The crustacean molt-inhibiting hormone (MIH) is released from the X-organ sinus gland complex and suppresses ecdysteroid synthesis by the Y-organ. In the present study, we have isolated a cDNA which encodes a MiH (Pej-SGP-IV) of the kuruma prawn Penaeus japonicus in order to study its expression and characterize the structure of its precursor. A cDNA fragment was isolated using RT-PCR with two degenerate oligonucleotide primers that were designed based on the peptide sequence of Pej-SGP-IV, and this fragment was used as a probe to screen an eyestalk cDNA library. In a positive cDNA clone (814 base pairs (bp)), an open reading frame of 315 bp was found; the conceptually translated protein consists of a putative signal peptide (28 residues) and Pej-SGP-IV (77 residues). In Northern blot analysis using a cDNA probe, specific hybridization to a transcript of 0.95 kb was seen in RNA extracted from the eyestalk but not from hepatopancreas, abdominal muscle, brain, thoracic ganglia or abdominal ganglia. The level of the Pej-SGP-IV mRNA in the eyestalk did not change significantly during the molt cycle.
In crustaceans, various neuropeptide hormones are released from the X-organ sinus gland complex which is located in the medulla terminalis of the eyestalk (reviewed by Keller, 1992). Several of these hormones, crustacean hyperglycemic hormone (CHH), gonad (vitellogenesis)-inhibiting hormone (GIH or VIH), molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH) are structurally related and thereby form a peptide family referred to as the CHH family (reviewed by Keller, 1992). The CHHs have been shown to regulate carbohydrate metabolism (Kegel et al., 1989; Yang et al., 1995, 1997). G!H inhibits the onset of vitellogenesis (Soyez et al., 1991) and MOIH inhibits methyl farnesoate (MF) synthesis by the mandibular organ (Wainwright et al., 1996).
MIHs have been isolated from the lobster Homarus americanus (Chang et al., 1990), the shore crab Carcinus maenas (Webster, 1991), the kuruma prawn P. japonicus (Yang et al., 1996) and the crayfish Procambarus clarkii (Nagasawa et al., 1996). MIHs are presumed to inhibit molting by suppressing secretion of the molting hormone ecdysteroids from the Y-organ except at the premolt stages when ecdysteroids are released to trigger molting (reviewed by Lachaise et al., 1993). Along these lines, the MIHs in C. maenas, P. japonicus and P. clarkii have been shown to suppress ecdysteroid secretion from the Y-organ in vitro (Webster and Keller, 1986; Terauchi et al., 1996; Yang et al., 1996). These observations have led to the postulate that the titer of the MIH in hemolymph decreases at the premolt stage. As an approach to address this postulate, we have isolated a cDNA encoding a precursor of the MIH (Pej-SGP-IV) from P. japonicus (Yang et al., 1996), and used this cDNA to study changes in levels of the Pej-SGP-IV mRNA in the eyestalk during the molt cycle. Levels of mRNA did not decrease significantly at the premolt stages, suggesting that the synthesis, modification and/or secretion of Pej-SGP-IV are regulated post-transcriptionally.
MATERIALS AND METHODS
Animals and molting stages
Live kuruma prawns P. japonicus caught in Hamanako Lake (Shizuoka, Japan) were purchased from a commercial source, and kept in 500 I tanks at the Fisheries Research Laboratory, Faculty of Agriculture, The University of Tokyo (Maisaka, Shizuoka, Japan). Adult prawns of both sexes were employed in the present study. Prawns were grouped the following five molting stages according to Drach and Tchernigovtzeff (1967) as modified by Hong (1977): early postmolt (stage A), late postmolt (B), intermolt (C), early premolt (D0-2) and late premolt (D3).
Isolation of RNA from the eyestalk, hepatopancreas, abdominal muscle, brain, thoracic ganglia, and abdominal ganglia
Total RNA was isolated from the eyestalk as described by Ohira et al. (1997). Preparation of total RNA from the hepatopancreas, abdominal muscle, brain, thoracic ganglia, and abdominal ganglia was performed as described for hepatopancreas total RNA according to Ohira et al. (1997). Poly(A)+ RNA was prepared from total RNA using Oligotex™-dT30 super (Roche Japan).
RT (reverse transcription)-PCR
Two degenerate oligonucleotide primers were designed based on the amino acid sequence of Pej-SGP-IV: primer 1 (the sense strand; 5′-GAYATHTAYAARAARGTNGT-3′) and primer 2 (the anti-sense strand; 5′-RAACCAYTCRTTRTARAARC-3′). These were derived from the sequences of amino acid positions 15-21 and 44-50, respectively (Fig. 1; Yang et al., 1996, 1997). cDNA was synthesized by annealing 1 pmol of primer 2 to 1 μg of the total RNA from the eyestalk and carrying out a RT reaction using Superscript™ reverse transcriptase (GIBCO BRL). The RT reaction product was mixed with 100 pmol of each of the two primers, and the following program was used for PCR amplification: 1 min (3 min only for the first cycle) at 94°C, 1 min at 50°C, 2 min at 65°C, for 40 cycles.
Screening of an eyestalk cDNA library
The product of the RT-PCR reaction was subcloned into the pCR™ll plasmid using TA Cloning® Kit (Invitrogen). The Eco Rl fragment (124 bp) of this plasmid was self-ligated using T4 DNA ligase to generate longer fragments, and labeled with [α-32P]dATP by using Multiprime™ DNA labeling systems (Amersham). Approximately 2.1 × 105 clones of an eyestalk cDNA library (Ohira et al., 1997) were screened with the labeled DNA. Hybridization was performed at 42°C in 50% formamide, 6 × SSPE (1.08 M NaCI, 60 mM NaH2P04, 6 mM EDTA, pH 7.4), 1% SDS, 10 μg/ml of denatured salmon sperm DNA. The radio-labeled cDNA probe was added to 5 × 105 cpm/ml. Following hybridization, filters were washed in 2 × SSPE and 0.1% SDS (2 × 15 min at room temperature), 1 × SSPE and 0.1% SDS (2x15 min at room temperature), 1 × SSPE and 0.1% SDS (15 min at42°C), and finally 0.5 × SSPE and 0.1% SDS (15 min at 42°C). Positive plaques were isolated in another round of screening, and the recombinant pBluescript SK-phagemids were rescued from the bacteriophage clones according to the instructions of the manufacturer (Stratagene). Dideoxy sequencing reactions were carried out using a Thermo Sequenase Cycle Sequencing Kit (Amersham), and electrophoresis and signal detection were done on an automatic sequencer SQ-3000/32 (HITACHI).
Northern blot analysis
RNA samples were quantitated by taking the OD260 of RNA solutions. Poly(A)+ RNAs (1 μig/lane) from the eyestalk (intermolt stage), hepatopancreas, abdominal muscle, brain, thoracic ganglia, and abdominal ganglia (indiscriminate molting stages) were run on 1.5% formaldehyde-agarose gels, transferred to a NYTRAN membrane (Schleicher & Schuell), and probed with a Pej-SGP-IV cDNA fragment (corresponding to nucleotides 1-643 in Fig. 1) which was radiolabeled with [α-32P]dATP. The activity of the probe in the hybridization buffer was 5 × 106 cpm/ml. The final wash of the membrane was done in 0.1 × SSPE and 0.1% SDS at 65°C. RNA ladders (0.16-1.77 and 0.24-9.4 kb purchased from GIBCO BRL) were used as molecular weight markers.
A Northern blot of poly(A)+ RNA of the eyestalk at the early postmolt (stage A), late postmolt (B), intermolt (C), early premolt (D0-2) and late premolt (D3) stages was probed, as described above, with the Pej-SGP-IVcDNA (corresponding to nucleotides 1-643 in Fig. 1), and subsequently with an RT-PCR product M108 (see below). Levels of hybridized transcripts were determined by using a Fujix BAS 1000 computerized densitometer scanner (Fuji Film).
M108 was generated by subjecting total eyestalk RNA to RT-PCR using two degenerate oligonucleotide primers which were derived from two conserved regions of the vertebrate and invertebrate actins (data not shown). An amplified sequence (396 bp) was conceptually translated into 132 amino acids, which exhibited 80-90% identity to actins in other arthropods (unpublished observation). The Eco Rl restriction sequence (GAATTC) was included in the two primers, and the Eco Rl fragment of a subcloned RT-PCR product was used as the probe for Northern hybridization.
RESULTS AND DISCUSSION
Total RNA isolated from the eyestalk was subjected to RT-PCR to obtain a cDNA fragment corresponding to amino acid position 15-50 of Pej-SGP-IV (Yang et al., 1996, 1997). The reaction products were subcloned and analyzed for nucleotide sequence. In one of the subclones, the conceptually translated sequence of the insert was identical to that of amino acid in positions 15-50 of Pej-SGP-IV (data not shown). This RT-PCR product that was considered likely to be part of a cDNA encoding Pej-SGP-IV, was therefore used as a probe to screen an eyestalk cDNA library. About 20 positive clones were identified, from which ten were randomly selected for further analysis.
Sequence analyses demonstrated that two of these clones encoded a Pej-SGP-IV precursor. We have also sequenced five of the remaining eight clones; these clones did not contain coding sequences for CHH family peptides (unpublished observation). The nucleotide sequence of one of the two which encoded a Pej-SGP-IV precursor is shown in Fig. 1. An open reading frame (ORF) of 315 bp was found, and this ORF was conceptually translated into a putative protein of 105 amino acid residues. The sequence of the 77 residues on the C-terminus side (Ser1-Gln77) was identical to that of Pej-SGP-IV. This segment was flanked N-terminally by Met−28-Ala−1 and followed by a stop codon. Segment Met-28-Ala-1 has a highly hydrophobic core (Trp−20-Var−12) and is therefore likely to constitute a signal peptide (Von Heijne, 1986). This cDNA also includes 5′ and 31 untranslated regions (51 and 437 bp, respectively). The 3′ region contains a consensus polyadenyla-tion signal (AATAAA) 10 bp upstream from the poly(A) tail (Fig. 1). The nucleotide sequence of the other positive clone was identical to the one shown in Fig. 1, except that it lacked the first 10 bp of the 5′ untranslated region.
In crustacean to date, cDNAs encoding the precursors of nine CHH family peptides have been cloned (Weidemann et al., 1989; Klein et al., 1993; De Kleijin et al., 1994a, b, 1995; Lee et al., 1995; Ohira et al., 1997): we aligned the amino acid sequence of the Pej-SGP-IV precursor against those of the nine precursors (Fig. 2A). In the Pej-SGP-IV precursor, no region was found which exhibited sequence similarity to CHH precursor-related peptides (CPRPs), which have been found in the precursors of type I CHH family peptides (see below). Thus, the result of the sequence analysis suggests that the Pej-SGP-IV precursor consists of only the signal peptide and mature hormone.
The CHH family peptides are grouped into two types (type I and II) based on two considerations: the extent of sequence similarity of the mature hormone and the presence/absence of a Gly residue at position 12 of the mature hormone (Yang et al., 1996). Results of the sequence analyses of the nine precursors have shown that the precursors of the two types have distinct organizations: the type I precursors consist of a signal sequence, CPRP and mature hormone, whereas the type II precursors lack a CPRP (Fig. 2B; De Kleijn and Van Herp, 1995). The result of the present study is consistent with the previous results, since CPRP was not found in the precursor of Pej-SGP-IV, a type II peptide (Fig. 2A).
In Northern blot analysis, a cDNA probe hybridized to a band in poly(A)+ RNA from the eyestalk, but not from hepato-pancreas, abdominal muscle, brain, thoracic ganglia or abdominal ganglia (Fig. 3). The autoradiogram was over-exposed to show that the Pej-SGP-IV mRNA was not detected in the latter five tissues. In a shorter exposure, hybridization was observed as a seemingly single band of approximately 0.95 kb.
We also investigated changes in levels of Pej-SGP-IV transcripts in the eyestalk during the molt cycle. The Pej-SGP-IV mRNA was detected on a Northern blot of poly(A)+ RNA from eyestalks at five different molting stages (Fig. 4A). The level of hybridization did not change significantly during the molt cycle, as the minimal level observed at the late postmoit stage was about half of the maximal level at the early postmoit and late premolt stages (Table 1). To ensure that the amounts of the loaded RNA samples were comparable, the filter was rehybridized with probe M108 which contained a partial coding sequence of an actin (or an actin homologue) in P. japonicus (see Materials and Methods). As shown in Fig. 4B, almost equal levels of hybridization was seen in all five samples.
Quantification of relative levels of the Pej-SGP-IV mRNA
Pej-SGP-IV has been shown to suppress secretion of ecdysteroids from the Y-organ in vitro (Terauchi et al., 1996). Pej-SGP-IV, therefore, is presumed to inhibit molting by suppressing secretion of ecdysteroids except for a premolt stage when ecdysteroids are released to trigger molting. In the present study, we did not observe any decrease in the level of Pej-SGP-IV transcripts at the early and late premolt stages. This observation suggests that the synthesis, modification and/or secretion of Pej-SGP-IV are regulated post-transcription-ally. However, the results of the present study do not exclude the possibility that transcription of the Pej-SGP-IV mRH A is repressed for a short duration prior to molting. The length of the duration of the premolt ecdysteroid surge has not been determined in P. japonicus. If the surge is brief, then levels of Pej-SGP-IV transcripts may decline for a short period, and individuals representing such a stage may not have been represented in the RNA samples (each of the five RNA samples are derived from 5-10 individuals).
It remains to be solved how synthesis, modification and/or secretion of Pej-SGP-IV are regulated, and how such regulation is related to secretion of ecdysteroids by the Y-organ in crustaceans.
The authors are grateful to Drs. M. Kono and K. Furukawa for use of the prawn rearing facilities. We also thank Dr. Marcy N. Wilder of Japan International Research Center for Agricultural Sciences for critical reading of this manuscript. This work was partly supported by Grants-in-Aid for Scientific Research (Nos. 07406009 and 08276204) from the Ministry of Education, Science, Sports and Culture of Japan.