Prediapause phase is a key period during which insects determine whether to develop into adults without interruption or to enter diapause at the pupal stage. Taking the latter path requires changes in the individual’s metabolic pathway to elevate energy reserves and produce cryoprotectants to defend against low temperature. However, little is known about the relationship between environmental signals and the expression of the diapause phenotype
. A major reason for this lack of knowledge is the fact that no obvious phenotypic changes can be observed in diapause-destined individuals during the prediapause phase. In this study, we combined proteomic and metabolomic approaches to investigate changes in the expression of proteins and metabolites between larval hemolymph from nondiapause- and diapauses-destined H. armigera. Significantly changed proteins and metabolites were identified and analyzed, and our results revealed important clues regarding the diapause induction and preparation in the prediapause phase. It is well known that the short daylength is the major factor behind the effective induction of diapause, while low temperature is an associated minor factor. We believe that the altered levels of proteins and metabolites in hemolymph are the result of a cooperative influence of photoperiod and temperature because we used short daylength and low temperature in our experiment.
Pyridoxine 5′-phosphate oxidase, an enzyme that is increased in the early stage of the sixth instar, catalyzes the biosynthesis of pyridoxal 5′-phosphate
. The vitamin B6 derivative pyridoxal 5′-phosphate acts as a coenzyme and participates in the biosynthesis of some neurotransmitters, such as serotonin, dopamine, and GABA
. Biogenic amine neurotransmitters in hemolymph, including serotonin and dopamine, have been shown to be involved in insect diapause in Mamestra brassicae and in Pieris brassicae[13, 14]. In the middle stage of the sixth instar, GABA was only detected in diapause-destined larval hemolymph. GABA is a major inhibitory neurotransmitter in insects. It has been reported that GABA and the GABA receptor antagonist picrotoxin influenced the incidence of diapause in Sarcophaga bullata. GABA was also found to repress diapause hormone (DH) secretion, leading to diapause in Bombyx mori. We speculate that the high concentration of GABA in diapauses-destined H. armigera larvae may be involved in inhibiting neurohormone secretion. This leads, in turn, to changes in larval metabolism and finally causes insects to enter diapause. Therefore, the increased pyridoxine 5′-phosphate oxidase in the diapause induction phase is most likely a response to the short daylength signal to regulate endogenous chemicals such as neurotransmitters for insect diapause.
The amino acid histidine is a precursor to histamine, and the histidine levels were dramatically decreased in the early stage of the sixth instar. There is much evidence to suggest that histamine acts as a neurotransmitter released from the photoreceptor synapse of insects and other arthropods. Histamine acts by directly gating chloride channels and inhibits the activity of central nervous circuits in arthropods
[17, 18]. Diapause is mostly induced in H. armigera by short daylength signals which are received by photoreceptors in the brain. Significant changes in the levels of histidine most likely indicate altered levels of the photoreceptor transmitter histamine, which is likely to participate in the molecular events of the diapause induction phase.
Diapause-destined individuals must store additional metabolic reserves such as fuel reserves and cryoprotectant molecules because adequate reserves are critical to allowing individuals to sustain a basic metabolism in the harsh environmental conditions that occur during diapause. The major energy resource glucose was maintained at a constant high level during the sixth instar in diapause-destined larvae. Fructose and pyruvate levels were also elevated in the middle stage of the sixth instar. In our previous study, glucose and pyruvate levels decreased in diapausing pupae whose metabolic activities were dramatically reduced
. Accordingly, accumulation of these glycolysis-related metabolites in diapause-destined larvae implies increased energy reserves in vivo. Interestingly, trehalose, as an important cryoprotectant and energy resource that usually accumulates at the pupal stage
, was heavily elevated in the early stage of the sixth instar.
On the other hand, there is significant evidence to suggest that synthesis of cryoprotectants is associated with insect diapause
. Glycerol and ribitol are important cryoprotectant molecules found in insects
. These two cryoprotectants were found to accumulate in the diapausing flesh fly, Sarcophaga crassipalpis, and in the rice stem borer, Chilo suppressalis. In this study, both compounds were found to be elevated in the middle stage of the sixth instar, showing that polyol concentrations in diapause individuals are closely correlated with cold resistance. Two major sugars, glucose and fructose, were also elevated in the sixth instar. These two sugars may also be used as precursors for the synthesis of cryoprotectants
Urea levels were increased in diapause-destined larval hemolymph in the middle stage of the sixth instar. On the other hand, the levels of ornithine, which serves as precursor of urea, were decreased. The concentrations of N-Acetylglutamate, an activator of carbamoyl phosphate synthetase in the urea cycle
, were also increased in diapause-destined larvae during the diapause preparation phase (the late sixth instar). It has been reported that the overwintering wood frog, Rana sylvatica, can accumulate additional urea, suggesting that the organic osmolyte also functions as a cryoprotectant such as glycerol in terrestrially hibernating amphibians
. Thus, urea in diapause-type insects may contribute to cold resistance.
Although both nondiapause- and diapause-destined larvae were reared at the same temperature (20°C) with different photoperiods, diapause-destined individuals could accumulate additional polyols and sugars. This result indicates that the synthesis of cryoprotectants and energy reserves is mainly attributable to short daylength conditions in this study, and the photoperiod is a key factor influencing the induction of diapause. Experiments in other insect species also showed this special linkage between photoperiod and cold-hardiness
Insects are vulnerable to fungal and bacterial infections during diapause. The innate immune system continues to function in diapausing individuals and plays an important role in preventing the invasion of the body by microorganisms
. Serine proteases participate in many aspects of invertebrate immunity, including hemolymph coagulation, antimicrobial peptide synthesis, and melanin synthesis
. The activity of these enzymes is regulated by serine protease inhibitors (serpins). Thus, serpins in insect hemolymph also function to protect individuals from infection
. The expression of serine protease-like protein 2, serpin 3a, and serpin was found to be increased in diapause-destined individuals. On the other hand, levels of Kazal-type inhibitor, hemolymph proteinase 18 (a serine proteinase identified in Manduca sexta hemolymph), and serine proteinase-like protein 1 were decreased in these individuals. Serine protease-like protein 2 and serpin, which accumulate at the late sixth instar of diapause-destined larvae, may be used in the long diapause phase to prevent invasion by microorganisms. This function has been reported for Nasonia vitripennis, Sesamia nonagrioides, Leptinotarsa decemlineata, Delia antiqua, and Culex pipiens, indicating that serine protease-like protein 2 and serpin are most likely closely linked to insect diapause. We speculate that the serine proteinase-like protein 1 may not be suitable to act in diapausing individuals at low temperature, and the expression of this protein is consequently decreased in the late sixth instar of diapause-destined larvae.
Two different C-type lectins exhibited significantly altered expression in the diapause preparation phase. The expression of C-type lectin 7 was increased at the late stage of the sixth instar in diapause-destined larvae, while the expression of another C-type lectin was decreased. C-type lectins are calcium-dependent carbohydrate-binding pattern recognition proteins. Lepidopteran C-type lectins have been shown to function in innate immune responses such as phagocytosis, prophenoloxidase activation, and hemocyte nodule formation
[34, 35]. C-type lectin 7, which accumulates at the late stage of the sixth instar in diapause-destined larvae, may be highly stable at low temperature and can be used by diapausing individuals. Pattern recognition proteins can recognize foreign invading microorganisms, which is the crucial first step in producing an immune response
. A pattern recognition protein, beta-1,3-glucan recognition protein 3 (βGRP3), was found to exhibit increased expression at the late stage of the sixth instar in diapause-destined larvae. Fungal infection can be recognized by βGRP through its binding to beta-1,3-glucan on the surface of fungal cell walls. Subsequently, βGRP may work in concert with serine proteases to trigger the prophenoloxidase activation pathway, which is an important defense against microbial invasion in insects
Previous studies on serine proteases, protease inhibitors and lectins in hemolymph have revealed their functions in immune response for morphogenesis. Because both diapause- and nondiapause-type individuals must undergo a molting process, high expression levels of proteases, protease inhibitors, and lectins in these two types of individuals are correlated with morphogenesis. However, high expression levels of these proteins only in diapause-type individuals may serve in immune responses to facilitate survival in adverse environmental conditions over the long diapause period.
One of the most common features in insect diapause is enhanced stress tolerance, which includes resistance to highly reactive chemical species, desiccation, ultraviolet radiation, salt, and other environmental or physiological stresses
. The expression of several stress tolerance-related proteins was significantly altered in the larval hemolymph of diapause-destined individuals. The expression of glutathione S-transferase (GST) was increased in the early stage of the sixth instar larvae. We showed that both GST mRNA and protein were heavily expressed in pupal brains of H. armigera at diapause initiation
[6, 39]. It has been reported that diapause hormone expressed at the larval stage of B. mori, as an intensified memory, can subsequently induce high expression of diapause hormone at the pupal stage
. Therefore, we speculate that GST expressed in the early stage of the sixth instar larvae may cause high GST expression at the pupal stage. GST has antioxidant activity and can protect cellular components of diapause individuals from oxidative damage, and thus, the accumulation of GST in diapause individuals is most likely involved in an oxidative stress response
. The levels of ferritin were also increased in the late stage of the sixth instar. High abundance of ferritin was also found in other diapause-destined insects such as N. vitripennis and Calanus finmarchicus, and in the pupal brains of H. armigera at diapause initiation
. It has been shown that the concentration of ferritin increases in response to stresses such as anoxia
. Ferritin serves as an important ion storage protein in hemolymph and functions in maintaining the iron balance. The accumulation of ferritin can prevent destructive oxidative reactions by mitigating iron toxicity
In addition, N-Acetylglucosamine (GlcNAc) levels were found to be elevated in diapause-destined larvae in the late stage of the sixth instar. GlcNAc is the monomeric unit of the polymer chitin, which is one of the major components of insect cuticle
. Cuticle structural components are enriched in the diapause preparation phase to enhance waterproofing and to promote survival
. Furthermore, increased pupal cuticle protein was observed during early adult diapause in the mosquito C. pipiens. Elevated levels of GlcNAc most likely contribute to a harder cuticle in diapause individuals, improving tolerance to winter stresses and defenses against microbial invasion.
Mevalonic acid was increased in the early stage of the sixth instar. Mevalonic acid is a precursor to the mevalonate pathway, which is the early step of juvenile hormone (JH) synthesis in insects
. JH participates in the regulation of insect development, metamorphosis, diapause, and reproduction
[1, 49, 50]. To date, no convincing experimental evidence exists to support the notion that JH plays a role in the induction of pupal diapause. Although major pulses of JH activity with a rhythmicity of 24 h were unique to pupal diapause-destined individuals in the flesh fly S. crassipalpis, the function of JH in the onset of pupal diapause requires further research.
An imaginal disc growth factor-like protein showed significantly increased expression in the middle stage of the sixth instar. Imaginal disc growth factors (IDGFs) are soluble growth factors that promote cell proliferation in imaginal discs. Several IDGFs have been identified from the fat body of Drosophila; these growth factors are secreted and transported to target tissues through hemolymph
. Thus, the IDGFs that accumulate in diapause-destined larval hemolymph may be used for the development of imaginal discs when diapause is broken.
A number of other proteins and metabolites showed significantly altered expression in diapause-destined larvae. These include sulfatase, endoU protein, protein disulfide isomerase, glycine, phosphorylethanolamine, and ribonic acid. Further research is required to reveal the roles of these proteins and metabolites in the regulation of diapause.