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Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 5 6
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Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species
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Yanqun Liu a,b,⇑, Qirui Zhang b, David L. Denlinger b,⇑
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a b
Department of Sericulture, College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, China Departments of Entomology and Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 5 February 2015 Accepted 9 February 2015 Available online xxxx Keywords: KK-42 Pupal diapause Antheraea pernyi Helicoverpa zea Sarcophaga crassipalpis
a b s t r a c t The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause. Ó 2015 Published by Elsevier Ltd.
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1. Introduction
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Chemical and environmental tools that can be used to manipulate the expression of insect diapause not only provide insights into physiological mechanisms regulating diapause but also potentially offer methods for manipulating laboratory colonies to facilitate rearing or for disrupting field populations as a tool for pest management (Denlinger, 2008). For example, a number of chemical agents are highly effective in breaking diapause, e.g. mild acids for embryonic diapause of the silkworm Bombyx mori (cf., Yamamoto et al., 2013), hexane for pupal diapause in the flesh fly Sarcophaga crassipalpis (Denlinger et al., 1980); several agents do not exert an immediate diapause-terminating effect but may shorten diapause (e.g. juvenile hormone in S. crassipalpis (Denlinger et al., 1988)) or delay its termination (e.g. cyclic AMP
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⇑ Corresponding author at: Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 43210, USA (Y. Liu). Tel.: +1 614 292 6425; fax: +1 614 292 2030. E-mail addresses: [email protected] (Y. Liu), [email protected] (D.L. Denlinger).
in flesh flies (Denlinger and Wingard, 1978)). In addition, a number of agonists and antagonists of diapause hormone have been developed as potent manipulators of pupal diapause in heliothine moths (Zhang et al., 2011). Among agents known to alter diapause responses is KK-42 (1benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole), an imidazole derivative (Fig. 1A). This chemical functions as an insect growth regulator that can cause precocious metamorphosis in the silkworm B. mori (Kuwano et al., 1985), the European corn borer Ostrinia nubilalis (Gelman et al., 1995), the flesh fly Sarcophaga bullata (Darvas et al., 1989), and induces abnormal metamorphosis in the locust Locusta migratoria (Roussel et al., 1989) and in the Hawaiian cockroach Diploptera punctata (Pratt et al., 1990). KK42 has also been reported to break embryonic diapause in the Japanese oak silkworm Antheraea yamamai (Suzuki et al., 1989; Kuwano et al., 1991) and the gypsy moth Lymantria dispar (Suzuki et al., 1993; Lee and Denlinger, 1996), and to reduce the incidence of embryonic diapause in progeny of B. mori that are programmed to produce diapausing embryos (Wu et al., 1996). Whether KK-42 influences insect diapauses that occur in stages other than the embryo remains unknown. In the experiments we
http://dx.doi.org/10.1016/j.jinsphys.2015.02.003 0022-1910/Ó 2015 Published by Elsevier Ltd.
Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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report here we used two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and one species of Diptera, the flesh fly S. crassipalpis, to investigate whether KK-42 can also influence pupal diapause. We chose A. pernyi as one of the model insects to address this issue because it is not only a classic organism for studying diapause regulation (Williams and Adkisson, 1964a,b; Sauman and Reppert, 1996; Wang et al., 2013a,b), but it is also an economically important insect used for silk production and as a human food source in China (Liu et al., 2010). A. pernyi enters a facultative pupal diapause when larvae are reared under short daylengths and low temperature. This diapause can be averted under long day conditions (Williams and Adkisson, 1964a), or by exposure to long daylengths after storage at a low temperature (Takeda et al., 2011). The corn earworm is a member of the Helicoverpa/Heliothis complex, a worldwide group of noteworthy crop pests. Like other members of this pest complex, H. zea enters a facultative diapause as a pupa in the winter in response to short daylengths and low temperature of autumn (Phillips and Newsom, 1966). Pupal diapause of H. zea can be terminated within just a few days by high temperature at 27 °C (Meola and Adkisson, 1977) or with an injection of ecdysteroids or diapause hormone (Zhang et al., 2008, 2009). In contrast to A. pernyi, neither photoperiod nor chilling have an effect on termination of pupal diapause in this species (Roach and Adkisson, 1971). The flesh fly also enters pupal diapause in response to short daylengths and, like H. zea, breaks diapause upon the elevation of temperature (Denlinger, 1972). Like pupal diapauses in other insects (Denlinger et al., 2005), pupal diapause in the flesh fly appears to be the result of a shut-down of the brain-prothoracic gland axis (Zdarek and Denlinger, 1975). Pupal diapause in the flesh fly can be terminated not only by high temperature but also with a topical application of hexane (Denlinger et al., 1980) or with an injection of ecdysteroids (Zdarek and Denlinger, 1975). Our experiments on A. pernyi and H. zea provide the first evidence that KK-42 can retard the termination of pupal diapause when applied to diapausing pupae. Additionally, we show that KK-42 can be administered to the final instar larvae of H. zea and S. crassipalpis and thus increase the incidence of pupal diapause. Our results are consistent with a KK-42 mode of action that impairs and/or retards the production or action of ecdysteroids associated with diapause onset and termination. The fact that KK-42 is an agent that can readily penetrate the cuticle suggests it may have utility as a disruptor of the diapause response.
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2. Materials and methods
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2.1. Chemicals
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KK-42 was synthesized as described (Kuwano et al., 1985) by Dr. Xinghai Li (Department of Pesticide Science, Plant Protection College, Shenyang Agricultural University, Shenyang, China). Purity of the synthesized sample was determined by HPLC to be
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approximately 90%, and its activity to induce precocious metamorphosis in third instar larvae of B. mori was confirmed by topical application (Fig. 1B). KK-42 was dissolved in acetone and stored at 20 °C.
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2.2. Culture of Antheraea pernyi
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The univoltine strain of A. pernyi used in this experiment was reared on oak trees in the field at the Sericultural Experiment Station of Henan Province, Nanyang, China, until cocoon formation. Larvae pupated in early June, 2014 and within 2 weeks were sent to the Denlinger laboratory under U.S. Department of Agriculture permit No. P526P-14-01794. Pupae were held at 23 °C with a photoperiod of LD 9:15 until used in experiments. The 20-Hydroxyecdysone (20-E) used to terminate pupal diapause was from Sigma (St. Louis, MO).
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2.3. Culture of Helicoverpa zea
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The strain of H. zea used in this study was obtained from the insectary at North Carolina State University (Raleigh, NC) in 2009 and was continuously maintained in a controlled-environment room in our laboratory as described (Zhang et al., 2011). Larvae were reared in 32-cell rearing trays and fed an artificial diet (BioServ). Non-diapausing individuals were generated by rearing at 25 °C with a photoperiod of LD 15:9. Diapausing pupae were induced by transferring early third instar larvae to 21 °C at LD 8:16 until pupation, conditions that yielded a pupal diapause incidence of 60%. Migration of the eyespots was used as an easy and reliable indictor of diapause status (Phillips and Newsom, 1966): eyespots of diapausing pupae remain in the middle of the eye during pupal diapause but migrate to the edge of the eye at the onset of development and finally disappear.
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2.4. Culture of Sarcophaga crassipalpis
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A recently-established colony (collected in 2014) of S. crassipalpis was maintained as described (Denlinger, 1972). To generate pupal diapause, adults, larvae and pupae were reared at 25 °C under short-day conditions (LD 9:15).
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2.5. Bioassay for diapause termination in pupae of A. pernyi
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A KK-42 solution (4 ll) was injected into 18-week-old diapausing pupae of A. pernyi. Controls were injected with 4 ll acetone. Mean fresh body weights for male and female pupae were 6.6 and 8.8 g per pupa, respectively. The needle of the microsyringe (Hamilton) used for injections was inserted into the first intersegmental membrane of the pupal abdomen. Injected pupae were kept at 25 °C at LD 15:9, conditions that prompt the termination of diapause in this species. Some injected pupae were also stored at 25 °C under a short day photoperiod (LD 9:15). Three replicates
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Fig. 1. Structure of KK-42 and its activity in B. mori. (A) Structure of KK-42 and its CAS number. (B) KK-42 activity showing precocious pupation in B. mori. The smaller cocoons were produced using a 1 lg topical application of KK-42 to third instar larvae of B. mori; the larger controls were treated with acetone.
Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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of 12 pupae were evaluated for each test. Diapause status of the pupae was checked and success of adult emergence was recorded.
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2.6. Bioassays using H. zea
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Diapause termination assays in H. zea used diapausing pupae that were kept at 21 °C for 2 weeks after pupation to verify diapause status. Two-week-old diapausing pupae received a 2 ll topical application to the head of KK-42 dissolved in acetone; a topical application of acetone served as the control. Mean fresh body weight of pupae was 0.3 g. After treatment, pupae were kept at 22 °C or 30 °C, depending on the experiments, with a LD 8:16 photoperiod. Each experiment consisted of three replicates, with 12–16 individuals in each replicate. Diapause status of the pupae was checked at 2–3 days intervals following topical applications. To determine if KK-42 could affect the decision to enter diapause, both diapause-programmed (21 °C, LD 8:16) and nondiapause-programed (25 °C, LD 15:9) final instar larvae received a 2 ll topical application of KK-42 dissolved in acetone. Larvae were returned to their respective rearing conditions following treatment. Pupal diapause incidence was determined 7 days after pupation for nondiapause-programmed individuals and 2 weeks after pupation for diapause-programmed individuals. Each experiment consisted of three replicates, with 12–16 individuals per replicate.
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2.7. Bioassay using S. crassipalpis to evaluate diapause incidence
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Under our experiment conditions for culturing S. crassipalpis, larvae complete feeding and initiate wandering six days after larviposition. Three days after larviposition (early third instar) larvae were fed homogenized beef liver (15 g) containing KK-42.
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Acetone-treated liver served as the negative control, and imidazole-supplemented liver was used as a positive control (Denlinger, 1976). Larvae were allowed to complete their feeding on the supplemented liver for the remainder of their feeding period (2–3 days). Thirty individuals were tested in each of three replicates. The incidence of pupal diapause was investigated 30 days after pupariation, at which time nondiapause-programmed individuals had emerged as adults.
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2.8. Statistical analysis
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Student’s t test was performed using the online server http:// www.physics.csbsju.edu/stats/t-test.html. Data were subjected to an unpaired t test to generate two-tailed P values. A nonlinear trend line and the ED50 of 20-E for diapause termination was calculated using GraphPad Prism 5 (GraphPad Software).
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3. Results
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3.1. Development of a biomarker for diapause termination in A. pernyi
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Earlier experiments described a pigment-free region of cuticle (a transparent window) above the brain, a region that is critical for the detection of long daylengths that terminate diapause in this species (Williams and Adkisson, 1964b). When pupae of A. pernyi are in diapause this window remains transparent and the brain can be easily seen (Fig. 2A). We examined this window following injection of 20-E, a trigger for diapause termination, to identify possible markers for diapause termination. A range of 20-E doses (5–30 lg) were injected into diapausing pupae, and pupae subsequently placed at 23 °C, LD 9:15 successfully terminated diapause, with an ED50 of approximately 10 lg/pupa (Fig. 2B). By examining
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Fig. 2. Developmental marker and effects of KK-42 and 20-E on the diapausing pupae of A. pernyi. Error bars indicate SDs from three replicates; a total of 12 individuals were tested in each replicate. (A) The ‘‘window’’ above the brain in diapausing and developed pupae. The ‘‘window’’ of diapausing pupae remains transparent, revealing the brain, but this window becomes milky white at the outset of development and finally red as development proceeds. (B) Dose–response curve of 20-E for terminating pupal diapause of A. pernyi. The 20-E-treated diapausing pupae were placed at 23 °C and LD 9:15. Diapause status was checked 12 day after treatment, based on the coloration of the ‘‘window’’ over the brain. (C) Incidence of adult emergence (diapause termination) in pupae 24 days after treatment with KK-42, and (D) developmental status of pupae 60 days after treatment of diapausing pupae with KK-42. Eighteen-week-old diapausing pupae were injected with 100 lg of KK-42. Treated pupae were kept at 25 °C and LD 15:9. A very significant difference between the treated and acetone group is shown by asterisks (Student t test: ⁄⁄⁄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.)
Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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changes in the cuticular window we documented a progression of changes following diapause termination. The window gradually lost its transparency, became milk white at day 10 after treatment, and 17 days after diapause termination this region of the cuticle turned red and was completely opaque. These developmental changes thus provided a reliable developmental marker to detect the timing of diapause termination in response to KK-42. 3.2. KK-42 delays diapause termination when applied to pupae of A. pernyi We injected 100 lg KK-42 into 18-week-old diapausing pupae to determine whether KK-42 could alter the timing of diapause termination in A. pernyi. Under short day conditions (conditions that prevent diapause termination), both KK-42-treated and control pupae remained in diapause for at least 60 days after treatment. However, under a long day photoperiod (the environmental cue for diapause termination), KK-42 altered the expected diapause response. All control pupae treated with acetone terminated diapause within 8 days, but none of the individuals treated with KK42 terminated diapause during this time interval. By 24 days, 50% of the KK-42 treated pupae still remained in diapause (Fig. 2C), indicating an extremely significant extension of the diapause period (Student’s t test: P < 0.001). While all pupae receiving an injection of acetone emerged as adults in 20–24 days, the first adults emerged from KK-42-treated individuals on day 27, 50% by day 39, and 100% by day 60 (Fig. 2D). The results suggested that, under diapause-terminating environmental conditions, KK-42 delays the termination of pupal diapause in A. pernyi but does not impede the eventual completion of adult differentiation and eclosion.
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3.3. KK-42 delays diapause termination in pupae of H. zea
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Two-week-old diapausing pupae of H. zea received a 50 lg topical application of KK-42 and were subsequently held at either 22 or 30 °C to evaluate the impact of this treatment on diapause. In this species, high temperature is used as an environmental signal for breaking diapause (Meola and Adkisson, 1977). At 22 °C (a condition that normally maintains diapause), both KK-42-treated and
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control pupae remained in diapause 7 day after treatment, but at 30 °C (a condition known to terminate diapause) an effect of KK42 was evident. Most control pupae (78%) treated with acetone developed within 7 days after treatment, but only 22% of pupae treated with KK-42 terminated diapause (Fig. 3A), a difference that was highly significant (t test: P < 0.001). A range of KK-42 doses from 0 to 100 lg were evaluated and a dose–response relationship was established (Fig. 3B). In this experiment, KK-42-treated pupae were placed at 30 °C, and diapause status was checked 5 days later. The response curve shown in Fig. 3B shows a progressive decline in diapause termination from 75% to 15% between 0 and 100 lg KK-42, with an ED50 of approximately 10 lg/pupa. These results indicate that the observed effect in preventing the termination of pupal diapause was dose-dependent and not the consequence of an excessive dose. The timing of diapause termination at 30 °C, LD 8:16 (Fig. 3C) indicated that all pupae treated with acetone alone terminated diapause within 13 days, but at that time only 60% of the pupae receiving 100 lg of KK-42 terminated diapause. All KK-42-treated pupae terminated diapause within 21 days, thus the effect did not permanently block diapause termination and adult eclosion, but simply delayed the timing of diapause termination. Among pupae kept at 22 °C and LD 8:16 for 30 day after treatment, 40% of controls broke diapause, but diapause was not broken in any of the KK-42 injected pupae (data not shown). This result indicates that the effect of KK-42 on pupal diapause is not temperature-dependent and can thus exert its effect at both high and low temperatures. Thus, results observed in pupae of H. zea were similar to our observations with pupae of A. pernyi. In both species KK-42 delayed the termination of pupal diapause.
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3.4. KK-42 increases pupal diapause incidence when applied to larvae of H. zea
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Rearing at 21 °C and LD 8:16 yielded a pupal diapause incidence in H. zea of approximately 90% at the first harvest day, but in order to lower the diapause incidence to approximately 60% we transferred larvae at the midpoint of the final larval instar to 25 °C
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Fig. 3. Effect of KK-42 on diapause termination in pupae of H. zea. Two-week-old pupae were applied topically with KK-42. Treated pupae were kept at 30 °C and LD 8:16. Error bars indicate SDs from three replicates; a total of 12–16 individuals were tested in each replicate. (A) Incidence of diapause termination in pupae of H. zea pupae, checked 7 days after KK-42 application. A very significant difference between the KK-42 and acetone groups is shown by asterisks (Student t test: ⁄⁄⁄P < 0.001). (B) Dose– response curve showing the effect of KK-42 in delaying pupal diapause termination in H. zea. Diapause status was checked 5 day after KK-42 application. (C) Development duration of diapausing pupae treated with different doses of KK-42. Diapause status was checked on the indicated days.
Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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and LD 15:9 for 1 day. Larvae in the final larval instar received a topical application of 50 lg KK-42, and the incidence of pupal diapause was determined (Fig. 4A). Controls receiving acetone had a diapause incidence of 62%. Topical application of KK-42 to the larvae increased the incidence of pupal diapause to 95%, an effect that was highly significant (t test: P < 0.001). A lower dose of KK-42 (5 lg) also showed a significant increase in the incidence of pupal diapause (77%; t test: P < 0.05). Duration of the final larval instar was also increased (1–4 days) by treating larvae with KK-42 (data not shown). Additionally, the effect of KK-42 was stage-dependent; the incidence of pupal diapause was higher when KK-42 was applied to larvae during the feeding stage rather than during the post-feeding stage (Fig. 4B). 3.5. KK-42 increases and imidazole decreases pupal diapause incidence when fed to larvae of S. crassipalpis To evaluate the effect of KK-42 on pupal diapause incidence in S. crassipalpis young third instar larvae (3 days after larviposition) were transferred to homogenized beef liver supplemented with KK-42 (Fig. 5A). Pupae reared as larvae on acetone-treated liver had a diapause incidence of 65%, whereas those fed a diet augmented with 20 lg KK-42 exhibited a significantly higher diapause incidence (85.5%) (t test: P < 0.01). Earlier results indicated that imidazole, a compound related to KK-42, reduces the incidence of pupal diapause in this fly (Denlinger, 1976), and we noted a similar reduction in diapause (18%) in our current experiments (Fig. 5B, t test: P < 0.001). Thus, KK-42 appears to boost the diapause incidence when administered to larvae programmed for diapause, while the related imidazole exerts the opposite effect. 3.6. KK-42 was not effective in inducing pupal diapause when applied to nondiapause-programmed larvae of H. zea We topically applied 50 lg KK-42 to final instar larvae of H. zea reared under environmental conditions that normally produce
Fig. 4. Diapause incidence of H. zea pupae when KK-42 was administered topically to larvae in the final larval instar at (A) different doses or (B) different stages. Larvae were reared at 21 °C and LD 8:16. Pupal diapause incidence was calculated 15 days after pupation. Error bars indicate SDs from three replicates; a total of 12–16 individuals were tested in each replicate. Differences between KK-42 and acetonetreated groups are shown by asterisks (Student t test: ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄ P < 0.001).
Fig. 5. The incidence of pupal diapause in S. crassipalpis when (A) KK-42 or (B) imidazole were incorporated into the larval diet of beef liver. Both adults and larvae were reared at 25 °C and LD 9:15. The pupal diapause incidence was calculated 30 days after pupariation. Error bars indicate SDs from three replicates; a total of 30 individuals were tested in each replicate. Differences between the treated and control groups are shown by asterisks (Student t test: ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001).
nondiapause individuals. All treated individuals emerged as adults without entering diapause, as did non-treated controls, and no extension of the pupal instar was observed, an observation consistent with a previous report (Wang et al., 1999). This same response was observed over a broad concentration range (5–100 lg) of KK42 applied to nondiapause-programmed larvae during the third and fourth instars. These results consistently indicated that KK-42 was not effective in inducing pupal diapause in H. zea if the larvae were reared under environmental conditions that do not normally promote diapause.
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4. Discussion
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Earlier studies demonstrated that KK-42 can terminate embryonic diapause in A. yamamai (Suzuki et al., 1989; Kuwano et al., 1991) and L. dispar (Suzuki et al., 1993; Lee and Denlinger, 1996), as well as reduce the incidence of embryonic diapause in B. mori when administered to the mother during her final larval instar (Wu et al., 1996). By contrast, our results show that KK-42 can delay termination of the pupal diapauses in A. pernyi and H. zea, and boost pupal diapause incidence when administered to larvae of H. zea and S. crassipalpis. Several chemicals are capable of affecting diapause, but they are typically effective on only a single species or diapause stage, thus KK-42 is rather unique in being capable of influencing diapause in both different species as well as different diapausing stages of development. How does KK-42 elicit what would appear to be opposite responses in these different diapauses? KK-42 appears to act by inhibiting ecdysteroid biosynthesis within the prothoracic gland (PG), without killing the cells (Yamashita et al., 1987; Kiuchi and Akai, 1988; Wang and Sehnal, 2001; Shiotsuki and Kuwano, 2004). KK-42 prevents pupal-adult development when administered to pupae of B. mori (Kadono-Okuda et al., 1987; Zhou et al., 1991), and thus impairment of ecdysteroid synthesis in KK-42 treated insects is consistent with the known role of ecdysteroids in promoting continuous development and terminating pupal
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Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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diapause (Denlinger, 2008). A drop in the ecdysteroid titer is critical for the onset of pupal diapause, and a rise in ecdysteroids is essential to re-initiate development when pupal diapause is terminated in both H. zea and S. crassipalpis (Zhang et al., 2009), and in the current study we provide evidence that ecdysteroids are also critical for pupal diapause termination in A. pernyi. Thus, the results we present here for pupal diapause are likely the result of KK-42 lowering the effective ecdysteroid titer in prediapausing pupae, shunting more into diapause and by retarding the synthesis of ecdysteroids at the end of diapause, thereby delaying the onset of adult development. We envision that, with time, the effect of KK-42 applied to diapausing pupae declines, allowing the activated prothoracic gland to regain the ability to produce ecdysteroids, as suggested by previous work (Fugo and Oshiti, 1988). The diapauseterminating effect of KK-42, at least in the embryonic diapause of L. dispar, is consistent with impairment of ecdysteroid production and/or action. In this case, the embryonic diapause is actually maintained by an elevated titer of ecdysteroids, and a drop in ecdysteroids is essential for the onset of development (Lee and Denlinger, 1996), thus diapause can be broken prematurely in L. dispar by using KK-42 to decrease the effective titer of ecdysteroids. These differing roles for ecdysteroids in embryonic and pupal diapause can thus explain how the same compound can influence these two types of diapause differently. Many, but not all, of the effects of KK-42 are consistent with impairment of ecdysteroid production or action. Such effects encompass a range of responses including precocious metamorphosis (Kuwano et al., 1985; Gelman et al., 1995; Darvas et al., 1989), abnormal metamorphosis (Roussel et al., 1989; Pratt et al., 1990), breakage of embryonic diapause (Suzuki et al., 1989, 1993; Kuwano et al., 1991; Lee and Denlinger, 1996), reduction in the incidence of embryonic diapause (Wu et al., 1996), as well as our current results with pupal diapause. KK-42 also exerts some effects that have been interpreted as juvenile hormone (JH) or antiJH related (Kuwano et al., 1985; Kiuchi et al., 1985; Gelman et al., 1995). It has also been proposed that KK-42 exerts anti-JH or antiecdysteroid effects by inhibiting cytochrome P-450-dependent monooxygenases (Jarvis et al., 1994; Unnithan et al., 1995), an enzyme involved in the final steps of JH and ecdysteroid synthesis. Other evidence has suggested that the molecular action of KK-42 is to induce JH esterase gene expression (Hirai et al., 2002), thus the possibility that KK-42 affects proteins related to regulation of JH cannot be excluded (Shiotsuki and Kuwano, 2004; Shiotsuki, 2012). Diapause hormone (DH) also plays a critical role in regulating embryonic diapause of B. mori (Yamashita, 1996) and pupal diapause in members of the Heliothis/Helicoverpa complex (Zhang et al., 2004, 2008, 2009), although the responses are opposite: in B. mori DH alters carbohydrate metabolism in the ovary of the mother and promotes diapause, while DH in the heliothine moths acts to terminate pupal diapause. Like prothoracicotropic hormone (PTTH), DH appears to act on the prothoracic gland of the heliothine moths to stimulate synthesis of ecdysteroids, thereby terminating pupal diapause (Zhang et al., 2004). Yet the actions of DH and PTTH are not identical; ecdysteroids can terminate diapause at any temperature, while DH is effective only at temperatures above 21 °C (Zhang et al., 2008). Thus, KK-42 could also be operating through DH to affect diapause, but this possibility remains to be tested. KK-42 also exhibits interesting biological effects beyond insects, such as promoting growth of shrimp Penaeus schmitti (Ning et al., 2007) and increasing survival of the oriental river prawn Macrobrachium nipponense (Wang et al., 2013a,b), possibly by activating prophenoloxidase, a key component of the crustacean immune defense system. Thus KK-42 is known to have widespread biological effects on diverse arthropods and their close relatives. Regardless of its precise molecular mode of action, KK-42 appears to bind to several different molecules (Shiotsuki and
Kuwano, 2004). A putative 220 kDa KK-42-binding protein has been obtained from the PG of pupae in B. mori (Shiotsuki and Kuwano, 2004), and a 45 kDa KK-42-binding protein was isolated from pharate first instar larvae of A. yamamai (Shimizu et al., 2002), as well as from post-diapause pupae of A. pernyi (Li et al., 2009; Liu et al., 2012). What is particularly attractive about KK-42 is that it is active when applied topically, thus KK-42 and related compounds have the potential to exert their effect without being injected or ingested. This raises the prospect that KK-42 and others in this family of compounds can be used to penetrate the insect cuticle and manipulate diapause and other developmental responses in an array of inset species.
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This work was supported in part by National Natural Science Foundation of China (No. 31372372) to YL, China Scholarship Fund (No. 201208210112) awarded to YL by the China Scholarship Council, and by USDA-NIFA Grant No. 2011-67013-30199 to DLD.
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Contents lists available at ScienceDirect
Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys 5 6
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Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species
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Yanqun Liu a,b,⇑, Qirui Zhang b, David L. Denlinger b,⇑
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a b
Department of Sericulture, College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang 110866, China Departments of Entomology and Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 43210, USA
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 5 February 2015 Accepted 9 February 2015 Available online xxxx Keywords: KK-42 Pupal diapause Antheraea pernyi Helicoverpa zea Sarcophaga crassipalpis
a b s t r a c t The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause. Ó 2015 Published by Elsevier Ltd.
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1. Introduction
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Chemical and environmental tools that can be used to manipulate the expression of insect diapause not only provide insights into physiological mechanisms regulating diapause but also potentially offer methods for manipulating laboratory colonies to facilitate rearing or for disrupting field populations as a tool for pest management (Denlinger, 2008). For example, a number of chemical agents are highly effective in breaking diapause, e.g. mild acids for embryonic diapause of the silkworm Bombyx mori (cf., Yamamoto et al., 2013), hexane for pupal diapause in the flesh fly Sarcophaga crassipalpis (Denlinger et al., 1980); several agents do not exert an immediate diapause-terminating effect but may shorten diapause (e.g. juvenile hormone in S. crassipalpis (Denlinger et al., 1988)) or delay its termination (e.g. cyclic AMP
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⇑ Corresponding author at: Department of Evolution, Ecology, and Organismal Biology, The Ohio State University, Columbus, OH 43210, USA (Y. Liu). Tel.: +1 614 292 6425; fax: +1 614 292 2030. E-mail addresses: [email protected] (Y. Liu), [email protected] (D.L. Denlinger).
in flesh flies (Denlinger and Wingard, 1978)). In addition, a number of agonists and antagonists of diapause hormone have been developed as potent manipulators of pupal diapause in heliothine moths (Zhang et al., 2011). Among agents known to alter diapause responses is KK-42 (1benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole), an imidazole derivative (Fig. 1A). This chemical functions as an insect growth regulator that can cause precocious metamorphosis in the silkworm B. mori (Kuwano et al., 1985), the European corn borer Ostrinia nubilalis (Gelman et al., 1995), the flesh fly Sarcophaga bullata (Darvas et al., 1989), and induces abnormal metamorphosis in the locust Locusta migratoria (Roussel et al., 1989) and in the Hawaiian cockroach Diploptera punctata (Pratt et al., 1990). KK42 has also been reported to break embryonic diapause in the Japanese oak silkworm Antheraea yamamai (Suzuki et al., 1989; Kuwano et al., 1991) and the gypsy moth Lymantria dispar (Suzuki et al., 1993; Lee and Denlinger, 1996), and to reduce the incidence of embryonic diapause in progeny of B. mori that are programmed to produce diapausing embryos (Wu et al., 1996). Whether KK-42 influences insect diapauses that occur in stages other than the embryo remains unknown. In the experiments we
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report here we used two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and one species of Diptera, the flesh fly S. crassipalpis, to investigate whether KK-42 can also influence pupal diapause. We chose A. pernyi as one of the model insects to address this issue because it is not only a classic organism for studying diapause regulation (Williams and Adkisson, 1964a,b; Sauman and Reppert, 1996; Wang et al., 2013a,b), but it is also an economically important insect used for silk production and as a human food source in China (Liu et al., 2010). A. pernyi enters a facultative pupal diapause when larvae are reared under short daylengths and low temperature. This diapause can be averted under long day conditions (Williams and Adkisson, 1964a), or by exposure to long daylengths after storage at a low temperature (Takeda et al., 2011). The corn earworm is a member of the Helicoverpa/Heliothis complex, a worldwide group of noteworthy crop pests. Like other members of this pest complex, H. zea enters a facultative diapause as a pupa in the winter in response to short daylengths and low temperature of autumn (Phillips and Newsom, 1966). Pupal diapause of H. zea can be terminated within just a few days by high temperature at 27 °C (Meola and Adkisson, 1977) or with an injection of ecdysteroids or diapause hormone (Zhang et al., 2008, 2009). In contrast to A. pernyi, neither photoperiod nor chilling have an effect on termination of pupal diapause in this species (Roach and Adkisson, 1971). The flesh fly also enters pupal diapause in response to short daylengths and, like H. zea, breaks diapause upon the elevation of temperature (Denlinger, 1972). Like pupal diapauses in other insects (Denlinger et al., 2005), pupal diapause in the flesh fly appears to be the result of a shut-down of the brain-prothoracic gland axis (Zdarek and Denlinger, 1975). Pupal diapause in the flesh fly can be terminated not only by high temperature but also with a topical application of hexane (Denlinger et al., 1980) or with an injection of ecdysteroids (Zdarek and Denlinger, 1975). Our experiments on A. pernyi and H. zea provide the first evidence that KK-42 can retard the termination of pupal diapause when applied to diapausing pupae. Additionally, we show that KK-42 can be administered to the final instar larvae of H. zea and S. crassipalpis and thus increase the incidence of pupal diapause. Our results are consistent with a KK-42 mode of action that impairs and/or retards the production or action of ecdysteroids associated with diapause onset and termination. The fact that KK-42 is an agent that can readily penetrate the cuticle suggests it may have utility as a disruptor of the diapause response.
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2. Materials and methods
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2.1. Chemicals
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KK-42 was synthesized as described (Kuwano et al., 1985) by Dr. Xinghai Li (Department of Pesticide Science, Plant Protection College, Shenyang Agricultural University, Shenyang, China). Purity of the synthesized sample was determined by HPLC to be
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approximately 90%, and its activity to induce precocious metamorphosis in third instar larvae of B. mori was confirmed by topical application (Fig. 1B). KK-42 was dissolved in acetone and stored at 20 °C.
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2.2. Culture of Antheraea pernyi
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The univoltine strain of A. pernyi used in this experiment was reared on oak trees in the field at the Sericultural Experiment Station of Henan Province, Nanyang, China, until cocoon formation. Larvae pupated in early June, 2014 and within 2 weeks were sent to the Denlinger laboratory under U.S. Department of Agriculture permit No. P526P-14-01794. Pupae were held at 23 °C with a photoperiod of LD 9:15 until used in experiments. The 20-Hydroxyecdysone (20-E) used to terminate pupal diapause was from Sigma (St. Louis, MO).
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2.3. Culture of Helicoverpa zea
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The strain of H. zea used in this study was obtained from the insectary at North Carolina State University (Raleigh, NC) in 2009 and was continuously maintained in a controlled-environment room in our laboratory as described (Zhang et al., 2011). Larvae were reared in 32-cell rearing trays and fed an artificial diet (BioServ). Non-diapausing individuals were generated by rearing at 25 °C with a photoperiod of LD 15:9. Diapausing pupae were induced by transferring early third instar larvae to 21 °C at LD 8:16 until pupation, conditions that yielded a pupal diapause incidence of 60%. Migration of the eyespots was used as an easy and reliable indictor of diapause status (Phillips and Newsom, 1966): eyespots of diapausing pupae remain in the middle of the eye during pupal diapause but migrate to the edge of the eye at the onset of development and finally disappear.
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2.4. Culture of Sarcophaga crassipalpis
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A recently-established colony (collected in 2014) of S. crassipalpis was maintained as described (Denlinger, 1972). To generate pupal diapause, adults, larvae and pupae were reared at 25 °C under short-day conditions (LD 9:15).
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2.5. Bioassay for diapause termination in pupae of A. pernyi
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A KK-42 solution (4 ll) was injected into 18-week-old diapausing pupae of A. pernyi. Controls were injected with 4 ll acetone. Mean fresh body weights for male and female pupae were 6.6 and 8.8 g per pupa, respectively. The needle of the microsyringe (Hamilton) used for injections was inserted into the first intersegmental membrane of the pupal abdomen. Injected pupae were kept at 25 °C at LD 15:9, conditions that prompt the termination of diapause in this species. Some injected pupae were also stored at 25 °C under a short day photoperiod (LD 9:15). Three replicates
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Fig. 1. Structure of KK-42 and its activity in B. mori. (A) Structure of KK-42 and its CAS number. (B) KK-42 activity showing precocious pupation in B. mori. The smaller cocoons were produced using a 1 lg topical application of KK-42 to third instar larvae of B. mori; the larger controls were treated with acetone.
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of 12 pupae were evaluated for each test. Diapause status of the pupae was checked and success of adult emergence was recorded.
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2.6. Bioassays using H. zea
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Diapause termination assays in H. zea used diapausing pupae that were kept at 21 °C for 2 weeks after pupation to verify diapause status. Two-week-old diapausing pupae received a 2 ll topical application to the head of KK-42 dissolved in acetone; a topical application of acetone served as the control. Mean fresh body weight of pupae was 0.3 g. After treatment, pupae were kept at 22 °C or 30 °C, depending on the experiments, with a LD 8:16 photoperiod. Each experiment consisted of three replicates, with 12–16 individuals in each replicate. Diapause status of the pupae was checked at 2–3 days intervals following topical applications. To determine if KK-42 could affect the decision to enter diapause, both diapause-programmed (21 °C, LD 8:16) and nondiapause-programed (25 °C, LD 15:9) final instar larvae received a 2 ll topical application of KK-42 dissolved in acetone. Larvae were returned to their respective rearing conditions following treatment. Pupal diapause incidence was determined 7 days after pupation for nondiapause-programmed individuals and 2 weeks after pupation for diapause-programmed individuals. Each experiment consisted of three replicates, with 12–16 individuals per replicate.
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2.7. Bioassay using S. crassipalpis to evaluate diapause incidence
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Under our experiment conditions for culturing S. crassipalpis, larvae complete feeding and initiate wandering six days after larviposition. Three days after larviposition (early third instar) larvae were fed homogenized beef liver (15 g) containing KK-42.
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Acetone-treated liver served as the negative control, and imidazole-supplemented liver was used as a positive control (Denlinger, 1976). Larvae were allowed to complete their feeding on the supplemented liver for the remainder of their feeding period (2–3 days). Thirty individuals were tested in each of three replicates. The incidence of pupal diapause was investigated 30 days after pupariation, at which time nondiapause-programmed individuals had emerged as adults.
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2.8. Statistical analysis
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Student’s t test was performed using the online server http:// www.physics.csbsju.edu/stats/t-test.html. Data were subjected to an unpaired t test to generate two-tailed P values. A nonlinear trend line and the ED50 of 20-E for diapause termination was calculated using GraphPad Prism 5 (GraphPad Software).
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3. Results
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3.1. Development of a biomarker for diapause termination in A. pernyi
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Earlier experiments described a pigment-free region of cuticle (a transparent window) above the brain, a region that is critical for the detection of long daylengths that terminate diapause in this species (Williams and Adkisson, 1964b). When pupae of A. pernyi are in diapause this window remains transparent and the brain can be easily seen (Fig. 2A). We examined this window following injection of 20-E, a trigger for diapause termination, to identify possible markers for diapause termination. A range of 20-E doses (5–30 lg) were injected into diapausing pupae, and pupae subsequently placed at 23 °C, LD 9:15 successfully terminated diapause, with an ED50 of approximately 10 lg/pupa (Fig. 2B). By examining
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Fig. 2. Developmental marker and effects of KK-42 and 20-E on the diapausing pupae of A. pernyi. Error bars indicate SDs from three replicates; a total of 12 individuals were tested in each replicate. (A) The ‘‘window’’ above the brain in diapausing and developed pupae. The ‘‘window’’ of diapausing pupae remains transparent, revealing the brain, but this window becomes milky white at the outset of development and finally red as development proceeds. (B) Dose–response curve of 20-E for terminating pupal diapause of A. pernyi. The 20-E-treated diapausing pupae were placed at 23 °C and LD 9:15. Diapause status was checked 12 day after treatment, based on the coloration of the ‘‘window’’ over the brain. (C) Incidence of adult emergence (diapause termination) in pupae 24 days after treatment with KK-42, and (D) developmental status of pupae 60 days after treatment of diapausing pupae with KK-42. Eighteen-week-old diapausing pupae were injected with 100 lg of KK-42. Treated pupae were kept at 25 °C and LD 15:9. A very significant difference between the treated and acetone group is shown by asterisks (Student t test: ⁄⁄⁄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.)
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changes in the cuticular window we documented a progression of changes following diapause termination. The window gradually lost its transparency, became milk white at day 10 after treatment, and 17 days after diapause termination this region of the cuticle turned red and was completely opaque. These developmental changes thus provided a reliable developmental marker to detect the timing of diapause termination in response to KK-42. 3.2. KK-42 delays diapause termination when applied to pupae of A. pernyi We injected 100 lg KK-42 into 18-week-old diapausing pupae to determine whether KK-42 could alter the timing of diapause termination in A. pernyi. Under short day conditions (conditions that prevent diapause termination), both KK-42-treated and control pupae remained in diapause for at least 60 days after treatment. However, under a long day photoperiod (the environmental cue for diapause termination), KK-42 altered the expected diapause response. All control pupae treated with acetone terminated diapause within 8 days, but none of the individuals treated with KK42 terminated diapause during this time interval. By 24 days, 50% of the KK-42 treated pupae still remained in diapause (Fig. 2C), indicating an extremely significant extension of the diapause period (Student’s t test: P < 0.001). While all pupae receiving an injection of acetone emerged as adults in 20–24 days, the first adults emerged from KK-42-treated individuals on day 27, 50% by day 39, and 100% by day 60 (Fig. 2D). The results suggested that, under diapause-terminating environmental conditions, KK-42 delays the termination of pupal diapause in A. pernyi but does not impede the eventual completion of adult differentiation and eclosion.
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3.3. KK-42 delays diapause termination in pupae of H. zea
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Two-week-old diapausing pupae of H. zea received a 50 lg topical application of KK-42 and were subsequently held at either 22 or 30 °C to evaluate the impact of this treatment on diapause. In this species, high temperature is used as an environmental signal for breaking diapause (Meola and Adkisson, 1977). At 22 °C (a condition that normally maintains diapause), both KK-42-treated and
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control pupae remained in diapause 7 day after treatment, but at 30 °C (a condition known to terminate diapause) an effect of KK42 was evident. Most control pupae (78%) treated with acetone developed within 7 days after treatment, but only 22% of pupae treated with KK-42 terminated diapause (Fig. 3A), a difference that was highly significant (t test: P < 0.001). A range of KK-42 doses from 0 to 100 lg were evaluated and a dose–response relationship was established (Fig. 3B). In this experiment, KK-42-treated pupae were placed at 30 °C, and diapause status was checked 5 days later. The response curve shown in Fig. 3B shows a progressive decline in diapause termination from 75% to 15% between 0 and 100 lg KK-42, with an ED50 of approximately 10 lg/pupa. These results indicate that the observed effect in preventing the termination of pupal diapause was dose-dependent and not the consequence of an excessive dose. The timing of diapause termination at 30 °C, LD 8:16 (Fig. 3C) indicated that all pupae treated with acetone alone terminated diapause within 13 days, but at that time only 60% of the pupae receiving 100 lg of KK-42 terminated diapause. All KK-42-treated pupae terminated diapause within 21 days, thus the effect did not permanently block diapause termination and adult eclosion, but simply delayed the timing of diapause termination. Among pupae kept at 22 °C and LD 8:16 for 30 day after treatment, 40% of controls broke diapause, but diapause was not broken in any of the KK-42 injected pupae (data not shown). This result indicates that the effect of KK-42 on pupal diapause is not temperature-dependent and can thus exert its effect at both high and low temperatures. Thus, results observed in pupae of H. zea were similar to our observations with pupae of A. pernyi. In both species KK-42 delayed the termination of pupal diapause.
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3.4. KK-42 increases pupal diapause incidence when applied to larvae of H. zea
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Rearing at 21 °C and LD 8:16 yielded a pupal diapause incidence in H. zea of approximately 90% at the first harvest day, but in order to lower the diapause incidence to approximately 60% we transferred larvae at the midpoint of the final larval instar to 25 °C
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Fig. 3. Effect of KK-42 on diapause termination in pupae of H. zea. Two-week-old pupae were applied topically with KK-42. Treated pupae were kept at 30 °C and LD 8:16. Error bars indicate SDs from three replicates; a total of 12–16 individuals were tested in each replicate. (A) Incidence of diapause termination in pupae of H. zea pupae, checked 7 days after KK-42 application. A very significant difference between the KK-42 and acetone groups is shown by asterisks (Student t test: ⁄⁄⁄P < 0.001). (B) Dose– response curve showing the effect of KK-42 in delaying pupal diapause termination in H. zea. Diapause status was checked 5 day after KK-42 application. (C) Development duration of diapausing pupae treated with different doses of KK-42. Diapause status was checked on the indicated days.
Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
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and LD 15:9 for 1 day. Larvae in the final larval instar received a topical application of 50 lg KK-42, and the incidence of pupal diapause was determined (Fig. 4A). Controls receiving acetone had a diapause incidence of 62%. Topical application of KK-42 to the larvae increased the incidence of pupal diapause to 95%, an effect that was highly significant (t test: P < 0.001). A lower dose of KK-42 (5 lg) also showed a significant increase in the incidence of pupal diapause (77%; t test: P < 0.05). Duration of the final larval instar was also increased (1–4 days) by treating larvae with KK-42 (data not shown). Additionally, the effect of KK-42 was stage-dependent; the incidence of pupal diapause was higher when KK-42 was applied to larvae during the feeding stage rather than during the post-feeding stage (Fig. 4B). 3.5. KK-42 increases and imidazole decreases pupal diapause incidence when fed to larvae of S. crassipalpis To evaluate the effect of KK-42 on pupal diapause incidence in S. crassipalpis young third instar larvae (3 days after larviposition) were transferred to homogenized beef liver supplemented with KK-42 (Fig. 5A). Pupae reared as larvae on acetone-treated liver had a diapause incidence of 65%, whereas those fed a diet augmented with 20 lg KK-42 exhibited a significantly higher diapause incidence (85.5%) (t test: P < 0.01). Earlier results indicated that imidazole, a compound related to KK-42, reduces the incidence of pupal diapause in this fly (Denlinger, 1976), and we noted a similar reduction in diapause (18%) in our current experiments (Fig. 5B, t test: P < 0.001). Thus, KK-42 appears to boost the diapause incidence when administered to larvae programmed for diapause, while the related imidazole exerts the opposite effect. 3.6. KK-42 was not effective in inducing pupal diapause when applied to nondiapause-programmed larvae of H. zea We topically applied 50 lg KK-42 to final instar larvae of H. zea reared under environmental conditions that normally produce
Fig. 4. Diapause incidence of H. zea pupae when KK-42 was administered topically to larvae in the final larval instar at (A) different doses or (B) different stages. Larvae were reared at 21 °C and LD 8:16. Pupal diapause incidence was calculated 15 days after pupation. Error bars indicate SDs from three replicates; a total of 12–16 individuals were tested in each replicate. Differences between KK-42 and acetonetreated groups are shown by asterisks (Student t test: ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄ P < 0.001).
Fig. 5. The incidence of pupal diapause in S. crassipalpis when (A) KK-42 or (B) imidazole were incorporated into the larval diet of beef liver. Both adults and larvae were reared at 25 °C and LD 9:15. The pupal diapause incidence was calculated 30 days after pupariation. Error bars indicate SDs from three replicates; a total of 30 individuals were tested in each replicate. Differences between the treated and control groups are shown by asterisks (Student t test: ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001).
nondiapause individuals. All treated individuals emerged as adults without entering diapause, as did non-treated controls, and no extension of the pupal instar was observed, an observation consistent with a previous report (Wang et al., 1999). This same response was observed over a broad concentration range (5–100 lg) of KK42 applied to nondiapause-programmed larvae during the third and fourth instars. These results consistently indicated that KK-42 was not effective in inducing pupal diapause in H. zea if the larvae were reared under environmental conditions that do not normally promote diapause.
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Earlier studies demonstrated that KK-42 can terminate embryonic diapause in A. yamamai (Suzuki et al., 1989; Kuwano et al., 1991) and L. dispar (Suzuki et al., 1993; Lee and Denlinger, 1996), as well as reduce the incidence of embryonic diapause in B. mori when administered to the mother during her final larval instar (Wu et al., 1996). By contrast, our results show that KK-42 can delay termination of the pupal diapauses in A. pernyi and H. zea, and boost pupal diapause incidence when administered to larvae of H. zea and S. crassipalpis. Several chemicals are capable of affecting diapause, but they are typically effective on only a single species or diapause stage, thus KK-42 is rather unique in being capable of influencing diapause in both different species as well as different diapausing stages of development. How does KK-42 elicit what would appear to be opposite responses in these different diapauses? KK-42 appears to act by inhibiting ecdysteroid biosynthesis within the prothoracic gland (PG), without killing the cells (Yamashita et al., 1987; Kiuchi and Akai, 1988; Wang and Sehnal, 2001; Shiotsuki and Kuwano, 2004). KK-42 prevents pupal-adult development when administered to pupae of B. mori (Kadono-Okuda et al., 1987; Zhou et al., 1991), and thus impairment of ecdysteroid synthesis in KK-42 treated insects is consistent with the known role of ecdysteroids in promoting continuous development and terminating pupal
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diapause (Denlinger, 2008). A drop in the ecdysteroid titer is critical for the onset of pupal diapause, and a rise in ecdysteroids is essential to re-initiate development when pupal diapause is terminated in both H. zea and S. crassipalpis (Zhang et al., 2009), and in the current study we provide evidence that ecdysteroids are also critical for pupal diapause termination in A. pernyi. Thus, the results we present here for pupal diapause are likely the result of KK-42 lowering the effective ecdysteroid titer in prediapausing pupae, shunting more into diapause and by retarding the synthesis of ecdysteroids at the end of diapause, thereby delaying the onset of adult development. We envision that, with time, the effect of KK-42 applied to diapausing pupae declines, allowing the activated prothoracic gland to regain the ability to produce ecdysteroids, as suggested by previous work (Fugo and Oshiti, 1988). The diapauseterminating effect of KK-42, at least in the embryonic diapause of L. dispar, is consistent with impairment of ecdysteroid production and/or action. In this case, the embryonic diapause is actually maintained by an elevated titer of ecdysteroids, and a drop in ecdysteroids is essential for the onset of development (Lee and Denlinger, 1996), thus diapause can be broken prematurely in L. dispar by using KK-42 to decrease the effective titer of ecdysteroids. These differing roles for ecdysteroids in embryonic and pupal diapause can thus explain how the same compound can influence these two types of diapause differently. Many, but not all, of the effects of KK-42 are consistent with impairment of ecdysteroid production or action. Such effects encompass a range of responses including precocious metamorphosis (Kuwano et al., 1985; Gelman et al., 1995; Darvas et al., 1989), abnormal metamorphosis (Roussel et al., 1989; Pratt et al., 1990), breakage of embryonic diapause (Suzuki et al., 1989, 1993; Kuwano et al., 1991; Lee and Denlinger, 1996), reduction in the incidence of embryonic diapause (Wu et al., 1996), as well as our current results with pupal diapause. KK-42 also exerts some effects that have been interpreted as juvenile hormone (JH) or antiJH related (Kuwano et al., 1985; Kiuchi et al., 1985; Gelman et al., 1995). It has also been proposed that KK-42 exerts anti-JH or antiecdysteroid effects by inhibiting cytochrome P-450-dependent monooxygenases (Jarvis et al., 1994; Unnithan et al., 1995), an enzyme involved in the final steps of JH and ecdysteroid synthesis. Other evidence has suggested that the molecular action of KK-42 is to induce JH esterase gene expression (Hirai et al., 2002), thus the possibility that KK-42 affects proteins related to regulation of JH cannot be excluded (Shiotsuki and Kuwano, 2004; Shiotsuki, 2012). Diapause hormone (DH) also plays a critical role in regulating embryonic diapause of B. mori (Yamashita, 1996) and pupal diapause in members of the Heliothis/Helicoverpa complex (Zhang et al., 2004, 2008, 2009), although the responses are opposite: in B. mori DH alters carbohydrate metabolism in the ovary of the mother and promotes diapause, while DH in the heliothine moths acts to terminate pupal diapause. Like prothoracicotropic hormone (PTTH), DH appears to act on the prothoracic gland of the heliothine moths to stimulate synthesis of ecdysteroids, thereby terminating pupal diapause (Zhang et al., 2004). Yet the actions of DH and PTTH are not identical; ecdysteroids can terminate diapause at any temperature, while DH is effective only at temperatures above 21 °C (Zhang et al., 2008). Thus, KK-42 could also be operating through DH to affect diapause, but this possibility remains to be tested. KK-42 also exhibits interesting biological effects beyond insects, such as promoting growth of shrimp Penaeus schmitti (Ning et al., 2007) and increasing survival of the oriental river prawn Macrobrachium nipponense (Wang et al., 2013a,b), possibly by activating prophenoloxidase, a key component of the crustacean immune defense system. Thus KK-42 is known to have widespread biological effects on diverse arthropods and their close relatives. Regardless of its precise molecular mode of action, KK-42 appears to bind to several different molecules (Shiotsuki and
Kuwano, 2004). A putative 220 kDa KK-42-binding protein has been obtained from the PG of pupae in B. mori (Shiotsuki and Kuwano, 2004), and a 45 kDa KK-42-binding protein was isolated from pharate first instar larvae of A. yamamai (Shimizu et al., 2002), as well as from post-diapause pupae of A. pernyi (Li et al., 2009; Liu et al., 2012). What is particularly attractive about KK-42 is that it is active when applied topically, thus KK-42 and related compounds have the potential to exert their effect without being injected or ingested. This raises the prospect that KK-42 and others in this family of compounds can be used to penetrate the insect cuticle and manipulate diapause and other developmental responses in an array of inset species.
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Acknowledgements
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This work was supported in part by National Natural Science Foundation of China (No. 31372372) to YL, China Scholarship Fund (No. 201208210112) awarded to YL by the China Scholarship Council, and by USDA-NIFA Grant No. 2011-67013-30199 to DLD.
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Please cite this article in press as: Liu, Y., et al. Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species. Journal of Insect Physiology (2015), http://dx.doi.org/10.1016/j.jinsphys.2015.02.003
