of JH application on thoracic development in winged aphids, the morphological features of winged aphids, winged/wingless intermediates with undeveloped thoracic structures and juvenilized individuals through supernumerary molt, which were induced by JH application were carefully examined under a scanning electron microscope (JSM-5510LV, JEOL, Japan) (cf. Miura et al., 2004). In addition to the thoracic parts, the morphology of the nymphal cauda differed from those in adults and was used as an index of juvenilization (Lees, 1980). Aphid samples were fixed in FAA solution (formalin:ethanol:acetic acid = 6:16:1), before being passed through increasing concentrations of ethanol and then finally rinsed in t-butanol. Samples were then freezedried (ES-2030 Hitachi Freeze Dryer, Japan) and coated with gold (E-1010 Ion Sputter, Hitachi). 3. Results 3.1. Transition in JH III titer during the postembryonic development of the winged/wingless lines To investigate the correlation between JH titer and wing-morph differentiation, changes in JH III titer during postembryonic development were examined in winged and wingless aphid lines. JH III titer was significantly different across the five postembryonic time points [younger instar (1st–2nd), third instar, fourth instar, fifth instar just after the imaginal molt (5th E), and fifth instar after the onset of larviposition (5th L)], and between the winged/wingless morphs (two-way factorial ANOVA followed by the Tukey–Kramer test, P < 0.01 for the time points, the winged/wingless morphs, and the interaction between the time points and morphs; Fig. 1). Comparisons of JH III titer between the winged and wingless morphs at each time point during postembryonic development revealed that, at the third instar, the wingless nymphs had significantly higher JH III titer than the winged nymphs (Fig. 1, winged: N = 10, wingless: N = 10, one-way ANOVA followed by the Tukey– Kramer test, P < 0.01), although there was no significant difference
Fig. 1. JH III titers in winged and wingless lines during postembryonic development. Columns and bars represent the average JH III titers and standard errors in winged (WD) and wingless (WL) lines, respectively. Numbers at the top right of the graph are the P-values from a two-way factorial ANOVA for assessing the effect of JH III titers on nymphal or adult stages (S), wing morph (M) and stage wing morph (S M). Different letters on bars denote significant differences among instars (two-way factorial ANOVA followed by the Tukey–Kramer test, P < 0.01). Asterisk () indicates significant difference between winged and wingless lines (one-way ANOVA followed by the Tukey–Kramer test, P < 0.01). The fifth instar was subdivided into the period just after the imaginal molt (5th E) and the period after the initiation of larviposition (5th L). At the third instar, JH levels in winged and wingless lines were significantly different.
between the winged and wingless lines at all the other stages, i.e. younger instar, fourth instar, fifth instar just after the imaginal molt, and fifth instar after the onset of larviposition (one-way ANOVA followed by the Tukey–Kramer test, P > 0.05). Since it was initially impossible to distinguish the winged and wingless morphs at the first or second instar stage based on external morphology, the winged samples at younger instars probably contained some wingless nymphs. Under high-density conditions, 83.8 ± 0.12% (mean ± SE) of the offspring were winged, while no winged aphids were produced under the low-density conditions. Thus, the observed difference in JH level between the winged and wingless samples at this stage may have been underestimated.
3.2. Effects of JH III treatment on wing-morph differentiation To clarify the role of high JH titer in wingless nymphs during the latter instar stages, JH III was topically applied to winged nymphs at the third and fourth instars. The survival rate of third instar aphids treated with 0.05, and 0.25 and 0.5 lg of JH III was 66% (N = 33), 32% (N = 16), and 20% (N = 10), respectively (N = 50 for each concentration category). The survival rate of fourth instar aphids treated with 0.25 and 0.5 lg of JH III was 72.5% (N = 29) and 42.5% (N = 17), respectively (N = 40 for each concentration category). For the control treatment (acetone only) the survival rates were 80% (N = 50) and 85% (N = 20) at the third and fourth instars, respectively. Some of the third-instar aphids that survived the treatment with JH III underwent the normal fourth molt and imaginal molts and started larviposition, while others became juvenilized individuals and exhibited supernumerary molting. The aphids that had normal imaginal molts, had undeveloped wings of various sizes that lacked a typical sheeted structure (Fig. 2A–C). However, the cauda showed a complex structure typical of that found in adults. These aphids were referred to as winged/wingless intermediates. Although the undeveloped wings of these wingless intermediates were similar to the wing pads of the fourth-instar nymphs in the winged line (Fig. 2G), they had joints at the base and were separated from the body trunk (white arrows in Fig. 2A and C). In addition, at the anterior margin of the forewing was a ridge that formed a wing fold (black arrowhead in Fig. 2B). In the aphids treated with 0.05 lg of JH III, 40% of survivors (13 individuals) possessed these undeveloped wings after normal fourth and imaginal molts (Fig. 3A). The juvenilized individuals with the supernumerary molts possessed nymphal cauda even after the fifth molt, which would normally correspond to the imaginal molt in aphids. Most of these juvenillized individuals entered another molt during which they died. The aphids that proceed to the sixth molt exhibited various states of undeveloped wings, and most had an adult cauda (Fig. 2D). Some of these aphids underwent a seventh molt (Fig. 2F), but very few of the aphids proceeding to the sixth and seventh molts started larviposition. As the dose of JH III increased, the proportion of aphids showing undeveloped wings without any supernumerary molts decreased. Instead the proportion of aphids that underwent supernumerary molts and that had undeveloped wings increased (Fig. 3A). No wingless phenotypes were induced by the JH III treatment. The JH III treatment at the fourth instar had more moderate effects on wing development. After the topical application of 0.5 lg of JH III, approximately half of the survivors (N = 9) became winged morphs with curly wings; however, a similar phenotype was also observed in the control aphids treated with acetone only (Fig. 3B). Although curly, these wings were completely sheet-like. In addition, a few of the aphid treated JH III at the fourth instar had undeveloped wings, and were similar to the intermediate
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Fig. 2. Scanning electron micrographs showing morphological changes associated with JH III treatment in aphids. (A–C) Aphids treated with JH III at the third instar. The aphids underwent fourth and fifth molts and started larviposition. Although the cauda is adult-like (I-J), aphids have undeveloped wings (B), which are separated from the body trunk (white arrows in A and C) and have structures on their margins (black arrowhead in B). (D and E) Sixth-instar aphid treated with JH III at the third instar stage. Aphid has already finished the supernumerary molt and possesses undeveloped wings (E) with structures on their margins (black arrowhead in E). The wings are separated from the body trunk (white arrow in D). (F) Sixth-instar aphid undergoing a seventh molt. The old cuticle is already split (white arrowhead). (F) Normal winged aphid at the fourth instar. (G) Normal winged aphid at the adult instar. Note the cauda is cone shaped .(H) Normal wingless aphid at the fourth instar. (I) Normal wingless aphid at the adult instar. Note the cauda is long and thin.
aphids (Fig. 3B). All of them started larviposition without supernumerary molts.
4. Discussion To clarify the role of JH in wing polyphenism, the transition in JH III titer during postembryonic development was investigated in winged and wingless lines of the vetch aphid. The LC–MS measurements showed that JH III levels increased at the third instar, decreased at the fourth instar, and were highest during the adult period after starting larviposition. It was unexpected that JH levels were lower in the fourth instar rather than the third instar, because several studies in aphids suggest that the third instar is JH-sensitive period for adult determination, in which JH titer would falls below the threshold in general (Corbitt and Hardie, 1985; Lees, 1980; Nijhout, 1994). Because we used aphids collected randomly at the third and fourth instar from the stock populations for the LC–MS analysis, it is possible that we overlooked the difference of JH titers during specific period in the third instar, which would be responsible for the adult determination. In addition, an increase in the JH titer was observed in wingless nymphs at the third instar. As in previous studies (Lees, 1980), the treatment of winged nymphs at the third instar with JH III inhibited normal wing development and caused the induction of ‘‘winged/wingless intermediates’’ as well as individuals that underwent supernumerary molts with malformed wings. Consequently, JH III is considered to play a role in winged/wingless differentiation as well as nymphal/adult determination at the third instar (Fig. 4). We also demonstrated that the proportion of individuals undergoing supernumerary molting after treatment with JH III increased in a dose-dependent manner (Fig. 3). Thus, it is possible that the threshold for the nymphal/adult determination is higher than that for the winged/wingless differentiation
(Fig. 4A). In the aphids treated with higher concentration of JH III, JH III titer may get above the threshold for adult determination, leading the supernumerary molting. Alternatively, it is also possible that the sensitive period for wined/wingless differentiation is different from that for the nymphal/adult determination during the third instar (Fig. 4B). If the sensitive period for adult determination is later stage during the third instar than the sensitive window for winged/wingless differentiation, topical applications of higher JH III will induce higher proportion of the supernumerary molting. The dual roles of JH III in winged/wingless differentiation and nymphal/adult determination may explain why the winged and wingless morphs showed only slight differences in JH III titers at the third instar. Further analyses for the detailed time schedule of JH III action will be necessary to reveal the pleiotropic roles of JH in the postembryonic development. Several studies have described the postembryonic development of the flight apparatus in winged and wingless aphids lines (Ganassi et al., 2005; Ishikawa and Miura, 2009; Ishikawa et al., 2008; Tsuji and Kawada, 1987). At the third instar, wing primordia start to develop rapidly and the epithelia proliferate and become folded into a complicated shape in the winged line. In the wingless line, however, the wing primorida were completely degenerated until the second instar (Ishikawa et al., 2008). In the present study, JH III application at the third instar of the winged line produced undeveloped wings of different sizes, possibly because the amount of JH incorporated into aphid bodies may have varied. In the butterfly Precis coenia, JH inhibits the growth and differentiation of the wing imaginal disc (Miner et al., 2000). Thus, in aphids lowering the JH III titer at the third instar may be important for wing growth in the winged line. A decrease in JH titer is also known to induce programmed cell death during metamorphosis in both of hemimetabolous and holometabolous insects (Mane-Padros et al., 2010). However, there was no difference in the JH III titers of the winged and wingless aphid lines at the first to second instars when
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Fig. 3. JH III treatment in third- and fourth-instar nymphs in the winged line. Effects of JH III treatment at the third instar (A) and fourth instar (B). Bars represent the proportion of individuals exhibiting various morphological changes in response to JH treatment. Numbers in parentheses show sample sizes. JH III treatment at the third instar severely affected the development of thoracic parts and the imaginal molt in winged aphids (A). However, the effects of JH III treatment on development at the fourth instar were less apparent (B).
the wing primordia have completely degenerated in the wingless line. Thus, JH might not be involved in degeneration of the wing primordia in the wingless nymphs. To understand the specific roles of JH in wing-morph differentiation, further studies including experimentally blocking the JH signaling pathway in wingless nymphs is necessary. Precocenes have been used for functional analyses on the JH actions in insects since they were reported to act as an anti-JH agent by destroying the JH synthesizing organ, corpora allata (Staal, 1986). In the case of aphid wing polyphenism, however, the previous works provide contradictory results (Braendle et al., 2006; Gao and Hardie, 1996; Hales and Mittler, 1981; Hardie et al., 1995; Sutherland, 1969). For example, although Precocene-II treatment on mother aphids induces the winged morph production, Precocene-III treatment on presumptive wingless aphids at the first and second instar inhibits the winged-morph differentiation and promotes precocious adult development (Gao and Hardie, 1996; Hardie et al., 1995). The inconsistent results of precocene treatments presumably derived from divergent effects of precocenes. The effectiveness and sensitive periods for destroying the corpora allata are varied among different taxa, and different derivatives (Staal, 1986). Because the present study suggests that the slight difference of JH titer in a specific sensitive period may have an important role in the wing polyphenism, alternative methods which can control JH titer finely are required to investigate the function of JH in winged/wingless differentiation. In this study, we showed that JH III was involved in aphid wing polyphenism. In crickets, there is a strong correlation between the
Fig. 4. Temporal pattern in JH titers during the postembryonic development of winged and wingless morphs. The solid black line and the dashed black line represent the JH titer in the winged (WD) and wingless (WL) lines, respectively. The solid red line and the dashed red line show possible JH-titer transition in aphids exposed to high and low concentrations of JH at the third instar, respectively.
levels of juvenile hormone esterase (JHE), JH titers, and winglength polyphenism (Zera and Denno, 1997). In the reproductive polyphenism in the pea aphid, JH III titers also show a negative correlation with gene expression levels of JHE (Ishikawa et al., 2012b). Thus, differences in genes involved in JH metabolism such as JHE are considered to be candidates for upstream cascade regulation of wing-morph differentiation. In aphids, RNA interference was successfully performed in a few studies (Jaubert-Possamai et al., 2007; Mao and Zeng, 2012; Mutti et al., 2006). In addition, recently, genetic knockouts methods using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN) have been reported as alternative options for functional analysis of genes in non-model insects (Watanabe et al., 2012). Thus, these technologies can be applied to investigate the involvement of genes related to JH metabolism in wing-morph differentiation in future studies. Acknowledgments We are grateful to S. Koshikawa and R. Cornette for their valuable comments on the study and assistance in various experiments. We also express our great appreciation to Y. Okumura, Y. Nakagawa, and K. Ogawa for their assistance with maintaining aphid stocks. This work was partly supported by Grants-in-Aid for Young Scientists (No. 21677001) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2013. 02.004.
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Iwanaga, K., Tojo, S., 1986. Effects of juvenile hormone and rearing density on wing dimorphism and oocyte development in the brown planthopper, Nilaparvata lugens. Journal of Insect Physiology, 32. Jaubert-Possamai, S., Le Trionnaire, G., Bonhomme, J., Christophides, G.K., Rispe, C., Tagu, D., 2007. Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum. BMC Biotechnology 7, 63. Lees, A.D., 1966. The control of polymorphism in aphids. Advances in Insect Physiology 3, 207–277. Lees, A.D., 1980. The development of juvenile hormone sensitivity in alatae of the aphid Megoura viciae. Journal of Insect Physiology 26, 143–151. MacKay, A., Wellington, W.G., 1977. Maternal age as a source of variation in the ability of an aphid to produce dispersing forms. Researches on Population Ecology 18, 195–209. Mane-Padros, D., Cruz, J., Vilaplana, L., Nieva, C., Urena, E., Belles, X., Martin, D., 2010. The hormonal pathway controlling cell death during metamorphosis in a hemimetabolous insect. Developmental Biology 346, 150–160. Mao, J., Zeng, F., 2012. Feeding-based RNA interference of a gap gene is lethal to the pea aphid, Acyrthosiphon pisum. PLoS One 7, e48718. Miner, A.L., Rosenberg, A.J., Nijhout, H.F., 2000. Control of growth and differentiation of the wing imaginal disk of Precis coenia (Lepidoptera: Nymphalidae). Journal of Insect Physiology 46, 251–258. Miura, T., 2005. Developmental regulation of caste-specific characters in socialinsect polyphenism. Evolution & Development 7, 122–129. Miura, T., Koshikawa, S., Machida, M., Matsumoto, T., 2004. Comparative studies on alate wing formation in two related species of rotten-wood termites: Hodotermopsis sjostedti and Zootermopsis nevadensis (Isoptera, Termopsidae). Insectes Sociaux 51, 247–252. Miyazaki, M., 1987. Forms and morphs of aphids. In: Minks, A.K., Harrewijn, P. (Eds.), Aphids, Their Biology, Natural Enemies and Control. Elsevier, Amsterdam, pp. 27–50. Müller, C.B., Williams, I.S., Hardie, J., 2001. The role of nutrition, crowding and interspecific interactions in the development of winged aphids. Ecological Entomology 26, 330–340. Mutti, N.S., Park, Y., Reese, J.C., Reeck, G.R., 2006. RNAi knockdown of a salivary transcript leading to lethality in the pea aphid, Acyrthosiphon pisum. Journal of Insect Science 6, 38. Nijhout, H.F., 1994. Insect Hormones. Princeton University Press, Princeton. Nijhout, H.F., 2003. Development and evolution of adaptive polyphenisms. Evolution & Development 5, 9–18. Nijhout, H.F., Wheeler, D.E., 1982. Juvenile hormone and the physiological basis of insect polymorphisms. The Quarterly Review of Biology 57, 109–133. Staal, G.B., 1986. Anti juvenile hormone agents. Annual Review of Entomology 31, 391–429. Sutherland, O.R.W., 1969. The role of crowding in the production of winged forms by two strains of the pea aphid, Acyrthosiphon pisum. Journal of Insect Physiology 15, 1385–1410. Schwartzberg, E.G., Kunert, G., Westerlund, S.A., Hoffmann, K.H., Weisser, W.W., 2008. Juvenile hormone titres and winged offspring production do not correlate in the pea aphid, Acyrthosiphon pisum. Journal of Insect Physiology 54, 1332– 1336. Tagu, D., Sabater-Munoz, B., Simon, J.-C., 2005. Deciphering reproductive polyphenism in aphids. Invertebrate Reproduction & Development 48, 71–80. Tsuji, H., Kawada, K., 1987. Development and degeneration of wing buds and indirect flight muscles in the pea aphid (Acyrthosiphon pisum (Harris)). Japanese Journal of Applied Entomology and Zoology 31, 247–252. Watanabe, T., Ochiai, H., Sakuma, T., Horch, H.W., Hamaguchi, N., Nakamura, T., Bando, T., Ohuchi, H., Yamamoto, T., Noji, S., Mito, T., 2012. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nature Communications 3, 1017. Wilkinson, T.L., Ishikawa, H., 2000. Injection of essential amino acids substitutes for bacterial supply in aposymbiotic pea aphids (Acyrthosiphon pisum). Entomologia Experimentalis Et Applicata 94, 85–91. Zera, A.J., Denno, R.F., 1997. Physiology and ecology of dispersal polymorphism in insects. Annual Review of Entomology 42, 207–230. Zera, A.J., Strambi, C., Tiebel, K.C., Strambi, A., Rankin, M.A., 1989. Juvenile hormone and ecdysteroid titres during critical periods of wing morph determination in Gryllus rubens. Journal of Insect Physiology 35, 501–511.
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Juvenile hormone titer and wing-morph differentiation in the vetch aphid Megoura crassicauda Asano Ishikawa 1, Hiroki Gotoh, Taisuke Abe, Toru Miura ⇑ Laboratory of Ecological Genetics, Graduate School of Environmental Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan
a r t i c l e
i n f o
Article history: Received 15 October 2012 Received in revised form 8 February 2013 Accepted 11 February 2013 Available online 19 February 2013 Keywords: Wing polyphenism Phenotypic plasticity Flight-apparatus development
a b s t r a c t Most aphids exhibit wing polyphenism, in which winged and wingless females are produced depending on aphid densities. Although juvenile hormone (JH) has been implicated in the regulation of aphid wing polyphenism, relatively few studies examining the direct relationship between JH titer and resultant wing morphs have been undertaken. We therefore investigated the relationship between JH III titer and the development of wing morphs in the vetch aphid Megoura crassicauda during postembryonic development. JH III measurements by liquid chromatography–mass spectrometry (LC–MS) revealed that, at the third instar, presumptive wingless nymphs had significantly higher JH III titers than winged nymphs. In winged nymphs at the third instar, JH III application inhibited wing development resulting in the appearance of winged/wingless intermediates as well as juvenilized individuals with supernumerary molting. These results suggest that JH III plays an important role in wing-morph differentiation during postembryonic development. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Juvenile hormone (JH) is one of the most important hormones, which play the various pleiotropic physiological roles in insect life cycles such as metamorphosis, reproduction, diapause, and the regulation of polyphenism in which different phenotypes are produced depending on environmental stimuli (Hartfelder and Emlen, 2005; Nijhout, 1994, 2003). In crickets, JH and its degradative enzyme, juvenile hormone esterase (JHE), are responsible for morph determination in brachypterous (short-winged) and macropterous (long-winged) morphs (Zera and Denno, 1997; Zera et al., 1989). In several coleopterans, such as dung beetles and stag beetles, JH regulates the dimorphic growth of male horns and/or mandibles in response to nutritional conditions (Emlen and Nijhout, 1999; Gotoh et al., 2011). In addition, JH also plays important roles in the regulation of caste differentiation in social insects (Miura, 2005; Nijhout, 1994; Nijhout and Wheeler, 1982). The role of JH in insect wing development has been investigated in many taxa, including the Orthoptera (Zera and Denno, 1997; Zera et al., 1989), Lepidoptera (Miner et al., 2000), and some Hemiptera (Iwanaga and Tojo, 1986). The most widely accepted hypothesis of endocrine control of wing-morph determination is that high JH titer during a critical developmental period either
⇑ Corresponding author. Tel./fax: +81 11 706 4524. E-mail address: [email protected] (T. Miura). Present address: Ecological Genetics Laboratory, Center for Frontier Research, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan. 1
0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.02.004
completely or partially blocks the normal development of wings, flight muscles, and structures related to the flight apparatus, producing brachypterous or wingless phenotypes (Hardie and Lees, 1985a; Nijhout, 1994; Zera and Denno, 1997). Most aphid species (Family Aphididae) exhibit two types of polyphenism; wing polyphenism and reproductive polyphenism in their annual life cycles (Dixon, 1998; Miyazaki, 1987). From spring to summer, aphids produce vast numbers of clonal offspring through parthenogenetic reproduction. Wing polyphenism refers to the production of winged and wingless adult females in response to the environmental conditions, such as population density, host-plant quality, and presence of natural enemies, during the parthenogenetic generations. On the other hand, reproductive polyphenism refers to the change of reproductive modes between parthenogenesis and sexual reproduction. At the beginning of fall, the males and sexual oviparous females that are produced in response to short-day length mate and produce overwintering eggs (Müller et al., 2001; Tagu et al., 2005). Most aphids alter their reproductive modes from parthenogenesis to sexual reproduction depending on seasonal conditions, and JH III plays a crucial role in this reproductive polyphenism (Hardie et al., 1985; Ishikawa et al., 2012b). Although the involvement of JH in the wing polyphenism in aphids has been studied since the 1960s, relatively little definitive information exists on the nature of the endocrinological mechanisms involved (Braendle et al., 2006). Some aphid species, such as the vetch aphid Megoura crassicauda and the pea aphid Acyrthosiphon pisum change their wing types trans-generationally
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depending on aphid densities encountered by the mother aphids. In these species, the determination and differentiation of wing morphs occur at completely different times in aphid development (Dixon, 1998). Specifically, morph determination occurs during embryogenesis in the maternal ovarian cavity and morph differentiation (i.e. wing development/degeneration) occurs during postembryonic development. Numerous studies have investigated the effect of JH on wing-morph determination in aphids, and some of them showed that JH and JH analogs (JHAs) exhibited the potential to inhibit wing development in winged aphids (Lees, 1966, 1980). However, Schwartzberg et al. (2008) found no correlation between JH titer in maternal hemolymph and winged- and wingless-morph production. It would seem to exclude a role for JH in winged/wingless determination, but not differentiation. In contrast, relatively few studies have examined wing-morph differentiation. In the vetch aphid, the topical application of JH on winged nymphs induced winged/wingless intermediates with undeveloped thoracic structures and juvenilized individuals with supernumerary molting, with sensitivity to JH being most apparent in the third instar (Lees, 1966, 1980). However, the effects of changes in JH titer during postembryonic development as well as the extent to which JH is correlated with wing-morph differentiation have not yet been clarified. We therefore investigated the transition in JH III titer during postembryonic development in winged and wingless morphs of the vetch aphid M. crassicauda. In addition, to confirm whether the topical application of JH III would induce individuals that were intermediate between winged and wingless aphids, JH III was topically applied to winged nymphs and the resulting morphs were compared by examining under a scanning electron microscope. 2. Materials and methods 2.1. Insects Mcr1 strain of vetch aphid M. crassicauda was collected in Japan and used as the focal species in this study because, by manipulating density conditions, winged and wingless aphids can easily be induced in the parthenogenetic viviparous generations (Ishikawa et al., 2012a; Ishikawa and Miura, 2009; Lees, 1966). Stock aphid populations were maintained for several generations under longday conditions (16L:8D, 20 °C) in tubes (diameter: 2.5 cm, height 10 cm) containing vetch seedlings (Vicia faba L.) growing on wet vermiculite (Wilkinson and Ishikawa, 2000). The postembryonic stages of the focal aphid species comprise four nymphal instars and a fifth adult instar (Blackman, 1987). As in a previous study (Ishikawa and Miura, 2007), hind-tibia length was used to discriminate nymphal instars. 2.2. Induction of winged and wingless morphs To analyze the relationship between JH titer and morph differentiation, we induced winged and wingless aphids by manipulating density, and measured JH titers in both wing morphs during postembryonic development. Since the effect of rearing conditions lasts for over two or three generations in the pea aphid (MacKay and Wellington, 1977), the stock aphids used in this study were maintained under low-density conditions (one wingless aphid per seedling) over three generations before beginning the density treatments. Since winged and wingless nymphs exhibited very similar external morphologies at the first or second instars (Ishikawa and Miura, 2009), we measured JH titers of aphids produced by mothers that were exposed high- and low-density conditions as described previously (Ishikawa et al., 2012). In the high-density
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treatment, 15–20 adult females at day 1 after the imaginal molt were placed on a single, approximately 1 cm-high vetch seedling for 48 h. In the low-density treatment, one adult aphid at day 1 after the imaginal molt was kept on a 1 cm-high vetch seedling for 48 h. During the 48 h, mother aphids started to produce progeny that had reached the first or second instar. The mother aphids were then removed from the seedlings and the offspring were collected as ‘‘younger-instar nymphs’’. At that time, more than fifty aphid nymphs were reared to confirm the efficacy of wing induction based on the wing-bud morphology, which was recognizable from the third instar (Ishikawa et al., 2008). In addition to the younger-instar nymphs, winged and wingless morphs at the third to fifth instars (based on wing-bud morphology), were collected from the stock aphid population. 2.3. JH quantification To investigate the correlation between JH titer and wing-morph differentiation, we quantified the JH titers of winged and wingless aphids during postembryonic development; ‘‘younger-instar’’, third, and fourth-instar nymphs, and fifth-instar adults. The physiology of winged aphids changes markedly during adulthood; the well-developed flight muscles that are present immediately after the imaginal molt are degraded to provide energy for developing ovaries before larviposition (Ishikawa and Miura, 2009). Thus, fifth-instar aphids (adults) were subdivided into two groups, i.e. the stage immediately after the imaginal molt (5th E), and the stage after the onset of larviposition (5th L). After collection, individual aphids were frozen and stored at 80°C until the JH assay. Replicated samples (each replicate derived from 60 first or second instars, ten third and fourth instars, and five fifth-instar adults) were prepared for each of the ten categories (winged: N = 5, wingless: N = 3, at the younger instar; winged: N = 10, wingless: N = 10, at the third instar; winged: N = 10, wingless: N = 7, at the fourth instar; winged: N = 4, wingless: N = 5, at the fifth instar just after the imaginal molt; and winged: N = 5, wingless: N = 5, at the fifth instar after the onset of larviposition). JH III extraction and quantification was performed as described previously (Ishikawa et al., 2012b). To determine whether differences in JH secretion existed among categories, a Peritz test followed by a Kruskal–Wallis test was performed. 2.4. Topical application of JH III To clarify the role of low JH III titers in differentiation of the winged morph, JH III was topically applied on winged nymphs at the third and fourth instars following the method of (Hardie and Lees, 1985b). Since from the third instar onward, we were able to distinguish winged lines from wingless lines based on the external morphology of the wing buds (Ishikawa and Miura, 2009), JH III treatments on the winged nymphs were performed at the third and fourth instar stage of development. Third- and fourth-instar winged nymphs were collected from the mother aphids maintained under high-density conditions. For the third-instar aphids, 0.05, 0.25 or 0.5 lg of JH III (P93%; Sigma–Aldrich, St. Louis, MO) diluted in 0.5 ll of acetone (i.e. 0.1, 0.5 or 1.0 lg/ll) was placed on the abdomen of the aphid (Hardie and Lees, 1985b). A total of 15 individuals were used for each concentration. For the fourth instar aphids, 0.25 lg or 0.5 lg of JH III diluted in 0.5 ll of acetone (i.e. 0.5 or 1.0 lg/ll) was applied to the abdomen of each aphid. A total of 20 individuals were used for each concentration. As controls, 0.5 ll of acetone without any JH III was applied to the abdomens of third and fourth instars of winged aphids. All of the aphids were kept on new vetch seedlings for several days and their thoracic structures were observed. Any aphids that died before the imaginal molt were excluded from the analysis.
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2.5. Scanning electron microscopy To examine the effect of JH application on thoracic development in winged aphids, the morphological features of winged aphids, winged/wingless intermediates with undeveloped thoracic structures and juvenilized individuals through supernumerary molt, which were induced by JH application were carefully examined under a scanning electron microscope (JSM-5510LV, JEOL, Japan) (cf. Miura et al., 2004). In addition to the thoracic parts, the morphology of the nymphal cauda differed from those in adults and was used as an index of juvenilization (Lees, 1980). Aphid samples were fixed in FAA solution (formalin:ethanol:acetic acid = 6:16:1), before being passed through increasing concentrations of ethanol and then finally rinsed in t-butanol. Samples were then freezedried (ES-2030 Hitachi Freeze Dryer, Japan) and coated with gold (E-1010 Ion Sputter, Hitachi). 3. Results 3.1. Transition in JH III titer during the postembryonic development of the winged/wingless lines To investigate the correlation between JH titer and wing-morph differentiation, changes in JH III titer during postembryonic development were examined in winged and wingless aphid lines. JH III titer was significantly different across the five postembryonic time points [younger instar (1st–2nd), third instar, fourth instar, fifth instar just after the imaginal molt (5th E), and fifth instar after the onset of larviposition (5th L)], and between the winged/wingless morphs (two-way factorial ANOVA followed by the Tukey–Kramer test, P < 0.01 for the time points, the winged/wingless morphs, and the interaction between the time points and morphs; Fig. 1). Comparisons of JH III titer between the winged and wingless morphs at each time point during postembryonic development revealed that, at the third instar, the wingless nymphs had significantly higher JH III titer than the winged nymphs (Fig. 1, winged: N = 10, wingless: N = 10, one-way ANOVA followed by the Tukey– Kramer test, P < 0.01), although there was no significant difference
Fig. 1. JH III titers in winged and wingless lines during postembryonic development. Columns and bars represent the average JH III titers and standard errors in winged (WD) and wingless (WL) lines, respectively. Numbers at the top right of the graph are the P-values from a two-way factorial ANOVA for assessing the effect of JH III titers on nymphal or adult stages (S), wing morph (M) and stage wing morph (S M). Different letters on bars denote significant differences among instars (two-way factorial ANOVA followed by the Tukey–Kramer test, P < 0.01). Asterisk () indicates significant difference between winged and wingless lines (one-way ANOVA followed by the Tukey–Kramer test, P < 0.01). The fifth instar was subdivided into the period just after the imaginal molt (5th E) and the period after the initiation of larviposition (5th L). At the third instar, JH levels in winged and wingless lines were significantly different.
between the winged and wingless lines at all the other stages, i.e. younger instar, fourth instar, fifth instar just after the imaginal molt, and fifth instar after the onset of larviposition (one-way ANOVA followed by the Tukey–Kramer test, P > 0.05). Since it was initially impossible to distinguish the winged and wingless morphs at the first or second instar stage based on external morphology, the winged samples at younger instars probably contained some wingless nymphs. Under high-density conditions, 83.8 ± 0.12% (mean ± SE) of the offspring were winged, while no winged aphids were produced under the low-density conditions. Thus, the observed difference in JH level between the winged and wingless samples at this stage may have been underestimated.
3.2. Effects of JH III treatment on wing-morph differentiation To clarify the role of high JH titer in wingless nymphs during the latter instar stages, JH III was topically applied to winged nymphs at the third and fourth instars. The survival rate of third instar aphids treated with 0.05, and 0.25 and 0.5 lg of JH III was 66% (N = 33), 32% (N = 16), and 20% (N = 10), respectively (N = 50 for each concentration category). The survival rate of fourth instar aphids treated with 0.25 and 0.5 lg of JH III was 72.5% (N = 29) and 42.5% (N = 17), respectively (N = 40 for each concentration category). For the control treatment (acetone only) the survival rates were 80% (N = 50) and 85% (N = 20) at the third and fourth instars, respectively. Some of the third-instar aphids that survived the treatment with JH III underwent the normal fourth molt and imaginal molts and started larviposition, while others became juvenilized individuals and exhibited supernumerary molting. The aphids that had normal imaginal molts, had undeveloped wings of various sizes that lacked a typical sheeted structure (Fig. 2A–C). However, the cauda showed a complex structure typical of that found in adults. These aphids were referred to as winged/wingless intermediates. Although the undeveloped wings of these wingless intermediates were similar to the wing pads of the fourth-instar nymphs in the winged line (Fig. 2G), they had joints at the base and were separated from the body trunk (white arrows in Fig. 2A and C). In addition, at the anterior margin of the forewing was a ridge that formed a wing fold (black arrowhead in Fig. 2B). In the aphids treated with 0.05 lg of JH III, 40% of survivors (13 individuals) possessed these undeveloped wings after normal fourth and imaginal molts (Fig. 3A). The juvenilized individuals with the supernumerary molts possessed nymphal cauda even after the fifth molt, which would normally correspond to the imaginal molt in aphids. Most of these juvenillized individuals entered another molt during which they died. The aphids that proceed to the sixth molt exhibited various states of undeveloped wings, and most had an adult cauda (Fig. 2D). Some of these aphids underwent a seventh molt (Fig. 2F), but very few of the aphids proceeding to the sixth and seventh molts started larviposition. As the dose of JH III increased, the proportion of aphids showing undeveloped wings without any supernumerary molts decreased. Instead the proportion of aphids that underwent supernumerary molts and that had undeveloped wings increased (Fig. 3A). No wingless phenotypes were induced by the JH III treatment. The JH III treatment at the fourth instar had more moderate effects on wing development. After the topical application of 0.5 lg of JH III, approximately half of the survivors (N = 9) became winged morphs with curly wings; however, a similar phenotype was also observed in the control aphids treated with acetone only (Fig. 3B). Although curly, these wings were completely sheet-like. In addition, a few of the aphid treated JH III at the fourth instar had undeveloped wings, and were similar to the intermediate
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Fig. 2. Scanning electron micrographs showing morphological changes associated with JH III treatment in aphids. (A–C) Aphids treated with JH III at the third instar. The aphids underwent fourth and fifth molts and started larviposition. Although the cauda is adult-like (I-J), aphids have undeveloped wings (B), which are separated from the body trunk (white arrows in A and C) and have structures on their margins (black arrowhead in B). (D and E) Sixth-instar aphid treated with JH III at the third instar stage. Aphid has already finished the supernumerary molt and possesses undeveloped wings (E) with structures on their margins (black arrowhead in E). The wings are separated from the body trunk (white arrow in D). (F) Sixth-instar aphid undergoing a seventh molt. The old cuticle is already split (white arrowhead). (F) Normal winged aphid at the fourth instar. (G) Normal winged aphid at the adult instar. Note the cauda is cone shaped .(H) Normal wingless aphid at the fourth instar. (I) Normal wingless aphid at the adult instar. Note the cauda is long and thin.
aphids (Fig. 3B). All of them started larviposition without supernumerary molts.
4. Discussion To clarify the role of JH in wing polyphenism, the transition in JH III titer during postembryonic development was investigated in winged and wingless lines of the vetch aphid. The LC–MS measurements showed that JH III levels increased at the third instar, decreased at the fourth instar, and were highest during the adult period after starting larviposition. It was unexpected that JH levels were lower in the fourth instar rather than the third instar, because several studies in aphids suggest that the third instar is JH-sensitive period for adult determination, in which JH titer would falls below the threshold in general (Corbitt and Hardie, 1985; Lees, 1980; Nijhout, 1994). Because we used aphids collected randomly at the third and fourth instar from the stock populations for the LC–MS analysis, it is possible that we overlooked the difference of JH titers during specific period in the third instar, which would be responsible for the adult determination. In addition, an increase in the JH titer was observed in wingless nymphs at the third instar. As in previous studies (Lees, 1980), the treatment of winged nymphs at the third instar with JH III inhibited normal wing development and caused the induction of ‘‘winged/wingless intermediates’’ as well as individuals that underwent supernumerary molts with malformed wings. Consequently, JH III is considered to play a role in winged/wingless differentiation as well as nymphal/adult determination at the third instar (Fig. 4). We also demonstrated that the proportion of individuals undergoing supernumerary molting after treatment with JH III increased in a dose-dependent manner (Fig. 3). Thus, it is possible that the threshold for the nymphal/adult determination is higher than that for the winged/wingless differentiation
(Fig. 4A). In the aphids treated with higher concentration of JH III, JH III titer may get above the threshold for adult determination, leading the supernumerary molting. Alternatively, it is also possible that the sensitive period for wined/wingless differentiation is different from that for the nymphal/adult determination during the third instar (Fig. 4B). If the sensitive period for adult determination is later stage during the third instar than the sensitive window for winged/wingless differentiation, topical applications of higher JH III will induce higher proportion of the supernumerary molting. The dual roles of JH III in winged/wingless differentiation and nymphal/adult determination may explain why the winged and wingless morphs showed only slight differences in JH III titers at the third instar. Further analyses for the detailed time schedule of JH III action will be necessary to reveal the pleiotropic roles of JH in the postembryonic development. Several studies have described the postembryonic development of the flight apparatus in winged and wingless aphids lines (Ganassi et al., 2005; Ishikawa and Miura, 2009; Ishikawa et al., 2008; Tsuji and Kawada, 1987). At the third instar, wing primordia start to develop rapidly and the epithelia proliferate and become folded into a complicated shape in the winged line. In the wingless line, however, the wing primorida were completely degenerated until the second instar (Ishikawa et al., 2008). In the present study, JH III application at the third instar of the winged line produced undeveloped wings of different sizes, possibly because the amount of JH incorporated into aphid bodies may have varied. In the butterfly Precis coenia, JH inhibits the growth and differentiation of the wing imaginal disc (Miner et al., 2000). Thus, in aphids lowering the JH III titer at the third instar may be important for wing growth in the winged line. A decrease in JH titer is also known to induce programmed cell death during metamorphosis in both of hemimetabolous and holometabolous insects (Mane-Padros et al., 2010). However, there was no difference in the JH III titers of the winged and wingless aphid lines at the first to second instars when
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Fig. 3. JH III treatment in third- and fourth-instar nymphs in the winged line. Effects of JH III treatment at the third instar (A) and fourth instar (B). Bars represent the proportion of individuals exhibiting various morphological changes in response to JH treatment. Numbers in parentheses show sample sizes. JH III treatment at the third instar severely affected the development of thoracic parts and the imaginal molt in winged aphids (A). However, the effects of JH III treatment on development at the fourth instar were less apparent (B).
the wing primordia have completely degenerated in the wingless line. Thus, JH might not be involved in degeneration of the wing primordia in the wingless nymphs. To understand the specific roles of JH in wing-morph differentiation, further studies including experimentally blocking the JH signaling pathway in wingless nymphs is necessary. Precocenes have been used for functional analyses on the JH actions in insects since they were reported to act as an anti-JH agent by destroying the JH synthesizing organ, corpora allata (Staal, 1986). In the case of aphid wing polyphenism, however, the previous works provide contradictory results (Braendle et al., 2006; Gao and Hardie, 1996; Hales and Mittler, 1981; Hardie et al., 1995; Sutherland, 1969). For example, although Precocene-II treatment on mother aphids induces the winged morph production, Precocene-III treatment on presumptive wingless aphids at the first and second instar inhibits the winged-morph differentiation and promotes precocious adult development (Gao and Hardie, 1996; Hardie et al., 1995). The inconsistent results of precocene treatments presumably derived from divergent effects of precocenes. The effectiveness and sensitive periods for destroying the corpora allata are varied among different taxa, and different derivatives (Staal, 1986). Because the present study suggests that the slight difference of JH titer in a specific sensitive period may have an important role in the wing polyphenism, alternative methods which can control JH titer finely are required to investigate the function of JH in winged/wingless differentiation. In this study, we showed that JH III was involved in aphid wing polyphenism. In crickets, there is a strong correlation between the
Fig. 4. Temporal pattern in JH titers during the postembryonic development of winged and wingless morphs. The solid black line and the dashed black line represent the JH titer in the winged (WD) and wingless (WL) lines, respectively. The solid red line and the dashed red line show possible JH-titer transition in aphids exposed to high and low concentrations of JH at the third instar, respectively.
levels of juvenile hormone esterase (JHE), JH titers, and winglength polyphenism (Zera and Denno, 1997). In the reproductive polyphenism in the pea aphid, JH III titers also show a negative correlation with gene expression levels of JHE (Ishikawa et al., 2012b). Thus, differences in genes involved in JH metabolism such as JHE are considered to be candidates for upstream cascade regulation of wing-morph differentiation. In aphids, RNA interference was successfully performed in a few studies (Jaubert-Possamai et al., 2007; Mao and Zeng, 2012; Mutti et al., 2006). In addition, recently, genetic knockouts methods using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALEN) have been reported as alternative options for functional analysis of genes in non-model insects (Watanabe et al., 2012). Thus, these technologies can be applied to investigate the involvement of genes related to JH metabolism in wing-morph differentiation in future studies. Acknowledgments We are grateful to S. Koshikawa and R. Cornette for their valuable comments on the study and assistance in various experiments. We also express our great appreciation to Y. Okumura, Y. Nakagawa, and K. Ogawa for their assistance with maintaining aphid stocks. This work was partly supported by Grants-in-Aid for Young Scientists (No. 21677001) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2013. 02.004.
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