Background

Insect diapause, a pause and an alteration in developmental programme, is a biological process characterized by diminished oxygen consumption and lower metabolic rate [1]. It is an incredibly interesting phenomenon in developmental biology and aging in which insects suddenly alter their O2 consumption, become dormant and extend their life span [2,3,4]. The tropical tasar silkworm, Antheraea mylitta Drury (Lepidoptera:Saturniidae), an insect of sericulture importance, exhibits well-defined pupal diapause. A. mylitta has distinct developmental stages in its life cycle i.e. egg, larva, pupa (inside cocoon) and the moth stage. Eggs are hatched into young worms called larvae. The process of larval development has five stages also known as instars (Ist, IInd, IIIrd, IVth and Vth instar). The larval stage is the only feeding stage in its entire life cycle. Larva, in its final (Vth) instar, attains full growth, stops feeding and spins a protective covering around itself popularly known as cocoon. It then changes into pupa, which exhibits facultative diapause [5] possibly to cope with the extremes of environment and shortage of food. A. mylitta exhibits three distinct patterns of life cycle with reference to the time duration of one generation, popularly referred to as voltinism. The voltine varities (uni-, bi- and tri-voltine i.e. one, two and three generations in a year) are conspicuous and distinct in relation to the duration of the pupal phase. In case of trivoltine strain, the pupae developing from larvae of the first two generations (1st and 2nd) are generally non-diapausing (NDP) in nature and have a comparatively short pupal period of 15–21 days. In contrast, the pupae developing from the larvae of 3rd generation are destined for diapause with an extended pupal life spanning around 160 days. It is pertinent to mention here that despite a differential duration of pupal period, individuals from both the generations achieve the same level of morphogenesis at the end of pupal development. Cascades of cellular events during pupal development are associated with the cellular redox balance, the balance between oxidants and antioxidants [6]. Reactive oxygen species (ROS) like superoxide radicals (O−), hydroxyl radicals (OH·) and hydrogen peroxides (H2O2) are common oxidants and inflict oxidative damages on important biomolecules of the cell when they cross their physiological limits. Lipid peroxidation (LPx) and protein carbonyl (PC), two important oxidative damaged products, have been linked with increased ROS level and extension of life span in some insect model [7]. Under normal conditions, the presence of antioxidant defense system protects the cell from ROS-mediated oxidative assaults. The antioxidant defense system consists of an array of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) along with some low molecular weight components like reduced glutathione (GSH) and ascorbic acid (ASA) [8]. In insects, ROS, particularly H2O2, serve as a key regulatory molecule not only in various physiological processes, it also plays an important role in the regulation of cell differentiation, development and diapause [9, 10]. H2O2 exhibits a dual role in the cellular environment: by its oxidative properties it acts as a prooxidant and through its function acts as a second messenger. Though the involvement of ROS in the process of development has been well documented, most of the studies are limited to in vitro effects of ROS or based on the findings from cell culture [11] and far fewer experiments have been carried out on developing embryos or larvae. Albeit a few reports suggest the involvement of oxidants and antioxidants in the process of development and diapause of insects [12,13,14], we have little understanding of the pupal development of tropical tasar silkworm, particularly in relation to redox active components and diapause.

However, studies on this species during pupal development in DP and NDP generations suggest a marked difference in oxygen consumption [15, 16]. Oxygen utilization is associated with ROS production and oxidative process [17]. The present experiment is, therefore, designed to ascertain the level of H2O2, oxidative damaged products (LPx and PC) and relative levels of antioxidant protection in two important tissues i.e. hemolymph (HL) and fat body (FB) of Antheraea mylitta pupa from DP and NDP generations having identical chronological age (10 days old pupae) with different physiological states. In this study we hypothesize that the dynamics of ROS metabolism and status of antioxidants in response to differential O2 consumption should be distinct and strategic in DP and NDP generations of pupae, which differ in their patterns of development and ageing.

Methods

Cocoons containing live pupae of tasar silk worm (A. mylitta) were collected from identical host plants (Terminalia arjuna) of the same age group maintained in the State government sericulture field located at Baripada (21° 56′ 6 N, 86° 43′ 17 E), Odisha, India. The pupae collected from the 2nd and 3rd generations served as NDP and DP groups, respectively. Ten days old pupae from both the generations were used in this experiment.

Tissue preparation

HL was collected from the healthy pupa in an ice chilled microcentrifuge tube coated with 0.03% phenylthiourea (PTU) to prevent melanization. Then the pupae were sacrificed, FB was collected and washed in physiological saline (0.67%), blotted dry and weighed in monopan balance. A 10% (w/v) homogenate was prepared in ice cold 50 mM phosphate buffer, pH 7.4 containing 0.1 mM EDTA and a pinch of phenylmethane sulphonyl fluoride (PMSF) using glass teflon mechanical homogenizer. Fat body homogenates were centrifuged at 10,000 x g for 20 min at 4 °C and the post-mitochondrial supernatant was collected. The HL samples were centrifuged at 3000 x g for 10 min at 4 °C to get rid of hemocytes. Endogenous hydrogen peroxide (H2O2) content and catalase (CAT) activity were measured in the samples immediately after centrifugation. The remaining supernatant was kept at − 50 °C for further analyses. All measurements were done in triplicate.

Measurement of hydrogen peroxide (H2O2) content

The hydrogen peroxide content in HL and post-mitochondrial fraction of FB was determined spectrophotometrically using horse radish peroxidase and H2O2 as standard according to the method employed by Pick & Keisari [18]. In brief, in each tube, 1.7 ml of phosphate buffer (50 mM pH 7.4), 0.1 ml of phenol red solution and 50 μl of horse radish peroxidase (50 units) were taken and incubated for 5 min at room temperature. Next 0.1 ml sample was added to it followed by immediate addition of 50 μl of NaOH (1 N) to stop the reaction. H2O2 content was expressed as nmoles H2O2/mg protein.

Oxidative damaged products

Measurement of lipid peroxidation (LPx)

The level of LPx was determined by monitoring the formation of malondialdehyde (MDA) according to the method of Ohkawa et al. [19]. A 10% (w/v) homogenate of the FB was prepared in 1.15% KCl and centrifuged at 1000 x g for 10 min at 4 °C to remove cell debris. Suitably diluted HL fraction and 1000 x g supernatant of FB (having around 500 μg protein) were used for the estimation of MDA. All samples were treated with 0.02% butylated hydroxytoluene to prevent endogenous oxidation. The amount of MDA formed was calculated from the extinction coefficient of MDA i.e. 1.56 × 105 M − 1 cm− 1 [20] and was expressed as nmol MDA formed/ mg protein.

Measurement of protein carbonyl content (PC)

Protein carbonyl content was estimated according to the method adopted by Levine et al. [21]. This assay was used to determine the amount of oxidatively modified proteins through the detection of carbonyl groups that react with 2,4-dinitrophenyl hydrazine (DNPH) to form hydrazone derivatives, whose concentration was determined spectrophotometrically at 366 nm using the complementary blank. The carbonyl content was determined from the molar absorption coefficient of 22,000 M -1 cm − 1 and expressed as nmol PC/mg protein.

Activities of antioxidant enzymes

Assay of superoxide dismutase (SOD) activity

For the measurement of total SOD activity, 0.4 ml of post-mitochondrial supernatant containing approximately 10–15 mg of protein was passed through a 2 ml column of sephadex G-25 and the elute was used for the activity assay according to the method of Das et al. [22] as described earlier [23]. SOD activity was expressed as units/mg protein.

Assay of catalase (CAT) activity

The post-mitochondrial supernatant was used directly for assay of catalase activity following the decrease in absorbance of H2O2 at 240 nm [24]. Calculation was done by taking the extinction coefficient of H2O2 as 43.6 M − 1 cm -1. Activity was expressed as pkat/mg protein (1pkat = 10− 12 katal). One katal is defined as the amount of enzyme that transforms one mole of substrate per second.

Assay of glutathione S-transferase (GST) activity

The column-passed samples were used for the assay of glutathione S-transferase activity adopting the method of Habig & Jakoby [25]. In brief, 2.6 ml of 50 mM potassium phosphate buffer pH 6.5, 0.2 ml of 90 mM GSH and 0.1 ml of sample (160 μg of protein for FB and 200 μg for HL) were added to prepare the reaction mixture. The reaction was initiated by adding 0.1 ml of 30 mM CDNB (1-chloro-2,4-dinitrobenzene) to it. Absorbance was recorded at 340 nm at 1 min interval till 6 min. Calculation was done by taking the extinction coefficient of CDNB i.e. 9.61 mM − 1 cm− 1. Activity was expressed as nmol CDNB conjugate formed/min/mg protein.

Estimation of reduced glutathione (GSH)

The supernatant from the FB homogenate and HL fractions isolated earlier were incubated with 5% (w/v) sulfosalicylic acid in ice and centrifuged at 1000 x g for 10 min. GSH content in the tissue samples was determined using DTNB (5,5′-Dithiobis-2-nitrobenzoic acid) according to the method of Ellman [26] and expressed as μmol/ml (for HL) or μmol/g tissue wet weight (for FB).

Estimation of ascorbic acid (ASA)

HL and FB samples were incubated with 5% (w/v) trichloroacetic acid (TCA) in ice and centrifuged at 1000 x g for 10 min. The supernatant was taken for the estimation of ASA using folin phenol reagent and ascorbic acid as standard according to the method adopted by Jagota & Dani [27] and ASA content of the tissue was expressed as μg/ml (for HL) or μg/g tissue wet weight (for FB).

Protein content

Protein content of the samples for the assay of oxidative damaged products and antioxidant enzymes was estimated according to the method of Lowry et al. [28] using bovine serum albumin as standard.

Statistical analysis

All data are reported as means ± S.E.M. for n = 6 samples. Data were analysed using t-test and considered as statistically significant when at least p < 0.05 between the DP and NDP generations as well as between two tissues of an identical group. Details of the statistical analysis have been included in the Additional file 1.

Results

H2O2 content and oxidative damaged products (Fig. 1a, b and c)

The level of H2O2 and oxidative damaged products like LPx (MDA content) and PC levels in HL and FB tissues of the pupae from DP and NDP generations of A. mylitta were found to be different in two different groups. FB tissues of the pupa from NDP generation have significantly higher level of H2O2 than those of their DP counterpart. In contrast, MDA and PC contents were found to be higher in both the tissue samples of the pupae from DP generation than the NDP sample.

Fig. 1
figure 1

a-c H2O2 content and oxidative damaged products. Level of H2O2 (a), MDA (b) and PC content (c) (nmol/mg protein) in the hemolymph (HL) and fat body (FB) of the pupae from nondiapausing (NDP) and diapausing (DP) generations. Bar represents mean ± S.E.M. (n = 6). Bars having different lower and upper case letters indicate significant difference in the measured parameters in HL and FB, respectively. Differences in the mean value have been considered statistically significant with a confidence interval of 95% and significant level of 5%

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Activities of antioxidant enzymes (Fig. 2a, b and c)

Higher specific activity of SOD was observed in both HL and FB tissues of the pupae from DP generation than those of the NDP group. Compared to NDP generation, CAT activity, exclusively detected in FB, was also significantly higher in the DP generation. It may be noted here that CAT activity was below the level of detection in HL under identical assay conditions. On the other hand, comparatively less GST activity was recorded in the pupal tissues (both HL and FB) of DP generation.

Fig. 2
figure 2

a-c Activities of antioxidant enzymes. Activities of SOD (a) (units/ mg protein), CAT (b) (pkat/mg protein) and GST (c) (nmol CDNB conjugate formed/min/mg protein) in the hemolymph (HL) and fat body (FB) of the pupae from nondiapausing (NDP) and diapausing (DP) generations. Bar represents mean ± S.E.M. (n = 6). Bars having different lower and upper case letters indicate significant difference in the measured parameters in HL and FB, respectively. ND:Not detected. Differences in the mean value have been considered statistically significant with a confidence interval of 95% and significant level of 5%

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Non-enzymatic antioxidants (Fig. 3a and b)

GSH content exhibited tissue specificity. Though its level in HL was found to be equal in both the categories of pupae, for FB it was significantly more in NDP generation. Comparatively low level of ASA was recorded in both the tissues of the pupa destined for diapause than the NDP one.

Fig. 3
figure 3

a-b Non-enzymatic antioxidants. Content of GSH (a) in the hemolymph (HL) (μmol/ml) and fat body (FB) (μmol/g tissue wet wt.); and ascorbic acid (b) in the HL (μg/ml) and FB (μg/g tissue wet wt.) of pupae from nondiapausing (NDP) and diapausing (DP) generations. Bar represents mean ± S.E.M. (n = 6). Bars having different lower and upper case letters indicate significant difference in the measured parameters in HL and FB, respectively. Differences in the mean value have been considered statistically significant with a confidence interval of 95% and significant level of 5%

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Discussion

Findings of this study indicate that the metabolism of ROS, the level of oxidative damaged products and status of antioxidant protection are different in the pupae destined for diapause compared to NDP group. In case of DP generation, 10 days old pupae are approaching the preparatory phase of diapause. O2 consumption is low, therefore, a low level of endogenous ROS is presumed than in their NDP counterpart. Lower H2O2 content in diapause-destined pupal tissues compared with NDP pupa, as observed in the present study, substantiate our presumption and also corroborate the findings of Zhao et al. [9, 29] in B. mori and Jena et al. [30] in A. mylitta. Zhao and coworkers have documented low level of H2O2 and higher activity of catalase in the eggs of B. mori during the initiation of diapause. We have also observed a higher catalase activity in the FB of diapause destined pupae. Shen et al. [31] observed that exogenous treatment of H2O2 could prevent initiation of diapause in B. mori. In a similar study on Artemia, Robbins et al. [32] further confirmed that supplementation of H2O2 in the incubating medium (sea water) terminates diapause in Artemia cysts. Above observations and our present findings indicate that low H2O2 is implicated in the initiation of diapause. In contrast, 10 days old pupae of NDP generation are in an active phase of development, leading to the termination of pupal life within the next 5–10 days. NDP pupae have been reported to exhibit higher O2 consumption than the DP counterpart [15, 16]. As O2 utilization is associated with the level of ROS production [17], the higher amount of H2O2 in the tissues of NDP pupae, observed in this study, is obvious. Though a few reports indicate a reciprocal relation between higher respiratory activity and mitochondrial ROS release [33,34,35,36], further studies are required to establish the findings in this insect model with simultaneous measurement of O2 consumption, metabolic rate and subcellular ROS generation in a single platform. Furthermore, we observed that despite a higher H2O2 level in the NDP pupae, the activities of SOD and CAT (exclusively observed in FB) were low in the same group. It is, therefore, assumed that the sequential actions of SOD and CAT may not be a major determining factor for H2O2 pool in this insect, as usually believed [37, 38]. Zhao and Shi [10] in their study on the metabolism of H2O2 in B. mori, indicated the existence of alternate mechanisms for the control of H2O2 in silkworm. Previously we also observed that CAT inhibition by aminotrizole in the pupal fat body of this insect did not result in the accumulation of H2O2 in the tissue concerned [39], which further substantiate the present findings.

Irrespective of tissues, MDA content and PC values were higher in the pupae of DP generation in comparison to those in the NDP group. Changes in fatty acid compositions of membrane lipids, i.e., towards more unsaturation [40] and extra storage of tissue lipids for sustained use [41], as a preparatory change for diapause may be the reasons for the susceptibility of the tissues to LPx. High PC value in the diapause-destined pupae can be due to high levels of LPx, as interaction of LPx intermediates with side chains of amino acids is a determining factor for protein carbonylation [42]. We presume that more oxidative damaged proteins are associated with lifespan extension of the pupae committed for diapause as reported earlier [7, 43]. High SOD and CAT activities in the pupae destined for diapause may be a strategy for extended life span, as observed earlier by Orr & Sohal [44] in SOD and CAT overexpressed Drosophila melanogaster, by Sim & Denlinger [45] in the diapausing mosquito, Culex pipiens and also by Tasaki et al. [46] in the long lived queens of the termite, Reticulitermes speratus. Lower GST activity and GSH levels observed in the pupae of this group are in good agreement with the findings of Meng et al. [47] in B. mori eggs. Zhao et al. [48] also observed low GSH/GSSG ratio as an important factor for diapause determination in the bivoltine strain of B. mori. In contrast, higher GST activity and GSH content in NDP pupae observed in this study correspond to the findings of Jovanović-Galović et al. [6] with reference to the nondiapausing larvae and pupae of European corn borer, Ostrinia nubilalis. GSTs are involved in the process of detoxification in insects [49], regulate their metamorphosis [6] and metabolize lipid peroxides without exhibiting any activity towards H2O2 [50, 51]. Higher GST activity in the pupae of NDP generation, therefore, can be linked with the attenuation of oxidative damage as reported earlier [50, 52] and to early metamorphosis of the pupae concerned [6].

The contents of nonenzymatic antioxidants (GSH and ASA), higher in the NDP group compared to DP generation, seems to be a strategic antioxidant response for the protection of insect tissues from oxidative damage [53]. Higher levels of GSH may also be correlated with the rapid development of pupae in NDP generation, as GSH has been reported to augment cell division and differentiation [54]. Similarly lower level of ASA in the diapause-destined pupal tissues corroborates the findings of Dmochowska-Ślęzak et al. [14] in diapausing Red mason bees, Osmia bicornis.

Conclusion

In summary, we observed that the fate of differential O2 consumption and consequently the ROS metabolism are different in these two groups of pupae as evident from the low H2O2 pool in the diapause-destined pupa. In this study we propose that low H2O2 content in diapause-destined pupa is associated with initiation of dormancy as reported earlier [8, 28, 30], along with higher activities of frontline antioxidant enzymes like SOD and CAT for enhanced oxidative resistance during extended lifespan [44, 46]. In contrast, higher GST activity and GSH content in the NDP group can be linked to rapid pupal development and an early metamorphosis (Fig. 4). Besides, higher levels of GSH and ASA content in NDP pupal tissues indicate a compensatory response to low SOD and CAT activities for the maintenance of redox status. Several mechanisms like changes in glutathione redox cycle [48], low H2O2 content [30], activation of extracellular regulated kinase (ERK) [55] and enhanced antioxidant protection [46] have been proposed as determining factors for dormancy and for an extended lifespan of diapause. However, ROS detoxification mechanism is well-known for the maintenance of homeostasis under such conditions.

Fig. 4
figure 4

Proposed mechanism of reactive oxygen species-mediated oxidative resistance and life span extension during pupal development of Antheraea mylitta

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The present findings, based on a naturally occurring phenomenon, in this silkworm species along with the previous reports brace up the conceptual framework for interdependence of ROS, quiescence, oxidative stress tolerance and extended lifespan. We envisage that future investigations should be carried out with an integrated approach for a comprehensive understanding of the above interactions and a possible crosstalk of the aforesaid components during insect development and diapause.