Animals

Cold-treated eggs of the silk moth Bombyx mori (Lepidoptera) were supplied from the National Institute of Agricultural, Science and Technology (Suwon, Korea) and hatched to the first instar larvae about 10 days after the onset of incubation at the temperature of 27–28°C and relative humidity of 60–70%. Larvae were reared on an artificial diet under a long-day photoperiod regimen (17-hr light and 7-hr dark). They were normally metamorphosed to pupae and then to adults.

RNA isolation and cDNA synthesis

Total RNA was isolated from the 3rd instar larval midguts using the TRI reagent-RNA isolation reagent (Molecular Research Center, INC) (Sambrook and Russell, 2001). About 20 midguts of the 3rd instar larvae were homogenized in the TRI reagent with several strong strokes and then placed it at room temperature for 5 min. For the RNA elution 0.2% chloroform was added. The mixture of homogenized midguts was again placed at room temperature for 5 min and subsequently centrifuged with 12,000xg at 4°C for 15 min. The supernatant was transferred into a new 1.5ml-eppendorf tube and then the isopropanol was added. Centrifugation was again performed with 12,000xg at 4°C for 5 min and the supernatant was discarded. The pellet was washed in 75% ethanol while gently flicking it and then dissolved in the TE buffer containing 10mM Tris-Cl, pH 8.0 and 1mM EDTA.

The cDNA for the templates of the reverse transcriptase-PCR (RT-PCR) was synthesized using a SMART cDNA synthesis kit (Clontech). Degenerate primers were designed, as described in Taylor et al. (1996). Four primers for RT-PCR (AT1:5′-CTTCAAGAACGTCGAAATGATG-3′; AT2:5′-AATGATGACYGCNAGAGGCTTC-3′; AT 3:5′-GCAACAGCGACCCACGCG-3′;AT4:5′-CTTTCAGATTTAAACCACG AC-3′; Y=A,T,G,C; N=C,G) and two primers for 5′-RACE PCR (primer A:5′-CCACG AACCTGGCGAAGTC-3′; primer B:5′-CGAATGCACCGGACGTCTTG-3′) were used.

PCR and cDNA cloning

The RT-PCR reactions were performed with the murine leukemia virus (MLV)-reverse transcriptase (BRL) in Taq polymerase buffer (Takara) supplemented to a final concentration of 3.5mM MgCl₂, 2.5mM dNTPs and 0.2M of each primer in 50 μl. The mixtures were overlaid with mineral oil and then amplified in a thermal cycler (Perkin Elmer) for 30 cycles (95°C for 1 min; 57°C for 1 min; 72°C for 90 s), preceded by a 4-min denaturation step at 94°C and followed by a 5-min final extension at 72°C. The electrophoresis was performed on the Mupid-21 Kit (50V). The DNA was eluted from the sliced band with a DNA Purification Kit (UltraCleanTM 15, MO BIO). After determination of the concentration of the PCR product, ligation and transformation reactions were performed with a pGEM-T Easy Vector (Promega) System. The Miniprep kit (Wizard, Promega) was used only for sequencing.

Autosequencing for cDNA and searching for the similarity

PCR was performed in the following mixtures; Big Dye, Primer T7, SP6 1 pmol each, plasmid DNA, bringing to total 10 μl on the GeneAmp PCR system 9700 (PE Applied Biosystem). A 1.1kb cDNA clone encoding the Mas-AT was analyzed with a 310 Genetic Analyzer (ABI Prism, Perkin-Elmer). The cDNA sequence similarity searching was performed using the BLAST from the NCBI data base through internet access.

PCR for 5′-RACE

As a full length of Bommo-AT cDNA was not detected in the RT-PCR, two new oligonucleotides (antisense primer A: 5′-CCACGAACCTGGCGAAGTC-3′; antisense primer B: 5′-CGAATGCACCGGACGTCTTG-3′) were designed based on the B. mori cDNA sequence to find the 5′ end of Bommo-AT cDNA by 5′-RACE. The 5′-RACE procedure was performed using a 5′-RACE system for the RACE kit (SMART, Clontech). PCR was conducted under the conditions of a cycle of 94°C for 2 min, 30 cycles of 94°C for 1 min, 59°C for 2 min, and 72°C for 2 min.

Northern blotting

Sense and antisense allatotropin RNA probes were synthesized from the cDNA clone. The probes were transcribed with a digoxigenin-11-UTP RNA labeling mix (Boehringer-Mannheim) using T7 RNA polymerase (Promega). Total RNAs were isolated from the midgut, head, integument, fat body and silk gland of the 3rd instar larva and electrophoresed with 5μg/ml total RNAs per lane. After electrophoresis, the gel was equilibrated in 20XSSC for 2X15 min to remove the formaldehye. The RNA was blotted in the gel by capillary transfer overnight at room temperature with 20XSSC. The probe was prehybridized in a solution at 68°C for 1.5 hr, denatured in boiling water for 10 min, and then immediately placed on the ice. After the discard of the prehybridization solution and then the addition of the hybridization solution containing the DIG-labeled probe, the membrane was washed twice in 2XSSC containing 0.1% SDS at room temperature (15 minX2). Another wash of the membrane continued in 0.5XSSC containing 0.1% SDS at room temperature (15 minX2). The membrane was equilibrated in washing buffer and blocked by gently agitating it in the blocking solution for 30-60 min. The blocking solution was removed and the membrane was incubated in the antibody solution for 30 min. The anti-DIG-POD solution was added to the blocking solution and then mixed gently. The membrane was washed to remove the unbound antibody, followed by equilibration in a detection buffer for 2 min. After the pour-off of the detection buffer and the addition of the color substrate solution to the membrane, the membrane was washed in distilled water and then a photo of the desired spots or bands was taken with a polaroid camera.

Wholemount immunocytochemistry

Tissue preparation and wholemount immunocytochemistry were performed according to Lee et al. (1998b) and Kim et al. (1998). Following anaesthetic treatment of the third instar larvae at 4°C for 1 hr, foreguts, midguts and hindguts were isolated in 0.1M sodium phosphate buffer (pH 7.4) (PB) and then fixed in 4% paraformaldehyde (PFA) in 0.1M PB for 5 hr at 4°C. The fixed tissues were immersed in 0.01M phosphate-buffered saline (PBS) with 1% Triton X-100 at 4°C overnight. Blockage of peroxidase activity was performed in 10% methanol in 3% H₂O₂ for 25 min. Washes in 0.1M Tris-HCl buffer (pH 7.6–8.6) containing 1% Triton X-100 and 4% NaCl were followed by incubation with a primary antiserum (anti-Mas-AT, Veenstra et al., 1994), diluted to 1: 1,500 in dilution buffer (0.01M PBS with 1% Triton X-100 and 10% normal goat serum), for 4-5 d while shaking gently. After washes in 0.01M PBS with 1% Triton X-100, tissues were incubated in peroxidaseconjugated swine anti-rabbit IgG (DAKO), diluted to 1:200 for 2 d at 4°C. Following preincubation in 0.03% diaminobenzidine (DAB, Sigma) in 0.05M Tris-HCl buffer for 1 hr at 4°C, tissues were treated with 0.03% DAB in 0.05M Tris-HCl buffer containing 0.01% H₂O₂ for 5-10 min. After being rinsed in a 0.05M Tris-HCl buffer, tissues were mounted in glycerin, examined and photographed with a Zeiss interference microscope. As a specificity control, immunocytochemistry was performed on whole foreguts, midguts and hindguts of larvae with anti-Mas-AT preincubated with 50 nmol synthetic Mas-AT per ml diluted antiserum (diluted to 1:1,500) for 24 hr.

Characterization of Bommo-AT cDNA

Bommo-AT cDNA was synthesized and two PCR products were made. Using a vector system, recombinant DNA was produced and the desirable trasnformation was properly carried out. The complete Bommo-AT cDNA sequence consists of the 1196 nucleotides with 390 coding sequences (Fig. 1). It contains 57 nucleotides of 5′untranslated region upstream of a single open reading frame beginning at position 58 and ending at position 448 with the translation stop codon TAA. The 3′untranslated region that follows is A/T-rich (60%) and extends for 749 nucleotides. The 3′untranslated region contains a 20-nucleotide poly-A tail and a consensus sequence directing polyadenylation (AATAAA) beginning at position 1156 and 15 bases upstream. The encoded Bommo-AT peptide located between Arg³⁶ and Gly⁵⁰ is identical to the sequence of Mas-AT (Kataoka et al., 1989) and to that predicted from Mas-AT cDNA (Taylor et al., 1996). The Bommo-AT peptide is flanked by two potential endoproteolytic cleavage sites of Arg³⁶ and Lys⁵¹-Arg⁵²), and ends with a glycine residue, the signal for carboxy-terminal amidation by peptidyl-glycine-α-amidating monooxygenase (Eipper et al., 1992). Endoproteolytic cleavage of these sites would result in the production of two additional peptides of 15 and 78 amino acids. This suggests that the Bommo-AT is a secreted peptide after processing from a large precursor peptide. The basic organization of the Bommo-AT peptide precursor, which consists of 130 amino acid sequence, is similar to that of two moths, M. sexta with 84% amino acid identity (Taylor et al., 1996) and P. unipunctata with 81% amino acid identity (Truesdell et al., 2000) (Fig. 2). However, the amino acid identity of the AT peptide precursor between B. mori and Aedes aegypti decreases to less than 70%.

Fig. 1

Nucleotide sequence of the Bommo-AT cDNA and deduced amino acid sequence of the AT peptide precursor. The sequences are numbered at the right. The amino acid sequence for the Mas-AT peptide is shown in bold type. Two proteolytic cleavage sites are surrounded by thick rectangles. Glycine residue required for amidation is single underlined. Potential polyadenylation signal (AATAAA, Whale and Keller, 1992) is shown bold underlined. RT-PCR oligonucleotide primer sequences are underlined with arrows and 5′-RACE primer sequences are surrounded by rectangles.

Fig. 2

Comparison of AT precursor peptides from B. mori, M. sexta (Taylor et al., 1996) and P. unipuncta (Truesdell et al., 2000). The common homology from three moths is shown in shaded bold-type letters. Translated portions are conserved with high homology.

Detection of AT gene expression by northern blotting

For investigation of the expression of the Bommo-AT gene in the tissues, northern blotting was performed using an RNA probe. The total RNAs isolated from the midgut, head, integument, fat body and silk gland of the third instar larva were investigated. The midgut showed the strongest expression of the AT gene, while there was no detection in the fat body and silk gland (Fig. 3). The Bommo-AT mRNA was also included in both head and integument, with its lowest level in the integument and a mild level in the head.

Fig. 3

Northern analysis for expression of Bommo-AT gene in the tissues. The amount of total RNA is 5μg/lane. The total RNAs in lanes 1-5 are from integument (1), head (2), midgut (3), fat body (4), and silk gland (5), respectively. The Bommo-AT gene is strongly expressed in the midgut with mild expression in the head and lowest expression in the integument, but the gene is not expressed in the fat body and silk gland.

Mas-AT-immunoreactivity in the midgut

There were Mas-AT-immunoreactive cells (Fig. 4A, B, arrowheads) of the endocrine type in the midguts of the third instar larvae, but not in the foreguts or hindguts. Labeled axons could also be detected in the wall of the midguts (Fig. 4A, arrows).

Fig. 4

Mas-AT-immunoreactivity in the larval midgut (whole-mount preparations). A. Midgut of third instar larva showing Mas-AT-immunore-active cells (arrowheads) and nerve fibers (arrows). B. Another midgut of third instar larva showing two intensively-stained cells.

Structure of Bommo-AT cDNA

It has been demonstrated that in B. mori the Mas-AT neuropeptide is produced in the brain and ventral ganglia (suboesophageal, thoracic and abdominal ganglia)(Park et al., 2001). The B. mori brains contain this neuropeptide in the larval stages with no Mas-AT neuropeptide during the pupal and adult stages. In the brain of early larval life, very limited number of cells (3 pairs) produce the Mas-AT. However, a larger number of Mas-AT-immunoreactive cells (10 pairs) are located in the brain of late larval stages. The larval midgut, which has been used for molecular characterization of Bommo-AT cDNA, contains a much larger number of the AT-immunoreactive cells than the larval brain, although expression patterns of Mas-AT mRNA in the midguts at various developmental stages remains to be investigated.

The Bommo-AT is identical to the AT identified in M. sexta (Talyor et al., 1996) and P. unipuncta (Truesdell, et al., 2000), although the pattern of the AT mRNA transcription or translation appears to vary greatly between B. mori and other moths. The Bommo-AT cDNA has very conserved sequences to that of M. sexta (Taylor et al., 1996) and P. unipuncta (Truesdell, et al., 2000) with 84% and 81% amino acid identity, respectively. However, it has smaller conserved sequences to the mosquito A. aegypti (Veenstra and Costes, 1999) with less than 70% amino acid identity.

The Mas-AT within the Bommo-AT precursor peptide is flanked by two potential endoproteolytic cleavage sites of Arg³⁶ and Lys⁵¹-Arg⁵², and it has also an end by a glycine residue, the signal for carboxy-terminal amidation by peptidyl-glycine-α-amidating monooxygenase (Eipper et al., 1992), as in M. sexta and P. unipuncta. Endoproteolytic cleavage of these sites would result in the production of two additional peptides of 15 and 78 amino acids. As with many other neuropeptides (Sossin et al., 1989), therefore, it appears that Mas-AT is translated from a transcript and derived from a polyprotein (Taylor et al., 1996).

Expression of Bommo-AT gene in the tissues

Northern blotting was performed to investigate the expression of the AT gene in the cells of midgut, head, integument, fat body and silk gland of third instar larva of B. mori, using a RNA probe which was synthesized by the RTPCR. The Bommo-AT gene was expressed in the midgut, head and integument, whereas there was no detection in the fat body and silk gland (Fig. 3). The midgut showed the strongest expression of the AT gene, with the lowest expression in the integument and mild expression in the head. The function of Bommo-AT in the midgut cells remains to be determined, but it could not be excluded that the AT might function as a myoregulatory peptide in the midgut. It is suggested that Bommo-AT mRNA expressed in the head is made for stimulation of JH biosynthesis by the CA. However, functional characteristics of the Bommo-AT made in the integument cells remains to be investigated.