Hormonal Control of Diapause

10  Hormonal Control of Diapause D.L. Denlinger Ohio State University, Columbus, OH, USA G.D. Yocum and J.P. Rinehart USDA-ARS Red River Valley Agricultural Research Center, Fargo, ND, USA © 2012 Elsevier B.V. All Rights Reserved

Summary A critical feature of the insect life cycle is the ability to shut down development and enter a period of diapause during inimical seasons. This chapter briefly discusses features of the seasonal environment that program this developmental arrest and examines endocrine processes that preside over this decision. Although many links in the pathway leading from reception of environmental signals to expression of the diapause phenotype remain poorly known, recent advances have defined new elements. Ecdysteroids,

juvenile hormones, and the neuropeptides that regulate their synthesis and release are well-known contributors to diapause regulation, but new roles for insulin signaling and newly discovered roles for additional neuropeptides have expanded our understanding of the regulatory schemes controlling diapause. Numerous physiological attributes are common to diapauses in different species and developmental stages, yet the diapause phenotype appears to have evolved numerous times using divergent molecular mechanisms.

10.1.  Introduction 10.2.  The Preparative Phase 10.2.1.  Making the Decision to Enter Diapause 10.2.2.  The Brain as Photoreceptor and Programmable Center 10.2.3.  Transduction of the Environmental Signals 10.3.  Endocrine Regulators 10.3.1.  Hormonal Basis for Embryonic Diapause 10.3.2.  Hormonal Basis for Larval and Pupal Diapauses 10.3.3.  Hormonal Basis for Adult Diapause 10.4.  Parallels to Other Dormancies

10.1.  Introduction The evolution of diapause is arguably one of the most critical events bolstering the success of insects. Having the capacity to periodically shut down development has enabled insects and their arthropod relatives to invade environments that are seasonally hostile. Indeed, very few environments permit continuous insect development. Even in the tropics, where temperatures throughout the year may be compatible with ectotherm development, seasonal patterns of rainfall drive cycles of plant growth that favor insects with the ability to periodically become dormant. Diapause is an arrest in development accompanied by a major shutdown in metabolic activity. Unlike a simple quiescence that is an immediate response to an unfavorable environmental condition, diapause is a genetically programmed response that occurs at a specific stage for each species. Sometimes the insect will enter diapause at this particular stage in each generation regardless of the DOI: 10.1016/B978-0-12-384749-2.10010-X

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environmental conditions it receives — a developmental program referred to as obligatory diapause. Much more frequently, the decision to enter diapause is determined by environmental factors, usually daylength, received by that individual or its mother at an earlier developmental stage. This is referred to as facultative diapause. Development can effectively be interrupted at many points, as evidenced by the rich diversity of developmental stages used by different species for diapause. Embryonic diapauses have been documented for nearly all possible stages of embryonic development, ranging from early blastoderm formation to pharate larvae. Larval diapauses are most common in late instars, but early instar diapauses are not uncommon. Pupal diapause is well documented, and a few species diapause as fully developed pharate adults, but, not surprisingly, there appear to be no instances where development is halted midway through pharate adult development. The most prevalent cases of adult

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diapause involve newly emerged adults that have not yet reproduced, but there are also examples of diapause interrupting periods of reproduction. Most commonly, insects enter diapause only once during their life cycle, but some species, especially those living at higher latitudes, may extend their development over several years, spending successive winters in diapauses of different stages. Some long-lived adults may also go through multiple diapauses to coordinate egg laying with favorable conditions. Diapause is programmed well in advance of its onset, which implies that there is a pre-diapause period that can be used to prepare the insect for the special conditions that lie ahead. Hence it is common for insects destined for diapause to have a somewhat prolonged period of pre-diapause development, attain a larger size, accumulate additional lipid reserves, and add additional waterproofing to their cuticle. Distinct coloration, often to match the insect’s overwintering environment, may accompany the entry into diapause. Insects entering a winter diapause frequently undergo metabolic adjustments to enhance cold hardiness. In some cases the cold hardiness is a component of the diapause program, but in other cases cold hardiness is invoked by a different set of ­environmental cues. The term diapause falsely suggests a period of arrest in which development comes to a complete halt. Diapause should not be regarded as a simple stop and restart of development. Andrewartha (1952) originally coined the term “diapause development” to refer to the ongoing progression of events that occurs during diapause and eventually results in its termination. It is evident that diapause is a dynamic process, as evidenced by characteristic changes in the utilization of energy reserves, systematic changes in patterns of oxygen consumption, changes in responsiveness to environmental stress and hormones, and distinct patterns and changes in gene expression. A progression of events must transpire before diapause can be terminated. In some cases, development is not completely halted. There are several examples in which developmental processes continue during diapause, albeit at much slower rates; for example, ovarian maturation during adult diapause in the mosquito Culex pipiens (Readio et al., 1999) and embryonic development during diapause in the pea aphid Acyrthosiphon pisum (Shingleton et  al., 2003). In some cases there can be profound differences even within the body of a single individual. For example, during pupal diapause in the tobacco hornworm, Manduca sexta, most tissues of the body are arrested, but the testicular stem cells continue to undergo mitotic division (Friedlander and Reynolds, 1992). These unique events of diapause, coupled with pre-diapause distinctions in diapause-­programmed individuals, suggest that it is most appropriate to view diapause as an alternate developmental pathway. Several comprehensive reviews cover select features of diapause including environmental (Tauber et  al., 1986;

Danks, 1987) and molecular (Denlinger, 2002; MacRae, 2010; Williams et al., 2010) regulation, circadian mechanisms involved in timekeeping (Saunders, 2002, 2010a,b), photoreceptors and clock genes (Goto et al., 2010; Kostal, 2011), the dynamics of diapause (Kostal, 2006), cold tolerance associated with diapause (Denlinger and Lee, 2010), impact of climate change (Bale and Hayward, 2010), and energy management during diapause (Hahn and Denlinger, 2007; 2011). The goal of this chapter is to complement the other reviews by highlighting major discoveries relevant to the hormonal control of diapause that have occurred subsequent to our previous reviews (Denlinger, 1985; Denlinger et al., 2005). Earlier findings are briefly summarized in this chapter, with emphasis on new developments.

10.2.  The Preparative Phase During the preparative phase, the insect must make the decision to enter diapause (and sometimes how long to remain in diapause) and make the accompanying alterations that will result in the sequestration of additional energy reserves and waterproofing agents as well as initiate specific behavioral programs needed to find protected sites appropriate for overwintering. Some species, such as the Monarch butterfly, Danaus plexippus, engage in long-distance flights to reach their overwintering site, but more commonly, insects programmed for diapause migrate to closer, buffered sites. When diapause is viewed as an alternate developmental program, these phases unique to diapause, even if they occur prior to diapause, must also be viewed as part of the diapause syndrome. These early distinctions underscore the fact that we cannot simply focus on the endocrine events that dictate the onset and termination of diapause. A comprehensive view of diapause also calls for an understanding of the control mechanisms regulating these early events, but regrettably we know very little about the events regulating early phases of this developmental pathway. For example, what endocrine signals direct a diapause-programmed insect to sequester more lipid reserves or coat its cuticle with additional waterproofing hydrocarbons? These observations suggest that the set points for lipid accumulation and synthesis of cuticular hydrocarbons differ in diapause- and non-diapause-destined individuals, distinctions most likely regulated by the endocrine system. The amounts of energy stores and the composition of those stores differ between diapause- and non-diapause-­ programmed individuals. A number of potential regulators of pre-diapause energy storage have been proposed (Hahn and Denlinger, 2011), including insulin signaling and its sister pathway, target of rapamycin (TOR) signaling, but few experiments have probed those pathways in relation to diapause.

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Figure 1  Sequence of signals required for the programming of pupal diapause in flesh flies (Sarcophaga). Reproduced with permission from Denlinger (1985).

10.2.1.  Making the Decision to Enter Diapause

For an obligate diapause, the decision to enter diapause is hard-wired. Diapause will be entered at a specific stage regardless of the environmental conditions. This is an arrangement common to species that have a single generation each year, such as the gypsy moth and the Cecropia silkmoth, although in both of these examples a few individuals fail to enter diapause. By contrast, a facultative diapause enables the insect to complete several generations per year, and the individual “decides” its own developmental fate based on the environmental cues it perceives. The plasticity of this arrangement offers flexibility and enables the insect to more closely coordinate development with the appropriate seasons. An additional variation of this theme is when the individual does not directly decide and the decision is made by the mother. The decision to diapause should not be viewed as a simple “yes/no” decision made at a single moment in time; it is the culmination of a series of events, as exemplified for the flesh fly example shown in Figure 1. This example shows that the programming of diapause can be averted at many points along the way, right up to the time that diapause is normally manifested. For the program to be enacted, all of the previous conditions must be fulfilled. Thus, the diapause program can be aborted at the last minute, but attempts to induce diapause by offering the correct signals only during the later phases of development are not sufficient to induce diapause. 10.2.1.1.  Reading the environmental cues  Short daylength, a reliable harbinger of winter, is the most common cue used for the programming of an overwintering diapause (Figure 2). The precision of this environmental token makes it a seasonal indicator of unparalleled reliability. The critical photoperiod, the transition point between photoperiods that elicit diapause and those that elicit non-diapause development, is usually quite sharp, implying that insects can readily distinguish daylength differences as small as 15 min (Denlinger, 1986). Insects that enter a summer diapause may use long daylengths instead of short daylengths for the programming of diapause (Masaki, 1980), and there

Figure 2  Critical photoperiod for diapause induction in populations of Sarcophaga bullata from Illinois (40° 15’N) and Missouri (38° 30’N) at 25°C. Reproduced with permission from Denlinger (1972).

are numerous examples of photoresponse curves showing restricted portions of the curve to be diapause inductive. Daylength appears to be measured as a threshold character, and only a few examples suggest that insects are capable of actually measuring changes in daylength (Tauber et al., 1986). The widespread importance of photoperiodism in regulating diapause induction is reflected by the fact that it is used almost universally by insects in all geographic areas excluding areas within approximately 5 degrees of the equator. Diapause is still prevalent in the equatorial region, but other cues such as temperature, rainfall, or plant volatiles replace photoperiod as the cue for diapause induction in that region (Denlinger, 1986). With a few exceptions, temperature also influences the decision to enter diapause. While it may act alone as the environmental cue used for diapause induction in some tropical species, temperature usually acts in concert with the photoperiod. As shown in Figure 3a, low temperature commonly increases the diapause incidence observed at short daylength without influencing the critical photoperiod, but in some cases the critical photoperiod

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(a)

(b)

Figure 4  The relationship between the photosensitive stage and the stage of diapause in three insect examples. Reproduced with permission from Denlinger (1985).

Figure 3  Low temperature can impact the photoperiodic response curve in two ways. (a) In some cases, the critical photoperiod remains the same but the incidence of diapause at short daylengths is higher. (b) In other instances, the critical photoperiod is also shifted toward longer daylengths at low temperature. Reproduced with permission from Denlinger (2001).

will shift toward longer daylengths at low temperature (Figure 3b). Under natural conditions, insects are simultaneously subjected to thermoperiods as well as photoperiods. Thermoperiod can sometimes substitute for a photoperiodic signal, and it presumably does so by linking to a common mechanism (van Houten and Veerman, 1990). Other factors contributing to the diapause decision include food presence (Fielding, 1990), food quality (Hahn and Denlinger, 2011; Liu et al., 2011), plant volatiles (Ctvrtecka and Zdarek, 1992), pheromones (Kipyatkov, 2001), and crowding (Desroches and Huignard, 1991; Saunders et al., 1999). The photosensitive stage, the stage that receives the photoperiodic cues used to program diapause, usually occurs fairly far in advance of the actual diapause stage, as noted in Figure 4. The photosensitive stage may be as brief as four days as observed in Sarcophaga crassipalpis, or it may encompass much of the pre-diapause period as noted in M. sexta. In the extreme example noted in the silkmoth Bombyx mori, the photosensitive period actually occurs in the previous generation. If diapause is to be programmed by daylength, it is imperative that the correct photoperiodic signals are received during this photosensitive period. At other times during development the length of the day is of no consequence for the programming of diapause. Thus, insects that rely on short days to program their diapause need to answer two questions: (1) Was the day short? and (2) How many short days were received? Answering the first question requires a sophisticated clock that can measure daylength with precision, and answering the second question requires a counter capable of summing the number of short days that comprise the photosensitive period (Saunders, 2002, 2010a).

10.2.1.2.  Letting mother decide  In a number of parasitic Hymenoptera, higher Diptera, and some Lepidoptera it is the mother that decides the diapause fate of her progeny. This arrangement may be particularly useful when the mother has access to environmental cues not readily accessible to her progeny. Most of these cases involve the mother determining the diapause status of her eggs. The embryos that enter diapause are often in an early stage of development and are not yet equipped with the sophisticated neural systems required to receive and respond to photoperiodic cues. Bombyx mori is in this category, and since quite a bit is known about diapause in this species it will be discussed separately (see Section 10.3.1.1.). In other cases, however, the mother influences the diapause fate of her progeny in their larval (e.g., Nasonia vitripennis) or pupal (e.g., S. bullata) stages. This clearly implies that the mother is not simply exerting some direct effect on her progeny as might be expected in an embryonic diapause, but that she is initiating an effect expressed at a much later stage of development. In spite of the intriguing nature of these maternal effects, very little is known about the mechanisms that regulate these transgenerational effects. In the flesh fly S. bullata a critical message that influences the maternal effect is transferred from the brain of the female to her ovary sometime between pupariation and shortly after adult emergence (Rockey et  al., 1989). Gamma aminobutyric acid (GABA) and octopamine are possibly involved in the transfer of information from mother to progeny (Webb and Denlinger,1998). Differential display of mRNA revealed a transcript unique to the ovaries of females that expresses the maternal effect, but the function of the protein it encodes is still unknown (Denlinger, 1998). How the mother can influence the diapause fate of her offspring offers an intriguing, and still unanswered, problem in development. This sort of transgenerational transfer of information (Ho and Burggren, 2010) appears to be ripe for investigating a potential epigenetic mechanism of regulation.

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10.2.2.  The Brain as Photoreceptor and Programmable Center

The conspicuous visual centers, the compound eyes and ocelli, sometimes function as the organs used for perception of photoperiodic information, but extraretinal reception of the light signal is equally common. Although there is no perfect correlation between the organs used for photoreception and phylogeny (Numata et al., 1997; Goto et al., 2010), some trends are emerging. For example, extraretinal reception seems to be particularly common among the Lepidoptera, whereas reliance upon both compound eyes and direct brain photoreception is evident among the Hemiptera, Coleoptera, and Diptera. In some cases both types of photoreception operate within the same species (Goto et al., 2010). In cases of extraretinal reception, blackening or ablating the compound eyes has no impact on the insect’s ability to respond to photoperiod. A translucent cuticular window or lightened regions of cuticle sometimes overlay the brain, facilitating transfer of light to extraretinal receptors within the brain. In classic experiments with the giant silkmoth, Antheraea pernyi, Williams and Adkisson (1964) elegantly demonstrated that the brain is the direct recipient of the light signal. This species terminates pupal diapause in response to long daylength, and by using a divided photoperiod chamber that offered diapause-terminating conditions to one half of the body and diapause-maintaining conditions to the other half, they were able to demonstrate that diapause can be broken only when the portion of the body containing the brain is exposed to diapause-­terminating conditions. This is true even when the brain is transplanted to the abdomen of the pupa. In yet another elegant demonstration of the brain’s role in photoreception, Bowen et  al. (1984) demonstrated that the brain of M. sexta, when exposed in vitro to short days, is capable of eliciting diapause when implanted into a debrained pupa. Likewise, larval brain–subesophageal ganglion complexes of B. mori can be photoperiodically programmed for diapause in vitro and elicit a diapause response when re-implanted into larvae (Hasegawa and Shimizu, 1987). A number of more recent cases also demonstrate a role for the compound eyes (Goto et  al., 2010). Females of the fly Protophormia terraenovae (Shiga and Numata, 1997), the cricket Modicogryllus siamensis (Sakamoto and Tomioka, 2007), and the bugs Plautia stali (Morita and Numata, 1999), Graphosoma lineatum (Nakamura and Hodkova, 1998), and Poecilocoris lewisi (Miyawaki et  al., 2003) cannot distinguish short days from long days after surgical removal of the compound eyes. Painting the compound eyes of the bug Riptortus ­clavatus with phosphorescent paint effectively extends the photoperiod, resulting in termination of diapause

(Numata and Hidaka, 1983). By contrast, painting the head region above the pars intercerebralis fails to terminate diapause, suggesting that the compound eyes are the dominant photoreceptors for this species. Not all ommatidia of the compound eye are involved in the photoperiodic response of R. clavatus. By selectively removing ommatidia from various regions of the compound eyes, it is evident that only ommatidia in the central region of the compound eye are involved in this response (Morita and Numata, 1997b). Evidence from circadian rhythms suggests that in some cases (e.g., P.  terraenovae) both retinal and extraretinal pathways contribute to rhythm entrainment within the same species (Hamasaka et  al., 2001). This also appears to be true for photoperiodism in some species such as P. stali (Morita and Numata, 1999). The diapause program resides within the brain and can be transferred from one individual to another by transplantation of the programmed brain, as demonstrated in the embryonic diapause of B. mori (Hasegawa and Shimizu, 1987), pupal diapause of M. sexta (Safranek and Williams, 1980; Bowen et  al., 1984), and S. crassipalpis (Giebultowicz and Denlinger, 1986), and adult diapause of the linden bug Pyrrhocoris apterus (Hodkova, 1992). In M. sexta the duration of diapause is also programmed within the brain prior to the onset of diapause. In this species, the number of short days the larva receives determines the length of the pupal diapause. Short days throughout produce a high incidence of diapause, but such a diapause is of short duration. By contrast, exposure to only a few short days yields a low incidence of diapause, but a diapause of long duration. Brain transplantation experiments show that information about the duration of the diapause can be transplanted along with the brain (Denlinger and Bradfield, 1981). Precisely which cells within the brain are involved in photoreception and storage of diapause information remains unknown in most cases, but future studies pinpointing photoreceptor pigments and specific clock genes involved in photoperiodism should reveal the correct target sites for this critical information. In the giant silkmoth A. pernyi, the response is localized within the dorsolateral region of the brain, the same region containing the lateral neurosecretory cells that produce prothoracicotropic hormone (PTTH), the neuropeptide that regulates diapause. Thus the cells involved in photoreception and the effector region that produces PTTH are together in the same region of the brain (Sauman and Reppert, 1996a). In the vetch aphid, Megoura viciae, antibodies directed against invertebrate and vertebrate opsins and phototransduction proteins consistently label an anterior ventral neuropil region of the protocerebrum (Gao et  al., 1999). Identification of this region is consistent with previous experiments using localized illumination and microlesions to identify the photoreceptor region.

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10.2.3.  Transduction of the Environmental Signals

When photoperiod is the primary cue leading to the induction of diapause, several key molecular components are essential. The first key component is a photoreceptor capable of distinguishing between the photophase and scotophase. Secondly, the insect must have the capacity to measure the length of each photophase (or scotophase), and thirdly, the insect must be able to sum the number of short (or long) days it has seen. Although our understanding of the molecular events involved in the transduction of the environmental signals remains in its infancy, several recent studies have proven insightful, and the early literature offers valuable clues about the processes involved. As discussed previously, the photoreceptive organ, and in some cases candidate cells, have been identified for several diapause models. Action spectra that elicit diapause have been described for several species, and in most cases the blue-green range of the visual spectrum is most sensitive to diapause induction (Truman, 1976), indicating that any putative photoreceptor involved in the diapause response must have features consistent with these early observations. How temperature exerts its effect on the diapause response is poorly understood. One attractive model proposed by Saunders (1971) for flesh flies suggests that low temperature simply increases the number of short days the larvae receive. In this system, 14 short days are required for diapause induction, and lowering the temperature retards development so a larger portion of the population experiences the required number of short days. Although this model is attractive for species with a long photosensitive period, such a system seems less likely to operate in other insects (including some closely related flesh fly species) that have a very short photosensitive period. In some cases, thermoperiod can completely substitute for photoperiod as an environmental cue. Adult diapause is induced in the predatory mite Amblyseius potentillae by a short-day photoperiod or by rearing under constant darkness with a thermoperiod of 16 h at 15°C and 8 h at 27°C (van Houten et  al., 1987). Exposing A. potentillae to short-day photoperiods or to short-day thermoper­ iods either concurrently or in succession is successful for inducing diapause, suggesting that a single physiological mechanism is integrating photoperiodic and thermoper­ iodic information to regulate diapause (van Houten and Veerman, 1990). The argument that a single mechanism is involved is further strengthened by the observation that mites reared on a diet deficient in carotenoids/vitamin A (see Section 10.2.3.1.) lose their ability to respond to either photoperiod or thermoperiod (van Houten et  al., 1987). By contrast, carotenoids/vitamin A are essential for the photoperiod response but not for the thermoperiodic response in the cabbage butterfly, Pieris brassicae (Claret,

1989). Clearly, species differences must again be evident in the integration of photoperiodic and thermoperiodic information. 10.2.3.1.  Cryptochromes and opsins  One of the most important quests in diapause research is to identify the photoreceptor pigment involved in this response. A number of proteins have been identified, but most appear to be involved in visual perception. Only a few are candidates for involvement in photoperiodism, especially given the blue light sensitivity of the diapause response and the common involvement of extraretinal light reception. One such candidate is the flavin-based blue light photoreceptor cryptochrome (CRY). This protein has been directly linked to the circadian pacemaker of the fruit fly Drosophila, acting to daily reset the clock upon initiation of the photophase as detailed in the next section. Null mutants of CRY exhibit poor synchronization during light cycling, whereas rhythmicity is present in constant darkness (Stanewsky et al., 1998). These features support a role for CRY in entrainment of the circadian clock by photoperiod. The possible role of CRY in diapause induction has yet to be established, although cry expression is upregulated in adult S. crassipalpis when the flies are reared at short as opposed to long daylengths (Goto and Denlinger, 2002). Another group of proteins linked to photoperiodism are the carotenoid/vitamin A associated opsin photoreceptors. A causal link with diapause has been established by rearing insects on carotenoid-free diets. Although several species, including Drosophila melanogaster (Zimmerman and Goldsmith, 1971) and B. mori (Shimizu et  al., 1981), retain circadian behavioral patterns when reared on a carotenoid-free diet, the induction of diapause can be affected in a number of species, including adults of the predatory mite A. potentillae (van Zon et  al., 1981; Veerman et al., 1985) and spider mite Tetranychus ­urticae (Bosse and Veerman, 1996), embryonic diapause of B. mori (Shimizu and Kato, 1984), and pupal diapause of P. brassicae (Claret, 1989; Claret and Volkoff, 1992). The opsin proteins have been linked to photoperiodism as well. Melanopsin has been implicated in circadian mechanisms in vertebrates. In mice, null mutants of melanopsin retain circadian rhythmicity in constant darkness, but lack photoperiodic entrainment (Panda et al., 2002; Ruby et al., 2002). Opsin 5 has also recently been identified as a deep brain photoreceptor functioning as an extraretinal receptor that regulates seasonal reproduction in birds (Nakane et  al., 2010). Similar work has yet to be conducted in insects, although boceropsin, a novel opsin expressed specifically in the central nervous system (CNS), has been proposed as a photoreceptor protein in B. mori (Shimizu et  al., 2001). A comparison of the phenotypes of null mutants of opsin- and carotenoid-deficient ­silkworms will be insightful.

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10.2.3.2.  Clock genes  Regardless of the type of photoreceptor involved in diapause, the photoreceptor must be functionally connected to a timekeeping mechanism to exert its effect. Whether the clock mechanism that controls circadian rhythms utilizes the same timekeeping mechanism involved in photoperiodic time measurement (i.e., diapause) remains controversial (Emerson et  al., 2008, 2009a; Goto et  al., 2010; Saunders, 2010a,b; Kostal, 2011). The molecular basis for circadian timekeeping is particularly well described in D. melanogaster (Stanewsky, 2002; Price, 2004; HelfrichForster, 2009). In Drosophila, the clock is composed of a multiple component autoregulatory feedback loop. The best described components involve the interactive oscillations of the proteins period (PER), timeless (TIM), clock (CLK), and cycle (CYC). In this system, the proteins PER and TIM heterodimerize to enter the nucleus and transcriptionally repress expression of CLK and CYC. Conversely, CLK and CYC act to transcriptionally upregulate both PER and TIM. Thus, when levels of the proteins PER and TIM are high, they repress transcription of CLK and CYC. As levels of CLK and CYC diminish, transcription of PER and TIM are decreased until they are low enough to no longer repress CLK and CYC. Once the levels of CLK and CYC are restored, PER and TIM will again be transcribed. The genes in this autoregulatory loop, in addition to several others including vrille, doubletime, and CREB2, create two interacting phases of protein oscillations (PER with TIM and CLK with CYC), creating a circadian rhythm of gene expression. The connection of this circadian pacemaker to photoperiod is attained through the interaction of the proteins TIM and CRY. TIM is degraded in the photophase through the action of CRY, “resetting the clock” according to the daily light cycle. Although most of these genes appear to be conserved, it is evident that details of the clock mechanism may differ among species. For example, in A. pernyi, PER apparently does not enter the nucleus (Sauman and Reppert, 1996b). These major clock genes have been studied intensely in association with circadian rhythms, yet it is not absolutely clear whether diapause uses the same timekeeping system. Although much evidence points to a role of circadian rhythms in the programming of diapause (Saunders, 2002, 2010b), several arguments defend the position that diapause does not have a circadian basis (Veerman, 2001; Veerman and Veenendaal, 2003). Even if diapause does have a circadian basis it is not clear whether the same clock genes and response mechanisms are involved in both circadian rhythmicity and photoperiodism (diapause). Several experiments suggest two distinct mechanisms. In both spider mites (Veerman and Veenendaal, 2003) and the blow fly Calliphora vicina (Saunders and Cymborowski, 2003), it is possible to segregate the photoperiodic response mechanism from the mechanism governing daily

behavioral rhythms. Experiments utilizing mutants suggest that distinct mechanisms may operate in these two systems. per null mutants of D. melanogaster are arrhythmic, yet they retain the ability to enter adult diapause (Saunders et al., 1989; Saunders, 1990). Similarly, in the drosophilid Chymomyza costata, mutants with reduced rhythmicity continue to enter larval diapause (Lankinen and Riihimaa, 1992), and per null mutants retain the capacity for diapause as well (Shimada, 1999; Kostal and Shimada, 2001). It is possible that there is enough redundancy in the system so that knocking out a single gene does not render the timekeeping mechanism involved in photoperiodism inoperative, and in per mutants, TIM could be degraded directly through CRY as nicely pointed out by Bradshaw and Holzapfel (2007). The involvement of clock genes in diapause is likely as suggested by several types of experiments. In S. ­bullata, the circadian system that regulates adult eclosion and diapause appear to be linked: a mutant that lacks rhythmicity also fails to enter diapause (Goto et al., 2006). In S. ­crassipalpis, period, cycle, clock, and cryptochrome show little differences in expression patterns when the flies are reared under short day or long day. However, the expression of timeless is greatly suppressed in long days as compared to short days (Goto and Denlinger, 2002b), suggesting a possible role for tim in the mechanism distinguishing short and long days. Although the developmental arrest in D. melanogaster is modest by comparison to other species and is probably more akin to quiescence (Emerson et al., 2009b), several lines of evidence point to the involvement of tim in this dormancy (Tauber et al., 2007; Sandrelli et al., 2007), as well as the diapause of the pitcher plant mosquito, Wyeomyia smithii (Mathias et al., 2005). In addition, RNAi directed against tim elicited a modest effect in reducing diapause in C. costata (Pavelka et  al., 2003), which suggests a possible role for tim as a component of the photoperiodic timekeeping mechanism. A role for tim in the diapause of C. costata is further bolstered by the interesting observation that, in the non-diapausing mutant, the protein TIM is not present in the two neurons in each brain hemisphere that contain this protein in wild-type flies (Stehlik et al., 2008). Collectively, these results underscore the importance of tim in the diapause of this species. Recent RNAi results also yielded a particularly interesting effect on diapause in the bug, R. pedestris: RNAi directed against per caused the bug to avert diapause when reared under a diapauseinducing photoperiod, while RNAi directed against cycle induced diapause under a diapause-averting photoperiod (Ikeno et al., 2010). A minimal interpretation of these profound effects is that both of these well-known clock genes participate in the diapause decision. In this experiment, altering the expression patterns of these two clock genes affected diapause as well as the circadian pattern of cuticle deposition, a nice demonstration that the same genes

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are involved in both of these timekeeping mechanisms. It is difficult, as pointed out by Bradshaw and Holzapfel (2010), to clearly distinguish between functions for the clock genes in photoperiodism versus pleiotropic effects that may be acting on sister pathways. Results are emerging that implicate a number of the clock genes in the programming of diapause, yet there are also sufficient reports in the literature suggesting that the gene interplay described so elegantly for the control of circadian rhythms of behavior in D. melanogaster may not be fully relevant to diapause induction. Resolution of these disparities is pivotal for understanding diapause induction at the molecular level.

10.3.  Endocrine Regulators 10.3.1.  Hormonal Basis for Embryonic Diapause

Embryonic diapause is common among the Orthoptera, Hemiptera, Lepidoptera, and Diptera (especially Aedes mosquitoes) with occasional reports from other orders as well. The diverse stages in which embryonic diapause occurs suggest a rich diversity of regulatory schemes. Hormonal regulation has been examined only in a few species of Lepidoptera, and it is evident that no single regulatory scheme prevails. The most intensely studied embryonic diapause is that of B. mori (Yamashita and Hasegawa, 1985; Yamashita, 1996). Classic experiments, begun in the 1950s by Fukuda (1951, 1952) and Hasegawa (1952), set the stage for a wealth of papers documenting the regulatory mechanism controlling the early embryonic diapause in this commercially important species. Recent work has provided major contributions to the hormonal, biochemical, and molecular characterization of diapause in B. mori (Yamashita et al., 2001). Diapause in B. mori differs from that of most other species. While both temperature and daylength are key components contributing to the diapause decision, the specifics are reversed as compared to other diapause models. High temperature and long daylength prompt diapause induction, while low temperature and short daylength lead to non-diapause development. Diapause induction in this species is under strict maternal control. The photosensitive stage occurs far in advance of the actual diapause stage: exposure of developing female embryos and larvae to diapause-inducing conditions cause the adult female to produce eggs that are destined to enter diapause. This effect is mediated by diapause hormone (DH), a neuropeptide released by the female adult during her period of egg maturation, which acts upon the ovarioles and leads to production of diapause-destined eggs. After being oviposited, the silkworm embryos enter a diapause characterized by G2 cell cycle arrest (Nakagaki et al., 1991). Once diapause has been established, this arrest can be broken after the embryos have been chilled for 1–3 months at 5°C,

after which development can be reinitiated by exposure to higher temperatures. As early as 1926, ovary transplantation experiments indicated that the factor controlling diapause induction was blood-borne (Umeya, 1926). Later studies by Fukuda and Hasegawa reported that the brain and subesophageal ganglion controlled the release of DH, and bioassays performed by injecting non-diapausetype individuals with subesophageal ganglion and brain extracts showed DH activity in these tissues. 10.3.1.1.  Diapause hormone and its action in Bombyx mori  Although the activity of DH was established quite early, purification and sequencing of the peptide progressed slowly. An initial characterization was made from extracts of 15,000 isolated brain–subesophageal ganglion complexes (Hasegawa, 1957). Further analysis, including an assessment of amino acid composition, was made from extracts of 1 million isolated male adult heads (Isobe et  al., 1973). A final push for determining the amino acid sequence began in 1987, when extracts from 100,000 subesophageal ganglia generated enough material for sequencing and finally yielded a 24 amino acid peptide (Imai et al., 1991). Subsequent cloning of the cDNA for DH confirmed the amino acid sequence (Figure 5) and led to further characterization of the hormone (Sato et al., 1992, 1993). DH is a member of the FXPRL-amide peptide family, sharing the same C-terminus five amino acid sequence with a variety of hormones including pheromone biosynthesis activating neuropeptide (PBAN) and pyrokinin. Although a sequence of PRL-NH2 is the minimum structure required for DH activity (Imai et  al., 1998), substantially greater activity is noted from the C-terminal pentapeptide. By synthesizing a series of DH analogs of different lengths, it was determined that two other regions of the peptide in the center and near the N-terminus, as well as the C-terminal amide group, also contributed to biological activity (Saito et al., 1994; Suwan et al., 1994). Cloning of the cDNA for DH resulted in an 800 bp nucleic acid sequence with a 192 amino acid open reading frame, much larger than the 24 amino acid DH peptide (Sato et al., 1992, 1993). Analysis of the deduced amino acid sequence revealed that not only DH, but several additional neuropeptides, including PBAN and three other members of the FXPRL neuropeptide family are encoded TDMKDESDRGAHSERGALWFGPRL- NH2

PBAN

DH

24 a.a.

7 a.a. 17 a.a.

33 a.a.

NH2 8 a.a.

Figure 5  Amino acid sequence of diapause hormone (DH) from Bombyx mori and organization of the precursor polyprotein including locations of DH, PBAN, and three additional subesophageal ganglion neuropeptides. Adapted from Sato et al. (1993).

438  10: Hormonal Control of Diapause

in the single open reading frame (Figure 5). The resulting polyprotein is a prohormone that is post-translationally processed to excise the active peptides. The presence of PBAN and other neuropeptides in the DH prohormone stresses the importance of post-translational control of hormone release, and helps to explain the formerly perplexing data indicating the presence of DH activity from a wide variety of developmental stages, from both sexes, and from both diapausing and non-diapausing individ­ uals. For instance, it is now assumed that the large peak of activity seen in developing pharate adults (Sonobe et al., 1977) can be attributed to the synthesis of PBAN as the moth completes its maturation and prepares for pheromone biosynthesis. Genomic characterization of DH has also been achieved: Southern blotting reveals a single copy of DH-PBAN in the Bombyx genome, and linkage analysis suggests that it is located on chromosome 11 (Pinyarat et al., 1995). The gene contains six exons separating five introns with DH encoded by the first two exons. The region upstream of the open reading frame includes an ecdysone response element and five homeodomain TAAT binding core domains (Xu et al., 1995a). Even in the absence of purified hormone or the DH clone, early researchers were able to characterize DH control mechanisms and the sites of DH synthesis and release. Early transplantation experiments demonstrated DH activity in the subesophageal ganglion, and histological studies from Fukuda’s laboratory identified a single pair of cells in the subesophageal ganglion as the likely site of DH release. More sensitive molecular methods have largely confirmed these results. Real-time PCR results indicate that DH is exclusively expressed in the subesophageal ganglion with no DH expression in the brain, thoracic or abdominal ganglia, or non-neural tissues (Sato et  al., 1994). In  situ hybridization studies illustrate that expression is concentrated in 12 cells grouped into three clusters along the midline of the subesophageal ganglion (Sato et  al., 1994). Surgical manipulation indicates that the different clusters are responsible for different activities of the prohormone. Females retaining only the two cells in the posterior cluster continue to be capable of inducing diapause in their progeny, while females with only

(a)

(b)

Figure 6  Localization of neurosecretory cells containing diapause hormone (DH) within the subesophageal ganglion of (a) diapause and (b) non-diapause type adults of Bombyx mori. Release of DH leads to a decrease of staining in diapause-type individuals. Arrows indicate the somata containing DH. Adapted from Sato et al. (1998).

the six cells in the medial cluster retain PBAN function. Removal of the anterior cluster of four cells does not substantially affect either function, suggesting that this cluster is involved in the function of other portions of the prohormone (Ichikawa et al., 1996). Immunohistochemical studies using antibodies recognizing the FXPRL-amide further support the identification of cells with DH activity. Of the 12 cells in the subesophageal ganglion that exhibit antigenicity, only two show decreased staining in diapause-type as compared to non-diapause-type females (Figure 6), suggesting that the peptide is being released (Sato et al., 1998; Kitagawa et al., 2005). Immunocytochemistry combined with intracellular Lucifer Yellow injections reveals that axons from all three clusters pass through the brain and into the corpus cardiacum (Ichikawa et al., 1995), the known site of DH release. While early studies showed DH activity in larval, pupal, and adult stages (Hasegawa, 1964), more sensitive techniques led to further resolution of DH expression patterns. Use of RT-PCR determined that DH-PBAN transcription is controlled by both development and temperature (Xu et  al., 1995b; Morita et  al., 2004). When B. mori is raised under diapause-inducing conditions of 25°C, multiple expression peaks are noted throughout development: the first during embryogenesis, one during both the fourth and fifth instars, and two during pupal and adult development. In contrast, when the insects are reared under a non-diapausing temperature of 15°C, only the final peak of gene expression, during pupal and adult development, is observed (Figure 7). This final transcription peak is likely associated with PBAN, rather than DH, activity.

(a)

(b)

Figure 7  Levels of DH-PBAN mRNA in (a) diapause and (b) non-diapause type Bombyx mori during the course of development. L4, L5, fourth and fifth instars; P, pupa; A, adult. Adapted from Xu et al. (1995a).

10: Hormonal Control of Diapause  439

The neural control of DH expression and release has also been characterized. Both in  vivo and in  vitro studies show that DH transcription is under control of the neurotransmitter dopamine. Dopamine levels in both the hemolymph and brain–subesophageal ganglion complexes of diapause-type individuals are significantly higher than in their non-diapause-type counterparts during the late larval and early pupal stages (Noguchi and Hayakawa, 2001), correlating with an increase in dopamine decarboxylase expression. Furthermore, experiments in which DOPA is fed to non-diapause-type larvae during the final larval instar or injected into non-diapause-type pupae resulted in the production of diapausing eggs by nondiapause-type females. Additionally, pupal injections of DOPA elevate DH mRNA in the brain–subesophageal ganglion complexes. In vitro studies further confirm the role of dopamine, as DH mRNA levels in isolated brain– subesophageal ganglion complexes from non-diapausetype pupae are elevated within 2 h after addition of either DOPA or dopamine to the medium. Expression of the dopamine receptors is not affected by diapause status and is concentrated in the mushroom bodies, suggesting that they contribute to the pathways leading to DH production (Mitsumasu et  al., 2008). Transcription factors involved in regulation of DH include a member of the pituitary homeobox gene family (Shiomi et al., 2007), as well as at least two members of the POU transcription factor family (Xu et al., 1995b; Zhang et al., 2004a). Neuronal control of DH is not restricted to the transcriptional level. The release of DH from the subesophageal ganglion is under control of GABAergic neurons. Culturing isolated brain–subesophageal complexes from diapause-egg-producing females in the presence of the inhibitory neurotransmitter GABA causes a substantial reduction in release of DH, while incubation with picrotoxin, the GABA inhibitor, promotes DH release from the cultured brain–subesophageal complexes (Hasegawa and Shimizu, 1990; Shimizu et al., 1997). Further insight into the control of DH release may be gleaned from data for PBAN, the release of which is under circadian control, with PBAN-releasing neurons firing during the scotophase of a photoperiodic cycle. This pattern persists with a periodicity of 24 h (free runs) even in constant darkness, confirming the circadian nature of this pattern (Tawata and Ichikawa, 2001). At this point, however, there is no direct evidence indicating a circadian pattern of DH release. The ovaries appear to be the sole target for DH. Many key components of the second messenger systems involved in DH signal transduction have been elucidated. The DH receptor has been identified as a 436 amino acid G-protein-coupled peptide, with affinity to several FXPRL-amides, but exhibiting highest affinity to DH (Homma et  al., 2006). Downstream components have been identified as well. In vitro experiments suggest

that early events, such as the upregulation of trehalase described later, are regulated by Ca2+ ion influx, since the addition of the chelating agent egtazic acid (EGTA) to the incubation medium suppresses DH activity, while the addition of Ca2+ restores this activity (Ikeda et  al., 1993). Later events may be mediated by cyclic nucleotide cascades. DH acts to reduce the concentration of cyclic GMP in diapause-destined eggs through the regulation of guanylate cyclase activity (Chen et  al., 1988; Chen and Yamashita, 1989). Downstream of DH, regulation of diapause appears to involve accumulation of sorbitol (Horie et al., 2000). A key indicator of DH activity has long been a metabolic switch, resulting in the accumulation of glycogen in diapause-destined eggs. This accumulation is due to the enzyme trehalase, which is transcriptionally upregulated after DH injection (Su et al., 1994). Accumulated glycogen is then converted to sorbitol, which acts as a classic cryoprotectant, lowering the supercooling point of the diapausing eggs (Chino, 1958). Upon diapause termination, an ERK/MAPK-mediated pathway signals the reconversion of sorbitol to glycogen (Fujiwara et al., 2006), which is subsequently used as an energy reserve. The accumulation of sorbitol not only offers cryoprotection but appears to exert a regulatory role as well (Horie et al., 2000). When diapause-destined eggs are dechorionated and cultured in Grace’s medium, diapause is averted. When sorbitol is added to the medium the ability to diapause is restored in a dose-dependent fashion. Substantial inhibition of development is noted at biologically relevant concentrations of sorbitol. Trehalose elicits similar inhibitory effects, but glycerol is less effective. Additionally, removal of sorbitol induces resumption of development, and addition of sorbitol to non-diapause-destined eggs leads to arrested development. Interestingly, the ERK/ MAPK pathway appears to also be involved in regulation of embryonic diapause in the false melon beetle, Atrachya menetriesi (Kidokoro et  al., 2006b), even though there is no evidence for DH activity in this or any other non-­ lepidopteran species. 10.3.1.2.  Other mechanisms for regulating embryonic diapause  There is no evidence to suggest that the regulatory mechanisms carefully documented in B.  mori are relevant to other forms of embryonic diapause, although DH has been implicated in the control of pupal diapause (see Section 10.3.2.). Embryonic diapauses are enormously diverse, as indicated by the wide range of stages at which development is arrested, and data suggest that the regulation is diverse as well. Consequently, a variety of regulatory mechanisms have been suggested for embryonic diapause as alternatives to what is seen in B. mori. The gypsy moth, Lymantria dispar, diapauses as a pharate first instar larva. Several lines of evidence indicate that this diapause is induced and maintained by an elevated

440  10: Hormonal Control of Diapause

ecdysteroid titer. Using synthesis of a 55 kDa gut protein — now known to be the immune protein hemolin (Lee et al., 2002) — as a distinct biomarker for diapause, Lee and Denlinger (1996, 1997) demonstrated that placing a ligature behind the prothoracic gland blocks hemolin synthesis, and co-culturing the gut with an active prothoracic gland promotes hemolin synthesis. Synthesis of hemolin can also be induced if an isolated abdomen is injected with 20E or the ecdysteroid agonist RH-5992. KK-42, an imidazole derivative thought to block ecdysteroid synthesis, averts diapause in this species (Suzuki et al., 1993; Bell, 1996; Lee and Denlinger, 1996), but this effect can be reversed with the application of ecdysteroids (Lee and Denlinger, 1997). Additionally, at the end of the mandatory chilling period, diapause can be readily terminated if pharate larvae are transferred to high temperatures, but this response can be prevented with exogenous ecdysteroids. Experiments with a mutant, non-diapausing strain of L. dispar also support a role for ecdysteroids, as suggested by the restoration of diapause when ecdysteroid concentrations are artificially boosted in the mutant (Lee et  al., 1997). Although ecdysteroids of maternal origin are known to be incorporated into eggs in other species (Lagueux et  al., 1981), at this late stage of embryonic development the ecdysteroids that control diapause are likely synthesized in the pharate larva’s prothoracic gland, which continuously synthesizes ecdysteroids throughout diapause but then shuts down synthesis as a first step in terminating diapause. A diapause-promoting effect for ecdysteroids may seem paradoxical, especially considering that a lack of ecdysteroids is the dominant feature of most larval and pupal diapauses (see Section 10.3.2.). Yet, it is well known that a drop in the ecdysteroid titer is an essential signal permitting the ongoing progression of development in other systems (Slama, 1980), and the gypsy moth appears to have exploited this function to bring about developmental arrest. The link between diapause induction and increased ecdysteroids is not exclusive to L. dispar. Metabolic depression has been linked to ecdysteroid peaks during embryogenesis in Schistocerca gregaria (Slama et al., 2000). Additionally, gene regulation during pre-diapause in the ground cricket Allonemobius socius suggests that this embryonic diapause is regulated in a similar manner (Reynolds and Hand, 2009), and increased ecdysone levels induce cell cycle arrest in cultured mosquito cells (Gerenday and Fallon, 2004), further suggesting a link between increased ecdysone and the development of diapause traits. Ironically, decreased ecdysteroid levels can also bring about embryonic diapause, as is the case for two locust species: the Australian plague locust Chortoicetes terminifera (Gregg et  al., 1987) and the migratory locust, Locusta migratoria (Tawfik et  al., 2002b). In both species, non-diapausing eggs contain approximately three times the amount of ecdysteroids as found in diapausing

eggs. In these species the diapauses occur early in embryonic development, and the ecdysteroids present are likely to be exclusively of maternal origin. Ecdysteroid titers increase in non-diapause eggs after the stage at which diapause normally occurs and in embryos that avert diapause in response to high temperature (Gregg et  al., 1987). Diapause termination also leads to an elevation in ecdysteroid titers (Tawfik et  al., 2002b), and exogenous application of ecdysteroids prompts diapause termination (Kidokoro et al., 2006a), thus the responses of these two locusts consistently point to diapause being caused by the absence of ecdysteroids, much akin to the endocrine scenario evident in most larval and pupal diapauses. Parallels between embryonic and adult diapause are evident as well, as exogenous application of juvenile hormone analogs has been shown to terminate diapause in the orthopteran Aulocara ellioti (Neumann-Visscher, 1976), as well in the aforementioned L. migratoria (Kidokoro et al., 2006a). The pharate first instar larval diapause of the silkmoth A. yamamai represents yet another intriguing regulatory scenario (Suzuki et al., 1990, 1993). The imidazole known to block ecdysteriod synthesis, KK-42, is highly efficacious for terminating diapause in intact pharate first instar larvae as well as in headless larvae. Still, diapause persists in an isolated head–thorax preparation, suggesting the presence of a repressive factor in the thorax. Isolated abdomens break diapause whether or not KK-42 is added. From these results, Suzuki et al. (1990) suggested the presence of a diapause-promoting factor produced in the mesothorax, and a development-promoting factor produced in the region of the second to fifth abdominal segments. During diapause the diapause-promoting factor prevails, halting development. Although the identities of these factors remain unknown, the effects cannot be mimicked by several of the obvious candidates: prothoracicotropic hormone, ecdysteroids, or JH. 10.3.1.3.  Associated changes in gene expression  Although considerable effort has been devoted to the identification of diapause-specific genes, the majority of these genes are likely involved in maintaining the diapause state and in enhancing stress resistance rather than being specifically involved in diapause regulation. One exception may be the genes regulating the G2 cell cycle arrest characteristic of diapause in B. mori (Nakagaki et al., 1991). Considerable interest has focused on the molecular basis of this cell cycle arrest. While there is little evidence for a connection between cyclin B transcription and diapause (Takahashi et  al., 1996), both cyclin dependent kinase 2 and cdc-2 related kinase are substantially downregulated in diapausing individuals and are thought to be involved in mediating the diapause cell cycle arrest (Takahashi et al., 1998). Another B. mori gene with possible regulatory implications is BmEts, a gene highly upregulated in early diapause but suppressed in a

10: Hormonal Control of Diapause  441

non-diapausing mutant strain (Suzuki et al., 1999). This gene is especially intriguing because, as the name suggests, BmEts belongs to the ETS gene family of transcription factors, which includes several genes such as Drosophila E74, a gene known to be important in the cellular ecdysone response. Additionally, the cold-induced gene Samui may be involved in the signal cascade of diapause termination. Downregulation of Samui occurs concurrently with activation of sorbitol dehydrogenase, the enzyme involved in converting sorbitol to glycogen (Moribe et al., 2001). As such it could be involved in removing the aforementioned sorbitol blockade to embryonic development. Recent suppressive subtractive hybridization studies on the embryonic diapauses of the cricket A. socius (Reynolds and Hand, 2009) and the Asian tiger mosquito Aedes albopictus (Urbanski et al., 2010a,b) have also identified genes with unique diapause expression profiles. Specific links between these genes and the neuroendocrine control of diapause remain to be identified, but several of the cricket genes upregulated in the pre-diapause phase are associated with ecdysteroid synthesis and signaling, suggesting a possible role for ecdysteroids in promoting the onset of this diapause. Concurrently, genes involved in yolk mobilization and/or metabolism are consistently downregulated in the cricket embryos, as one might anticipate for the dormant state. The diapause-associated genes identified in A. albopictus appear to be involved mainly in downstream physiological components of the diapause syndrome such as desiccation resistance. The A. ­albopictus study focused on transcripts derived from oocytes dissected from the mother, thus in this case, the genes affecting the progeny’s diapause are maternally derived. 10.3.2.  Hormonal Basis for Larval and Pupal Diapauses

Larval diapauses are best known from the Lepidoptera, but there are good examples of larval (or nymphal) diapauses in other orders as well. Diapause in the last larval instar is reported most frequently, but some larvae diapause occurs in earlier instars or as pre-pupae. Pupal diapause has been examined extensively in Lepidoptera and the higher Diptera, but appears to be rare in other taxa. It is the true pupal stage that is usually involved in diapause, but occasional reports indicate a diapause late in pharate adult development. Larval and pupal diapauses share much in common. Central to both is a failure to progress to the next metamorphic stage. This suggests that one of the key endocrine events regulating such a diapause is likely to be a failure of the prothoracic gland to produce the ecdysteroids needed to initiate the next molt. Invariably, this has proven to be a key element of larval and pupal diapauses. One might also predict a shutdown until the end of diapause in the production of PTTH (see Chapter 1). This may often be the case but not always. The other major

metamorphic hormone, juvenile hormone (JH), may or may not play a role, depending on the species. 10.3.2.1.  Role of the brain–prothoracic gland axis  The older literature is replete with experiments showing that activation of the brain is essential for terminating diapause. Of special significance are the classic experiments by Carroll Williams (1946, 1947, 1952) demonstrating that the brain of the giant silkworm Hyalophora cecropia, when activated by chilling, is capable of terminating diapause when implanted into a brainless pupa. These experiments were critical not only for understanding the mechanism of diapause but also for deciphering the endocrine basis for insect metamorphosis. One can now be more specific and attribute these brain effects not to the whole brain, but to the production of PTTH by a pair of neurosecretory cells in the pars intercerebralis of each brain hemisphere (Agui et  al., 1979; Sauman and Reppert, 1996). In the pupal diapause of M. sexta intracellular electrical recordings of the cells that produce PTTH show all the characteristics of silent cells: high voltage thresholds, low input resistance, and few spontaneous action potentials (Tomioka et al., 1995). Electrical differences between the PTTH cells of diapausing and non-diapausing pupae are already evident within one day after pupation. As diapause progresses in chilled pupae, the cells gradually display more spontaneous action potentials, and more excitatory postsynaptic potentials are observed. In spite of the early curtailment of neurophysiological activity in the PTTH cells, there are no corresponding ultrastructural changes that would appear to reflect the electrical silencing of the cells in early diapause (Hartfelder et al., 1994). One common method for monitoring PTTH activity is to culture prothoracic glands in  vitro and monitor ecdysone production when brains or brain extracts are added to the culture medium. When this has been done, low PTTH activity associated with larval and pupal diapause has been observed in some cases. In Diatraea ­grandiosella, a species that diapauses late in the final larval instar, PTTH activity is far lower in brains and brain extracts from diapause-destined larvae and diapausing larvae than in non-diapausing larvae (Yin et  al., 1985). Likewise, in the European corn borer, Ostrinia nubilalis, PTTH activity is lower in brains of diapausing larvae than in non-­diapausing brains (Gelman et al., 1992). Similar low levels of PTTH activity are noted in pupal diapause of the cabbage armyworm, Mamestra brassicae (Endo et al., 1997). Yet, brains from all of these species clearly show some PTTH activity, and pupal brain extracts from M. sexta (Bowen et al., 1984) and Sarcophagia ­argyrostoma (Richard and Saunders, 1987) actually have as much or more PTTH activity than brain extracts from non-­ diapausing pupae. Likewise, PTTH activity is higher in larvae of S. peregrina that are destined for pupal diapause

442  10: Hormonal Control of Diapause

than in larvae not destined for diapause (Moribayashi et al., 1992). These observations suggest that the hormone may be present during diapause but is simply not released. In Heliothis virescens, expression of transcript encoding PTTH persists at relatively high levels throughout larval development, but at the onset of wandering behavior, expression in larvae programmed for pupal diapause drops and remains low during diapause, while the ­transcript continues to be highly expressed in non-­ diapause-destined larvae and non-diapausing pupae (Xu and Denlinger, 2003). These observations suggest that, in this species, PTTH is regulated at the level of transcription. Thus, there may be species variation in the mechanisms of PTTH regulation. Either a halt in PTTH synthesis or a failure of PTTH release could result in a developmental shutdown. Several experiments suggest that high dopamine levels in the brain may be critical for diapause induction, possibly by preventing release of PTTH. High levels of dopamine in brains of diapause-destined larvae of C. costata (Kostal et al., 1998), diapause-destined pupae of M. ­brassicae (Noguchi and Hayakawa, 1997), and pupae of P. brassicae (Puiroux et al., 1990) suggest a causal relationship. In diapausing pupae of A. pernyi, a drop in brain dopamine is noted shortly before the surge of ecdysteroids that elicits diapause termination (Matsumoto and Takeda, 2002). But the most compelling evidence suggesting a role for dopamine is the fascinating observation that feeding L-DOPA to last instar larvae of M. brassicae reared under long daylengths (not diapause-inducing) elicits a diapause-like state in the pupae (Noguchi and Hayakawa, 1997). Other than DH in Bombyx, there are few examples of chemical agents capable of inducing diapause or a diapause-like state, thus this result with DOPA feeding is quite striking. A potential role is also discussed for dopamine in the induction of embryonic diapause of B. mori (Noguchi and Hayakawa, 2001; see Section 10.3.1.1.). Among genes that are upregulated by both short-day conditions and the feeding of DOPA in M. brassicae, the most interesting is an upregulated gene with high identity to a receptor for activated protein kinase C (RACK) from H. virescens (Uryu et al., 2003). In situ hybridization with a RACK probe reveals expression of this gene within a few cells in the medial protocerebral neuropil. As a member of the protein kinase C (PKC) signaling cascade, RACK has the potential to be an important gene contributing to transduction of the signals involved in diapause induction. Noguchi and Hayakawa (1997) proposed that elevation of dopamine may be the key to preventing PTTH release. Elevation of brain serotonin (5-hydroxytryptamine) has also been linked to diapause in several cases (Kostal et al., 1999; L’Helias et  al., 2001), but not in others (Puiroux et  al., 1990). In C. costata, depletion of serotonin does not alter the fly’s capacity to enter larval diapause (Kostal

et al., 1999), suggesting that the observed elevation of this monoamine is not essential for diapause induction. The shutdown in the brain–prothoracic gland axis is not exclusively the result of a failure of the PTTH cells to produce or release PTTH. The prothoracic gland also becomes refractory to PTTH at this time, providing a double assurance that the neuroendocrine system will not provide the signal needed to promote development. Assays for PTTH activity indicate that prothoracic glands dissected from diapausing individuals are refractory to stimulation, as shown in larvae of C. vicina (Richard and Saunders, 1987) and O. nubilalis (Gelman et al., 1992), and in pupae of M. sexta (Bowen et al., 1984) and S. argyrostoma (Richard and Saunders, 1987). This loss of competency can be rather rapid (Figure 8a). In S. ­argyrostoma the prothoracic glands lose their competency to produce ecdysone within a 1- to 2-day period at the onset of diapause (Richard and Saunders, 1987). In the larval diapause of C. vicina loss of competency is more gradual, occurring over a 6-day period, and even during diapause the gland maintains limited competency to produce ecdysone (Figure 8b). At diapause termination, prothoracic gland competency is restored quickly. When diapausing larvae of C. vicina are transferred from 11 to 25°C, the prothoracic gland regains competency to produce ecdysone within 24 h (Richard and Saunders, 1987). PTTH stimulation of the prothoracic gland in nondiapausing insects is mediated by cyclic AMP as a second messenger, but the diapausing glands of M. sexta (Smith et  al., 1986) and S. argyrostoma (Richard and Saunders, 1987) fail to respond to cyclic AMP or agents known to elevate cyclic AMP, implying that the block in gland function occurs beyond the stage of cyclic nucleotide action. Reports from several species of Lepidoptera indicate that cells of the prothoracic gland are greatly reduced in size during diapause, both the cytoplasm and nuclei are shrunken, and the mitochondria take on a swollen configuration (Denlinger, 1985). In S. crassipalpis, there is a slight reduction in the size of the cells in the prothoracic gland component of the ring gland, but this difference is not as conspicuous as noted in the Lepidoptera (Joplin et al., 1993). Results of an ultrastructural examination of the fly prothoracic gland during diapause are rather surprising. Instead of being inactive, the presence of extensive arrays of rough endoplasmic reticulum suggests that the glands remain active in protein synthesis. Pulse labeling of the ring gland further supports the contention that the gland continues to synthesize proteins during diapause. The function of this curious diapause activity remains unknown, but it would appear to be unrelated to ecdysone production since all evidence indicates that this function is shut down at this time. Although there appears to be species variation as to whether the synthesis or release of PTTH is regulated, the net effect, a shutdown in the synthesis of ecdysteroids, is

10: Hormonal Control of Diapause  443

(a)

(b)

Figure 8  Decline in the competency of ring glands from diapause-destined (a) Sarcophaga argyrostoma and (b) Calliphora vicina to synthesize ecdysone in response to incubation with prothoracicotropic hormone (PTTH) producing brains. As S. argyrostoma enters pupal diapause and as C. vicina enters larval diapause their ring glands become refractory to PTTH stimulation. Adapted from Richard et al. (1987).

consistent. Earlier literature provides numerous examples documenting low or undetectable ecdysteroid titers in larvae or pupae during diapause and an elevation in the ecdy­ steroid titer at diapause termination. More recent studies continue to provide evidence that this is the case; for example, in larval diapauses of O. nubilalis (Gelman and Woods, 1983; Gelman and Brents, 1984; Peypelut et al., 1990) and Omphisa fuscidentalis (Singtripop et al., 2002) and pupal diapauses of M. configurata (Bodnaryk, 1985), M. sexta (Friedlander and Reynolds, 1992), S. argyrostoma (Richard et al., 1987), and S. peregrina (Moribayashi et al., 1988). As discussed in Section 10.3.2.4., some larvae periodically undergo a stationary molt during diapause, and in these cases a burst of ecdysteroid synthesis may occur during diapause and prompt the larva to molt. The surge in ecdysteroids that normally occurs at the end of diapause may come as two distinct waves. In M. configurata the release of ecdysone into the hemolymph is followed

several days later by the appearance of 20E (Bodnaryk, 1985). Since these two peaks are nearly identical in size, they may simply represent the conversion of ecdysone to 20E. In O. nubilalis a small transient peak of 20E is followed several days later by a major peak of 20E (Peypelut et al., 1990). In this case, imaginal wing disc development is thought to be initiated by the small initial peak, but the later phases of post-diapause development require the sustained presence of 20E. Consistent with the concept that the absence of ecdy­ steroids is central to maintaining the diapause state is the observation that an application of exogenous ecdysteroids or ecdysteroid mimics can terminate diapause. Again, the older literature includes numerous examples supporting this response, and more recent work continues to document this effect; for example, larval diapauses of O. ­nubilalis (Gadenne et  al., 1990) and Diprion pini (Hamel et  al., 1998), and pupal diapauses of A.  mylitta (Mishra et  al.,

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2008), M. configurata (Bodnaryk, 1985), P.  brassicae (Pullin and Bale, 1989), and M. sexta (Friedlander and Reynolds, 1992; Sielezniew and Cymborowski, 1997; Champlin and Truman, 1998). What still remains unclear is how, at the molecular level, the brain becomes reactivated to initiate the process of promoting the PTTH synthesis and/or release that leads to ecdysteroid synthesis and diapause termination. Although the environmental events required for diapause termination have been well defined (Tauber et  al., 1986), the mechanism of activation still remains largely unknown. In the few cases that use photoperiod to terminate diapause, the response most likely utilizes the clock-gene neurons that are closely aligned to the neurons that produce PTTH (see Section 10.2.2.). Temperature is more commonly the environmental factor prompting the termination of diapause, and how temperature is perceived or the effects of temperature are accumulated is unclear. One element of the signaling pathway, however, has recently been identified in the pupal diapause of S. ­crassipalpis (Fujiwara and Denlinger, 2007). In this fly, high temperature or an application of hexane will terminate diapause, and within 10 min after hexane application, extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, is phosphorylated, a response that is also noted when diapause is terminated by transferring the flies from 20 to 25°C. An injection of ecdysteroids does not activate ERK nor is ERK activated within the ring gland, suggesting that ERK exerts its effect through stimulation of the brain, quite possibly as a component of the cascade transmitting environmental signals to the PTTH cells within the brain. Two other members of the MAPK family, p38 MAPK and JNK, showed no response at diapause termination. A signaling cascade involving ERK emerges as a potential mediator of the environmental signal, resulting ultimately in ecdysteroid synthesis and diapause termination. A similar role for ERK in the termination of embryonic diapause was noted earlier (see Section 10.3.1.1.). From the Drosophila literature, it is evident that Torso, a receptor tyrosine kinase, functions as the PTTH receptor and its activation by PTTH stimulates the ERK pathway (Rewitz et al., 2009; see also Chapter 1). 10.3.2.2.  The missing pieces  The role of the brain– prothoracic gland axis in diapause, as presented earlier, is reasonably cohesive. In a simplified version, the prothoracic gland fails to produce ecdysone because it has not been stimulated by PTTH from the brain. Yet, some pieces of information are not consistent with this simple story and remain unexplained. For H. cecropia the previous story is just fine. The brain, when chilled, becomes activated to release PTTH and this in turn prompts the prothoracic glands to secrete the ecdysteroids needed to initiate development. Without a brain, the pupa is locked into a permanent

Figure 9  Time during pupal diapause at which several species are no longer dependent on the brain for initiation of adult development. Adapted from Denlinger (1985).

diapause, but in several cases this is not true (Denlinger, 1985). In M. sexta, P. rapae, and A.  polyphemus removal of the brain during the first month or so of diapause will produce a permanent diapause, but in later stages removal of the brain has no impact (Figure 9). The extreme case is noted in Helicoverpa zea: brain removal within the first 4 h after pupation will cause a permanent diapause, but within 24 h the diapausing pupa is already independent of the brain. Clearly the H. cecropia model cannot explain these observations. Meola and Adkisson (1977) argued that PTTH exerts its effect very early in H. zea, and after receiving the PTTH signal, the prothoracic glands take on the role of the regulatory organ: high temperature (27°C) promotes ecdysteroid synthesis, while low temperature (21°C) prevents ecdysteroid synthesis. In these cases it is clear that one cannot simply explain the end of diapause as an event triggered by the brain’s release of PTTH. What is lacking is a good explanation of this peculiar interaction between the brain and prothoracic gland. What does it mean for the brain, presumably via PTTH, to stimulate the prothoracic gland so early and for the gland to delay the onset of ecdysteroid synthesis by many months? An alternative explanation may be that our traditional view of PTTH playing a role in promoting the synthesis of ecdysteroids is not correct. This scenario gains credence from recent results in Drosophila showing that ablation of the PTTH-producing cells had no effect on molting or metamorphosis (McBrayer et al., 2007), a result not too different from the diapause experiments showing that brain extirpation fails, in some cases, to block the termination of diapause. If, as shown in Drosophila, PTTH is not absolutely essential for stimulating ecdysteroid synthesis, the door is opened to considering alternative pathways for stimulating ecdysteroid synthesis. Several lines of evidence suggest that there may be differences in the way the prothoracic gland is activated in non-diapausing individuals and at the termination of diapause. In S. crassipalpis, cyclic AMP levels in the brain and ring gland are high in non-diapausing pupae and low in diapausing pupae at the onset of diapause, and diapause

10: Hormonal Control of Diapause  445

can easily be averted in the diapause-programmed pupae if cyclic AMP levels are artificially boosted around the time of expected diapause entry (Denlinger, 1985). This observation is compatible with other evidence suggesting that cyclic AMP is used as a second messenger for PTTH (see Chapter 1). One might then assume it would also be possible to employ this same tactic of cyclic AMP elevation to initiate development in a pupa that has been in diapause for some time. Surprisingly, this does not work because cyclic AMP injected along with ecdysteroids actually retards the termination of diapause. By contrast, cyclic GMP, which is ineffective in averting diapause, can readily break diapause by itself and will enhance the diapausebreaking effect of ecdysteroids (Denlinger and Wingard, 1978). Richard and Saunders (1987) were unable to stimulate ecdysteroid production with PTTH or cyclic nucleo­ tides in ring glands cultured from diapausing pupae of S.  argyrostoma or diapausing larvae of C. vicina, which also suggests that the glands are somehow activated in a different way at the termination of diapause. Although prothoracic glands dissected from C. vicina are competent to synthesize ecdysone within 24 h after a transfer of the diapausing larvae to a high temperature, brain–ring gland complexes cultured in vitro fail to gain competency when subjected to the same temperature switch (Richard and Saunders, 1987). This suggests that some additional component from another part of the body may contribute to reactivation of the prothor­ acic gland. The pieces of the fly puzzle include: cyclic GMP readily breaks diapause when injected in vivo, but fails to activate prothoracic glands cultured in vitro, and the brain–­prothoracic gland can regain competency in response to high temperature in  vivo, but not in  vitro. A  minimal interpretation of these observations is that some additional prothoracicotropic factor, acting through cyclic GMP, contributes to activation of the prothoracic gland at the termination of diapause. Although our best evidence points to the ecdysteroids as the major coordinators of diapause in larvae and pupae, other hormones can be expected to play roles as well. Roles for JH (see Section 10.3.2.4.) and DH (see Section  10.3.2.5.) are also well established, but others beyond these may also be implicated. For example, neuropeptide-like precursor 4 is uniquely expressed in association with diapause in flesh fly pupae (Li et al., 2009), but its function remains unknown. This potential regulatory agent, and others like it, suggest that we may not yet have identified all the players involved in regulating larval and pupal diapause. 10.3.2.3.  Ecdysone receptor and Ultraspiracle  Two proteins, Ecdysone receptor (EcR) and Ultraspiracle (USP), dimerize to form the functional receptor for ecdysone (see Chapter 5). 20-Hydroxyecdysone binds to this complex, which in turn binds directly to ecdysone

response elements to elicit specific gene action. The central role for ecdysteroids in the regulation of larval and pupal diapauses suggests that these proteins could contribute to the diapause control mechanism, but thus far few experiments have addressed this question. During pupal diapause of S. crassipalpis expression of EcR mRNA persists throughout diapause, but the expression of USP gradually declines at the onset of diapause and is undetectable after 20 days in diapause (Rinehart et al., 2001). USP transcripts again appear in late diapause (beginning on day 50) and are further upregulated 9 h after pupae are artificially stimulated to break diapause. This 9 h time frame coincides precisely with the rise in the ecdysteroid titer following hexane application. It is interesting to note that upregulation of the USP transcripts at day 50 coincides with the pupa’s increased sensitivity to injected ecdysteroids. This suggests the possibility that reappearance of the USP transcript is a preparatory step, perhaps a critical one, leading to the eventual termination of diapause. In the second instar larval diapause of Choristoneura fumiferana, genes encoding both isoforms of EcR are expressed throughout diapause, as is the gene encoding USP (Palli et al., 2001). However, the ecdysoneinducible transcription factors (CHR75A and CHR75B) and hormone receptor 3 (CHR3) are not present during diapause, but reappear at diapause termination. In M. sexta the transcript encoding EcR disappears at the onset of diapause and reappears when diapause is terminated (Fujiwara et al., 1995). In the larval diapause of the midge Chironomus tentans (Imhof et  al., 1993) and the bamboo borer O. fuscidentalis (Tatun et  al., 2008), EcR transcripts are detectable throughout diapause and an increase is noted at diapause termination. The patterns of USP mRNA expression are unknown for these two species. Although the database remains small at this point, the results do not suggest the emergence of a universal pattern in relation to diapause, but the presence or absence of these receptor proteins could very well be an important component of the regulatory scheme for diapause. Turning off the expression of one or both of these genes could promote diapause, while the presence of these gene products is likely to be essential for the resumption of development. 10.3.2.4.  Role of the corpora allata  The early views of larval and pupal diapause focused strictly on the brain–prothoracic gland axis, leaving little reason to suspect involvement of JH. This view was challenged by a set of experiments on D. grandiosella by Chippendale, Yin and their colleagues beginning in the early 1970s (Chippendale, 1977). What launched their work on JH was the surprising discovery that injection of ecdysteroids into a diapausing larva of D. grandiosella prompted a stationary larval molt rather than pupation. The stationary molt suggested the presence of JH because the

446  10: Hormonal Control of Diapause

larva would have been expected to pupate in the absence of JH. This suspicion of JH involvement was followed by verification that the hemolymph titer of JH remains high and the corpora allata (CA) are active during diapause. At the end of diapause, the JH titer drops, and the larva pupates. A similar regulatory scenario is evident in some of the Chilo stemborers. These two taxa both undergo stationary molts during diapause, and the only way to undergo a stationary molt rather than a progressive molt is to maintain a high JH titer. The high JH titer reported for larval diapause in the Mediterranean corn borer, Sesamia nonagrioides (Eizaguirre et al., 1998, 2005) and the yellow-spotted longicorn beetle, Psacothea hilaris (Munyiri and Ishikawa, 2004) suggests that they too have a diapause maintained by JH. Species that do not undergo stationary molts presumably do not need to maintain a high JH titer during diapause, and in a number of Lepidoptera larvae JH appears to play no role; for example, in O. nubilalis and Laspeyresia pomonella (Denlinger, 1985). In these species JH is actually high at the onset of diapause, but the titer drops to low levels shortly after diapause is initiated, and an injection of ecdysteroids elicits pupation, not a stationary molt. Why the JH titer is high at the onset of diapause is unclear, but it does not appear to be involved in the induction process. Application of JH to non-diapausing larvae at this stage does not cause the induction of larval diapause. An application of JH can terminate larval diapause in O. fuscidentalis (Singtripop et al., 2000), as reported previously for a number of other larval and pupal diapauses (Denlinger, 1985), and JH presumably exerts this effect by stimulating the prothoracic gland to produce ecdysone, as suggested by prothoracic gland transplantation studies in O. fuscidentalis (Singtripop et al., 2008). There is no evidence to suggest that JH by itself is the natural trigger for diapause termination. A combined role for JH with ecdy­ steroids is, however, likely in some cases. In flesh flies, JH by itself has no effect in terminating pupal diapause, but dramatic synergism is noted when JH is applied in concert with a small dose of ecdysteroid (Denlinger, 1979). When diapause is terminated naturally in flesh flies, a small peak of JH activity precedes the sustained elevation of the ecdysteroid titer, thus the combined application of JH and ecdysteroid likely is an effective mimic of the natural reactivation process. JH contributes to the regulation of metabolic events occurring during pupal diapause, but it does not appear to play a regulatory role in the induction or termination of diapause. In flesh flies, JH is involved in regulating metabolic cycles that persist during diapause (Denlinger et al., 1984). In these flies the metabolic rate is not constant; it occurs in 4- to 6-day cycles. These cycles are driven by JH. The hemolymph JH titer progressively increases during the trough of the cycle, reaches a critical point,

triggers a bout of high oxygen consumption, and then drops precipitously. The rapid decline in JH titer is aided by a rise in JH esterase (Denlinger and Tanaka, 1989). When the ring gland containing the CA is removed, the diapausing pupae become acyclic. Application of a high dose of JH analog to the pupae just before diapause will not alter the diapause fate of the pupa, but such pupae fail to exhibit oxygen consumption cycles and instead consume oxygen at a high rate throughout diapause. Pupae treated this way have a much shorter diapause, presumably because their high metabolic rate has prematurely exhausted their energy reserves. This conclusion assumes a mechanism used by the pupae to monitor and respond to depleted energy reserves (Denlinger et al., 1988; Hahn and Denlinger, 2011). 10.3.2.5.  A role for diapause hormone?  Diapause hormone is well known for its role in diapause induction in B. mori (see Section 10.3.1.), but attempts to induce diapause in other species with DH have failed. Thus, DH appeared to be a neuropeptide with some other primary function that was simply recaptured by B. mori as a diapause regulator (Denlinger, 1985). We now know, however, that a gene encoding a DH-like sequence is present and expressed in all Lepidoptera that have been examined, even those that lack an embryonic diapause. Diapause hormone and PBAN, along with several additional subesophageal ganglion neuropeptides (SGNPs), are encoded by a common precursor (see Section 10.3.1.1.). However, the function of this DH-like peptide in lepidopteran species other than B. mori has remained elusive. Does DH contribute to diapause in other species? DH-like peptides from H. virescens (Xu and Denlinger, 2003), H. armigera (Zhang et  al., 2004a,b) and H. zea (Zhang et al., 2008) appear to have no stimulatory effect on diapause induction, but quite surprisingly, diapausing pupae of these heliothine species readily respond to the DH-like peptides by terminating diapause. This could represent a non-specific effect, but non-amidated forms of DH and other injected proteins fail to terminate diapause, suggesting that the effect is specific for DH. The possibility that a drop in DH contributes to diapause induction is further supported by the observation that expression of the mRNA encoding DH-PBAN declines at the onset of larval wandering behavior and remains low throughout pupal diapause, whereas expression remains high in larvae and pupae not destined for diapause. When diapausing pupae of H. armigera are transferred to high temperatures to terminate diapause, the mRNA encoding DH-PBAN is quickly elevated (Wei et  al., 2005). In H. virescens, Northern blots indicate that the DH-PBAN message is most highly expressed in the subesophageal–ganglion complex (Xu and Denlinger, 2003), and this is supported by immunocytochemical evidence in H. armigera

10: Hormonal Control of Diapause  447

(Wei et  al., 2005). The hemolymph titer of DH-like peptide is also much higher in non-diapausing pupae than in diapausing ones (Wei et al., 2005). Injection of DH into diapausing pupae prompts a major increase in hemolymph ecdysteroids 3 to 7 days after DH injection. Current evidence from these noctuid moths is consistent with a drop in DH being essential for pupal diapause induction and DH elevation serving as a possible trigger for the ecdysteroid surge needed for diapause termination. As shown in Figure 5, a single gene encodes DH, PBAN, and three SGNPs of unknown function. There is well-documented cross reactivity between DH and PBAN, and even the SGNPs are capable of terminating pupal diapause in Helicoverpa assulta (Zhao et al., 2004) and H. armigera (Zhang et al., 2004b), suggesting similarities in the receptors for members of this family. Yet, differences in efficacy of these different neuropeptides in bioassays and more recent evidence on the structures of the receptors for DH and PBAN (Homma et al., 2006; Watanabe et al., 2007) indicate that these neuropeptides use distinctly different receptors. The 24-amino acid sequences of DH used by H. zea and H. virescens differ by only a single amino acid, but a greatly truncated version of the neuropeptide, C-terminal heptapeptide LWFGPRLa (the core sequence), is sufficient to terminate diapause (Zhang et al., 2008). Identification of the active components of DH led to the development of several hyperpotent agonists (Zhang et al., 2009) that could potentially disrupt diapause in this important group of agricultural pests. How and why the heliothine moths use both DH and ecdysteroids for the termination of pupal diapause is not yet clear. Both hormones, by themselves, are capable of terminating diapause when injected into diapausing pupae, yet the responses are distinct. While an injection of ecdysteroids will break diapause at any temperature, DH is ineffective at 18°C but will readily break diapause at temperatures of 21°C or higher (Zhang et al., 2008). The DH agonists that have been developed also show the same restricted range of temperature effectiveness as DH (Zhang et  al., 2009). Diapause hormone can stimulate the prothoracic glands of H. armigera to produce ecdy­ steroids (Zhang et al., 2004c; Liu et al., 2005), indicating that both PTTH and DH can promote steroidogenesis in heliothine prothoracic glands, presumably by activating the diazepam-binding inhibitor/acyl-CoA-binding pathway (Liu et  al., 2005). The temperature sensitivity of the DH response differs from that of the ecdysteroids, suggesting that the DH pathway for terminating diapause relies on high temperature and may trigger pupae to break diapause in response to high temperature. This scenario is consistent with the observation that the timing of diapause termination in heliothine moths is independent of the brain shortly after pupation (see Section 10.3.2.2.).

10.3.2.6.  Associated changes in gene expression  The endocrine mechanism that dictates the diapause fate can be viewed as a developmental switch that brings into play the upregulation and downregulation of huge suites of genes and their products as evidenced by recent large-scale studies on pupal diapause using microarrays (Emerson et al., 2010; Ragland et al., 2010), proteomics (Li et al., 2007; Cheng et  al., 2009; Chen et  al., 2010; Lu and Xu, 2010), and metabolomics (Michaud and Denlinger, 2007). A large-scale proteomics approach has also been used to probe the larval diapause of N. vitripennis (Wolschin and Gadau, 2009). Some genes, such as the clock genes discussed in Section 10.2.3.2., are upstream of the endocrine regulatory switch, while others are involved with downstream events set in motion by the endocrine program. Downstream physiological responses commonly linked to the diapause program include metabolic depression, cell cycle arrest, and enhanced stress tolerance. Transcriptomic (Ragland et  al., 2010) and metabolomic (Michaud and Denlinger, 2007) results with flesh flies suggest that pathways involving glycolysis and gluconeogensis are favored during pupal diapause. The expression patterns of several genes in the TCA cycle are altered by the diapause program, and the enrichment of genes involved in anaerobic metabolism during diapause is noted in flesh fly pupae as well as in diapausing larvae of the pitcher plant mosquito (Emerson et al., 2010). This suggests that such a shift to anaerobic metabolism may be a common downstream component of diapause in larvae and pupae as well as in adult dormancy in Drosophila melanogaster and in the dauer state of the nematode Caenorhabditis elegans (Ragland et al., 2010). Another related consequence of favoring glycolysis and gluconeogenesis is the generation of carbohydrates, polyols, and amino acids that are known to have cryoprotective functions. Diapausing insects are well known to have enhanced tolerance to low temperatures, desiccation, hypoxia, and oxidative stress, and gene expression that supports the acquisition of these forms of stress protection are commonly noted during diapause. The upregulation of genes encoding heat shock proteins is also an attribute of diapause in quite a few species, and suppression of these genes in flesh flies results in impaired cold tolerance (Rinehart et al., 2007). Cell cycle arrest is another feature central to diapause. In diapausing flesh fly pupae this arrest in the brain occurs at the Go/G1 stage and downregulation of the cell cycle regulator, proliferating cell nuclear antigen (pcna), appears to be critical for bringing about the arrest (Tammariello and Denlinger, 1998). This response is quite similar to that observed in the CNS of diapausing larvae of the fly C.  costata (Kostal et  al., 2009). A pentapeptide Yamamarin, extracted from diapausing pharate first instar larvae of the Japanese oak silkmoth A. yamamai, is a potent agent capable of arresting cell proliferation in

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insects and insect cell lines as well as rat leukemic cell lines (Sato et al., 2010). This peptide can also induce embryonic diapause in B. mori when injected into pupae of silkmoths not programmed for diapause. How widespread such an agent may be remains unknown, but clearly some mechanism for halting the cell cycle can be expected to be a component common to many forms of diapause. It is not currently clear how the endocrine system is linked to the cell cycle arrests noted earlier, but a role for ecdy­ steroids in regulating the cell cycle is suggested from cell culture studies (Gerenday and Fallon, 2004). Although most diapauses can be characterized by the same end points of metabolic depression, enhanced stress resistance, and cell cycle arrest, these end points can be attained by diverse tactics, and we can expect that different insect species will have targeted different components of the gene network to elicit a similar physiological response. We remain at a very early stage in sorting out what specifics genes are being regulated. 10.3.3.  Hormonal Basis for Adult Diapause

The central feature of adult diapause is a cessation in reproduction. For females this implies an arrest in oocyte development and in males the most conspicuous feature is their failure to mate with receptive females. The status of the testes is not a consistently reliable indicator of diapause in males (Pener, 1992): in some cases the testes are underdeveloped, while in other cases they may be full of mature sperm cysts. In both males and females the accessory glands are usually reduced in size during diapause. Although a period of long-distance flight may precede the entry into diapause, once adults arrive at their diapause site, flight muscles may degenerate (especially common in Coleoptera) for the duration of diapause and then regenerate when diapause is terminated. Adults remain fairly inactive in diapause, but some local movements may be noted. Mating may occur either before the entry into diapause or after diapause has been completed. When mating occurs prior to diapause, the males usually die without entering diapause and only the females bridge the diapause period. When both sexes diapause, mating usually occurs only after diapause has been terminated. Most early work on the hormonal control of adult diapause focused on JH. Pioneering work on adult diapause, initiated in the late 1950s by Jan de Wilde and his colleagues in the Netherlands, utilized the Colorado potato beetle as their model system (de Kort, 1990). They observed that the JH titer in diapause-programmed Leptinotarsa decemlineata decreases after adult emergence, remains low throughout diapause, and then rises after termination of diapause. Further evidence supporting a role for JH in adult diapause is provided by the responsiveness of diapausing adults to JH, histological studies of the CA, and experimental manipulations of the CA.

Application of synthetic JH and JH analogs to diapausing females prompts egg laying (although such periods are not sustained). The CA from diapausing L. ­decemlineata are small and inactive. Surgical removal of the CA induces a diapause-like state in long-day beetles (those not programmed for diapause): they stop feeding, leave the food, and burrow into the soil. Chemical destruction of the CA in long-day beetles with precocene II elicits the same diapause-like response. In non-diapausing adults, cauterizing the pars intercerebralis (the region of the brain that controls CA activity) also induces a diapause-like state. This same surgery performed with diapausing adults blocks the beetle’s ability to become reproductively active when placed under long daylengths (the environmental signal used to promote reproduction). Severing the axons between the pars intercerebralis and the CA does not appear to alter the diapause decision, suggesting that the CA in this species is under hormonal, rather than neural, control. In the early experimental years there would have been no reason to suspect a role for ecdysteroids as a regulator of adult diapause because the well-known larval source of ecdysteroid synthesis, the prothoracic gland, degenerates in adults. But the discovery in the late 1970s that adults can produce ecdysteroids from other tissues such as ovaries raised the possibility that ecdysteroids could also be involved in regulating reproductive events. In the Colorado potato beetle, the hemolymph ecdysteroid titer is nearly twice as high in diapause-programmed beetles as it is in non-diapause-programmed beetles, but the titer drops sharply a few days after adult eclosion. The functional significance of this elevated ecdysteroid titer associated with diapause has not been determined. The scheme for the hormonal control of adult diapause that has been developed from experiments in L. ­decemlineata is applicable to many species that diapause as adults, but there are also clear species differences. These differences include whether both sexes enter diapause, sexual distinctions in the environmental cues used for regulating diapause, and the formation of stable intermediate phenotypes that lie somewhere between fully sexually active and completely inactive. The regulatory mechanisms controlling the CA during adult diapause vary among species. The brain’s regulation of the CA can be either hormonal as in L. decemlineata or neural as in P. apterus, Tetrix undulata, and L. migratoria. Clear unifying themes are evident within adult diapause, yet speciesspecific distinctions are also apparent. 10.3.3.1.  Role of the corpora allatal  Research on the endocrine basis of adult diapause continues to focus primarily on the role of JH. In substantially more species we now have new or additional evidence that JH plays a major role; for example, the mosquito C. pipiens (Sim and Denlinger, 2008), the butterfly Speyeria idalia (Kopper

10: Hormonal Control of Diapause  449

et al., 2001), the black rice bug Scotinophara lurida (Cho et al., 2007), the stink bug P. stali (Kotaki et al., 2011), the moth Caloptilia fraxinella (Evenden et al., 2007), and the plum curculio Conotrachelus nenuphar (Hoffmann et al., 2007). Earlier results arguing a role for JHs in regulating the adult diapause of C. pipiens were based on the ability of JH applications to terminate diapause; more recently this evidence was supplemented by demonstrating that cultured CA dissected from diapausing females secrete very little JH (Figure 10), whereas those dissected from non-­diapausing females are quite active, especially during the first week after adult eclosion (Readio et  al., 1999). Females of C. pipiens that are allatectomized and then transferred to diapause-terminating conditions remain in a diapause-like state and fail to initiate the blood feeding noted in control groups. Finally, the argument for JH regulation of adult diapause is further strengthened by gene silencing experiments (Sim and Denlinger, 2008), as discussed in Section 10.3.3.2. as well as with experiments demonstrating that a knockdown of two ribosomal genes, S2 and S3a, causes a diapause-like arrest in C. ­pipiens that can be reversed with application of JH III (Kim and Denlinger, 2010; Kim et al., 2010). The CA, of course, does not function autonomously. Activity of the CA is coordinated by the brain, and the brain exerts its control through both hormonal and neuronal conduits. Active suppression of the CA by the brain during diapause has been demonstrated in the following insect orders: Coleoptera: L. decemlineata (Khan et  al., 1983, 1988); Diptera: P. terraenovae (Matsuo et al., 1997); Heteroptera, P. stali (Kotaki and Yagi, 1989), R. clavatus (Morita and Numata, 1997a), and P. apterus (Hodkova et al., 2001); and Orthoptera: L. migratoria (Okuda and Tanaka, 1997). In the diapause of P. terraenovae inhibition is maintained by neurons that originate in the pars lateralis

Figure 10  In vitro activity of the corpora allata (CA) from diapausing and nondiapausing females of the mosquito Culex pipiens on different days following adult emergence. Adapted from Readio et al. (1999).

and terminate within the CA (Shiga and Numata, 2000), but in several other species, including P. apterus (Hodkova, 1994), the inhibition appears to originate in the pars intercerebralis. The diapause program may actually influence the organization of the synapses. Rearing L. decemlineata throughout its life under short-day conditions boosts both the number and activity of neurosecretory synapses within the CA as compared to beetles reared under long-day conditions (Khan and Buma, 1985). Hormonal control of the CA is also evident in a number of species. CA activity in diapausing L. migratoria is suppressed by neuropeptide(s) stored in the corpora ­cardiaca (CC; Okuda and Tanaka, 1997). Neural regulation of the CA during diapause may not be as simple as the presence or absence of an allatostatin. Brain extracts from both diapausing and nondiapausing P. apterus stimulate increased JH production in CA from non-diapausing donors but have no effect on JH synthesis in CA from diapausing donors. This suggests that diapausing adults may lack receptors for allatotropins or the presence of an allato-inhibiting factor in the CA or CC that blocks the action of allatotropins (Hodkova et al., 1996). Extracts of the brain–subesophageal ganglionCC-CA complex from diapausing P. apterus inhibit the CA activity of P. stali, indicating that this neurosecretory complex from diapausing bugs may contain both allatoinhibiting and -stimulating factors (Hodkova et al., 1996). The JH hemolymph titer is a function of both JH synthesis and its degradation (de Kort and Granger, 1996). JH esterases (JHE) play a central role in regulating the JH titer in diapause-destined adults of L. decemlineata. There is an inverse relationship between the JH hemolymph titer and JHE activity in pre-diapausing adults, with peak esterase activity occurring just prior to diapause initiation. This peak in JHE activity contributes to the low JH titer noted during the first two days of adult life (Kramer, 1978). The effect of JH on JHE activity is indirect and appears to require a factor from the brain. JHE isolated from the hemolymph of fourth instar larvae of L. decemlineata is a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997a). cDNA clones of two different JHEs (A and B) have been isolated from fourth instar larvae (Vermunt et  al., 1997b, 1998). JHE-A appears to encode the protein first isolated from the hemolymph, whereas JHE-B encodes a protein that does not appear to be secreted into the hemolymph. Significant expression of the gene encoding JHE-A is present in brains of fourth instar larvae and fat body of both fourth instar larvae and short-day adults; low expression is noted in long-day adults (Vermunt et al., 1999). Treating diapause-destined day 1 adults with the JH analog pyriproxyfen strongly suppresses JHE-A expression. JHE-A sensitivity to pyriproxyfen decreases as beetles near diapause initiation. Vermunt et al. (1999) concluded that JHE activity in the hemolymph is critical for the initiation of diapause in the Colorado potato beetle. However, a role for JHEs in regulation of the JH titer appears not to

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be essential for some other cases of adult diapause. In the locusts Nomadacris japonica and L. migratoria there are no differences in JHE hemolymph activity between long- and short-day adults (Okuda et al., 1996). Although evidence supporting involvement of JH in reproductive diapause is compelling, results indicate that the absence of JH is not the exclusive cause of diapause induction and maintenance. Results, even in the Colorado potato beetle, suggest that lack of JH cannot account for all observations. Juvenile hormone application, at best, can stimulate only a short burst of oviposition in diapausing females, suggesting that the actual termination of diapause requires more than a single boost in the JH titer. This observation has usually been interpreted as the need for sustained stimulation of the CA by the brain (Denlinger, 1985). A minimal interpretation is that at the end of diapause the brain must become reactivated to promote JH synthesis by the CA. Brain activation clearly needs to precede activation of the CA and it is possible that the brain independently directs specific events associated with diapause termination. It is also possible that these events occur prior to activation of the CA. This is evident from the observation that adults of L. ­decemlineata actually dig their way out of the soil prior to activation of the CA (Lefevere et al., 1989). This suggests that activation of the CA may be the consequence of diapause termination, rather than the cause. The regulatory scheme for males is not always the same as that in females. In P. apterus (Hodkova, 1994) and P. ­terraenovae (Tanigawa et al., 1999), regulation of diapause in females clearly involves the CA, but the CA does not appear to be involved in males. Allatectomy has little effect on the manifestation of diapause in males; they respond to photoperiod and display mating behavior even in the absence of a CA. The inhibitory center regulating mating behavior resides in the brain’s pars intercerebralis, but this important inhibitory pathway does not involve the CA. When the CA are involved in regulating diapause, they presumably preside over the whole diapause syndrome, not just the regulation of reproduction. This appears to be the case in P. apterus, a species in which photoperiod dictates not only the reproductive status of the female but also the profile of phospholipid molecules in cell membranes (Hodkova et al., 2002). The unique winter pattern of phosphatidylethanolamines, associated with cold hardening, is programmed by short daylength, not low temperature. Non-diapausing females deprived of their CA assume the lipid profile normally associated with diapausing females. Similarly, earlier studies with the Colorado potato beetle demonstrated involvement of the CA in determining the status of flight muscles during diapause. The occurrence of reproductive arrest in insects is not confined to diapause. Adults of P. apterus have two distinct forms of reproductive arrest, a photoperiod-controlled diapause and a non-diapause-starvation reproductive

arrest. These two arrests are both controlled by the brain but by different mechanisms, resulting in distinct morphological changes in the corpus allatum (Hodkova et al., 2001). Experiments using transplanted brain–CA complexes and application of JH analogs have clearly demonstrated that JH terminates both forms of reproductive arrest (Hodkova and Socha, 2006; Socha, 2007; Socha and Sula, 2008). Delineating commonalities and differences in the various forms of reproductive arrest will be helpful in defining convergent mechanisms for shutting down development. Another intriguing connection between JH and adult diapause has been proposed by Hunt et  al. (2007) for Polistes wasps. In this scenario, accumulation of a storage protein, hexamerin 1, may dictate the JH titer, determining the diapause fate of the emerging females. Low levels of hexamerin are suggested to elevate JH, which in turn promotes the non-diapause phenotype that is primed to reproduce, while high levels of hexamerin generate a JH-deficient diapause phenotype that fails to reproduce until the following year. Although pieces of this regulatory scheme remain to be defined, if this scheme is verified, this would be an unusual case where the JH titer is dictated by the presence of hexamerins, rather than the more usual assumption that JH and ecdysteroids regulate production and accumulation of the hexamerins (Denlinger et al., 2005). Energy stores are enormously important for the success of diapause (Hahn and Denlinger, 2011), and the mechanism proposed for the paper wasps provides a nice link between nutrition and the endocrine mechanisms of diapause. 10.3.3.2.  Insulin signaling  Insulin signaling plays a critical role in dauer formation in the nematode C. elegans (Lee et  al., 2003), and the same appears to be true for reproductive arrest in D. melanogaster (Tatar et al., 2001; Allen, 2007) and the mosquito C. pipiens (Sim and Denlinger, 2008). The importance of insulin signaling in a wide range of insect regulatory functions (Wu and Brown, 2006; Brown et al., 2008; see Chapter 2) underscores its likely role in diapause, especially since diapause frequently involves decisions regarding nutrient management (Hahn and Denlinger, 2011), which is a key area regulated by the insulin pathway. Knocking down the insulin receptor in non-diapausing females of C. pipiens using RNAi results in an arrest in ovarian development that simulates diapause (Sim and Denlinger, 2008). Mosquitoes could be rescued from this developmental arrest with an application of JH or a JH analog, the endocrine trigger known to terminate diapause in this species. When dsRNA directed against forkhead transcription factor (FOXO), a gene downstream of insulin, was injected into diapausing females, the females failed to accumulate the huge fat depositions that characterize diapause. This suggests that a shut down

10: Hormonal Control of Diapause  451

in insulin signaling halts JH production and simultan­ eously prompts activation of the downstream gene FOXO, leading to the diapause phenotype, as summarized in Figure  11. Although most invertebrates are thought to have just one insulin-like receptor, numerous insulin-like peptides (ILPs) are known from insects. Among these ILPs, ILP-1 appears to be the one of greatest importance for diapause regulation in C. pipiens (Sim and Denlinger, 2009a). The activation of FOXO is thus proposed to lead to the dramatic shift in lipid metabolism associated with diapause (Sim and Denlinger, 2009b). The scenario presented in Figure 11 is consistent with the results currently on hand, but not all steps in the pathway between insulin signaling and JH are known at this time. Key evidence pointing to a role for insulin in the reproductive arrest of Drosophila is presented by Williams et  al. (2006). Genetic crosses of geographic strains of D. ­melanogaster exhibiting different potentials for ovarian arrest led to the isolation of Dp110, a gene that encodes phosphoinositol 3 OH kinase (PI3 K), a component of the insulin signaling pathway. Mutants with a Dp110 deletion have an elevated incidence of reproductive arrest, whereas increased expression of Dp110 in the nervous system elicits the opposite effect. Results from both C. pipiens and D. melanogaster are consistent with a model showing that reduced insulin signaling is linked to adult diapause. 10.3.3.3.  Role of ecdysteroids  The role of ecdysteroids in the regulation of adult diapause has been investigated most thoroughly in D. melanogaster, although this arrest appears to be a low-temperature induced halt, more akin to a quiescence than a photoperiodically induced diapause (Emerson et al., 2009b). The arrest occurs at the pre-vitellogenic stage of ovarian follicular development

A.

B.

Figure 11  Model for the role of insulin signaling and FOXO in regulation of adult diapause in the mosquito Culex pipiens. (A) Insulin signaling in non-diapausing females results in ovarian development and suppression of FOXO. (B) Insulin signaling in diapausing females suppresses the insulin signaling pathway, resulting in arrested development. FOXO is activated, which leads to fat hypertrophy and enhanced stress resistance. Adapted from Sim and Denlinger (2008, 2009a).

(Saunders et al., 1989). Yolk proteins (YPs) are present in the hemolymph of these females, but the oocytes fail to incorporate the YPs (Saunders et al., 1990). Ecdysteroids are proposed to activate endocytosis at termination of the arrest allowing initiation of vitellogenesis (Richard et al., 1998). The evidence for this intriguing scenario is compelling. During this reproductive arrest ovaries produce low level of ecdysteroids; the rate of synthesis begins to increase 4 h after transferring the flies to 25 °C to terminate the arrest, and reaches maximum levels by 12 h (Richard et  al., 1998). Injection of 20E terminates the arrest in a dose-dependent manner as measured by ovarian maturation (Richard et  al., 2001b). Yolk protein uptake by the oocytes is by a receptor-mediated endocytotic mechanism (Bownes et  al., 1993) that involves at least three key proteins. Clathrin coats the pit, and adaptin binds to both clathrin and the transmembrane YP receptor (Gao et  al., 1991; Smythe and Warren, 1991). Once the vesicle is internalized the vesicle breaks down, and the clathrin and adaptin are recycled (Gao et  al., 1991). During the arrest, clathrin, adaptin, and YP receptor are undetectable by immunostaining. Terminating the arrest by transferring flies to 25°C or injecting them with 20E induces expression of clathrin, adaptin, and the YP receptor in nurse cells adjacent to the oocytes (Richard et  al., 2001a). Incubating ovaries from females in this reproductive arrest with 20E fails to elicit expression of clathrin, adaptin, or the YP receptor, indicating that dormancy termination is not regulated by the ovaries and that some external factor is needed (Richard et al., 2001a). How can these ecdysteroid results be reconciled with the huge body of data suggesting that vitellogenesis in higher Diptera is mediated by JH? Indeed application of JH III or JHB3 breaks this arrest in D. melanogaster (Saunders et  al., 1990), but this response is an indirect effect caused by JH triggering the ecdysteroid-mediated initiation of vitellogenesis (Richard et al., 1998, 2001b). When flies are transferred to reproduction permissive conditions, it is the ecdysteroid titer that goes up quickly (within hours), while the JH titer remains low. Roles for ecdysteroids are suggested in a number of other species as well, but the results are sometimes conflicting. Significantly lower ecdysteroid titers are reported for diapausing females of L. migratoria than for nondiapausing females (Tawfik et  al., 2002a), a result that is consistent with D. melanogaster. The ecdysteroid titer in L. migratoria fluctuates between 22 and 40 ng ml−1 in diapausing females, whereas the titer reaches a peak of 354 ng ml−1 in 18- to 22-day old non-diapausing females. Treating diapausing females with three consecutive applications of JH III induces ovarian development and increases ovarian and hemolymph titers of ecdy­ steroids to levels similar to those noted in sexually active

452  10: Hormonal Control of Diapause

females (Tawfik et al., 2002a). This relationship between the ecdysteroid titer and diapause induction is, however, opposite of that seen in L. decemlineata (Briers and de Loof, 1981). In these beetles, high titers of ecdysteroids are noted in diapause-destined beetles shortly after adult eclosion, but the titers drop within the first week, whereas the titers are initially low in non-diapausing adults and then increase in females at the onset of reproduction. During diapause, the ecdysteroid titer gradually increases (Briers et  al., 1982). This may be essential for diapause termination. Interestingly, an injection of a combination of 20E and JH is much more effective in breaking diapause than either hormone by itself (Lefevere, 1989), a response also noted for pupal diapause termination in flesh flies (Denlinger, 1979). Lefevere et al. (1989) suggested that the ecdysteroid injection enhances diapause termination by blocking activation of JHE, facilitating elevation of the JH titer. A role for ecdysteroids has also been proposed for diapause in P. apterus (Sauman and Sehnal, 1997). Under long-day conditions, maturation of the male accessory glands and testes of P. apterus occur during the fourth nymphal instar. Injecting an ecdysteroid or implanting brain–subesophageal ganglion complexes from long-day nymphs into early fourth instar nymphs reared at short days induces sexual development in the normally immature reproductive tract. Sauman and Sehnal (1997) concluded that the pre-diapause physiology in the fourth instar nymph of P. apterus is regulated by ecdysteroids and neurohormones, an observation that is consistent with other reports showing a low ecdysteroid titer contributing to the diapause fate of the adult. Prothoracicotropic hormone, the neuropeptide that drives ecdysteroid production in the prothoracic gland of larvae and pupae, is generally considered not to play a role in adults because the prothoracic gland has degenerated by that time. Yet, ovarian-derived ecdysteroids are now well-known regulators of adult reproductive events, and a possible role for PTTH in adults cannot be discounted as evidenced by recent work on the mosquito C. pipiens (Zhang and Denlinger, 2011; see Chapter 1). Transcript abundance of the gene encoding PTTH drops quickly following adult emergence in non-diapausing female mosquitoes but persists at a high level for the first month in diapausing females. These results indicate that, at least at the transcript level, there are conspicuous PTTH distinctions between females programmed for diapause and nondiapause, suggesting possible differences in ecdysteroid titers as well. It seems unlikely that adult diapause will prove to be based simply on either a JH or ecdysteroid deficiency. In all of the earlier examples showing evidence for ecdysteroid involvement, JH also has been implicated, suggesting roles for both of these hormones in orchestrating the regulation of adult diapause.

10.3.3.4.  Associated changes in gene expression  As seen for larval and pupal diapauses the endocrine mechanisms controlling adult diapause set into motion complex changes in gene expression, as evidenced from a suppressive subtractive hybridization study in C. pipiens (Robich et al., 2007), a targeted gene study in P. apterus (Kostal et al., 2008), and differential display studies with L. decemlineata (Yocum et  al., 2009a,b). Thus far, the only large-scale genomic approach to adult diapause is based on the reproductive arrest noted in D. melanogaster, where a comparison was made between flies in arrested reproduction and those that terminated their arrest and initiated egg development (Baker and Russell, 2009). Such studies give a good perspective on the magnitude of gene expression changes associated with diapause. Numerous transcripts are altered in abundance. In the Drosophila study, 2786 transcripts were two- or threefold more abundant during diapause. As might be expected, genes involved in processes associated with follicle and egg maturation are downregulated, whereas many genes involved in response to environmental stress, lipid conservation, and protective structural modifications are upregulated. Among diapause-associated genes that may be particularly important for regulating reproduction are ribosomal proteins S2 (Kim and Denlinger, 2010) and S3a (Kim et al., 2010). Expression of both of these ribosomal proteins is shut down during early diapause in the mosquito C. pipiens, and RNAi directed against these genes results in a “diapause-like” ovarian state in mosquitoes programmed for continuous development. Both genes are known to play critical roles in regulating oogenesis in other insects, suggesting that their shutdown could be critical for the diapause response. Arrest in ovarian development observed in RNAi-treated females can be rescued with an application of JH III, the hormone known to break diapause in C. pipiens, indicating that these two ribosomal genes respond to JH and their shutdown may be an essential component for generating the diapause phenotype. Like the shutdown in insulin signaling discussed in Section 10.3.3.2., these genes are downregulated at specific times in early diapause and are clearly responsive to JH, the known endocrine terminator of adult diapause. Transcript levels of certain genes such as those encoding aldose reductase and sorbitol dehydrogenase change systematically during diapause in P. apterus and can serve as effective markers for the progression of diapause (Kostal et al., 2008). Likewise, several genes noted in L. decemlineata can be used as biomarkers to track the progression of diapause (Yocum et al., 2009a,b). As discussed in Section 10.2.3.2., clock genes have been examined in association with adult diapause in several species, but critical links between clock gene expression and downstream pathways, including the endocrine components that dictate the diapause fate, remain unknown.

10: Hormonal Control of Diapause  453

10.4.  Parallels to Other Dormancies Diverse endocrine mechanisms are capable of halting development at different developmental stages and in different species that enter diapause at the same stage. Sometimes the same net effect, diapause, is elicited by contrasting responses to the very same hormone. For example, a shutdown in ecdysteroid production is key to most pupal diapauses, but an elevation in ecdysteroids elicits diapause in pharate first instar larvae of the gypsy moth. Similarly, the absence of JH characterizes many adult diapauses, while some larval diapauses are maintained by a high JH titer. Progression through the different stages of embryonic and post-embryonic development and the initiation of reproduction by the adult is orchestrated by a well-defined series of endocrine events requiring periods of hormone release and other equally important phases of hormone absence. This requirement for precisely timed periods of hormone presence or absence to elicit uninterrupted development suggests that nearly any interruption in this normal sequence could lock the insect into a developmental arrest at the point of interruption. This is presumably why we observe diverse endocrine mechanisms causing the same developmental shutdown we recognize as diapause. This means that the endocrine control mechanism for halting development has evolved independently numerous times. Certainly there are taxa in which the diapause stage and the control mechanism are highly conserved. For example, all of the flesh fly species (Sarcophagidae) from around the temperate and tropical world that have a diapause rely on a pupal diapause that represents a shutdown in the brain–prothoracic gland axis, suggesting a highly conserved trait within this family. By contrast, different members of the Drosophilidae are known to enter diapause as larvae, pupae, or adults, suggesting that the endocrine basis for diapause has evolved several times within this family. The challenge to identify a unifying endocrine basis for diapause becomes even more difficult when one seeks parallels in the endocrine control mechanisms for dormancies in non-arthropods. A connection between the insulin-like receptor daf-2 in the production of dauer larvae of the nematode C. elegans (Kimura et al., 1997; Lee et al., 2003) and the involvement of a homologous receptor that influences JH synthesis in D. melanogaster (Tatar et al., 2001; Tatar and Yin, 2001) and C. pipiens (Sim and Denlinger, 2008) offers a tantalizing connection with some adult diapauses, but because JH is not an active player in many forms of insect diapause suggests that this connection will not be applicable to all insect diapauses. It is quite likely that many different non-arthropod species will have, like insects, tapped into diverse endocrine mechanisms to regulate development as well as developmental arrest. This argument, however, does not preclude the possibility that at the molecular level diapause and other forms of

dormancy may target the same or similar ­cellular processes to bring about an arrest in development. Several results already hint of some commonality to the molecular events associated with diapause. For example, the diapause-associated upregulation of genes encoding heat shock proteins was first noted in flesh fly pupae, but heat shock protein upregulation during insect diapause has now been documented in several pupal diapauses as well as embryonic, larval, and adult diapauses of other insects and in the dormancies of brine shrimp, nematodes, and some plants (Denlinger et al., 2001; Rinehart et al., 2007). This common feature represents a widespread contribution of heat shock proteins to cellular defense during diapause and/or a contribution to the mechanism for shutting down development. Although not all insects elevate heat shock proteins during diapause (Goto et  al., 1998), elevated stress responses of some sort are a component of most diapauses. Another feature that would appear to be an essential cellular event common to most diapauses is a shutdown of the cell cycle (Tammariello, 2001; Kostal et al., 2009). Again, there may be diversity in the phase of the cell cycle arrested (e.g., G0/G1 in pupae of S. crassipalpis and larvae of C. costata, G2 in pupae of M. sexta, and in embryos of B. mori) and in the cell cycle regulator that is targeted, but the overall targeting of the cell cycle is a likely ultimate target of whatever endocrine mechanism is utilized. Metabolic depression is yet another feature common to diapause, but again diverse mechanisms can be used to achieve this response. Diversity in the control mechanisms for diapause is most evident at the transcriptional level. In spite of common physiological end points such as metabolic depression, cell cycle arrest and enhanced stress tolerance in dauer larvae of C. elegans, adult dormancy in D. melanogaster, and pupal diapause in S. crassipalpis, there is remarkably little overlap in the genes generating the diapause response (Ragland et  al., 2010). This suggests that the key elements of diapause can be attained by different transcriptional strategies. This, in turn, suggests that we can expect equally diverse endocrine oversight directing diapause mechanisms. In summary, we argue that the diverse patterns of diapause, although sharing many common physiolog­ ical attributes, are attained through divergent molecular mechanisms that reflect an equal diversity of endocrine regulators. The ultimate physiological responses that characterize diapause can be turned on or off by influencing numerous targets in the gene network that generate the diapause phenotype.

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