Journal of Agricultural Science and Technology A 3 (2013) 935-947 Earlier title: Journal of Agricultural Science and Technology, ISSN

1939-1250

DAVID

PUBLISHING

Diapause Regulation in the Moth Sesamia nonagrioides (Lepidoptera: Noctuidae)

Anna Kourti
Department of Biotechnology, Laboratory of Molecular Biology, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece Received: September 22, 2013 / Published: December 20, 2013. Abstract: Insects enter in diapause in response to diverse environmental cues. During diapause, insects arrest their development and many genes are down-regulated while a small number of genes uniquely expressed at this time. This review aims to present available data regarding the regulation of diapause in the moth Sesamia nonagrioides (Lepidoptera: Noctuidae). Studying the transcriptional regulation of several genes (five heat shock proteins, two storage proteins and one juvenile hormone esterase) showed that these genes may play various roles in the diapause programming. The results show that SnoHsp19.5 gene was consistently expressed, while SnoHsp20.8 was down-regulated in deep diapause and was up-regulated at the termination of diapause. SnoHsc70 may play important roles in assisting protein conformation during specific stages of diapause. SnoHsp83 displays a similar pattern to SnoHsc70 under diapause conditions, when extra larval moults occur, indicating that could be involved in the developmental process that occurs between two moults. Expression of two SnoSP1 and SnoSP2 hexamerin genes was also observed throughout diapause. And the results lead us to the conclusion that larval diapause of S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins. In addition, the transcript level of the carboxylesterase SnoJHER was higher in non-diapausing larvae than in diapausing ones. During the fifth instar of the non-diapausing and the ninth instar of the diapausing larvae, SnoJHER mRNAs reached higher expression levels on the days close to each larval molt. Key words: Sesamia nonagrioides, diapause, heat shock proteins, storage proteins, juvenile hormone esterase.

1. Introduction
Diapause, a developmental program that has existed in metazoans for millions of years, is characterized by processes opposite to those of reproductive growth, such as the arrest or slowing of cell division in response to an immediate or anticipated stress, reduction of metabolism and enhancement of stress tolerance [1, 2]. Diapause is a more profound, endogenously and centrally mediated interruption that routes the developmental program away from direct morphogenesis into an alternative diapause program of succession of physiological events. The start of diapause usually precedes the advent of adverse conditions and the end of diapause
Corresponding author: Anna Kourti, associate professor, research field: insect molecular genetics. E-mail: akourti@aua.gr.

need not coincide with the end of adversity [3, 4]. Among different insect groups, diapause may occur at any stage of the development, although in every species the diapause stage is specific [5, 6]. Diapause is divided into overlapping stages encompassing induction, preparation, initiation, maintenance and termination, with the latter followed by post-diapause quiescence if environmental conditions are not conducive to growth [4]. The entry into and progression through diapause are mediated by molecular mechanisms similar to those which guide cellular reproductive growth, and include differential gene expression, post-transcriptional events, post-translational protein modifications and protein localization to specific regions within cells and organisms. The ability to execute these events in a coordinated manner implies the function of

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molecular switches that exert regulatory roles, directing development along the appropriate pathways. Identifying the switches and the extent to which they are shared between organisms are central questions in the study of diapause [7]. A typical indicator of diapause is the low rate of protein synthesis, while shortly before and after diapause this rate is significantly higher [8]. In many insect species, the onset of diapause is also correlated with the synthesis of selected proteins. In nondiapausing individuals those proteins are either not detectable or found in much lower concentrations [9, 10]. Such facts led researchers to hypothesize that diapause can be associated with a specific pattern of gene expression rather than a shutdown of genetic activity. Several of the diapause up-regulated genes that have been identified so far encode proteins that may be involved in stress responses or may contribute to the protection of the insect during its long period of dormancy. It is known that genes characteristics of reproductive growth are down-regulated during diapause, while other genes, often termed diapause-specific, are up-regulated, and the expression of still others is either not altered or changes intermittently [11]. The Mediterranean corn borer Sesamia nonagrioides (Lefbvre) is a Lepidopteran of the Noctuidae family that feeds mainly on maize, sugar cane and sorghum. It is found in almost all Mediterranean countries and is a multivoltine species. In the area of this study, Greece, 3-4 generations are completed per year. Fantinou et al. [12] and Stavrakis [13] reported that the pupation of the overwintering population in Greece takes place in April-May. It seems that the fourth generation is partial since some of the late progeny of the third generation will not make it through [14]. The importance of this fourth generation depends on the percentage of second-generation larvae that enter diapause. This state is determined genetically and is a response to a series of stimuli that announce

forthcoming adverse conditions for the insect. A short-day photoperiod induces larval diapause during the first and second instars, the effect of the photoperiod being modulated by the temperature and the quality of the nutrients [15, 16]. Diapausing larvae continue to develop and overwinter in the stubbles of maize. While nondiapausing larvae mostly molt to pupae after the sixth instar, diapausing larvae feed, move and molt with an indeterminate number of supernumerary molts [15, 16]. The insects in this study were obtained from an established laboratory colony of S. nonagrioides, derived from larvae collected in Kopais (latitude 3814, Central Greece) in 2007, maintained at 25 C 1 C and reared on an artificial diet [14]. The colony of nondiapausing insects was reared under long day (LD) conditions (16:8, light:dark) at 25 C, while diapausing larvae were reared under short day (SD) conditions (10:14, light:dark) at 25 C. In the following text, the ontogeny that includes diapause will be divided into three main phases: (1) pre-diapause: larvae grow through the 6th instar; (2) deep-diapause: during the deep-diapause phase, larvae of S. nonagrioides undergo supernumerary moults and increase their body weights until the 9th instar; (3) post-diapause: after diapause development, larvae remain under a post-diapause phase. Body weight decreased, larvae exhibit supernumerary moults and proceed to pupation. Larvae of S. nonagrioides under SD conditions exhibit 13 instars [17]. The molecular mechanisms underlying diapause has begun to emerge in a number of insect species, providing us with tantalizing directions for future research. This review presents available data regarding the regulation of diapause in the moth Sesamia nonagrioides (Lepidoptera: Noctuidae). Studying the transcriptional regulation of several genes involved in stress resistance and development (five heat shock proteins, two storage proteins and one juvenile hormone esterase (JHE)) showed that these genes may play various roles in the diapause programming.

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2. The Role of Heat Shock Proteins (Hsp) in the Larval Diapause of S. Nonagrioides
The level of stress tolerance achieved during diapause varies, along and with the degrees of morphogenesis behavioural modifications

contributing to survival under adverse conditions. Here the authors investigated the stress resistance of S. nonagrioides during diapause. Heat shock proteins (Hsps) comprise a diverse group of different classes of proteins present in almost all forms of life and show transient increased expression in response to a rapid increase in temperature [18, 19]. Many are present constitutively in cells, while others are induced when a cell or organism undergoes various types of environmental stress such as heat, cold, desiccation and oxygen deprivation [20, 21]. The Hsps function as molecular chaperons that protect cellular proteins during protein biosynthesis, including the recognition and binding of unfolded and non-native proteins [22, 23]. In insects there are four major heat-shock gene families: the small Hsp family with molecular masses ranging from 20-30 kDa, the Hsp60 family with molecular masses of approximately 60 kDa, the Hsp70 family with molecular masses of approximately 70 kDa and the Hsp90 family with higher masses [24]. In insects, Hsps are assumed to play a role in the regulation of diapause [25, 26]. During diapause, many genes are down-regulated and a small number of genes uniquely expressed at this time [7]. Some of the up-regulated genes encode Hsps. Genes encoding certain stress proteins (Hsp23 and Hsp70) are highly up-regulated during diapause, while others are either unaffected (Hsc70) or down-regulated (Hsp90) [4]. How Hsps may function in the long-term developmental arrest associated with diapause is unclear. Yocum et al. [25] suggested that Hsps may persist for long periods during diapauses. This is very interesting because extended expression of Hsps can lead to deleterious effects, including retardation and cessation of development [20].

The present review was undertaken to determine the molecular effects of diapause in S. nonagrioides larvae and Hsps genes (Hsp70, Hsc70, Hsp83, Hsp20.8 and Hsp19.5) were selected as model genes, as they are biomarkers of cellular stress activated by heat and cold shock [7, 17, 20]. In order to investigate the expression pattern of SnoHsps genes during diapause, RNA was isolated from the 5th instar non-diapausing or diapausing larvae and analyzed by semi-quantitative RT-PCR and real-time PCR assays [17, 27, 28]. 2.1 Small Hsp The up-regulation of sHsps appears to be common to diapause in species representing diverse insect orders, including Diptera, Lepidoptera, Coleoptera and Hymenoptera that occurs in different developmental stages (embryo, larva, pupa and adult) [29]. In contrast, recent studies propose that this is not the case in all insect species studied so far [30-32]. In order to unravel the potential contribution of sHsps transcripts to diapause of S. nonagrioides, two full-lengths cDNA sequences of S. nonagrioides were isolated, the SnoHsp19.5 and SnoHsp20.8 [17]. These genes were registered in GenBank with accession number EU668902 and DQ336356, respectively [17]. At 25 C, a non-stress temperature, the presence of mRNAs encoding sHsps is linked to the entire diapause period. The expression of SnoHsp19.5 gene begins with the onset of larval diapause, and the mRNAs are present until post-diapause. SnoHsp20.8 transcript levels were increased in pre-diapause, down-regulated during deep larval diapause and up-regulated in post-diapause stage. These results showed that SnoHsp19.5 and SnoHsp20.8 genes had different regulation regimes throughout the course of diapause. SnoHsp20.8 transcripts were relatively high during the initiation and termination phases of diapause [17]. This pattern of accumulation clearly defines the early stages of diapause as well as the culmination of the diapause process. Our

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Diapause Regulation in the Moth Sesamia nonagrioides (Lepidoptera: Noctuidae)

understanding of how diapause ends is still very incomplete. maintenance It is recognized culminate that in diapause may spontaneous

termination and resumption of direct development in many insects and mites which are under constant laboratory conditions, such as in the diapause of S. nonagrioides [4, 33]. The increase in expression level of SnoHsp20.8 could serve as a molecular marker of diapause termination in S. nonagrioides. These results show that the two genes may have distinctive roles in the initiation, continuation and termination of the dynamic process of diapause. This clear distinction in mRNA accumulation of the two genes in diapause and non-diapause larvae could indicate distinctive roles of the two genes during the process of diapause and development [17]. A possible role for small Hsps in diapause is the involvement in the regulation of the cell cycle arrest [34]. Denlinger et al. [24] suggested that small Hsps are involved in protecting organisms against low temperature stress and in the regulation of cell cycle arrest. The fact that sHsps are up-regulated during diapause in a variety of insect species, representing different diapause stages, suggests that sHsps are key players in the overwintering response of many insects [29]. By contrast, sHsp genes were not up-regulated in response to diapause in D. triauraria [34, 30], Lymantria dispar [35, 36], Lucilia sericata [31] and Chilo suppressalis [32]. Some sHsps are induced while others are suppressed. For the question why there are so many sHsps, in S. nonagrioides it seems that SnoHsp19.5 and SnoHsp20.8 have specific functions. Most likely, the different sHsps are interacting differently with specific groups of proteins. The results suggest that these two sHsps contribute to diapause and cold response in slightly different ways. The SnoHsp19.5 gene was consistently expressed in response to diapause, while SnoHsp20.8 gene was down-regulated in deep diapause and up-regulated at the termination of diapause (Table 1).

Table 1 Expression of five heat shock proteins, two storage proteins and one JHE genes in diapausing larvae of Sesamia nonagrioides. The diapausing insects were reared under short day (SD) conditions (10:14, light:dark) at 25 C. The ontogeny of diapause was divided into three main phases: (1) pre-diapause; (2) deep-diapause; (3) post-diapause. Genes SnoHsp19.5 SnoHsp20.8 SnoHsp70 SnoHsc70 SnoHsp83 SnoSP1 SnoSP2 SnoJHER Expression (or transcript) levels Pre Deep Post diapause diapause diapause low low low medium high low high low low high low low high high high medium low low medium low

2.2 SnoHsc/Hsp70 The relationship between diapause and Hsp70 has been studied in several insects. Hsp70 were found to be up-regulated during diapause in a number of species, including adults diapause of the flesh fly, Sarcophaga crassipalpis [26] pupal diapauses of the solitary bee, Megachile rotundata [37], larval diapause of the rice stem borer Chilo suppressalis [24], and embryonic diapause of the silkmoth Bombyx mori [38]. However, limited studies have reported on the expression of Hsc70 during insect diapause. In S. nonagrioides, the full Hsc70 and Hsp70 cDNA sequences were determined. The cDNA sequence of SnoHsc70 and SnoHsp70 were deposited in GenBank with accession number DQ004584 and EU430480, respectively [27]. At 25 C, SnoHsp70 transcripts were consistently low and there was almost no difference in the levels as a function of the developmental stage of non-diapause larvae or during diapause. The pattern of SnoHsp70 transcript accumulation remained similar in all stages of diapause [27]. Accumulated data from different species have resulted in contradictory conclusions. Hsp70 is not up-regulated as a function of diapause in Drosophila triauraria and Lucilia sericata [31, 34] while it is slightly up-regulated in the diapausing

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adults of Colorado potato beetle Leptinotarsa decemlineata [39] and is only expressed in the diapausing pharate first instar larva of the gypsy moth Lymantria dispar after exposure to low temperature [35]. In contrast, the SnoHsc70 gene was up-regulated during deep larval diapause. SnoHsc70 showed constitutive expression in pre-diapause and post-diapause. Transcript accumulation increased in deep-diapause, showed a gradient increase with a peak in 60-day-old larvae and thereafter a gradual decrease. These results showed that the SnoHsc70 transcript levels differed throughout the course of diapause (Table 1). The two members of 70 kDa heat shock protein family are differentially expressed during diapause in S. nonagrioides [27]. Only a few studies in insect diapause have reported on Hsc70. In S. crassipalpis the transcripts of Hsc70 showed no difference in levels throughout the course of diapause and remained the same in diapausing and nondiapausing individuals [26], and in M. rotundata, Hsc70 is a normal part of both the diapause and post-diapause developmental programs [37]. The results in S. nonagrioides show that the regulation of the SnoHsc70 is under strict developmental regulation. The latter, combined with results obtained under normal deep diapausing conditions, indicates that SnoHsc70 may play a crucial role during the diapause of S. nonagrioides, most likely assisting the low but vital protein proper conformation during this specific developmental period. Possibly SnoHsc70 transcripts during deep-diapause are hormonally controlled. Larval diapause is characterized by certain endocrine events, such as the presence of important juvenile hormone (JH) activity [40]. According to Eizaguirre et al. [41], JH is concerned in the maintenance of diapause in S. nonagrioides. In the presence of JH, insect tissues maintain their developmental status quo when exposed to ecdysteroids (the molting hormones), whereas in the absence of JH, tissues change their

commitment to that of the next metamorphic stage upon exposure to ecdysteroids [42, 43]. In M. sexta, the brain neuropeptide prothoracicotropic hormone (PTTH) stimulates a rapid increase in ecdysteroid hormone synthesis that is accompanied by general and specific increases in protein synthesis, including that of a 70 kDa cognate heat shock protein (Hsc70) [44]. In this species, protein and mRNA data suggest that Hsc70 could be involved in a negative feedback loop regulating assembly of the ecdysone receptor complex. SnoHsc70 could possibly be involved in a loop regulating the ecdysone receptor complex, as in M. sexta. The resolution of the function of Hsc70 in ecdysteroidogenesis or other cellular processes will require development of model systems in which Hsc70 levels can be readily manipulated under a variety of conditions. The results presented here for the corn stalk borer and other examples from the literature clearly demonstrate that the regulation of the heat shock response is not a simple on/off reaction but is finely tuned to developmental and environmental conditions. The differential expression of SnoHsc70 and SnoHsp70 during diapause (Table 1) indicates that individual members of the 70 kDa heat shock protein family may have varying roles in diapause of S. nonagrioides well. 2.3 SnoHsp83 Hsp90 proteins are essential molecular chaperones involved in signal transduction, cell cycle control, stress management, folding, degradation and transports of proteins. Hsp90 proteins have been found in a variety of organisms suggesting that they are ancient and conserved [45]. A unique feature of Hsp90 proteins is that they are constitutively expressed at substantial level during non-stress conditions [46]. The up-regulation of Hsp90 in response to heat shock is not as robust as for other heat shock proteins. In most cases, a 10-15 fold increase in protein levels can be expected when

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Diapause Regulation in the Moth Sesamia nonagrioides (Lepidoptera: Noctuidae)

comparing heat-stressed organisms to unstressed controls [46]. Hsp90 appears to have a specific function during recovery from stress, being especially important in the reactivation of stress-inactivated protein [47]. Hsp90 genes are also implicated in developmental regulation, such as morphological evolution and arrest of reproduction in drosophila [48], during the dauer stage of Chaenorhabditis elegans [49], the diapause of Chillo suppressalis and Dellia antiqu [32, 45]. Studies in D. triauraria and the blowfly (Lucillia sericata) have not found the evidence of Hsp90 involvement in diapause [30, 31], whereas in studies with Sarcophaga crassipalpis, Hsp90 transcripts are down-regulated during diapause [26]. The full Hsp83 cDNA sequences were determined in S. nonagrioides and were deposited in GenBank/EMBL/DDBJ with accession numbers DQ198859 [28]. SnoHsp83 showed constitutive expression in pre-diapause and post-diapause phase (Table 1). The transcript accumulation increased in deep-diapause period, showed a gradient increase with a peak at 60-day-old larvae and thereafter a gradual decrease. These results showed that the SnoHsp83 transcript levels differed throughout the course of diapause [28]. Hsp90 during diapause of S. nonagrioides could be regulated by ecdysteroids. In D. melanogaster, the presence of ecdysteroids leads to up-regulation of Hsp90 [49] and this gene is critical for generating functional ecdysone receptors [50]. In the C. elegans, dauer larvae are enriched in Hsp90 gene transcripts and their products could interact with steroid hormone receptors [49, 51]. The ecdysteroids titer rises before each extra larval molt in S. nonagrioides and a burst at the passing 8th to 9th instar [52] is at day 60 of our collection time [53]. Since S. nonagrioides larvae continue supernumerary molts during diapause, their physiological state cyclically changes. The pattern of the SnoHsp83 expression shows a period that could be correlated to the supernumerary molts during diapause or the cyclic

changes of ecdysteriod titre. The up-regulation of SnoHsp83, as well as that of SnoHsc70 could represent important molecular cues for corn borer diapause. It is very interesting that the two genes display a similar pattern when larvae are under diapause conditions. SnoHsc70 could possibly be involved in a loop regulating the ecdysone receptor complex [27], and SnoHsp83 and SnoHsc70 could be required for ecdysone activity. The up-regulation of the two genes in deep-diapause phase of S. nonagrioides is consistent with this possibility, when extra larval molts go through. These results might indicate that Hsp90 transcripts are regulated developmentally in diapausing larvae of S. nonagrioides.

3. The Role of Storage Proteins during Diapause of S. nonagrioides

Induction of diapause is correlated with many physiological, biochemical and behavioral traits. Among different insect groups, diapause may occur at any stage of development, although in every species the diapausing stage is specific [43, 53]. A typical indicator of diapause is the low rate of protein synthesis, while shortly before and after diapause this rate is significantly higher [9]. In many insect species, the onset of diapause is also correlated with the synthesis of selected proteins. A few previous studies have shown that diapause associated proteins may be hexameric storage proteins [54, 55]. In insects, before the initiation of metamorphosis, a characteristic family of proteins has been found in storage tissues such as the hemolymph and fat body. Most of these storage proteins are hemocyanin-related macromolecules and generally referred as hexamerins, because of the composition of six identical or similar subunits in the 72,000- to 90,000-dalton size range [56, 57]. Hexamerins have long been proposed to serve as a source of amino acids for tissue reconstruction during pupal development and have been shown to be a component of the sclerotizing

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system of the cuticle [58]. They also serve as an ecdysteroid carrier in the haemolymph and identified to date function in nutrient uptake and storage, but some are capable of binding the insect morphogenetic hormone JH [59-61]. Hexamerins are generally classified into three groups: (1) the arylphorins, polypeptides rich in the aryl groups tyrosine and phenylalanine; (2) methionine-rich proteins; (3) proteins which are neither rich in aromatic amino acids nor in methionine [62-64]. In Lepidoptera, there are three different hexamerin classes that have been diversified up to 290 MYA. In the higher extant Lepidoptera, there are two different types of methionin rich hexamerins that can be discriminated according to their amino acid composition [64]. Two complete hexamerin-cDNA clones were isolated in the corn stalk borer, the SnoSP1 and SnoSP2, and were deposited in GenBank/EMBL/DDBJ with accession numbers DQ004585 and DQ147770, respectively. The SnoSP1 belongs to the group of JH suppressible hexamerins [65] and the SnoSP2 falls into the class of hexameric [66]. Total RNA was isolated from fat body at different times and semi-quantitative RT-PCR and real-time PCR analyses were performed. The changes were examined in SnoSP1 and SnoSP2 transcripts in the fat bodies during pre-diapause, deep-diapause and post-diapause phases [65, 66]. 3.1 SnoSP1 Expression of SnoSP1 has been confirmed in both diapausing and non-diapausing periods. In non-diapausing insects, the abundance of SnoSP1 in fat body was found in high levels during the last larval stage and decreased gradually during the pupal stage. The persistence of SnoSP1 transcript through the early pupal stage suggests that mRNA might be stable or expressed during the pupal stage. The SnoSP1 transcripts were induced by short days (10L:14D). The expression of SnoSP1 was detected from the

beginning of diapause and increased until at least day 130 since hatching. The SnoSP1 mRNA expression levels were higher in the non-diapausing than the diapausing larvae and declined when the insect initiated metamorphosis [65]. SnoSP1 serves as amino acid reservoirs at the termination of diapause and is a distinct possibility during adult development. Larval diapause is characterized by certain endocrine events, such as the presence of important JH activity [67]. Many researchers suggest that high levels of JH are necessary only at the initiation phase of diapause rather than in the long lasting deep diapause of some Lepidoptera species [68, 69]. Thus we suggest that the absence of SnoSP1 expression into the first day of diapause period (pre-diapause) is caused by a relatively high level of juvenilizing factors (JH or its metabolites). After its decrease of concentration, SnoSP1 transcription begins. According to Eizaguirre et al. [41], diapausing larvae of S. nonagrioides have a higher JH titer in the hemolymph than non-diapausing larvae, which shows that JH is concerned in the maintenance of diapause in this species. The increase in the levels of the SnoSP1 mRNAs seems to be correlated with the commitment peak of ecdysteroids in the 5th instar (25 days since hatching) [65]. When the JH titer decreases (35 days since hatching), it allows the expression of SnoSP1 mRNAs (35 days since hatching). It is likely that when the larvae of last instar commit to become pupae, the JH titer decreases, which allows the expression of SnoSP1 mRNAs. This kind of JH suppression of larval specific proteins has been reported for related proteins, TniSP1 and TniSP2 [38, 68]. Since JH is generally considered necessary for maintaining the insect in the larval state, it is probable that the absence of JH allows the expression of SnoSP1 mRNA. Expression of SnoSP1 gene is observed throughout diapause, for as long as 130 days since hatching. The results confirm the importance of larval storage proteins (LSP) biosynthesis and finally lead us to the conclusion that the larval diapause of S. nonagrioides

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is associated with continuous accumulation of storage proteins. 3.2 SnoSP2

synthesis

and

In diapausing larvae, SnoSP2 transcripts appear in day 24 since hatching and increased two-fold in day 28. After pre-diapause phase, mRNA level was decreased gradually from the deep-diapause phase to the end of post-diapause phase until disappeared. In diapausing period, the transcript levels of SnoSP2 and SnoSP1 genes are almost the same, but the patterns are different. In the pre-diapause phase the induction of SnoSP2 was different and high levels were apparent, while the levels of SnoSP1 were very low. In the contrary, in the post-diapause phase, levels of SnoSP1 were apparent very high, while the levels of SnoSP2 declined and disappeared at the end of the phase. The level of SnoSP2 mRNA remained high until day 55 (deep-diapause) and started to decrease following the post-diapause phase, at the end of which disappeared. The up-regulation of SnoSP2 gene expression during this period indicates that it is associated with the onset of diapause [66]. The pattern of SnoSP2 mRNA is quite similar to the pattern of Lhp76 gene of Galleria mellonella, in relation to diapause and CfDAP2 gene of Choristoneura fumiferana [66]. During diapause, expression initiation of SnoSP2 and SnoSP1 genes was different (Table 1). Although expression of SnoSP2 lasted from the beginning of diapause until the last dayday 130, SnoSP1 transcripts were very low in larvae preparing to enter diapause and increased dramatically in those which were in diapause as well as in those terminate diapause. It has established that SnoSP1 gene is JH-suppressible [65]. The results do not confirm the above statement for the SnoSP2 gene. The same concentration of JHA is high enough to silence SnoSP1 gene, but SnoSP2 transcripts are still present [65]. There are a few probable explanations for this discrepancy. Different sensitivity of the two genes to JHA, different stability of the transcripts, or even

insensitivity of SnoSP2 to the hormone are possibilities. Larval diapause is characterized by certain endocrine events, such as the presence of important JH activity [40]. Many researchers suggest that high levels of JH are necessary only at the initiation phase of diapause rather than in the long lasting deep diapause of some Lepidoptera species [68, 69]. SnoSP2 mRNA levels did not change significantly in response to JHA application. Thus we suggest that the presence of SnoSP2 expression in the first day of diapause period (pre-diapause) is caused by a relatively high level of juvenilizing factors (JH or its metabolites). After its decrease of concentration, SnoSP2 transcription declined. According to Eizaguirre et al. [41], diapausing larvae of S. nonagrioides have a higher JH titer in the hemolymph than non-diapausing larvae, which shows that JH is concerned in the maintenance of diapause in this species. In S. nonagrioides, diapause programming includes an increase of JH titer in the hemolymph from about 20 nM to 50 nM in the 4th and 5th instar larvae (18-25 days since hatching). The JH titer drops to zero before pupation but remains around 20 nM during diapause. The increase in the levels of the SnoSP2 mRNAs seems to be correlated with the commitment peak of ecdysteroids in the 5th instar (25 days since hatching) [69]. When the JH titer decreases (day 35 since hatching), the expression of SnoSP2 mRNAs (35 days since hatching) also decreases. It is likely that when the larvae of last instar commit to become pupae, the JH titer decreases, which also happens in the expression of SnoSP2 mRNAs. Since JH is generally considered necessary for maintaining the insect in the larval state, it is probable that the presence of JH induces the expression of SnoSP2. To date, two hexamerin encoding genes, SnoSP1 and SnoSP2 were characterized in corn stalk borer. Expression of both hexamerin genes in S. nonagrioides is observed throughout diapause for as long as 130 days. The results confirm the importance of SnoSP1 and SnoSP2 biosynthesis and finally lead

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us to the conclusion that larval diapause of S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins. However, enhanced levels of SnoSP2, but not SnoSP1, were detected at the initiation of diapause. In contrast, levels of SnoSP1 were detected in deep-diapause phase and greatly increased during post-diapause phase. Our present data support different roles of SnoSP2 and SnoSP2 genes in diapause of S. nonagrioides.

4. The Role of the JHE (SnoJHER) in the Diapause of S. nonagrioides

Esterases are hydrolytic enzymes that cleave ester bonds in a diversity of biomolecules [70]. Many insect esterases have well-defined biological functions, such as those involved in xenobiotic, lipid, acetylcholine and JH metabolism. Carboxylesterases (COEs) are a multifunctional superfamily ubiquitous in all living organisms, including insects and other animals, plants and microbes [71]. Based on sequence similarity and substrate specificity, insect COE genes can be subdivided into eight subfamilies: -esterases, -esterases, JHEs, gliotactins, acetylcholinesterases, neurotactins, neuroligins and glutactin class [72]. JHE is a COE that has attracted great interest regarding its critical role in regulating larval to adult transition in insects and other arthropods. JHE hydrolyzes the key developmental and reproductive hormone JH, and partially regulates its titer [42]. JH plays a major role in the control of growth, development, metamorphosis, diapause and reproduction in insects [73]. JH is normally present at the time of increase in ecdysteroid titres for larval molts and ensures that larvae molt to the next larval stage (instar). However, at the time of final larval molt, JH disappears, allowing ecdysone to induce metamorphosis [42]. JHE is crucial for JH hemolymph titre reduction and thereby for the initiation of metamorphosis in diverse insects, but strong inhibition of JHE activity in S. nonagrioides larvae has no effect on the onset of metamorphosis [74].

A member of the COE superfamily has been identified in the corn stalk borer S. nonagrioides, SnoJHER, and was deposited in GenBank with accession number EU178813. The primary structure of the deduced amino acid sequence of the cDNA showed that the catalytic site of SnoJHER has a cysteine residue next to the catalytic serine (GQSCG), while most other described JHEs have alanine (GQSAG) at this position. The JHER COE in S. nonagrioides is structurally distinguishable from other JHEs based on certain residues at the active site [75]. In order to assess the developmental expression of SnoJHER under diapause conditions, mRNA levels were examined by semi-quantitative RT-PCR. In diapausing conditions (SD photoperiod), SnoJHER mRNAs showed a peak in 9th instar of deep-diapause. In the 9th instar, the SnoJHER mRNA levels were the lowest at the beginning of instar and increased gradually, reaching peak expression at the end, a day before the molt into the next instar (10th) (Table 1). During diapause (SD photoperiod), the SnoJHER mRNA levels for diapausing larvae were lower than non-diapausing larvae [75]. According to Schafellner et al. [74], the diapausing and non-diapausing larvae exhibited similar levels of JHE activity in S. nonagrioides. These levels of JHE activity did not coincide with SnoJHER mRNA levels observed during diapause. Despite the low levels of SnoJHER during the diapausing period, it seems that SnoJHER presents higher levels during the last day of 9th instar of diapause. This peak expression of SnoJHER in the 8th instar of diapause seems to be coincided with the ecdysteroid peaks in diapausing larvae of S. nonagrioides measured by Eizaguirre et al. [52]. According to Eizaguirre et al. [67], in S. nonagrioides larvae, the ecdysteroid titer rose before each extra larval moult and peak during the molting of the 8th to the 9th diapausing instar. Additionally, increasing the cycle number of the semi-quantitative RT-PCR to amplify low levels of SnoJHER expressed in

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diapausing 9th instars, showed that expression was the highest on the last day of the instar, prior to the molting to a 10th larval instar in diapause. This gene expression pattern was similar to that of non-diapausing 5th instars. Thus, the SnoJHER mRNA appeared to peak on the final day of both non-diapausing and diapausing larval-stage instars [75].

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5. Conclusions
In conclusion, the regulation and function of the various Hsp families during diapause of corn stalk borer could provide insights into the molecular mechanisms involved in the diapause of this species. The SnoHsp19.5 gene was consistently expressed in response to diapause, while SnoHsp20.8 gene was down-regulated in deep-diapause and up-regulated at the termination of diapause. The differential expression of SnoHsc70 and SnoHsp70 during diapause in response to high and low temperatures indicates that individual members of the 70 kDa heat shock protein family may well have varying roles in diapause of S. nonagrioides. The SnoHsp83 expression showed that it could be correlated to the supernumerary molts during diapause. The up-regulation of SnoHsp83, as well as that of SnoHsc70 could represent important molecular cues for corn borer diapause. Consistent with a function in metamorphosis, mRNAs of SnoSP1 and SnoSP2 were abundant during last larval stage, and were gradually depleted during pupal stage. Expression of both hexamerin genes in S. nonagrioides were observed throughout diapause, for as long as 130 days. The results confirm the importance of SnoSP1 and SnoSP2 biosynthesis and finally lead us to the conclusion that larval diapause of S. nonagrioides is associated with continuous synthesis and accumulation of storage proteins. Finally, the SnoJHER mRNA appeared to peak on the final day of both non-diapausing and diapausing larval-stage instars.

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