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Indian Journal of Experimental Biology Vol. 48, November 2010, pp. 1143-1151

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori Kangayam M Ponnuvel*, Geetha N Murthy, Arvind K Awasthi, Guruprasad Rao & Nanjappa B Vijayaprakash Seribiotech Research Laboratory, Carmelram Post, Kodathi, Bangalore 560 035, India Received 15 December 2009; revised 21 June 2010 Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp)genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis. Keywords: Bombyx mori, Diapause, Differential gene expression, Embryogenesis, Multivoltine

The silkworm Bombyx mori follows a system of hibernation adopted by insects during their life cycle1,2 entering into diapause at an early embryonic stage before dermal differentiation is completed to tide over unfavorable conditions. Induction of diapause in silkworm, involves external factors such as low temperature, short photoperiod and availability of coarse mulberry leaf. At the physiological level, the embryonic diapause is induced in B. mori by the diapause hormone (DH), secreted by the neurosecretory cells of suboesophageal ganglion (SG)3. In addition to scientific interest, the technological innovation in sericultural industry has allowed accumulation of much knowledge on diapause mechanisms for its application towards long-term egg preservation and controlled hatching of larvae4. _________ *Correspondent author Telephone: +91-80-28440651; Fax: +91-80-284401901 E-mail: [email protected]

Diapause expression is triggered by environmental signals such as photoperiod, temperature and food quality, which are transduced into hormones, in the neuroendocrine organs finally bringing about the altered phases from development to diapause or vice versa through structural and functional changes in the target organ4. It is characterized by reduced metabolic rate and simultaneous activation of selected associated metabolic pathways. The most common example is the accumulation of bio-molecules such as polyhydric alcohols that function as cryoprotectants so that freezing can either be avoided or tolerated5. Among many insects, diapause is seasonally regulated through production of polyols and other low molecular weight compounds that include glycerol, sorbitol, mannitol, trehalose and proline in response to short day length and low temperature over a period of days or weeks2,6.

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In insects, carbohydrate metabolism during embryonic development and embryonic diapause has been studied extensively from the viewpoint of cold hardiness and energy supply7. Several researchers have reported fluctuation in free sugars8, intermediary metabolites and activities of the rate limiting enzyme9 during embryonic diapause and development of Bombyx eggs. The Phosphofructokinase is a primary key enzyme that regulates the end product, either glycerol or trehalose, formed due to breakdown of glycogen during diapause10. At the initiation of diapause, activation of phosphorylase is related to the intense rate of glycogen breakdown within silkworm eggs. The flow of carbon released by activation of glycogen degradation may be routed to the pentose cycle because the activity of glucose-6-phosphate dehydrogenase (G6P-DH) is found to be significantly higher increasing with cold-acclimation in diapause adults, while, it was relatively low decreasing with cold-acclimation in non-diapause adults11. At the termination of diapause, sorbitol is converted into glycogen, which is utilized as a source of energy for embryogenesis12. This conversion is controlled by NAD sorbitol dehydrogenase (SDH1, EC1.1.1.14), induced at 5°C9,13. The appearance of NAD-SDH activity at the termination of diapause14 results in an increase in enzyme amount due to the activation of NAD-SDH gene at low temperature15. The diapause hormone induces expression of the Trehalase gene in ovaries16. Trehalase is a key enzyme responsible for accumulation of glycogen in diapause egg3 which is changed into sorbitol with the initiation of diapause and this in turn is an arresting factor in embryonic development17. Suzuki et al.18 indicated that BmEts encoded a novel ETS (transcription factor) family member which was strongly associated with embryonic diapause. The diapause initiation or termination was regulated by extracellular signal regulated kinase (ERK) and mitogen activated protein kinase (MAPK)19. During early embryogenesis in the silkworm B. mori, a RNA helicase like (RHL) protein has been reported which has an important role in the non-diapause stage of the silkworm20. Moribe et al.21 isolated a novel cold inducible gene, samui, which serves to transmit the 5°C signal for SDH expression in diapause eggs and to protect against cold injuries in non-diapause eggs. The molecular mechanism of diapause during the embryogenesis of the silkworms, B. mori has been studied22 where 24 individual cDNA transcripts were

expressed differentially in a total of 1,468 different cDNAs. Among those clones, mRNA transcript from cytochrome oxidase subunit I (COI) gradually increased in diapause-activated eggs during early embryogenesis. A common feature observed in diapausing insects including Diptera, Lepidoptera, Coleoptera and Hymenoptera orders was the up-regulations of heat shock proteins (Hsps) occurring at different development stages23. Hsps represent a ubiquitous component of the stress response in diverse organisms24, which suggest that they may play a role in the rapid increase in stress tolerance noted during early diapause. However, all of these inducible Hsps are up regulated in response to most types of environmental stress25 suggesting the possibility of specific roles for the different Hsps during diapause. In the flesh fly Sarcophaga crassipalpis, a number of diapause specific gene classes have been distinguished based on their expression pattern during early, mid and/or late diapause26. Changes in the expression of genes encoding Hsps represent one of the most conspicuous features of diapause in S. crassipalpis. Hsp 70 is up regulated during diapause in several insect species, by extreme temperature and desiccation in non-diapausing individuals27. Transcripts of Hsp 23 and Hsp 70 were found to be absent in unstressed non-diapause pupae and are expressed mostly in diapausing pupae28,29 while, Hsp 90 was down regulated during diapause30. The temperature-controlled expression of DH-PBAN gene is closely correlated to incidence of diapause, indicating that DH-PBAN gene expression is the initial event that leads to diapause induction4. The expression of the RHL mRNA is also associated with non-diapause or diapause termination and may play an important role in the developmental process20. However, other metabolic enzyme genes involved in diapause initiation or termination mechanism have not been studied in diapause and non-diapause eggs of silkworm B. mori. These genes may play a role in the diapause induction, initiation, as well as maintenance and termination process. Therefore, the present study has been undertaken to investigate the molecular aspects of genes involved in carbohydrate metabolism and heat shock protein response in diapause and nondiapause eggs during early embryogenesis. Materials and Methods Strain selected for the study—A multivoltine strain MW13 (Indian origin) was selected for the study. The

PONNUVEL et al.: DIFFERENTIAL GENE EXPRESSION IN EGGS OF SILKWORM

larvae were reared by standard rearing method31 up to third instar. The diapause eggs were obtained by rearing late stage (4th and 5th instars) larvae under low temperature (18ºC) and photoperiod (6L:18D) up to cocooning stage and the eggs were made to lay at normal room temperature (25ºC). RNA isolation—After oviposition, the diapause and non-diapause egg samples were collected from 6 to 48 h. Total RNA was extracted from the diapause and non-diapause eggs using TRIzol reagent (Invitrogen) and quantified by measuring the UV absorbance at 260 or 280 nm. The total RNA sample was denatured in formaldehyde, formamide and electrophoresed in 2.0% agarose gels. cDNA preparation—The first strand cDNA was synthesized utilizing RNA (2 µg) treated with 0.5 µl of DNase buffer and 0.5 µl of DNase (Invitrogen) for 15 min. Then, the reaction was terminated by heating at 75ºC for 10 min and 1 µl 10 mM dNTP, oligo (dT)18 (MWG India Pvt Ltd, Bangalore) was added followed by incubation at 65ºC for 5 min. Finally 1X reverse transcriptase buffer (4 µl), 5 mM DTT (1 µl) and M-MLV Superscript III reverse transcriptase (Invitrogen) (1 µl) was added to obtain a final volume of 20 µl. The reaction was terminated by heating at 75ºC for 10 min according to the superscript protocol. Identification of metabolic genes—The B. mori Tre (Acc No. D13763) sequence was obtained from the database and the forward primer (5'cgctgcttcattacgttcaa-3') and reverse primer (5'tgttcgggtttttcaaggac-3') was designed for cDNA sequence. Similarly, the SDH-2 gene cDNA (Acc No. DQ443393) was obtained and primers were designed for the cDNA sequence as forward (5'gatggtagcgagtgggaaaa-3') and reverse (5'gatagcccaagcaaggttca-3') primers. However, the sequences for the other genes such as GPase, PFK and G6P-DH were not reported in B. mori. Therefore, the heterologous gene sequence available in Drosophila melanogaster and Antheraea paukstadtorum were retrieved and used for identification of homologous corresponding gene sequences in the Bombyx silk database. The D. melanogaster GPase gene was homology searched with the silk database (http://morus.ab.a.utokyo.ac.jp) and the sequence (Acc No.NRPG1576) which showed the maximum homology with the Bombyx GPase gene was selected. Based on the GPase gene sequence, the forward (5'gatggtgatcgagaacatcg-3') and reverse (5'-

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caagctgcgagtttaaagtacaa-3') primers were designed. Similarly, PFK gene (Acc No. ovS302A08f) was obtained from the silk database and the forward (5'-atgaactccatgcacctagc-3') and reverse (5'-caatattttgattaccacgatgga-3') primers were designed. The A. paukstadtorum G6P-DH gene was also searched in silk database and the gene sequence (Acc No. fcaL-P16_F_O12) which showed homology with Bombyx G6P-DH was obtained. Subsequently, the forward (5'-aaccttttgccgcataacac-3') and reverse (5'- gctgacatcatcacgtccaa-3') primers were designed for the G6P-DH gene sequence. Identification of heat shock protein genes—The heat shock protein genes were analyzed for diapause and non-diapause egg and tissue samples. The B. mori Hsp 20.4 gene sequence was obtained from the database (Acc No. AF315318) and forward (5'ttttggccttgccttaacac-3') and reverse (5'ttcgctctggtccttgatct-3') primers were designed. Hsp 20.8 gene sequence (Acc No. AF315317) was searched from the database and forward (5'ctaaccccgaacgacatgct-3') and reverse (5'gatgtacccatcggcagtct-3') primers were designed. Similarly, Hsp 40 gene sequence (Acc No. AB206400) was obtained and forward (5'tcggacgatgacatcaagaa-3') and reverse (5'cccgggcgatatcttctaat-3') primers designed. The Hsp 70 gene was also retrieved (Acc No. DQ311189.1) and the forward (5'- gaacacactcgctgcacatc-3') and reverse (5'-gaggagtgcccaagatcgac-3') primers designed. Based on Hsp 90 cDNA (Acc.No. AB060275) sequence, the requisite forward (5'-ttcccagttcattggctacc-3') and reverse (5'-tcttgcgcttcttgttttca-3') primers were designed. Reverse transcription polymerase chain reaction (RT PCR) analysis—PCR amplification was performed in a 25 µl reaction mixture containing 2.0 µl 10 × reaction buffer (100 mM Tris-HCL, pH 8.3, 500 mM KCl), 0.2 mM dNTPs, 1.5 mM MgCl2, 66 ng of forward and reverse primers, 0.3 U of Taq DNA polymerase (MBI Fermentas) with 1 µl first strand cDNA as template. β actin forward (5'cactgaggctcccctgaac 3') and reverse (5' ggagtgcgtatccctcgtag 3') (Bangalore Genei) primers ware used as an internal standard. The PCR amplification was carried out under the following conditions: 94°C for 3 min followed by 30 cycles of 94°C for 30s, 50°C for 30s, 72°C for 2 min and a final extension of 7 min at 72°C.

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Semi quantitative measurement—The semi quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was adopted according to Goto and Kimura32 for quantification of mRNA. In this experiment, PCR reactions with 30 cycles were fixed for metabolic genes, Hsp genes and β actin. After the PCR, gel electrophoresis was carried out wherein equal volume of the PCR product of each sample was run in triplicate on 1.5% agarose gel. Further, the PCR products were detected under UV after staining with ethidium bromide, the intensity of stained products quantified by a Densito quantifying image analyzer (Syngene Gel documentation system, Madison, USA) and mean values calculated. β actin mRNA was quantified in the same manner for normalization. Results Metabolic enzyme gene expression—The expression of metabolic enzyme genes viz. phosphofructo kinase (PFK), sorbitol dehydrogenase (SDH-2), trehalase (Tre), glycogen phosphorylase (GPase) and glucose 6 phosphate dehydrogenase (G6P-DH) involved during early embryogenesis (6 to 48 h after oviposition) were analyzed in diapause induced and non-diapause eggs

of multivoltine silkworm B. mori kept at 25°C. All the genes recorded differential responses in non-diapause as well as diapause induced silkworm eggs (Fig. 1). Phosphofructo kinase gene (PFK)—PFK expression was low in diapause eggs at 6 h after oviposition, but revealed an increasing trend at 12, 18, 30, 36 and 48 h after oviposition with sharp declines at 24 and 42 h. In non-diapause eggs, there was a declining trend with higher expressions at 6, 30, 36 and 48 h that coincided with the expression level of diapause eggs (Figs 1 and 2a). Sorbitol dehydrogenase gene (SDH-2)—The SDH-2 gene did not get expressed till 18 h and 24 h in non-diapause and diapause eggs, respectively. The diapause induced eggs revealed a lower expression of the gene till 48 h after oviposition, while, comparatively, non-diapause eggs revealed a higher gene expression except at 48 h (Figs 1 and 2b). Trehalase gene (Tre)—The expression of Tre gene was lower in diapause induced eggs compared to non diapause eggs. Diapause induced eggs recorded a slight rise from 6-12 h after oviposition followed by a gradual decline up to 24 h and then a gradual rise up to 36 h, followed by a sharp decline at 42 h and a sharp rise at 48 h. On the other hand, in non-diapause

Fig.1—Gene expression patterns in non-diapause and diapause induced eggs 48 h after oviposition in silkworm Bombyx mori. The RNA from non-diapause and diapause induced eggs was isolated at 6 hourly intervals from 6 to 48 h and the gene products were run on 1.5% agarose gel. β actin was used as internal standard to confirm equal loading. Each sample was run in triplicate.

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Fig. 2—Gene expression patterns during embryonic development of non-diapause and diapause induced eggs. These eggs were kept at 25°C after oviposition and the RNA was isolated at 6 hourly intervals from 6 to 48 h after oviposition. a. PFK non-diapause ( ) and diapause induced ( ) b. SDH non-diapause ( ) and diapause induced ( ) c. Tre non-diapause ( ) and diapause induced ( ) d. GPase non-diapause ( ) and diapause induced ( ) e. G6P-DH ( ) non-diapause and diapause induced ( ) f. Hsp 19.8 non-diapause ( ) and diapause induced ( ) g. Hsp 20.4 non-diapause ( ) and diapause induced ( ) h. Hsp 40 non-diapause ( ) and diapause induced ( ) i. Hsp 70 non-diapause ( ) and diapause induced ( ) j. Hsp 90 non-diapause ( ) and diapause induced ( ). Each mRNA was quantified and normalized to β actin.

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eggs, Tre gene expression showed higher fluctuations between 18 and 36 h after which it declined till 48 h to coincide with the expression level of diapause induced eggs (Fig. 2c). Glycogen phosphorylase gene (GPase)—The nondiapause eggs revealed maximum expression of GPase gene with high fluctuations between 18 and 48 h after oviposition compared to diapause induced eggs which recorded an overall lower expression of the gene without many fluctuations. However, the expressions slightly decreased by 42 h and then increased by 48 h (Figs 1 and 2d). Glucose 6 phosphate dehydrogenase gene (G6PDH)—G6P-DH gene expression was observed with fluctuations in both non-diapause and diapause induced eggs over a period of 48 h after oviposition. Though maximum expression was observed in nondiapause eggs at 6 h after oviposition, diapause induced eggs revealed overall higher expression till 36 h after oviposition compared to non-diapause eggs. However, later in contrast to non-diapause eggs, the diapause induced eggs recorded a steep decrease at 42 h after oviposition and then a sudden increase at 48 h after oviposition (Figs 1 and 2e). Heat shock protein (Hsp) expression—Similar to metabolic enzyme genes, the heat shock protein genes were also analyzed after every 6 h following oviposition up to 48 h in the non-diapause and diapause induced eggs. Hsp 19.8: Both non-diapause and diapause eggs revealed similar trend in Hsp 19.8 gene expression. However, Hsp 19.8 gene revealed much lower expression levels in diapause induced eggs. Maximum expression was noticed at 6 h after oviposition in both non-diapause and diapause induced eggs after which both types of eggs revealed a sharp decline in gene expression. Similarly, both types of eggs recorded a sharp increase in expression level from 42 to 48 h after oviposition. No expression was observed at 18 h after oviposition in non-diapause eggs, while, in diapause induced eggs the gene did not express at 18 h as well as 24 h after oviposition (Figs 1 and 2f). Hsp 20.4: Expression levels of Hsp 20.4 did not vary much in non-diapause and diapause induced eggs except that, non-diapause eggs, exhibited sudden rise in expression level from 42 to 48 h (Figs 1 and 2g). Hsp 40: The expression levels of Hsp 40 gene revealed only slight variations from 6 to 48 h after oviposition in diapause induced eggs, compared to non-diapause eggs, which revealed major fluctuations

between 6 to 24 h after oviposition. A peak expression was observed at 6 h after oviposition with a sharp decline at 12 h followed by nil expression at 18 h and then a sharp rise at 24 h with slight variations till 48 h in non-diapause eggs (Figs 1 and 2h). Hsp 70: Among the Hsp genes, Hsp 70 gene revealed high expression levels in both non-diapause eggs and diapause induced eggs with a gradual decline towards 48 h after oviposition. However, in diapause induced eggs, the expression recorded a sharp rise from 24 to 36 h followed by a sharp decline leading to no expression at 42 h and increased expression at 48 h (Figs 1 and 2i). Hsp 90: Hsp 90 gene exhibited similar expression levels in diapause and non-diapause eggs up to 36 h after oviposition. Later, unlike non-diapause eggs which revealed gradual decline at 42 h after oviposition followed by a slight rise at 48 h, diapause induced eggs recorded sharp decline at 42 h followed by a sharp rise at 48 h to coincide with the expression level of non-diapause eggs (Figs 1 and 2j). Discussion Embryogenesis occurs in B. mori, in two phases viz. differentiation and organogenesis. Differentiation begins after oviposition and continues up to 4 days and organogenesis starts from 4th day and continues till hatching. The said two stages reveal distinct variations in energy metabolism of diapause and non-diapause silkworm eggs14. The embryonic development in non-diapause eggs incubated at 25°C proceeds continuously for 9.5 days until hatching33. In contrast, diapause eggs kept at 25°C reveal slow down of mitotic activities of embryonic cells and the cell division finally stops on 3rd day after oviposition34. It has been reported that, in B.mori, Tre, GPase, PFK, G6P-DH and SDH genes are expressed at different levels in various target tissues and are actively involved in the diapause process35. Further, the activity related to diapause process in eggs is completed by 24-36 h. In this backdrop, a study was taken up on analysis of the expression of these metabolic enzyme genes as well as Hsp genes in both non-diapause and diapause induced eggs at every 6 hour interval from 6 to 48 h after oviposition. The study revealed expression of all the metabolic enzyme and heat shock protein genes in both non-diapause and diapause induced eggs. However, GPase and Hsp

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19.8 genes were expressed at lower level in diapause induced eggs. Utilization of glycerol in the pentose phosphate pathway has been observed in freeze tolerant goldenrod gall fly, Eurosta solidaginis. However, when the temperature drops below 5°C, the expression of phosphofructokinase (PFK) is strongly inhibited, diverting the metabolism of insects from glycerol to sorbitol synthesis in one step process37,38. The present study indicated that in non-diapause eggs, the PFK expression started at a higher level in the early stage (6 h after oviposition), while in diapause induced eggs, it started at a low level. However, the PFK expression from 6 to 48 h was comparatively higher in diapause eggs indicating utilization of glycerol for energy to sustain metabolism in diapause conditions. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. Niimi et al.33 observed that the SDH mRNA being expressed within yolk cells of diapause eggs after chilling at 5°C for 40-50 days. They also postulated that the possible mechanisms includes activation of the SDH gene by factor(s) that appear only when diapause eggs are incubated at 5°C and the suppression of SDH gene expression by factor(s) present in diapausing eggs kept at 25°C. The present study revealed that, down regulation of SDH-2 gene in diapause induced eggs up to 24 h indicating that, the activity is regulated at transcriptional level. Further, the expression levels of sorbitol dehydrogenase coincided with that of PFK gene at 48 h after oviposition. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 h in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. Trehalase (Tre) is distributed in almost all tissues and organs of insects at various levels throughout the life cycle. Predominance of Tre gene was reported in the midgut throughout larval-pupal-adult development and far less in other tissues of silkworm head and

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cuticle. However, maximum expression was found in ovary and midgut16,36. In the present study covering a period of 6-48 h after oviposition, Tre expression was observed in both non-diapause and diapause induced eggs. However, the expression was at a low level in diapause induced eggs compared to non-diapausing eggs indicating lower Tre activity in diapause induced eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The diapause process requires less energy source which reflects lower activity of the gene. Yamashita et al.39 reported that glycogen phosphorylase (GPase) activation is triggered by anaerobic condition in diapause eggs as well as non-diapause eggs. Glycogen phosphorylase, glyceroldehyde-3-phosphatase and polyol dehydrogenase are all activated in the stem borer during seasonal acclimation40. Present study revealed higher expression of GPase gene in non-diapause eggs compared to diapause induced eggs. However, the increasing activity of the gene after 42 h suggests a possible up regulation in later stages of diapause process. Sonoda et al.41 on Hsp gene expression in diapausing larvae of Chilo suppressalis rice stem borer, revealed that, the Hsp 19.7 is not expressed in the diapausing larvae but was up regulated after cold acclimation41. However, Hwang et al.42 reported that the Hsp 20.8 gene was expressed at high level in diapausing eggs incubated at 25°C for 30 days after oviposition. In the pupal diapause of the flesh fly, S. crassipalpis, Hsp 70 gene is expressed immediately upon entry into diapause and remains up regulated until diapause is terminated29 and in non diapausing flesh flies, Hsp 70 is up regulated in response to desiccation45. In B. mori, Hsp 70 is synthesized in diapausing and non-diapausing egg development at the early germ-anlage stage43 and also after heat shock or acid treatment during embryogenesis except for two unaffected phases, the pre-blastodermic stages and deep diapause. The synthesis of Hsp 70 could be induced again immediately by the end of the reactivation phase at 5°C for 35 days44. Hsp 90 is not involved in diapause in B. mori up to nine days as it is expressed in both diapause and non-diapause eggs. Studies in Drosophila triauraria and the blowfly, Lucilia silicate, indicated no evidence of Hsp 90 involvement in diapause32. In contrast to this, Hsp 90 transcripts are down regulated during diapause in S. crassipalpis30 and up regulated in response to cold

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shock46 but not involved in diapause30. The current study revealed almost similar expression levels for most of the heat shock protein genes (Hsp 20.4, 40, 70, and 90) in both the eggs at all stages of embryonic development. This is in confirmation with the minor role of Hsp genes during early embryonic development in both non-diapause and diapause induced eggs as reported earlier35. However, Hsp 19.8 gene revealed very low expression levels in diapause induced eggs suggesting a very minor role of this gene compared to other Hsp genes. Saravanakumar et al.35 reported involvements of various genes in eggs of silkworm, B. mori and their up/down regulation during diapause over a period of 9 days after oviposition indicating that insect diapause is regulated by many genes expressed at various levels and time point, which have direct role or functioning as transcriptional activator. The present study provides an overview of the differential expression levels of specific genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition and major differences in gene expression between the eggs. The study also confirms the major role of metabolic enzyme genes and minor role of Hsp genes during early embryogenesis. Future investigations such as micro array based gene expression studies could reveal co-ordinated gene expression at different time points. In addition, probing of the specific role of each gene involved in diapause process through RNAi directed against the specific genes also would be useful to provide valuable inputs to understand the molecular mechanism involved in diapause process to develop efficient approaches for conservation of multivoltine silkworm races of B. mori. Acknowledgment This research work was supported by grants from Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/PR11397/PBD/19/192/2008 dated 30th June 2009). References 1 2

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Andrewartha H G, Diapause in relation to the ecology of insects, Biol Rev Cambridge Phil Soc, 27 (1952) 50. Lee R E, Principles of insect low temperature tolerance, in: Insect at low temperature edited by D L Denlinger (Chapman and Hall New York) 1991, 17. Yamashita O & Hasegawa K, Embryonic diapause, in Comprehensive insect physiology, biochemistry and pharmacology, (Pergamon Press, Oxford) 1985, 407.

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