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Hormonal regulation of the expression of two storage proteins in the larval fat body of the greater wax moth (Galleria mellonella)
Journal of Insect Physiology 49 (2003) 551–559 www.elsevier.com/locate/jinsphys
Hormonal regulation of the expression of two storage proteins in the larval fat body of the greater wax moth (Galleria mellonella) Jakub Godlewski a,∗, Barbara Kłudkiewicz b, Krystyna Grzelak b, Małgorzata Bere sewicz a, Bronisław Cymborowski a b
a Warsaw University, Department of Invertebrate Physiology, 1 Miecznikowa Street, 02-096 Warsaw, Poland Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawin´skiego Street, 02-106 Warsaw, Poland
Received 10 December 2002; received in revised form 4 February 2003; accepted 4 February 2003
Abstract During larval development of the greater wax moth, Galleria mellonella, genes of storage proteins LHP76 and LHP82 are tissueand stage-specifically expressed. In this study, hormonal regulation of this expression has been investigated in vivo. Messenger RNAs of the juvenile hormone (JH-suppressible) Lhp82 gene are present only during the feeding period of the final larval instar, suggesting that a high level of JH during earlier stages prevents its expression and that a small rise in JH titer observed on day 8 of the final larval instar is responsible for the rapid shut-off of its transcription. Application of 1µg of JH analog (fenoxycarb) specifically inhibits expression of Lhp82, whereas Lhp76 mRNAs remain at the same level. 20-hydroxyecdysone (20HE) does not exert any inhibitory effects on transcription of Lhp genes when injected in a dose of 0.5 or 1.5 µg per individual, regardless of larval age. However, the same dose of 20HE significantly lowers the rate of LHPs synthesis within the fat body and completely blocks secretion of LHPs into the hemolymph. Therefore, we propose that 20HE inhibits the synthesis of storage proteins and their secretion without altering the level of mRNAs. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Storage proteins; Hexamerins; Galleria mellonella; Juvenile hormone; 20-hydroxyecdysone; Stage-specific gene expression
1. Introduction In the majority of holometabolic insect species, synthesis of storage proteins is one of the most distinguishable molecular features during larval development. Nearly all of the storage proteins belong to the superfamily of hexameric larval hemolymph proteins (LHP) or hexamerins, named according to their composition of six identical subunits of 70–85 kDa. Hexamerins are synthesized within the fat body during larval life, secreted into the larval hemolymph and then taken up by fat body cells shortly before pupation. In some developmental stages, hexamerins accumulate in the hemo∗ Corresponding author. Present address: The Texas A&M University System Health Science Center, College of Medicine, Cardiovascular Research Institute, Division of Molecular Cardiology, 1901 South 1st Street, Bldg 162, Temple, TX 76504, USA. Tel.: +1-2547784811x1218; fax: +1-254-8996165. E-mail address: [email protected] (J. Godlewski).
lymph to extraordinarily high concentrations, reaching up to 50% of the total protein content (Telfer and Kunkel, 1991). Within the fat body of the pupa these proteins are stored and gradually broken down proteolitically in order to provide a pool of amino acids necessary for completion of adult differentiation during the non–feeding pupal stage (Haunerland, 1996). There are two major groups of storage proteins among lepidopteran species; arylphorins (so called due to their high content of aromatic amino acids phenyloalanine and tyrosine) and juvenile hormone–suppressible (JHsuppressible) storage proteins. The latter group is believed to be unique to Lepidoptera, whereas arylphorins are widely distributed throughout insects (Burmester, 1999). There are some additional types of hexamerins in some lepidopteran species, e.g. methionine-rich hexamerins, although they are not as abundant (Bean and Silhacek, 1989). Galleria mellonella (Lepidoptera: Pyralidae), the subject of this study, has four storage proteins called LHPs
0022-1910/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0022-1910(03)00026-X
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with numbers indicating their molecular mass as: LHP74, 76, 81, 82 (Miller and Silhacek, 1982). In this study we picked two that belong to each of the two major lepidopteran storage protein groups; LHP76, a typical arylphorin with Phe+Tyr content of 17.2% (Memmel et al., 1992) and LHP82, a JH–suppressible storage protein (Memmel et al., 1994). Many previous studies indicate that storage proteins are expressed in a stage-specific temporal manner, and strict tissue–specific regulation is observed (for review see Levenbook, 1985). In some cases, sex–specific mechanisms also are involved (Sakurai et al., 1988). As most hexamerins are regulated developmentally, certain signals are required for control of the expression of encoding genes. Insect hormones are believed to be key regulators of such developmental control. The major hormones in this regard are juvenile hormone (JH) and 20–hydroxyecdysone (20HE). JH is one of the most crucial factors controlling insect development and metabolism. Larval molting as well as larval metabolism demand high levels of JH, and high JH titres are, in fact, maintained throughout larval life (Rembold and Sehnal, 1987). The last larval instar however, is the first time during development that the JH concentration drops to an undetectable level, a phase called the metamorphosis–preparative phase (Riddiford, 1995). 20HE is a direct factor initiating ecdysis. During larval life, it is present in the hemolymph during short periods preceding each larval molt. In the last larval instar two peaks of 20HE are observed. A small peak in the second third of the instar, also called the commitment peak, ends the feeding period and induces 20HE-receptor gene expression (Karim and Thummel, 1992; Jindra et al., 1996; Minakuchi et al., 2002). A large, prepupal peak at the end of the instar directly triggers molting. In spite of many studies concerning the influences that these hormones exert on the expression of storage proteins conducted so far, there is still no comprehensive scheme of such regulation. It is clear that JH strongly inhibits the expression of JH–suppressible proteins, but it has no effect on arylphorins (Jones et al., 1993; Zheng et al., 2000; Cheon et al., 2002). The action of ecdysteroids on hexamerin expression is not clear. Some authors suggested a stimulating effect of 20HE on transcription in Drosophila melanogaster (Diptera)(Powell et al., 1984), while others have not observed such an influence, e.g. in Manduca sexta (Lepidoptera) (Caglayan and Gilbert, 1987). In Calliphora vicina (Diptera) translation, but not transcription, was suppressed by an elevated 20HE titer (Burmester and Scheller, 1995). The results of previous studies concerning the influence of hormones on storage proteins expression in G. mellonella were, in some aspects, contradictory to our preliminary results. For example, authors claimed that 20HE inhibits Lhp genes transcription (Ray et al., 1987),
but our results suggest that 20HE exerts post-transcriptional effects. To clarify these discrepancies, we present a more comprehensive review of hormonal regulation of the expression of storage proteins in G. mellonella.
2. Materials and methods 2.1. Insect rearing The greater wax moth G. mellonella (Lepidoptera, Pyralidae) was reared in constant darkness at 30 oC on a semi-artificial diet prepared as described by Sehnal (1966). Larvae of sixth instars preparing to ecdyse were recognized by head capsule slippage and selected for subsequent procedures. Newly molted last, seventh instar larvae were recognized by the size of the head capsule and general body pigmentation. Larvae within 0–12 h after ecdysis were regarded as day1 larvae. When G. mellonella larvae are reared at an optimal temperature of 30 oC, the seventh instar lasts approximately nine days, and pupation occurs on the tenth day, preceded by a short pharate pupal stage. 2.2. Hormonal treatment Larvae were injected intra-abdominally using a 10 µl syringe with 0.5 or 1.5 µg of 20HE (Sigma) per individual. Hormone was dissolved in Ringer solution (130 mM NaCl; 1.3 mM KCl; 1.3 mM CaCl2×2H2O; 2.3 mM Na2HCO3; pH 6.8) containing 10% ethanol. Fat body and hemolymph were collected prior to injection, 24, 48 and 72 h after injection. The same volume of Ringer solution containing 10% ethanol was injected as a control. Fenoxycarb [ethyl 2-(4-phenoxyphenoxy) ethylcarbamate] dissolved in acetone was applied topically using a pipette at a dose of 1 µg/individual. Fat body was collected both prior to application, and 24 and 48 h after application. The same volume of acetone was applied as a control. 2.3. In vivo labeling of fat body and hemolymph proteins Larvae after hormonal treatment were injected intraabdominally with 5 µCi of 14C-leucine (specific activity 8.88 MBq/mmol) using a 10 µl syringe. Fat body and hemolymph were collected after 3 h incubation with the isotope. Preparation of the 14C-leucine labeled protein sample was performed as described in section 2.4. Five microliters of radio-labeled protein extract was spotted on Whatman GF/C filter paper, precipitated with trichloroacetic acid (TCA) and counted on a LKB 1209 scintillation counter. Equal counts were then run on the gel.
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2.4. Preparation of fat body and hemolymph proteins Fat body was dissected into cold Ringer solution and rinsed twice with the buffer. Tissue was homogenized in 50 mM potassium phosphate, pH 8.0 buffer, containing 1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 1 mM EDTA and 0.01% 1-phenyl2-thiourea. After centrifugation of the crude extract for 5 min at 10,000g the supernatant was stored at ⫺20 oC. Hemolymph was collected with a pipette. Measured volumes were immediately diluted 1:9 with cold homogenization buffer and then were centrifuged at 10,000g for 5 min to remove cellular material. The samples of supernatant were stored at ⫺20 oC. Protein concentrations in the hemolymph were estimated by the Bradford method (1976), using bovine serum albumin as a standard. 2.5. Electrophoresis, Western blots and autoradiography Non–radioactive hemolymph proteins were separated by SDS–PAGE and blotted (Towbin et al., 1979) onto polyvinylidene difluoride membranes. SDS–PAGE was carried out on a 10% running gel under denaturing conditions, until the tracking dye (bromophenol blue) migrated to the end of the gel. Anti-arylphorin from Manduca sexta (rabbit, polyclonal, 1:12,500) was used as a primary antibody. As a secondary antibody, we used anti-rabbit antibody, alkaline phosphatase conjugated (mouse, monoclonal, 1:25,000). Secondary antibodies were detected during a colorimetric reaction with nitro blue tetrazolium and 5-bromo 4-chloro 3-indolyl phosphate. Acrylamide gels, containing 14C-labeled fat body and hemolymph proteins, were treated with ENHANCE reagent (New England Nuclear corp.) and then dried in vacuum. Dried gels were exposed to X-ray film (Kodak). The fluorograms were developed after appropriate exposure at ⫺70 oC (usually 5–10 days). 2.6. RNA isolation 2.6.1. Acid guanidium thiocyanate phenol-chloroform method Freshly dissected fat bodies (peripheral and perivisceral layer separately), midguts, Malpighian tubules, head capsules, larval gonads and whole larvae were rinsed with cold Ringer solution and homogenized in TRI Reagent (1 ml/100 mg) (Molecular Research Center). Total RNA was prepared by an acid guanidium thiocyanate phenol-chloroform method (Chomczyn´ ski and Sacchi, 1987) and finally dissolved in FORMAZOL (Molecular Research Center). The samples were kept at ⫺20 °C until further use.
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2.6.2. Lithium chloride–urea method Freshly dissected silk glands were rinsed with cold Ringer solution and homogenized. Total RNA was prepared by the lithium chloride–urea method (Auffray and Rougeon, 1980) and finally dissolved in FORMAZOL. The samples were stored at ⫺20 °C until further use. 2.7. Construction and labeling of cDNA probes One microgram of total RNA extracted from fat body of larvae (day 5 of seventh instar) served as a template for probe synthesis using the RT–PCR One Step System (Boehringer-Mannheim) as described previously (Godlewski et al., 2001). Its identity with a template was confirmed by DNA sequencing. The probes were labeled with digoxygenin by random priming using DIG High Prime Labeling and Detection Kit (BoehringerMannheim) according to the manufacturer’s instructions. 2.8. Northern blotting Two micrograms of total RNA from fat body of larvae from desired stage and age was denatured and subjected to electrophoresis in a 0.8% agarose gel containing 2.2 M formaldehyde in 3-N-morpholino-propane-sulfonic acid buffer. Following electrophoresis, gels were rinsed in 20×SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) and then transferred to positively charged nylon membranes (Roche) according to manufacturer’s instructions. Prior to loading, RNA concentrations were determined spectrophotometrically at 260 nm. Ethidium bromide staining checked equal loading and successful transfer. Filters were baked at 120 °C for 30 min, prehybridized in 5×SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, 2% blocking agent for 30 min and then hybridized in the same solution containing DIG-labeled cDNA probe (25 ng/ml, 2.5 ml/cm2 of membrane) overnight at 68 °C. Filters were then washed twice with 2×SSC, 0.1% SDS at room temperature for 5 min and subsequently twice with 0.1×SSC, 0.1% SDS at 68 °C for 15 min. For detection of specific signal, filters were incubated with an alkaline phosphatase conjugated anti-DIG antibody and reacted with CSPD as chemiluminescence substrates followed by fluorography. All procedures were as described in the manufacturer’s protocols. 2.9. DNA sequence analysis DNA sequences of the Lhp genes, including 1.2–1.3 kb upstream non-coding regions (Memmel et al., 1992; Memmel et al., 1994) were screened (using GENPRO 4.20 software) for presence of ecdysone response elements (EcREs), using either consensus sequence derived from published EcREs or storage protein-associated EcRE from D. melanogaster (Antoniewski et al.,
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1995). Lhp76 accesion number: M73793; Lhp82 accesion number: L21997. 3. Results 3.1. Tissue–specific expression of Lhp genes Tissue specificity was tested on day 5 larvae of the seventh larval instar. Total RNA from several tissues and parts of the body was extracted and probed with Lhp76 and Lhp82 specific cDNA. Hybridization signals were detected only in fat body samples (both peripheral and perivisceral) and in whole body homogenates (Fig. 1). 3.2. Expression pattern of Lhp genes during post– embryonic development Lhp76 transcripts were present throughout larval life from at least the fourth instar to the end of the seventh instar. They were also detectable in freshly molted pupae but not in older ones. Lhp82 mRNA was found only in samples from seventh instar larvae. Transcripts were first detected approximately 24 h after larval ecdysis and disappeared in pharate pupae. No transcripts were detected in pupae. Transcripts of neither gene were found in pharate adults (Fig. 2). 3.3. Influence of juvenile hormone analogue (fenoxycarb) on expression of Lhp genes The JH analogue fenoxycarb was topically applied to day 5 larvae of the seventh larval instar. Acetone treat-
Fig. 1. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from different tissues, (C) Lhp82 probe hybridized to RNA obtained from different tissues (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. RNA from: (1) whole larva body homogenate, (2) midgut, (3) Malpighian tubules, (4) head capsule, (5) silk glands, (6) perivisceral fat body, (7) peripheral fat body, (8) larval gonads.
ment was performed as a control. Fenoxycarb treatment did not affect the level of Lhp76 mRNA, but Lhp82 transcripts were barely detectable 24 h after treatment and disappeared completely after another 24 h. Application of pure acetone had no effect on either transcript (Fig. 3). 3.4. Influence of 20-hydroxyecdysone on expression of Lhp genes Injection of 0.5 µg of 20HE into seventh instar larvae had no effect on the level of Lhp76 mRNA in the fat body, regardless of the time of treatment. No influence was observed on day 1, day 4 or day 7 larvae of the seventh instar. Similarly, application of the hormone did not exert any effect on the level of Lhp82 transcripts in the fat body when day 4 or day 7 larvae were used in the investigation. On the other hand, 20HE injected into day 1 larvae of the seventh instar blocked initiation of transcription of the Lhp82 gene for at least 3 days, when compared to non–treated individuals. Application of Ringer solution containing 10% of ethanol had no effect on either transcript (Fig. 4). Sequence analysis of upstream non-coding DNA, associated with both Lhp genes within a distance of approximately 1.2–1.3 kb, did not reveal presence of EcREs (data not shown). 3.5. Influence of 20-hydroxyecdysone on synthesis of LHP proteins Post–transcriptional effects of 20HE on LHP protein synthesis were observed by in vivo labeling of newly synthesized proteins with radioisotope and by Western blotting. The first experiment monitored synthesis within fat body cells and the subsequent appearance of the proteins in the hemolymph. In vivo labeling experiments showed that synthesis of a group of proteins with molecular masses corresponding to LHPs in the fat body was markedly diminished 24 h after 20HE application, and previous levels of synthesis were restored after another 24 h (Fig. 5A). On the other hand, secretion of the proteins into the hemolymph (Fig. 5B) was blocked completely for at least 48 h. The protein recognized by an antibody against arlyphorin of another Lepidoptera species, Manduca sexta, was previously shown to be the actual G. mellonella arylphorin (LHP76) by N–terminus sequencing (Godlewski et al., 2001). In Fig. 6 we demonstrate that LHP76 level in the hemolymph decreases dramatically between 24 and 48 h after 20HE treatment.
4. Discussion The detailed analysis of Northern blots described here shows that transcription of the Lhp76 and Lhp82 genes,
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Fig. 2. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from the fat body of different developmental stages, (C) Lhp82 probe hybridized to RNA obtained from the fat body of different developmental stages, (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. RNA from the fat body of: (1) larvae of the fourth instar, (2) larvae of the fifth instar; larvae of the sixth instar: (3) day 2, (4) day 3; larvae of the seventh instar: (5) immediately after molt, (6) day 1, (7) day 2, (8) day 3, (9) day 4, (10) day 5, (11) day 6, (12) day 7, (13) day 8, (14) day 9, (15) pharate pupae; pupae: (16) immediately after molt, (17) day 1, (18) day 4; (19) pharate adults.
Fig. 3. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from the fat body of larvae of the seventh instar, (C) Lhp82 probe hybridized to RNA obtained from the fat body of larvae of the seventh instar, (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. Larvae (day 5) were treated with pure acetone or 1 µg of fenoxycarb dissolved in acetone. Nontreated larvae: (1) day 5, (2) day 6, (3) day 7; acetone treated larvae: (4) 24 h after treatment, (5) 48 h after treatment; fenoxycarb treated larvae: (6) 24 h after treatment, (7) 48 h after treatment.
as well as synthesis of encoded proteins described previously (Godlewski et al., 2001), is strictly developmentally regulated, both spatially (Fig. 1) and temporally (Fig. 2). The same data however, revealed that both genes, though closely related functionally, are regulated in distinctively different manners. Little is known about mechanisms of tissue specificity (for review see Harshman and James, 1998). In G. mellonella expression of the genes encoding both storage proteins is strictly limited to fat body tissue, although in other lepidopteran species, e.g. Hyphantria cunea, transcripts of storage proteins were found in Malpighian tubules and gonads in addition to fat body (Cheon et al., 2002). Stage specificity, as well as differential patterns of expression for the duration of one stage, indicates that certain developmental triggers that start and inhibit
Fig. 4. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. Equal loading of RNA was ensured by spectrophotometric analysis and ethidium bromide staining of the gel. NT—non-treated larvae, RS—larvae treated with Ringer solution (10% ethanol), 20HE—larvae treated with 0.5 µg of 20HE dissolved in Ringer solution (10% of ethanol); (A) larvae treated as day 1 of the seventh instar; (1,4,7) 24 h after treatment; (2,5,8) 48 h after treatment; (3,6,9) 72 h after treatment; (B) larvae treated as day 4 of the seventh instar; (10,13,16) 24 h after treatment; (11,14,17) 48 h after treatment; (12,15,18) 72 h after treatment; (C) larvae treated as day 7 of the seventh instar; (19,22,25) 24 h after treatment; (20,23,26) 48 h after treatment; (21,24,27) 72 h after treatment.
genes expression must be present. In insects, hormones are the most common and most versatile developmental cues. When the course of expression of the Lhp genes is compared to the endogenous levels of the two most
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Fig. 5. Autoradiogram of proteins synthesized by the fat body of larvae of the seventh instar (A) and detected in their hemolymph (B). Larvae were injected with 0.5 µg of 20HE dissolved in Ringer solution (10% ethanol) as day 5 and subsequently for 3 h with 5 µCi of 14Cleucine: (2,6) prior to treatment; (3,7) 24 h after treatment; (4,8) 48 h after treatment, (1,5) radioactive standard proteins in kDa. Equal amounts of radioactive material (2000 cpm) were loaded per lane.
Fig. 6. Western blot analysis of changes in abundance of LHP76 protein after injection of 0.5 µg of 20HE dissolved in Ringer solution (10% of ethanol), into the hemolymph of the seventh instar larvae. Larvae were injected as day 5. Each lane was loaded with hemolymph samples containing 20 µg of protein. Proteins were probed with antibody against α and β arylphorin of Manduca sexta. Molecular weight standards in kDa and LHP76 are indicated. Non-treated larvae: (1) day 6, (2) day 7; Ringer solution (10% ethanol) treated larvae: (3) 24 h after treatment, (4) 48 h after treatment; 20HE treated larvae: (5) 24 h after treatment, (6) 48 h after treatment.
important hormones in G. mellonella hemolymph, 20HE (Sehnal et al., 1981) and JH (Rembold and Sehnal, 1987), it is apparent that endogenous peaks of neither JH nor 20HE are likely to affect the level of Lhp76 mRNA. However, the level of Lhp82 mRNA was negatively correlated with the JH titer. During early larval instars (up to the sixth instar), JH levels remain high and thus most likely suppress Lhp82 gene expression. Approximately 24 h after the beginning of the seventh instar, JH levels decline to undetectable amounts, and expression of Lhp82 is initiated immediately thereafter. In the course of the seventh instar Lhp82 is silenced after day 8, a time that coincides with the small, commitment peak of 20HE and a minute peak of JH. Many researchers confirmed the latter, by different means of JH detection such as RIA (Plantevin et al., 1984), chromatography (Rembold and Sehnal, 1987) and bio–assays (S´ mietanko et al., 1989).
Therefore, Lhp82 mRNA seems to be negatively regulated by JH level. To confirm this, we investigated the effect of JH on the expression of Lhp genes. In those experiments we used fenoxycarb, a compound that exerts JH–like effects on numerous physiological processes in invertebrates (Davey and Gordon, 1996; Dean and Meola, 1997; Dhadialla et al., 1998; Nates and McKenney, 2000). Application of fenoxycarb on Lhp82– expressing individuals led to a rapid shut–down of its mRNA. Fenoxycarb did not affect Lhp76 expression in any way, a finding which confirms that lepidopteran arylphorins are not sensitive to juvenilizing treatments nor to JH of endogenous origin (Jones et al., 1993; Zheng et al., 2000). Since JH is among the most pleiotropic hormones ever known, the mode of its action is exceptionally complex. It has been shown that JH can regulate gene expression by multiple mechanisms including induction of transcription factors (Zhang et al., 1996) and stimulation of second messengers system (Yamamoto et al., 1988; Sevala and Davey, 1993); for reviews see Harshman and James (1998) and Davey (2000). Studies on the effects exerted by JH on JH–suppressible storage protein genes in Trichoplusia ni (Lepidoptera) concluded that, besides suppression of transcription itself, stability of the corresponding mRNAs is greatly reduced as well (Jones et al., 1993). This may be similar to the case in G. mellonella. A comparison of Lhp mRNAs abundance with 20HE level does not suggest any noteworthy impact of that hormone on the expression of Lhp genes. Sequence analysis of upstream non-coding DNA, associated with both Lhp genes within a distance of approximately 1.2– 1.3 kb, did not reveal presence of ecdysone response elements (EcREs), neither consensus sequence derived from published EcREs nor storage protein-associated EcRE from D. melanogaster (data not shown). In D. melanogaster, EcRE of storage protein gene Lsp-2 is situated at a –75 upstream position relative to the transcription initiation site (Antoniewski et al., 1995). Thus, most probably 20HE has no effect on Lhp gene expression at the transcriptional level. To clarify this we performed a series of experiments using animals of different ages. This may be a crucial criterion since it has been shown that the level of 20HE, which varies greatly during the course of aging, is a key factor stimulating expression of its own receptor, EcR, in both Diptera (Karim and Thummel, 1992; Imhof et al., 1993) and Lepidoptera (Jindra et al., 1996; Hiruma et al., 1997) species. It has been also shown that in the absence of hormone, dimerization of EcR with its partner USP, which is necessary for appropriate function, is weak (Spindler et al., 2001). In these experiments, we used day 1, day 4 and day 7 larvae of the last instar. The level of 20HE is very low in day 1 and day 4 larvae, however day 1 larvae of the seventh instar show quite high concentrations of the hormone several hours before, during
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day 3 larvae of the sixth instar (Sehnal et al., 1981). On the other hand, day 7 individuals have been already exposed to a small, EcR synthesis-inducing peak of ecdysteroids. Larvae injected with 0.5 µg 20HE did not alter their expression of the Lhp76 gene, regardless of the age of the treatment. Injection of a higher dose 1.5 µg 20HE likewise had no effect (not shown). Similar results, with one significant exception, have been obtained in the case of Lhp82 (Fig. 4). The exception is the absence of Lhp82 mRNAs in day 1, 20HE treated individuals, until at least 48 h after treatment. What might cause such a delay in one-day old larvae, while older ones were 20HE-insensitive? Possibly the inhibition of JH–suppressible Lhp82 gene expression can be triggered by a juvenilizing effect of the ecdysteriod treatment. It has been shown that high levels of ecdysteroids mimic the action of JH, possibly by activating the insects’ own corpora allata, or by acting synergistically with a low level of endogenous JH (Willis, 1974). Also in larvae of G. mellonella treated with 20HE, a dose–dependent increase of allatotropic activity and subsequent rise of JH titer has been observed (Muszyn´ ska-Pytel et al., 1992). Why then was the juvenilizing effect of the ecdysteroid treatment observed only in one-day old larvae? The course of action of JH esterase during the seventh instar may provide an answer. During the first three days of the instar JH esterase activity remains at a basal level of less than 10 nmol/min/ml, while starting from day four it elevates rapidly reaching 80 nmol/min/ml (Hwang-Hsu et al., 1979; Szołajska, 1991). In previous studies we showed that synthesis of the LHP group is silenced after the feeding period of the seventh instar (Kłudkiewicz et al., 1996; Godlewski et al., 2001). However mRNAs of the Lhp genes (at least Lhp76) were present until the very end of the instar or even longer (Fig. 2), hence the presence of 20HE, both endogenous and exogenous in origin did not inhibit transcription of the Lhp genes. To understand the nature of the LHPs disappearance in that period of development we performed two experiments assessing the influence of 20HE on synthesis of LHPs in the fat body and on their appearance in the hemolymph. Injection of 20HE into LHP-synthesizing, feeding larvae caused a temporary decline of their synthesis rate in the fat body, lasting approximately 24 h. At the same time, however, secretion of LHP protein into the hemolymph was blocked entirely for more than two days (Fig. 5). The latter result has been confirmed by screening of hemolymph proteins using LHP76-recognizing antibody (Fig. 6), which revealed that the amount of LHP76 declined rapidly between 24 and 48 h after ecdysteroid treatment. It has been shown that ecdysteroids promote synthesis of storage protein-specific receptor and thus support the process of their being taken into the fat body (Burmester and Scheller, 1995; Burmester et al., 1999), therefore
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blocking of the secretion of LHP by high concentrations of 20HE is credible. The disappearance of the LHP76 that already exists in the hemolymph is most probably due to re-adsorption by the fat body, however rapid turnover of the protein in the hemolymph cannot be excluded. Our observation that 20HE operates on the translational rather than the transcriptional level to inhibit synthesis of LHP is supported by research on dipteran species, in which high levels of in vitro translatable LSP2 mRNA remain detectable after pupariation in D. melanogaster (Powell et al., 1984; Lepesant et al., 1986). Also, application of 20HE in vivo specifically inhibits translation of storage proteins without altering their mRNA levels in Calliphora vicina (Burmester et al., 1995). Previous reports of regulation of LHP expression by hormones (Ray et al., 1987) claimed that 20HE inhibits transcription of Lhp genes. Our results do not support that claim. The difference may be caused by the significantly higher dose of hormone used by Ray and coworkers (5 µg of hormone instead of 0.5 µg used in this study). We believe that such a high dose is physiologically irrelevant. Methods employed by Ray and co-workers were also less sensitive (dot-blot) and RNA was obtained from very few time-points of development. Our analysis is supported by investigation of proteins and sequence analysis, which revealed that no EcREs were found in upstream non-coding regions of either Lhp gene that was investigated.
Acknowledgements The authors are grateful to Dr Elisabeth Willott for the anti–arylphorin antibody of Manduca sexta and to Jonathan N.E. Day, for critical reading of the manuscript. The State Committee for Scientific Research (KBN, Poland) supported this work by grant no.3P04C00622.
References Antoniewski, C., O’Grady, M.S., Edmondson, R.G., Lassieur, S.M., Benesˇ, H., 1995. Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene. Molecular Genetics and Genomics 249, 545–556. Auffray, C., Rougeon, T., 1980. Purification of mouse immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. European Journal of Biochemistry 107, 303–314. Bean, D.W., Silhacek, D.L., 1989. Changes in the titer of the female predominant storage protein (81K) during larval and pupal development of the Galleria mellonella. Archives of Insect Biochemistry and Physiology 10, 333–348. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
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Burmester, T., 1999. Evolution and function of the insect hexamerins. European Journal of Entomology 96, 213–235. Burmester, T., Scheller, K., 1995. Ecdysteroid-mediated uptake of arylphorin by larval fat bodies of Calliphora vicina: involvement and developmental regulation of arylphorin binding proteins. Insect Biochemistry and Molecular Biology 25, 700–706. Burmester, T., Matzner, U., Scheller, K., 1995. Effect of 20HE on synthesis and uptake of arylphorin by the larval fat body of Calliphora vicina. European Journal of Entomology 92, 217–227. Burmester, T., Antoniewski, C., Lepesant, J.-A., 1999. Ecdysone— regulation of synthesis and processing of fat body protein 1, the larval serum protein receptor of Drosophila melanogaster. European Journal of Biochemistry 262, 49–55. Caglayan, S.H., Gilbert, L.I., 1987. In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control. Comparative Biochemistry and Physiology B 87, 989–997. Cheon, H.-M., Hwang, S.-J., Kim, H.-J., Jin, B.R., Chae, K.-S., Yun, C.-Y., Seo, S.-J., 2002. Two juvenile hormone suppressible storage proteins may play different roles in Hyphantria cunea Drury. Archives of Insect Biochemistry and Physiology 50, 157–172. Chomczyn´ ski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156–159. Davey, K.G., 2000. The modes of action of juvenile hormones: some question we ought to ask. Insect Biochemistry and Molecular Biology 30, 663–669. Davey, K.G., Gordon, D.R., 1996. Fenoxycarb and thyroid hormones have JH-like effects on the follicle cells of Locusta migratoria in vitro. Archives of Insect Biochemistry and Physiology 32, 613– 622. Dean, S.R., Meola, R.W., 1997. Effect of juvenile hormone and juvenile hormone mimics on sperm transfer from the testes of the male cat flea (Siphonaptera: Pulicidae). Journal of Medical Entomology 34, 485–488. Dhadialla, T.S., Carlson, G.R., Le, D.P., 1998. New insecticides with ecdysteroidal and juvenile hormone activity. Annual Review of Entomology 43, 545–569. Godlewski, J., Kłudkiewicz, B., Grzelak, K., Cymborowski, B., 2001. Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause. Journal of Insect Physiology 47, 759–766. Harshman, L.G., James, A.A., 1998. Differential gene expression in insects: transcriptional control. Annual Review of Entomology 43, 671–700. Haunerland, N.H., 1996. Insect storage proteins: gene families and receptors. Insect Biochemistry and Molecular Biology 26, 755–765. Hiruma, K., Bocking, D., Lafont, R., Riddiford, L.M., 1997. Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta. General and Comparative Endocrinology 107, 84–97. Hwang-Hsu, K., Reddy, G., Krishna Kumaran, A., 1979. Correlations between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleria mellonella. Journal of Insect Physiology 25, 105–111. Imhof, M.O., Rusconi, S., Lezzi, M., 1993. Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanegaster ecdysteroid receptor (dEcR). Insect Biochemistry and Molecular Biology 23, 115–124. Jindra, M., Malone, F., Hiruma, K., Riddiford, L.M., 1996. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wing of the tobacco hornworm, Manduca sexta. Developmental Biology 180, 258–272. Jones, G., Venkatarman, V., Manczak, M., Schelling, D., 1993. Juven-
ile hormone action to suppress gene transcription and influence message stability. Developmental Genetics 14, 323–332. Karim, F.D., Thummel, C.S., 1992. Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO Journal 11, 4083–4093. Kłudkiewicz, B., Godlewski, J., Grzelak, K., Cymborowski, B., Lassota, Z., 1996. Influence of low temperature on the synthesis of some Galleria mellonella proteins. Acta Biochimica Polonica 43, 639–644. Lepesant, J.-A., Maschat, F., Kejzlarova-Lepesant, J., Benesˇ, H., Yanicostas, C., 1986. Developmental and ecdysteroid regulation of gene expression in the larval fat body of Drosophila melanogaster. Archives of Insect Biochemistry and Physiology 3, 133–141. Levenbook, L., 1985. Insect storage proteins. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 10. Pergamon Press, Oxford, pp. 307–346. Memmel, N.A., Trewitt, P., Silhacek, D., Krishna Kumaran, A., 1992. Nucleotide sequence and structure of the arylphorin gene from Galleria mellonella. Insect Biochemistry and Molecular Biology 22, 333–342. Memmel, N.A., Trewitt, P.M., Grzelak, K., Rajaratnam, V.S., Kumaran, A.K., 1994. Nucleotide sequence, structure and developmental regulation of Lhp82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella. Insect Biochemistry and Molecular Biology 24, 133–144. Miller, S.G., Silhacek, D.L., 1982. Identification and purification of storage proteins in tissues of the greater wax moth Galleria mellonella. Insect Biochemistry 12, 277–292. Minakuchi, C., Nakagawa, Y., Kiuchi, M., Tomita, S., Kamimura, M., 2002. Molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis. Insect Biochemistry and Molecular Biology 32, 999–1008. Muszyn´ ska-Pytel, M., Mikołajczyk, P., Pszczo´ łkowski, M.A., Cymborowski, B., 1992. Juvenilizing effect of ecdysone mimic RH 5849 in Galleria mellonella larvae. Experientia 48, 1013–1017. Nates, S.F., McKenney, C.L. Jr., 2000. Growth, lipid class and fatty acid composition in juvenile mud crabs (Rhithropanopeus harrisii) following larval exposure to fenoxycarb, insect juvenile hormone analog. Comparative Biochemistry and Physiology C 127, 317– 325. Plantevin, G., De Reggi, M., Nardon, C., 1984. Changes in ecdysteroid and JH titres in the hemolymph of Galleria mellonella larvae and pupae. General and Comparative Endocrinology 56, 218–230. Powell, D., Sato, J.D., Brock, H.W., Roberts, D.B., 1984. Regulation of synthesis in the larval serum proteins of Drosophila melanogaster. Developmental Biology 102, 206–215. Ray, A., Memmel, N.A., Krishna Kumaran, A., 1987. Developmental regulation of the larval hemolymph protein genes in Galleria mellonella. Roux’s Archives of Developmental Biology 196, 414–420. Rembold, H., Sehnal, F., 1987. Juvenile hormones and their titer regulation in Galleria mellonella. Insect Biochemistry 17, 997–1001. Riddiford, L.M., 1995. Hormonal regulation of gene expression during lepidopteran development. In: Goldsmith, M.R., Wilkins, A.S. (Eds.), Molecular Model Systems in the Lepidoptera. Cambridge University Press, Cambridge, pp. 293–322. Sakurai, H., Fujii, T., Izumi, S., Tomino, S., 1988. Complete nucleotide sequence of gene for sex-specific storage protein of Bombyx mori. Nucleic Acids Research 16, 7717–7718. Sehnal, F., 1966. Kritisches Studium der Bionomie und Biometric der in Verschiedenen Lebensbedingungen Gezuchteten Waschmotte Galleria mellonella (Lepidoptera). Zeitschrift fu¨ r wissenschaftliche Zoologie 174, 53–82. Sehnal, F., Maroy, P., Mala, J., 1981. Regulation and significance of ecdysteriod titre fluctuations in lepidopterous larvae and pupae. Journal of Insect Physiology 27, 535–544. Sevala, V.L., Davey, K.G., 1993. Juvenile hormone dependent phos-
J. Godlewski et al. / Journal of Insect Physiology 49 (2003) 551–559
phorylation of a 100kDa polypeptide is mediated by protein kinase C in follicle cells of Rhodnius prolixus. Invertebrate Reproduction and Development 22, 189–193. S´ mietanko, A., Wis´niewski, J., Cymborowski, B., 1989. Effect of low rearing temperature on development of Galleria mellonella larvae and their sensitivity to juvenilizing treatment. Comparative Biochemistry and Physiology A 92, 163–169. Spindler, K.-D., Przibilla, S., Spindler-Barth, M., 2001. Moulting hormones of arthropods: molecular mechanisms. Zoology 103, 189– 201. Szołajska, E., 1991. Hydrolytic degradation of juvenile hormones in hemolymph and corpora allata of Galleria mellonella. Acta Biochimica Polonica 38, 321–333. Telfer, W., Kunkel, J., 1991. The function and evolution of insects’ storage hexamers. Annual Review of Entomology 36, 205–228. Towbin, H., Staehelin, T., Gordon, A., 1979. Electrophoretic transfer
559
of proteins from poliacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 76, 4350–4354. Willis, J.H., 1974. Morphogenetic action of insect hormones. Annual Reviews of Entomology 19, 97–115. Yamamoto, K., Chadarevian, A., Pelligrini, A., 1988. Juvenile hormone action mediated in male accessory glands of Drosophila by calcium and kinase. Science 239, 916–919. Zhang, J., Saleh, D.S., Wyatt, G.R., 1996. Juvenile hormone regulation of an insect gene: a specific transcription factor and a DNA response element. Molecular and Cellular Endocrinology 122, 15–20. Zheng, Y., Sugie, H., Tojo, S., 2000. Juvenile hormone titre and hormonal regulation of storage protein synthesis in the common cutworm, Spodoptera litura. Entomological Science 3, 9–18.
Hormonal regulation of the expression of two storage proteins in the larval fat body of the greater wax moth (Galleria mellonella) Jakub Godlewski a,∗, Barbara Kłudkiewicz b, Krystyna Grzelak b, Małgorzata Bere sewicz a, Bronisław Cymborowski a b
a Warsaw University, Department of Invertebrate Physiology, 1 Miecznikowa Street, 02-096 Warsaw, Poland Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawin´skiego Street, 02-106 Warsaw, Poland
Received 10 December 2002; received in revised form 4 February 2003; accepted 4 February 2003
Abstract During larval development of the greater wax moth, Galleria mellonella, genes of storage proteins LHP76 and LHP82 are tissueand stage-specifically expressed. In this study, hormonal regulation of this expression has been investigated in vivo. Messenger RNAs of the juvenile hormone (JH-suppressible) Lhp82 gene are present only during the feeding period of the final larval instar, suggesting that a high level of JH during earlier stages prevents its expression and that a small rise in JH titer observed on day 8 of the final larval instar is responsible for the rapid shut-off of its transcription. Application of 1µg of JH analog (fenoxycarb) specifically inhibits expression of Lhp82, whereas Lhp76 mRNAs remain at the same level. 20-hydroxyecdysone (20HE) does not exert any inhibitory effects on transcription of Lhp genes when injected in a dose of 0.5 or 1.5 µg per individual, regardless of larval age. However, the same dose of 20HE significantly lowers the rate of LHPs synthesis within the fat body and completely blocks secretion of LHPs into the hemolymph. Therefore, we propose that 20HE inhibits the synthesis of storage proteins and their secretion without altering the level of mRNAs. 2003 Elsevier Science Ltd. All rights reserved. Keywords: Storage proteins; Hexamerins; Galleria mellonella; Juvenile hormone; 20-hydroxyecdysone; Stage-specific gene expression
1. Introduction In the majority of holometabolic insect species, synthesis of storage proteins is one of the most distinguishable molecular features during larval development. Nearly all of the storage proteins belong to the superfamily of hexameric larval hemolymph proteins (LHP) or hexamerins, named according to their composition of six identical subunits of 70–85 kDa. Hexamerins are synthesized within the fat body during larval life, secreted into the larval hemolymph and then taken up by fat body cells shortly before pupation. In some developmental stages, hexamerins accumulate in the hemo∗ Corresponding author. Present address: The Texas A&M University System Health Science Center, College of Medicine, Cardiovascular Research Institute, Division of Molecular Cardiology, 1901 South 1st Street, Bldg 162, Temple, TX 76504, USA. Tel.: +1-2547784811x1218; fax: +1-254-8996165. E-mail address: [email protected] (J. Godlewski).
lymph to extraordinarily high concentrations, reaching up to 50% of the total protein content (Telfer and Kunkel, 1991). Within the fat body of the pupa these proteins are stored and gradually broken down proteolitically in order to provide a pool of amino acids necessary for completion of adult differentiation during the non–feeding pupal stage (Haunerland, 1996). There are two major groups of storage proteins among lepidopteran species; arylphorins (so called due to their high content of aromatic amino acids phenyloalanine and tyrosine) and juvenile hormone–suppressible (JHsuppressible) storage proteins. The latter group is believed to be unique to Lepidoptera, whereas arylphorins are widely distributed throughout insects (Burmester, 1999). There are some additional types of hexamerins in some lepidopteran species, e.g. methionine-rich hexamerins, although they are not as abundant (Bean and Silhacek, 1989). Galleria mellonella (Lepidoptera: Pyralidae), the subject of this study, has four storage proteins called LHPs
0022-1910/03/$ - see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0022-1910(03)00026-X
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with numbers indicating their molecular mass as: LHP74, 76, 81, 82 (Miller and Silhacek, 1982). In this study we picked two that belong to each of the two major lepidopteran storage protein groups; LHP76, a typical arylphorin with Phe+Tyr content of 17.2% (Memmel et al., 1992) and LHP82, a JH–suppressible storage protein (Memmel et al., 1994). Many previous studies indicate that storage proteins are expressed in a stage-specific temporal manner, and strict tissue–specific regulation is observed (for review see Levenbook, 1985). In some cases, sex–specific mechanisms also are involved (Sakurai et al., 1988). As most hexamerins are regulated developmentally, certain signals are required for control of the expression of encoding genes. Insect hormones are believed to be key regulators of such developmental control. The major hormones in this regard are juvenile hormone (JH) and 20–hydroxyecdysone (20HE). JH is one of the most crucial factors controlling insect development and metabolism. Larval molting as well as larval metabolism demand high levels of JH, and high JH titres are, in fact, maintained throughout larval life (Rembold and Sehnal, 1987). The last larval instar however, is the first time during development that the JH concentration drops to an undetectable level, a phase called the metamorphosis–preparative phase (Riddiford, 1995). 20HE is a direct factor initiating ecdysis. During larval life, it is present in the hemolymph during short periods preceding each larval molt. In the last larval instar two peaks of 20HE are observed. A small peak in the second third of the instar, also called the commitment peak, ends the feeding period and induces 20HE-receptor gene expression (Karim and Thummel, 1992; Jindra et al., 1996; Minakuchi et al., 2002). A large, prepupal peak at the end of the instar directly triggers molting. In spite of many studies concerning the influences that these hormones exert on the expression of storage proteins conducted so far, there is still no comprehensive scheme of such regulation. It is clear that JH strongly inhibits the expression of JH–suppressible proteins, but it has no effect on arylphorins (Jones et al., 1993; Zheng et al., 2000; Cheon et al., 2002). The action of ecdysteroids on hexamerin expression is not clear. Some authors suggested a stimulating effect of 20HE on transcription in Drosophila melanogaster (Diptera)(Powell et al., 1984), while others have not observed such an influence, e.g. in Manduca sexta (Lepidoptera) (Caglayan and Gilbert, 1987). In Calliphora vicina (Diptera) translation, but not transcription, was suppressed by an elevated 20HE titer (Burmester and Scheller, 1995). The results of previous studies concerning the influence of hormones on storage proteins expression in G. mellonella were, in some aspects, contradictory to our preliminary results. For example, authors claimed that 20HE inhibits Lhp genes transcription (Ray et al., 1987),
but our results suggest that 20HE exerts post-transcriptional effects. To clarify these discrepancies, we present a more comprehensive review of hormonal regulation of the expression of storage proteins in G. mellonella.
2. Materials and methods 2.1. Insect rearing The greater wax moth G. mellonella (Lepidoptera, Pyralidae) was reared in constant darkness at 30 oC on a semi-artificial diet prepared as described by Sehnal (1966). Larvae of sixth instars preparing to ecdyse were recognized by head capsule slippage and selected for subsequent procedures. Newly molted last, seventh instar larvae were recognized by the size of the head capsule and general body pigmentation. Larvae within 0–12 h after ecdysis were regarded as day1 larvae. When G. mellonella larvae are reared at an optimal temperature of 30 oC, the seventh instar lasts approximately nine days, and pupation occurs on the tenth day, preceded by a short pharate pupal stage. 2.2. Hormonal treatment Larvae were injected intra-abdominally using a 10 µl syringe with 0.5 or 1.5 µg of 20HE (Sigma) per individual. Hormone was dissolved in Ringer solution (130 mM NaCl; 1.3 mM KCl; 1.3 mM CaCl2×2H2O; 2.3 mM Na2HCO3; pH 6.8) containing 10% ethanol. Fat body and hemolymph were collected prior to injection, 24, 48 and 72 h after injection. The same volume of Ringer solution containing 10% ethanol was injected as a control. Fenoxycarb [ethyl 2-(4-phenoxyphenoxy) ethylcarbamate] dissolved in acetone was applied topically using a pipette at a dose of 1 µg/individual. Fat body was collected both prior to application, and 24 and 48 h after application. The same volume of acetone was applied as a control. 2.3. In vivo labeling of fat body and hemolymph proteins Larvae after hormonal treatment were injected intraabdominally with 5 µCi of 14C-leucine (specific activity 8.88 MBq/mmol) using a 10 µl syringe. Fat body and hemolymph were collected after 3 h incubation with the isotope. Preparation of the 14C-leucine labeled protein sample was performed as described in section 2.4. Five microliters of radio-labeled protein extract was spotted on Whatman GF/C filter paper, precipitated with trichloroacetic acid (TCA) and counted on a LKB 1209 scintillation counter. Equal counts were then run on the gel.
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2.4. Preparation of fat body and hemolymph proteins Fat body was dissected into cold Ringer solution and rinsed twice with the buffer. Tissue was homogenized in 50 mM potassium phosphate, pH 8.0 buffer, containing 1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 1 mM EDTA and 0.01% 1-phenyl2-thiourea. After centrifugation of the crude extract for 5 min at 10,000g the supernatant was stored at ⫺20 oC. Hemolymph was collected with a pipette. Measured volumes were immediately diluted 1:9 with cold homogenization buffer and then were centrifuged at 10,000g for 5 min to remove cellular material. The samples of supernatant were stored at ⫺20 oC. Protein concentrations in the hemolymph were estimated by the Bradford method (1976), using bovine serum albumin as a standard. 2.5. Electrophoresis, Western blots and autoradiography Non–radioactive hemolymph proteins were separated by SDS–PAGE and blotted (Towbin et al., 1979) onto polyvinylidene difluoride membranes. SDS–PAGE was carried out on a 10% running gel under denaturing conditions, until the tracking dye (bromophenol blue) migrated to the end of the gel. Anti-arylphorin from Manduca sexta (rabbit, polyclonal, 1:12,500) was used as a primary antibody. As a secondary antibody, we used anti-rabbit antibody, alkaline phosphatase conjugated (mouse, monoclonal, 1:25,000). Secondary antibodies were detected during a colorimetric reaction with nitro blue tetrazolium and 5-bromo 4-chloro 3-indolyl phosphate. Acrylamide gels, containing 14C-labeled fat body and hemolymph proteins, were treated with ENHANCE reagent (New England Nuclear corp.) and then dried in vacuum. Dried gels were exposed to X-ray film (Kodak). The fluorograms were developed after appropriate exposure at ⫺70 oC (usually 5–10 days). 2.6. RNA isolation 2.6.1. Acid guanidium thiocyanate phenol-chloroform method Freshly dissected fat bodies (peripheral and perivisceral layer separately), midguts, Malpighian tubules, head capsules, larval gonads and whole larvae were rinsed with cold Ringer solution and homogenized in TRI Reagent (1 ml/100 mg) (Molecular Research Center). Total RNA was prepared by an acid guanidium thiocyanate phenol-chloroform method (Chomczyn´ ski and Sacchi, 1987) and finally dissolved in FORMAZOL (Molecular Research Center). The samples were kept at ⫺20 °C until further use.
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2.6.2. Lithium chloride–urea method Freshly dissected silk glands were rinsed with cold Ringer solution and homogenized. Total RNA was prepared by the lithium chloride–urea method (Auffray and Rougeon, 1980) and finally dissolved in FORMAZOL. The samples were stored at ⫺20 °C until further use. 2.7. Construction and labeling of cDNA probes One microgram of total RNA extracted from fat body of larvae (day 5 of seventh instar) served as a template for probe synthesis using the RT–PCR One Step System (Boehringer-Mannheim) as described previously (Godlewski et al., 2001). Its identity with a template was confirmed by DNA sequencing. The probes were labeled with digoxygenin by random priming using DIG High Prime Labeling and Detection Kit (BoehringerMannheim) according to the manufacturer’s instructions. 2.8. Northern blotting Two micrograms of total RNA from fat body of larvae from desired stage and age was denatured and subjected to electrophoresis in a 0.8% agarose gel containing 2.2 M formaldehyde in 3-N-morpholino-propane-sulfonic acid buffer. Following electrophoresis, gels were rinsed in 20×SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) and then transferred to positively charged nylon membranes (Roche) according to manufacturer’s instructions. Prior to loading, RNA concentrations were determined spectrophotometrically at 260 nm. Ethidium bromide staining checked equal loading and successful transfer. Filters were baked at 120 °C for 30 min, prehybridized in 5×SSC, 0.1% N-lauroyl sarcosine, 0.02% SDS, 2% blocking agent for 30 min and then hybridized in the same solution containing DIG-labeled cDNA probe (25 ng/ml, 2.5 ml/cm2 of membrane) overnight at 68 °C. Filters were then washed twice with 2×SSC, 0.1% SDS at room temperature for 5 min and subsequently twice with 0.1×SSC, 0.1% SDS at 68 °C for 15 min. For detection of specific signal, filters were incubated with an alkaline phosphatase conjugated anti-DIG antibody and reacted with CSPD as chemiluminescence substrates followed by fluorography. All procedures were as described in the manufacturer’s protocols. 2.9. DNA sequence analysis DNA sequences of the Lhp genes, including 1.2–1.3 kb upstream non-coding regions (Memmel et al., 1992; Memmel et al., 1994) were screened (using GENPRO 4.20 software) for presence of ecdysone response elements (EcREs), using either consensus sequence derived from published EcREs or storage protein-associated EcRE from D. melanogaster (Antoniewski et al.,
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1995). Lhp76 accesion number: M73793; Lhp82 accesion number: L21997. 3. Results 3.1. Tissue–specific expression of Lhp genes Tissue specificity was tested on day 5 larvae of the seventh larval instar. Total RNA from several tissues and parts of the body was extracted and probed with Lhp76 and Lhp82 specific cDNA. Hybridization signals were detected only in fat body samples (both peripheral and perivisceral) and in whole body homogenates (Fig. 1). 3.2. Expression pattern of Lhp genes during post– embryonic development Lhp76 transcripts were present throughout larval life from at least the fourth instar to the end of the seventh instar. They were also detectable in freshly molted pupae but not in older ones. Lhp82 mRNA was found only in samples from seventh instar larvae. Transcripts were first detected approximately 24 h after larval ecdysis and disappeared in pharate pupae. No transcripts were detected in pupae. Transcripts of neither gene were found in pharate adults (Fig. 2). 3.3. Influence of juvenile hormone analogue (fenoxycarb) on expression of Lhp genes The JH analogue fenoxycarb was topically applied to day 5 larvae of the seventh larval instar. Acetone treat-
Fig. 1. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from different tissues, (C) Lhp82 probe hybridized to RNA obtained from different tissues (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. RNA from: (1) whole larva body homogenate, (2) midgut, (3) Malpighian tubules, (4) head capsule, (5) silk glands, (6) perivisceral fat body, (7) peripheral fat body, (8) larval gonads.
ment was performed as a control. Fenoxycarb treatment did not affect the level of Lhp76 mRNA, but Lhp82 transcripts were barely detectable 24 h after treatment and disappeared completely after another 24 h. Application of pure acetone had no effect on either transcript (Fig. 3). 3.4. Influence of 20-hydroxyecdysone on expression of Lhp genes Injection of 0.5 µg of 20HE into seventh instar larvae had no effect on the level of Lhp76 mRNA in the fat body, regardless of the time of treatment. No influence was observed on day 1, day 4 or day 7 larvae of the seventh instar. Similarly, application of the hormone did not exert any effect on the level of Lhp82 transcripts in the fat body when day 4 or day 7 larvae were used in the investigation. On the other hand, 20HE injected into day 1 larvae of the seventh instar blocked initiation of transcription of the Lhp82 gene for at least 3 days, when compared to non–treated individuals. Application of Ringer solution containing 10% of ethanol had no effect on either transcript (Fig. 4). Sequence analysis of upstream non-coding DNA, associated with both Lhp genes within a distance of approximately 1.2–1.3 kb, did not reveal presence of EcREs (data not shown). 3.5. Influence of 20-hydroxyecdysone on synthesis of LHP proteins Post–transcriptional effects of 20HE on LHP protein synthesis were observed by in vivo labeling of newly synthesized proteins with radioisotope and by Western blotting. The first experiment monitored synthesis within fat body cells and the subsequent appearance of the proteins in the hemolymph. In vivo labeling experiments showed that synthesis of a group of proteins with molecular masses corresponding to LHPs in the fat body was markedly diminished 24 h after 20HE application, and previous levels of synthesis were restored after another 24 h (Fig. 5A). On the other hand, secretion of the proteins into the hemolymph (Fig. 5B) was blocked completely for at least 48 h. The protein recognized by an antibody against arlyphorin of another Lepidoptera species, Manduca sexta, was previously shown to be the actual G. mellonella arylphorin (LHP76) by N–terminus sequencing (Godlewski et al., 2001). In Fig. 6 we demonstrate that LHP76 level in the hemolymph decreases dramatically between 24 and 48 h after 20HE treatment.
4. Discussion The detailed analysis of Northern blots described here shows that transcription of the Lhp76 and Lhp82 genes,
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Fig. 2. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from the fat body of different developmental stages, (C) Lhp82 probe hybridized to RNA obtained from the fat body of different developmental stages, (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. RNA from the fat body of: (1) larvae of the fourth instar, (2) larvae of the fifth instar; larvae of the sixth instar: (3) day 2, (4) day 3; larvae of the seventh instar: (5) immediately after molt, (6) day 1, (7) day 2, (8) day 3, (9) day 4, (10) day 5, (11) day 6, (12) day 7, (13) day 8, (14) day 9, (15) pharate pupae; pupae: (16) immediately after molt, (17) day 1, (18) day 4; (19) pharate adults.
Fig. 3. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. (A) Lhp76 probe hybridized to RNA obtained from the fat body of larvae of the seventh instar, (C) Lhp82 probe hybridized to RNA obtained from the fat body of larvae of the seventh instar, (B, D) ethidium bromide staining of 18S rRNA band from corresponding gels. Larvae (day 5) were treated with pure acetone or 1 µg of fenoxycarb dissolved in acetone. Nontreated larvae: (1) day 5, (2) day 6, (3) day 7; acetone treated larvae: (4) 24 h after treatment, (5) 48 h after treatment; fenoxycarb treated larvae: (6) 24 h after treatment, (7) 48 h after treatment.
as well as synthesis of encoded proteins described previously (Godlewski et al., 2001), is strictly developmentally regulated, both spatially (Fig. 1) and temporally (Fig. 2). The same data however, revealed that both genes, though closely related functionally, are regulated in distinctively different manners. Little is known about mechanisms of tissue specificity (for review see Harshman and James, 1998). In G. mellonella expression of the genes encoding both storage proteins is strictly limited to fat body tissue, although in other lepidopteran species, e.g. Hyphantria cunea, transcripts of storage proteins were found in Malpighian tubules and gonads in addition to fat body (Cheon et al., 2002). Stage specificity, as well as differential patterns of expression for the duration of one stage, indicates that certain developmental triggers that start and inhibit
Fig. 4. Hybridization of cDNA probes to Northern blots. 2 µg of total RNA were hybridized with DIG-labeled probes. Equal loading of RNA was ensured by spectrophotometric analysis and ethidium bromide staining of the gel. NT—non-treated larvae, RS—larvae treated with Ringer solution (10% ethanol), 20HE—larvae treated with 0.5 µg of 20HE dissolved in Ringer solution (10% of ethanol); (A) larvae treated as day 1 of the seventh instar; (1,4,7) 24 h after treatment; (2,5,8) 48 h after treatment; (3,6,9) 72 h after treatment; (B) larvae treated as day 4 of the seventh instar; (10,13,16) 24 h after treatment; (11,14,17) 48 h after treatment; (12,15,18) 72 h after treatment; (C) larvae treated as day 7 of the seventh instar; (19,22,25) 24 h after treatment; (20,23,26) 48 h after treatment; (21,24,27) 72 h after treatment.
genes expression must be present. In insects, hormones are the most common and most versatile developmental cues. When the course of expression of the Lhp genes is compared to the endogenous levels of the two most
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Fig. 5. Autoradiogram of proteins synthesized by the fat body of larvae of the seventh instar (A) and detected in their hemolymph (B). Larvae were injected with 0.5 µg of 20HE dissolved in Ringer solution (10% ethanol) as day 5 and subsequently for 3 h with 5 µCi of 14Cleucine: (2,6) prior to treatment; (3,7) 24 h after treatment; (4,8) 48 h after treatment, (1,5) radioactive standard proteins in kDa. Equal amounts of radioactive material (2000 cpm) were loaded per lane.
Fig. 6. Western blot analysis of changes in abundance of LHP76 protein after injection of 0.5 µg of 20HE dissolved in Ringer solution (10% of ethanol), into the hemolymph of the seventh instar larvae. Larvae were injected as day 5. Each lane was loaded with hemolymph samples containing 20 µg of protein. Proteins were probed with antibody against α and β arylphorin of Manduca sexta. Molecular weight standards in kDa and LHP76 are indicated. Non-treated larvae: (1) day 6, (2) day 7; Ringer solution (10% ethanol) treated larvae: (3) 24 h after treatment, (4) 48 h after treatment; 20HE treated larvae: (5) 24 h after treatment, (6) 48 h after treatment.
important hormones in G. mellonella hemolymph, 20HE (Sehnal et al., 1981) and JH (Rembold and Sehnal, 1987), it is apparent that endogenous peaks of neither JH nor 20HE are likely to affect the level of Lhp76 mRNA. However, the level of Lhp82 mRNA was negatively correlated with the JH titer. During early larval instars (up to the sixth instar), JH levels remain high and thus most likely suppress Lhp82 gene expression. Approximately 24 h after the beginning of the seventh instar, JH levels decline to undetectable amounts, and expression of Lhp82 is initiated immediately thereafter. In the course of the seventh instar Lhp82 is silenced after day 8, a time that coincides with the small, commitment peak of 20HE and a minute peak of JH. Many researchers confirmed the latter, by different means of JH detection such as RIA (Plantevin et al., 1984), chromatography (Rembold and Sehnal, 1987) and bio–assays (S´ mietanko et al., 1989).
Therefore, Lhp82 mRNA seems to be negatively regulated by JH level. To confirm this, we investigated the effect of JH on the expression of Lhp genes. In those experiments we used fenoxycarb, a compound that exerts JH–like effects on numerous physiological processes in invertebrates (Davey and Gordon, 1996; Dean and Meola, 1997; Dhadialla et al., 1998; Nates and McKenney, 2000). Application of fenoxycarb on Lhp82– expressing individuals led to a rapid shut–down of its mRNA. Fenoxycarb did not affect Lhp76 expression in any way, a finding which confirms that lepidopteran arylphorins are not sensitive to juvenilizing treatments nor to JH of endogenous origin (Jones et al., 1993; Zheng et al., 2000). Since JH is among the most pleiotropic hormones ever known, the mode of its action is exceptionally complex. It has been shown that JH can regulate gene expression by multiple mechanisms including induction of transcription factors (Zhang et al., 1996) and stimulation of second messengers system (Yamamoto et al., 1988; Sevala and Davey, 1993); for reviews see Harshman and James (1998) and Davey (2000). Studies on the effects exerted by JH on JH–suppressible storage protein genes in Trichoplusia ni (Lepidoptera) concluded that, besides suppression of transcription itself, stability of the corresponding mRNAs is greatly reduced as well (Jones et al., 1993). This may be similar to the case in G. mellonella. A comparison of Lhp mRNAs abundance with 20HE level does not suggest any noteworthy impact of that hormone on the expression of Lhp genes. Sequence analysis of upstream non-coding DNA, associated with both Lhp genes within a distance of approximately 1.2– 1.3 kb, did not reveal presence of ecdysone response elements (EcREs), neither consensus sequence derived from published EcREs nor storage protein-associated EcRE from D. melanogaster (data not shown). In D. melanogaster, EcRE of storage protein gene Lsp-2 is situated at a –75 upstream position relative to the transcription initiation site (Antoniewski et al., 1995). Thus, most probably 20HE has no effect on Lhp gene expression at the transcriptional level. To clarify this we performed a series of experiments using animals of different ages. This may be a crucial criterion since it has been shown that the level of 20HE, which varies greatly during the course of aging, is a key factor stimulating expression of its own receptor, EcR, in both Diptera (Karim and Thummel, 1992; Imhof et al., 1993) and Lepidoptera (Jindra et al., 1996; Hiruma et al., 1997) species. It has been also shown that in the absence of hormone, dimerization of EcR with its partner USP, which is necessary for appropriate function, is weak (Spindler et al., 2001). In these experiments, we used day 1, day 4 and day 7 larvae of the last instar. The level of 20HE is very low in day 1 and day 4 larvae, however day 1 larvae of the seventh instar show quite high concentrations of the hormone several hours before, during
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day 3 larvae of the sixth instar (Sehnal et al., 1981). On the other hand, day 7 individuals have been already exposed to a small, EcR synthesis-inducing peak of ecdysteroids. Larvae injected with 0.5 µg 20HE did not alter their expression of the Lhp76 gene, regardless of the age of the treatment. Injection of a higher dose 1.5 µg 20HE likewise had no effect (not shown). Similar results, with one significant exception, have been obtained in the case of Lhp82 (Fig. 4). The exception is the absence of Lhp82 mRNAs in day 1, 20HE treated individuals, until at least 48 h after treatment. What might cause such a delay in one-day old larvae, while older ones were 20HE-insensitive? Possibly the inhibition of JH–suppressible Lhp82 gene expression can be triggered by a juvenilizing effect of the ecdysteriod treatment. It has been shown that high levels of ecdysteroids mimic the action of JH, possibly by activating the insects’ own corpora allata, or by acting synergistically with a low level of endogenous JH (Willis, 1974). Also in larvae of G. mellonella treated with 20HE, a dose–dependent increase of allatotropic activity and subsequent rise of JH titer has been observed (Muszyn´ ska-Pytel et al., 1992). Why then was the juvenilizing effect of the ecdysteroid treatment observed only in one-day old larvae? The course of action of JH esterase during the seventh instar may provide an answer. During the first three days of the instar JH esterase activity remains at a basal level of less than 10 nmol/min/ml, while starting from day four it elevates rapidly reaching 80 nmol/min/ml (Hwang-Hsu et al., 1979; Szołajska, 1991). In previous studies we showed that synthesis of the LHP group is silenced after the feeding period of the seventh instar (Kłudkiewicz et al., 1996; Godlewski et al., 2001). However mRNAs of the Lhp genes (at least Lhp76) were present until the very end of the instar or even longer (Fig. 2), hence the presence of 20HE, both endogenous and exogenous in origin did not inhibit transcription of the Lhp genes. To understand the nature of the LHPs disappearance in that period of development we performed two experiments assessing the influence of 20HE on synthesis of LHPs in the fat body and on their appearance in the hemolymph. Injection of 20HE into LHP-synthesizing, feeding larvae caused a temporary decline of their synthesis rate in the fat body, lasting approximately 24 h. At the same time, however, secretion of LHP protein into the hemolymph was blocked entirely for more than two days (Fig. 5). The latter result has been confirmed by screening of hemolymph proteins using LHP76-recognizing antibody (Fig. 6), which revealed that the amount of LHP76 declined rapidly between 24 and 48 h after ecdysteroid treatment. It has been shown that ecdysteroids promote synthesis of storage protein-specific receptor and thus support the process of their being taken into the fat body (Burmester and Scheller, 1995; Burmester et al., 1999), therefore
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blocking of the secretion of LHP by high concentrations of 20HE is credible. The disappearance of the LHP76 that already exists in the hemolymph is most probably due to re-adsorption by the fat body, however rapid turnover of the protein in the hemolymph cannot be excluded. Our observation that 20HE operates on the translational rather than the transcriptional level to inhibit synthesis of LHP is supported by research on dipteran species, in which high levels of in vitro translatable LSP2 mRNA remain detectable after pupariation in D. melanogaster (Powell et al., 1984; Lepesant et al., 1986). Also, application of 20HE in vivo specifically inhibits translation of storage proteins without altering their mRNA levels in Calliphora vicina (Burmester et al., 1995). Previous reports of regulation of LHP expression by hormones (Ray et al., 1987) claimed that 20HE inhibits transcription of Lhp genes. Our results do not support that claim. The difference may be caused by the significantly higher dose of hormone used by Ray and coworkers (5 µg of hormone instead of 0.5 µg used in this study). We believe that such a high dose is physiologically irrelevant. Methods employed by Ray and co-workers were also less sensitive (dot-blot) and RNA was obtained from very few time-points of development. Our analysis is supported by investigation of proteins and sequence analysis, which revealed that no EcREs were found in upstream non-coding regions of either Lhp gene that was investigated.
Acknowledgements The authors are grateful to Dr Elisabeth Willott for the anti–arylphorin antibody of Manduca sexta and to Jonathan N.E. Day, for critical reading of the manuscript. The State Committee for Scientific Research (KBN, Poland) supported this work by grant no.3P04C00622.
References Antoniewski, C., O’Grady, M.S., Edmondson, R.G., Lassieur, S.M., Benesˇ, H., 1995. Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene. Molecular Genetics and Genomics 249, 545–556. Auffray, C., Rougeon, T., 1980. Purification of mouse immunoglobulin heavy chain messenger RNAs from total myeloma tumor RNA. European Journal of Biochemistry 107, 303–314. Bean, D.W., Silhacek, D.L., 1989. Changes in the titer of the female predominant storage protein (81K) during larval and pupal development of the Galleria mellonella. Archives of Insect Biochemistry and Physiology 10, 333–348. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
558
J. Godlewski et al. / Journal of Insect Physiology 49 (2003) 551–559
Burmester, T., 1999. Evolution and function of the insect hexamerins. European Journal of Entomology 96, 213–235. Burmester, T., Scheller, K., 1995. Ecdysteroid-mediated uptake of arylphorin by larval fat bodies of Calliphora vicina: involvement and developmental regulation of arylphorin binding proteins. Insect Biochemistry and Molecular Biology 25, 700–706. Burmester, T., Matzner, U., Scheller, K., 1995. Effect of 20HE on synthesis and uptake of arylphorin by the larval fat body of Calliphora vicina. European Journal of Entomology 92, 217–227. Burmester, T., Antoniewski, C., Lepesant, J.-A., 1999. Ecdysone— regulation of synthesis and processing of fat body protein 1, the larval serum protein receptor of Drosophila melanogaster. European Journal of Biochemistry 262, 49–55. Caglayan, S.H., Gilbert, L.I., 1987. In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control. Comparative Biochemistry and Physiology B 87, 989–997. Cheon, H.-M., Hwang, S.-J., Kim, H.-J., Jin, B.R., Chae, K.-S., Yun, C.-Y., Seo, S.-J., 2002. Two juvenile hormone suppressible storage proteins may play different roles in Hyphantria cunea Drury. Archives of Insect Biochemistry and Physiology 50, 157–172. Chomczyn´ ski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156–159. Davey, K.G., 2000. The modes of action of juvenile hormones: some question we ought to ask. Insect Biochemistry and Molecular Biology 30, 663–669. Davey, K.G., Gordon, D.R., 1996. Fenoxycarb and thyroid hormones have JH-like effects on the follicle cells of Locusta migratoria in vitro. Archives of Insect Biochemistry and Physiology 32, 613– 622. Dean, S.R., Meola, R.W., 1997. Effect of juvenile hormone and juvenile hormone mimics on sperm transfer from the testes of the male cat flea (Siphonaptera: Pulicidae). Journal of Medical Entomology 34, 485–488. Dhadialla, T.S., Carlson, G.R., Le, D.P., 1998. New insecticides with ecdysteroidal and juvenile hormone activity. Annual Review of Entomology 43, 545–569. Godlewski, J., Kłudkiewicz, B., Grzelak, K., Cymborowski, B., 2001. Expression of larval hemolymph proteins (Lhp) genes and protein synthesis in the fat body of greater wax moth (Galleria mellonella) larvae during diapause. Journal of Insect Physiology 47, 759–766. Harshman, L.G., James, A.A., 1998. Differential gene expression in insects: transcriptional control. Annual Review of Entomology 43, 671–700. Haunerland, N.H., 1996. Insect storage proteins: gene families and receptors. Insect Biochemistry and Molecular Biology 26, 755–765. Hiruma, K., Bocking, D., Lafont, R., Riddiford, L.M., 1997. Action of different ecdysteroids on the regulation of mRNAs for the ecdysone receptor, MHR3, dopa decarboxylase, and a larval cuticle protein in the larval epidermis of the tobacco hornworm, Manduca sexta. General and Comparative Endocrinology 107, 84–97. Hwang-Hsu, K., Reddy, G., Krishna Kumaran, A., 1979. Correlations between juvenile hormone esterase activity, ecdysone titre and cellular reprogramming in Galleria mellonella. Journal of Insect Physiology 25, 105–111. Imhof, M.O., Rusconi, S., Lezzi, M., 1993. Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homologous to the Drosophila melanegaster ecdysteroid receptor (dEcR). Insect Biochemistry and Molecular Biology 23, 115–124. Jindra, M., Malone, F., Hiruma, K., Riddiford, L.M., 1996. Developmental profiles and ecdysteroid regulation of the mRNAs for two ecdysone receptor isoforms in the epidermis and wing of the tobacco hornworm, Manduca sexta. Developmental Biology 180, 258–272. Jones, G., Venkatarman, V., Manczak, M., Schelling, D., 1993. Juven-
ile hormone action to suppress gene transcription and influence message stability. Developmental Genetics 14, 323–332. Karim, F.D., Thummel, C.S., 1992. Temporal coordination of regulatory gene expression by the steroid hormone ecdysone. EMBO Journal 11, 4083–4093. Kłudkiewicz, B., Godlewski, J., Grzelak, K., Cymborowski, B., Lassota, Z., 1996. Influence of low temperature on the synthesis of some Galleria mellonella proteins. Acta Biochimica Polonica 43, 639–644. Lepesant, J.-A., Maschat, F., Kejzlarova-Lepesant, J., Benesˇ, H., Yanicostas, C., 1986. Developmental and ecdysteroid regulation of gene expression in the larval fat body of Drosophila melanogaster. Archives of Insect Biochemistry and Physiology 3, 133–141. Levenbook, L., 1985. Insect storage proteins. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 10. Pergamon Press, Oxford, pp. 307–346. Memmel, N.A., Trewitt, P., Silhacek, D., Krishna Kumaran, A., 1992. Nucleotide sequence and structure of the arylphorin gene from Galleria mellonella. Insect Biochemistry and Molecular Biology 22, 333–342. Memmel, N.A., Trewitt, P.M., Grzelak, K., Rajaratnam, V.S., Kumaran, A.K., 1994. Nucleotide sequence, structure and developmental regulation of Lhp82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella. Insect Biochemistry and Molecular Biology 24, 133–144. Miller, S.G., Silhacek, D.L., 1982. Identification and purification of storage proteins in tissues of the greater wax moth Galleria mellonella. Insect Biochemistry 12, 277–292. Minakuchi, C., Nakagawa, Y., Kiuchi, M., Tomita, S., Kamimura, M., 2002. Molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis. Insect Biochemistry and Molecular Biology 32, 999–1008. Muszyn´ ska-Pytel, M., Mikołajczyk, P., Pszczo´ łkowski, M.A., Cymborowski, B., 1992. Juvenilizing effect of ecdysone mimic RH 5849 in Galleria mellonella larvae. Experientia 48, 1013–1017. Nates, S.F., McKenney, C.L. Jr., 2000. Growth, lipid class and fatty acid composition in juvenile mud crabs (Rhithropanopeus harrisii) following larval exposure to fenoxycarb, insect juvenile hormone analog. Comparative Biochemistry and Physiology C 127, 317– 325. Plantevin, G., De Reggi, M., Nardon, C., 1984. Changes in ecdysteroid and JH titres in the hemolymph of Galleria mellonella larvae and pupae. General and Comparative Endocrinology 56, 218–230. Powell, D., Sato, J.D., Brock, H.W., Roberts, D.B., 1984. Regulation of synthesis in the larval serum proteins of Drosophila melanogaster. Developmental Biology 102, 206–215. Ray, A., Memmel, N.A., Krishna Kumaran, A., 1987. Developmental regulation of the larval hemolymph protein genes in Galleria mellonella. Roux’s Archives of Developmental Biology 196, 414–420. Rembold, H., Sehnal, F., 1987. Juvenile hormones and their titer regulation in Galleria mellonella. Insect Biochemistry 17, 997–1001. Riddiford, L.M., 1995. Hormonal regulation of gene expression during lepidopteran development. In: Goldsmith, M.R., Wilkins, A.S. (Eds.), Molecular Model Systems in the Lepidoptera. Cambridge University Press, Cambridge, pp. 293–322. Sakurai, H., Fujii, T., Izumi, S., Tomino, S., 1988. Complete nucleotide sequence of gene for sex-specific storage protein of Bombyx mori. Nucleic Acids Research 16, 7717–7718. Sehnal, F., 1966. Kritisches Studium der Bionomie und Biometric der in Verschiedenen Lebensbedingungen Gezuchteten Waschmotte Galleria mellonella (Lepidoptera). Zeitschrift fu¨ r wissenschaftliche Zoologie 174, 53–82. Sehnal, F., Maroy, P., Mala, J., 1981. Regulation and significance of ecdysteriod titre fluctuations in lepidopterous larvae and pupae. Journal of Insect Physiology 27, 535–544. Sevala, V.L., Davey, K.G., 1993. Juvenile hormone dependent phos-
J. Godlewski et al. / Journal of Insect Physiology 49 (2003) 551–559
phorylation of a 100kDa polypeptide is mediated by protein kinase C in follicle cells of Rhodnius prolixus. Invertebrate Reproduction and Development 22, 189–193. S´ mietanko, A., Wis´niewski, J., Cymborowski, B., 1989. Effect of low rearing temperature on development of Galleria mellonella larvae and their sensitivity to juvenilizing treatment. Comparative Biochemistry and Physiology A 92, 163–169. Spindler, K.-D., Przibilla, S., Spindler-Barth, M., 2001. Moulting hormones of arthropods: molecular mechanisms. Zoology 103, 189– 201. Szołajska, E., 1991. Hydrolytic degradation of juvenile hormones in hemolymph and corpora allata of Galleria mellonella. Acta Biochimica Polonica 38, 321–333. Telfer, W., Kunkel, J., 1991. The function and evolution of insects’ storage hexamers. Annual Review of Entomology 36, 205–228. Towbin, H., Staehelin, T., Gordon, A., 1979. Electrophoretic transfer
559
of proteins from poliacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences USA 76, 4350–4354. Willis, J.H., 1974. Morphogenetic action of insect hormones. Annual Reviews of Entomology 19, 97–115. Yamamoto, K., Chadarevian, A., Pelligrini, A., 1988. Juvenile hormone action mediated in male accessory glands of Drosophila by calcium and kinase. Science 239, 916–919. Zhang, J., Saleh, D.S., Wyatt, G.R., 1996. Juvenile hormone regulation of an insect gene: a specific transcription factor and a DNA response element. Molecular and Cellular Endocrinology 122, 15–20. Zheng, Y., Sugie, H., Tojo, S., 2000. Juvenile hormone titre and hormonal regulation of storage protein synthesis in the common cutworm, Spodoptera litura. Entomological Science 3, 9–18.