Endocrine signals that break diapause

The most obvious and best understood factors that break diapause are the hormones that naturally regulate diapause. Although there are exceptions, most types of diapause are the consequence of a hormone deficiency (Denlinger et al., Reference Denlinger, Yocum, Rinehart and Gilbert2012). Most larval and pupal diapauses are initiated and persist as long as environmental cues prevent synthesis and/or release of ecdysteroids from the prothoracic gland, while most adult diapauses are the consequence of a failure of the corpora allata to produce a juvenile hormone (JH). Thus, the developmental hiatus can usually be broken when ecdysteroids are injected into diapausing larvae or pupae, and adult diapauses are routinely terminated by the application of JH. Many examples demonstrate the efficacy of such hormone treatments for diapause termination, but such methods are not without challenges. The major challenge is finding a dose that is neither too small nor too large, and successful breaking of diapause may require being able to sustain an elevated hormone titer for several days. Dosages of ecdysteroids that are too high can lead to hyperecdysonism, a condition in which development is initiated but not appropriately synchronized, leading to generation of abnormalities that prevent successful completion of development through to adulthood (Williams, Reference Williams1968, Ždárek and Denlinger, Reference Ždárek and Denlinger1975). A more moderate dose can be chosen to prompt immediate diapause termination with no adverse side effects. Doses that are too small will not break diapause immediately but may shorten the duration of diapause, as noted in the flesh fly S. crassipalpis (Ždárek and Denlinger, Reference Ždárek and Denlinger1975). In some examples using JH to terminate adult diapause, the hormone prompts the start of reproductive events such as egg laying, but the response is not always sustained and the adult reverts to a diapause-like state, as noted in the Colorado potato beetle Leptinotarsa decemlineata (Schooneveld et al., Reference Schooneveld, Sanchez and de Wilde1977). But, by tailoring and fine-tuning the dosage and duration of application to the test insect, diapause can usually be successfully broken using commercially available hormones or hormone analogs.

JH does not immediately terminate pupal diapause in the flesh fly S. crassipalpis, as do the ecdysteroids, but it can halve the duration of diapause if applied to third instar larvae just prior to diapause entry (Denlinger, Reference Denlinger, Bhaskaran, Friedman and Rodriquez1981). This effect is likely due to the elevated metabolic rate noted in such pupae (Denlinger et al., Reference Denlinger, Shukla and Faustini1984), an effect causing a more rapid depletion of energy reserves and consequently diapause termination (Hahn and Denlinger, Reference Hahn and Denlinger2011).

One of the more unusual endocrine diapause terminators is the neuropeptide diapause hormone (DH), a hormone first described for its action as an inducer of embryonic diapause in the commercial silkmoth Bombyx mori (Yamashita, Reference Yamashita1966). Curiously, this same neuropeptide terminates pupal diapause in species within the moth genera Heliothis and Helicoverpa, a large complex of agricultural pests (Xu and Denlinger, Reference Xu and Denlinger2003). A number of DH analogs have been generated, some which are as much as 50× more potent than DH in breaking diapause (Zhang et al., Reference Zhang, Nachman, Kaczmarek, Zabrocki and Denlinger2011). Although advances have been made in developing DH analogs capable of penetrating the insect cuticle (Zhang et al., Reference Zhang, Nachman, Kaczmarek, Kierus, Zabrocki and Denlinger2015), this still remains a formidable challenge. Attempts to incorporate DH and its analogs into food have thus far been unsuccessful in preventing or breaking diapause, thus injection still remains the best way to administer the neuropeptide, a feature that has obvious drawbacks for large scale use. Recent success in fusing DH with the protein transduction domain (PTD) of the human immunodeficiency virus-1 transactivator of transcription (TAT) provides a tool that prevents rapid degradation of DH in the insect gut, thus allowing new possibilities for oral administration of DH (Zhou et al., Reference Zhou, Li, Yuan, Zhang and Qu2015). Although oral administration of TAT-PTD-DH retards larval development, it has not so far been successful in altering the diapause response.

Like other insects with a pupal diapause, diapause in Heliothis/Helicoverpa can also be terminated with ecdysteroids, but the actions of ecdysteroids and DH are not identical. Ecdysteroids can break diapause at any temperature, but DH is effective only at temperatures of 21 °C or higher (Xu and Denlinger, Reference Xu and Denlinger2003). Diapause termination by ecdysteroids and DH share some common RNA and microRNA responses but also some distinctions (Reynolds et al., Reference Reynolds, Nachman and Denlinger2019), suggesting that different pathways may evoke mechanisms leading to the same developmental response.

Mediators of the endocrine signaling cascade can also be expected to trigger diapause termination. For example, cyclic nucleotides, especially cyclic AMP (cAMP) and cyclic GMP (cGMP), function as second messengers used by neuropeptides to deliver the message from membrane receptors to the nucleus within the cell. Such second messengers can thus be expected to mediate the action of brain-based neuropeptides such as prothoracicotropic hormone, allatostatin or allatoinhibins in activating or inhibiting endocrine glands such as the prothoracic gland or corpora allata. cGMP, for example, can terminate pupal diapause in the flesh fly S. crassipalpis, and co-injection of cGMP with 20-hydroxyecdysone is more potent than either agent alone (Denlinger and Wingard, Reference Denlinger and Wingard1978). cGMP co-injected with 20-hydroxyecdysone elicits a similar diapause-terminating effect on pupal diapause in the Bertha armyworm Mamestra configurata (Bodnaryk, Reference Bodnaryk1978). In both of these examples, cAMP is ineffective as a diapause terminator, but curiously cAMP is highly effective in breaking pupal diapause in the silkmoths Hyalophora cecropia and Antheraea peryni (Berry, Reference Berry1981). Such mediators, along with their analogs and antagonists, are potent manipulators of diapause, but the fact that there are species difference in the response highlights the diversity that may be expected. Many of the chemical triggers that can terminate diapause, as discussed below, also likely target one of the links in the endocrine cascade.

Synthetic analogs of juvenile hormone, especially methoprene and fenoxycarb, have been used successfully in a small-scale field trial to disrupt adult diapause of the apple blossom weevil Anthonomus pomorum (Ždárek et al., Reference Ždárek, Ctvrtecka, Hovorka and Koštál2000). Diapausing females exposed to methoprene show elevated locomotory activity, accompanied by a decrease in dry weight, elevated water content, depletion of carbohydrate stores, a decrease in cold hardiness and ultimately 100% mortality within four weeks.

Chemical triggers that break diapause

An array of chemical agents have been used to break diapause, as summarized in table 1. The earliest reports of such agents, summarized by Lees (Reference Lees1955), come from French and Japanese experiments with the commercial silkmoth Bombyx mori (Henneguy, Reference Henneguy1904; Kogure, Reference Kogure1933). Brushing or immersion of diapausing silkmoth eggs in warm dilute hydrochloric or sulfuric acids is quite effective in breaking diapause in this species, a technique enabling continuous propagation of this important commercial species. Diapausing eggs of the grasshopper Melanoplus differentialis treated with xylol and then supplied with water promptly initiate development (Slifer, Reference Slifer1946). In this species, the natural event responsible for terminating diapause appears to be access to water, and the addition of xylol destroys the waterproof coating on the chorion, allowing the diapausing embryo to access the water trigger.

Table 1. Non-hormonal chemical agents capable of breaking or preventing diapause

Vapors of xylol or carbon tetrachloride are capable of breaking larval diapause in the sugarbeet webworm Loxostege sticticalis (Pepper, Reference Pepper1973), and vapors or topical applications of a range of organic solvents are potent terminators of pupal diapause in the flesh fly S. crassipalpis (Denlinger et al., Reference Denlinger, Campbell and Bradfield1980). This effect on fly pupae was discovered by accident when acetone was used as a carrier for the application of juvenile hormone (Ždárek and Denlinger, Reference Ždárek and Denlinger1975). A more systematic survey of related solvents revealed that alkanes such as hexane, pentane, heptane, isooctane and cyclohexane are all extremely active, as is the alkene hexene and diethyl ether (Denlinger et al., Reference Denlinger, Campbell and Bradfield1980). A number of other chemicals also successfully break diapause in the flies (e.g. linoleic acid, dichloromethane, benzene), but mortality occurs during pharate adult development or around the time of adult emergence from the puparium, making them less useful as diapause terminators. Hexane is among the most effective agents that both breaks diapause and allows successful adult eclosion and subsequent reproduction. Hexane can be applied either topically, by immersion or by vapor exposure. As little as a 2 μl topical application to the surface of the pupa, a 15 min immersion of the intact puparium, or a 4 h exposure of the intact puparium to hexane vapors is adequate to elicit the effect. Within 15 min of a topical application of hexane the rate of oxygen consumption spikes dramatically (100-fold increase) in the fly pupae and then continues at a high rate consistent with the onset of development. Hexane has proven to be a valuable tool for generating detailed timelines of molecular events surrounding diapause termination and the onset of development in flesh flies. Head-ligated pupae or pupae that have been debrained do not respond to hexane, implying that the brain and/or prothoracic gland are essential for coordinating the response. Topical application of solvents is not effective in terminating pupal diapause in the tobacco hornworm Manduca sexta, but injection of either hexane or acetone is modestly effective (Denlinger et al., Reference Denlinger, Campbell and Bradfield1980). Hexane vapors are also modestly effective in breaking the obligate adult diapause of the red mason bee Osmia bicornis, but it is effective only on males and only after they have been chilled for several months (Scott Hayward, personal communication).

Urea is a potent diapause terminator in eggs of the cricket Teleogryllus commodus (Hogan, Reference Hogan1961), as is an application of gaseous ammonia (Hogan, Reference Hogan1964). High concentrations of urea are effective in breaking diapause in an embryo that has already been in diapause for some time, but much lower concentrations of urea can avert diapause if administered shortly after the diapause-programmed embryo has been deposited. Injection of ammonium acetate breaks larval diapause in the European corn borer Ostrinia nubilalis (Beck et al., Reference Beck, Shane and Garland1969). How urea and the ammonia compounds work is unclear, but ammonium acetate boosts excretion, suggesting a possible diapause link to the excretory system.

Pyruvate terminates pupal diapause in the cotton bollworm H. armigera (Wang et al., Reference Wang, Geng, Guan and Xu2018). Low pyruvate is linked to diapause initiation, while high pyruvate is associated with nondiapause development or the resumption of development at the termination of diapause. Simulating diapause termination by injecting pyruvate into diapausing pupae is effective in breaking diapause, a result suggesting an intriguing role for pyruvate in diapause regulation.

Overwintering diapause in queens of the bumblebee Bombus terrestris can be readily broken with CO₂ narcosis, a treatment that prompts egg laying within one week after treatment (Röseler, Reference Röseler1985). As discussed later, CO₂ narcosis not only breaks diapause but can be used to avert diapause entry as well (Amsalem and Grozinger, Reference Amsalem and Grozinger2017). It is not clear whether narcosis acts directly on the corpora allata (Larrere et al., Reference Larrere, Lavenseau, Tsei and Couillaud1993), but at some point JH synthesis is needed to initiate egg laying. Regardless of the mode of action, the utility of this technique allows year-round laboratory rearing, a feature that has greatly facilitated use of this species for experimentation (e.g. Amsalem et al., Reference Amsalem, Galbraith, Cnaani, Teal and Grozinger2015).

Anoxia, brought on by either CO₂ or N₂, is also effective as a diapause terminator in embryos of the false melon beetle Atrachya menetriesi, but in this case, the anoxia treatment works only on embryos that have already been chilled for 50 days at 7.5 °C (Kidokoro and Ando, Reference Kidokoro and Ando2006), suggesting perhaps that the treatment accelerates post-diapause development rather than actually terminating diapause. Another mechanism for breaking embryonic diapause in A. menetriesi is to place the diapausing eggs on filter paper saturated with mercuric chloride or other mercury compounds, at a concentration of approximately 5 ppm (Kurihara and Ando, Reference Kurihara and Ando1969). Dipping eggs in a mercuric chloride solution is equally effective (Kidokoro et al., Reference Kidokoro, Iwata, Takeda and Fujiwara2006).

In an insect relative, the brine shrimp Artemia franciscana, embryonic diapause normally requires freezing and/or desiccation for diapause termination, but the natural termination process can be short-circuited with a 4-h exposure to either hydrogen peroxide (H₂O₂) or nitric oxide (NO) (Robbins et al., Reference Robbins, Van Stappen, Sorgeloos, Sung, MacRae and Bossier2010). NO is effective at a lower concentration, but H₂O₂ results in more of the embryos breaking diapause.

The imidazole derivative KK-42 (1-benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole) is an effective diapause terminator in several insect species. KK-42 (Suzuki et al., Reference Suzuki, Minagawa, Kumagai, Naya, Endo, Osana and Kuwano1990), as well as numerous other imidazole compounds (Kuwano et al., Reference Kuwano, Fujisawa, Suzuki and Eto1991), are highly effective in breaking the pharate first-instar diapause of the wild silkmoth, Antheraea yamamai, presumably by suppressing the ecdysteroid titer. The topical application of imidazoles offers a simple tool that can be used to propagate this commercially important species without the delay of diapause. Diapause in the pharate first-instar larvae of the gypsy moth L. dispar can also be terminated promptly with KK-42 (Suzuki et al., Reference Suzuki, Nakamura, Yanbe, Kurihara and Kuwano1993, Bell, Reference Bell1996, Lee and Denlinger, Reference Lee and Denlinger1996). The gypsy moth diapause is maintained by a high ecdysteroid titer and a drop in the titer is required for diapause termination (Lee et al., Reference Lee, Valaitis and Denlinger1997). KK-42 likely exerts its diapause terminating effect by lowering the effective concentration of ecdysteroids in the embryo, thus providing an endocrine trigger for diapause termination and subsequent hatching of the larva. Having a tool such as KK-42 that works on the gypsy moth is especially attractive because this species has an obligate diapause that is otherwise difficult to terminate without a long time delay. Pupal diapause of H. armigera can also be broken with KK-42 (Wang et al., Reference Wang, Gong and Qin1999), but this response is not as easily explained. If KK-42 acts solely to suppress the ecdysteroid titer, one would not expect this to result in the termination of a pupal diapause. Attempts to break pupal diapause of the flesh fly S. crassipalpis with KK-42 have not been successful (Liu et al., Reference Liu, Zhang and Denlinger2015), thus this agent, like many diapause terminators, exerts its effect only within a narrow taxonomic range of species.

Physical triggers that break diapause

As with chemical triggers reported to break diapause, a number of physical manipulations are also documented as diapause terminators (table 2). Electric shocks have been known for quite some time to trigger diapause termination in embryos of the commercial silkmoth (Kogure, Reference Kogure1933). Although we still cannot say for certain how electric shocks work, they possibly disrupt electrical properties in membranes encapsulating neurosecretory products, thus prompting premature discharge of neuropeptides that stimulate development. Early reports on larval diapauses of the chloropid fly Lipara lucens (Varley and Butler, Reference Varley and Butler1933) and blow fly Lucilia sericata (Roubaud, Reference Roubaud1922) also show the efficacy of pricking and singeing as a trigger for diapause termination. The physical injury usually boosts the metabolic rate, and in diapausing pupae of the saturniid A. polyphemus injury from pricking is capable of breaking diapause (McDaniel and Berry, Reference McDaniel and Berry1967), an effect not noted in diapausing pupae of another saturniid, H. cecropia, or the flesh fly S. argyrostoma (Denlinger et al., Reference Denlinger, Willis and Fraenkel1972).

Table 2. Physical manipulations capable of breaking or preventing diapause

Light at night

Photoperiodic cues dominate in determining the timing of diapause entry (Danks, Reference Danks1987, Denlinger, Reference Denlinger2002). Most temperate latitude species rely on short daylength to program diapause entry. It is thus possible to use long daylengths in the laboratory to prevent diapause in insects with a facultative diapause, and several field trials have been at least partially successful in preventing diapause entry in Lepidoptera by using artificial light to either extend daylength in the field (Ankersmit, Reference Ankersmit1968, Hayes et al., Reference Hayes, Sullivan, Oliver and Schechter1970, Schechter et al., Reference Schechter, Hayes and Sullivan1971, Berlinger and Ankersmit, Reference Berlinger and Ankersmit1976) or by using pulses of light at strategic times during the night (Barker et al., Reference Barker, Cohen and Mayer1964, Hayes et al., Reference Hayes, Cawley, Sullivan, Adler and Schechter1974). Diapause can be prevented in the Kanzawa spider mite Tetranychus kanzawai using a 1-h light pulse with a light intensity of 2.5 kJm⁻² at wavelengths of 350–1050 nm in mid-scotophase (Shah et al., Reference Shah, Suzuki, Ghazy, Amano and Ohyama2011), a treatment that would likely prove too costly for field application. The growing problem of light pollution at night (Gaston, Reference Gaston2018) is suspected to be a potential problem affecting diapause and other life-history traits as demonstrated in the noctuid moth M. brassicae (van Geffen et al., Reference van Geffen, vanGrunsven, van Ruijven and Berendse2014). Issues of light intensity and photoperiod duration can also alter the efficacy of parasitoids used in greenhouses to combat whiteflies (Zilahi-Balogh et al., Reference Zilahi-Balogh, Shipp, Cloutier and Brodeur2006).

Hormonal, chemical and physical factors that can prevent diapause

When administered at the onset of diapause, the insect hormones that can break diapause, as discussed above, are also capable of preventing entry into diapause. Thus, ecdysteroids delivered at low levels can prevent most larval or pupal diapauses, DH and its analogs administered at this time can prevent entry into pupal diapause in the heliothine moths, and JH can prevent diapause entry in most adults.

Although pupal diapauses are primarily regulated by the absence of ecdysteroids, application of either an ecdysteroid or a JH analog can prevent pupal diapause in the flesh fly S. crassipalpis (Denlinger, Reference Denlinger1976). A rather high dose (10 μg) of the JH analog methoprene applied topically to newly ecdysed third instar (final) larvae prevents entry into pupal diapause. An ecdysteroid injection of 1 μg into post-fed third instar larvae averts pupal diapause and an even lower dose (0.01 μg) is effective if injected into young pupae immediately before the onset of diapause. Diapause can also be prevented in S. crassipalpis by injecting cholera toxin into larvae 24 h before pupariation (Denlinger, Reference Denlinger1976). Cholera toxin is an activator of adenylate cyclase, thus the elevated levels of cAMP generated likely trigger this effect, mimicking the action of a second messenger system that mediates the action of prothoracicotropic hormone or other neuropeptides involved in promoting continuous development. Other agents that are effective in averting pupal diapause when injected into post-feeding third instar larvae of S. crassipalpis include imidazole and pilocarpine, although neither are as effective as cholera toxin (Denlinger, Reference Denlinger1976).

Carbon dioxide narcosis, a treatment that breaks adult diapause in the bumble bee B. impatiens can also prevent diapause entry, allowing the queen bumble bee to completely bypass diapause and initiate egg laying (Amsalem and Grozinger, Reference Amsalem and Grozinger2017). This treatment for preventing diapause appears to be devoid of negative side effects that might impair flight, stress responses, or longevity. The effect appears to be a response to the carbon dioxide rather than the absence of oxygen, but it is still unclear how the effect is mediated.

Another manipulation effective in averting pupal diapause in the flesh fly S. crassipalpis is to rear larvae on a diet supplemented with deuterium oxide (Rockey and Denlinger, Reference Rockey and Denlinger1983). Deuterium oxide, heavy water, alters circadian cycles and thus likely averts diapause by preventing accurate discrimination between long and short daylengths. An injection of mescaline sulfate or lysergic acid diethylamide (LSD) into larvae of Pieris brassicae during their photosensitive period prevents entry into pupal diapause (Vuillaume and Berkaloff, Reference Vuillaume and Berkaloff1974). The effectiveness of these psychodysleptics is restricted to the photosensitive stage, suggesting the effect may be achieved by disrupting measurement of daylength or transduction of the daylength cue. Injection of ouabain, an inhibitor of active transport of certain inorganic cations, into adult females of B. mori prevents the female from laying diapausing eggs (Takeda and Hasegawa, Reference Takeda and Hasegawa1975), an effect possibly blocking the release of DH from the subesophageal ganglion. Dimethyl sulfoxide (DMSO) is also a potent preventer of diapause in B. mori (Yamamoto et al., Reference Yamamoto, Mase and Sawada2013), but the active window is rather brief. For DMSO to prevent diapause it must be administered within 24 h of oviposition. DMSO is as effective in diapause prevention and termination in B. mori eggs as is the long-time standard used for this purpose, HCl.

Once the programming of diapause has been completed, there are relatively few physical manipulations that can reverse the outcome. In the flesh fly S. crassipalpis, 5 days of wet treatment, desiccation for 3 days on silica gel, exsanguination (removing 15 μl of blood), inflation with air, starvation, cold shock and a 1-day heat shock at 33 °C are all ineffective in preventing the onset of pupal diapause (Denlinger, Reference Denlinger1976). Heating post-fed, third instar larvae at 33 °C for 2 days, however will avert pupal diapause (Denlinger, Reference Denlinger1976), as will heat treatment immediately after pupariation (Gibbs, Reference Gibbs1975). Also, if the wet treatment continues beyond 5 days, until the larvae are actually forced to pupariate in a wet substrate, pupal diapause incidence drops approximately 50% (Denlinger et al., Reference Denlinger, Giebultowicz, Adedokun, Sehnal, Zabza and Denlinger1988). Both high temperatures and a wet environment are conditions that a diapausing pupa would not be able to survive for very long, thus these unfavorable conditions apparently trigger a fail-safe reversal of the diapause program. One additional manipulation that is effective is to physically shake the larvae: fly larvae mounted on an oscillating platform shaker (160 oscillations min⁻¹) enter diapause at a significantly lower rate than undisturbed controls (Denlinger, Reference Denlinger, Bhaskaran, Friedman and Rodriquez1981).