Expression of a gene that encodes pheromone biosynthesis activating neuropeptide in the central nervous system of corn earworm, Helicoverpa zea

Insect Biochemistry and Molecular Biology 28 (1998) 373–385

Expression of a gene that encodes pheromone biosynthesis activating neuropeptide in the central nervous system of corn earworm, Helicoverpa zea Peter W.K. Ma, Douglas C. Knipple, Wendell L. Roelofs

*

Department of Entomology, NYS Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Received 15 October 1997; accepted 23 January 1998

Abstract Expression of the pheromone biosynthesis activating neuropeptide (Hez-PBAN) gene in the central nervous system of larval, pupal and adult Helicoverpa zea was studied using Northern hybridization analyses, reverse-transcriptase–polymerase chain reaction (RT-PCR), in situ hybridization histochemistry, and whole-mount immunocytochemistry. Northern hybridization experiments demonstrated the presence of a 0.8-Kb Hez-PBAN transcript in the subesophageal ganglion in adults, pupae and day 0–1 final instar larvae. In the subesophageal ganglion, Hez-PBAN mRNA was localized by in situ hybridization histochemistry to the mandibular, maxillary and labial cell clusters. Whole-mount immunocytochemical studies showed that these three cell clusters also possess PBAN-like immunoreactivity. Low levels of Hez-PBAN mRNA were revealed in other parts of the central nervous system, including the brain, thoracic ganglia and abdominal ganglia by RT-PCR. In day 0–1 final instar larvae, these low levels of Hez-PBAN mRNA were localized by in situ hybridization histochemistry to a pair of ventral midline neurons in each thoracic ganglion and some abdominal ganglia. PBAN-like immunoreactivity was also detected in these neurons. This study shows that in H. zea, the HezPBAN gene is expressed predominantly in the subesophageal ganglion, and at relatively low levels in other parts of the central nervous system.  1998 Elsevier Science Ltd. All rights reserved Keywords: PBAN; In situ hybridization histochemistry; Northern blotting; RT-PCR; Immunocytochemistry

1. Introduction Pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of pheromone biosynthesis in many lepidopterous insects (Raina, 1993). The PBAN in Helicoverpa zea (Hez-PBAN) is a 33-amino-acid peptide containing an amidated carboxyterminus (Raina et al., 1989). Cloning and sequencing of the Hez-PBAN cDNA revealed the preprohormone structure of the Hez-PBAN precursor protein (Ma et al., 1994). The 191-amino-acid prepro-Hez-PBAN contains several putative endoproteolytic processing sites that flank five structurally related peptide sequences, each having a similar C-terminal pentapeptide sequence FXP(K or R)Lamide (where X = G, S or T). The presence of these five peptides in the subesophageal ganglion (SEG) and the corpora cardiaca, a neurohemal organ,

* Corresponding author. 0965-1748/98/$19.00  1998 Elsevier Science Ltd. All rights reserved PII: S 0 9 6 5 - 1 7 4 8 ( 9 8 ) 0 0 0 0 9 - 5

was confirmed by a HPLC-ELISA profiling study on extracts of the brain-SEG complex of H. zea adult males and females (Ma et al., 1996). Besides regulation of pheromone biosynthesis, peptide sequences encoded in prepro-Hez-PBAN appear to serve multiple physiological functions. According to Matsumoto et al. (1990), cuticular melanization in lepidopterous insects appear to be regulated by PBAN or hormone(s) that belong to the PBAN peptide family. This conclusion was later confirmed in another insect, Pseudaletia separata, in which cuticular melanization is induced by an 18amino-acid neuropeptide termed Pss-pheromonotropin (later termed Pss-melanization and reddish coloration hormone or Pss-MRCH) (Matsumoto et al., 1992a). Furthermore, Pss-MRCH was shown to induce embryonic diapause in Bombyx mori (Matsumoto et al., 1992b). In B. mori, embryonic diapause is regulated by a 23-aminoacid neuropeptide Bom-diapause hormone (Bom-DH) isolated from the SEG (Imai et al., 1991). Both BomDH and Pss-MRCH contain the C-terminal FXPRLam-

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ide motif, and bear striking sequence similarity to PBAN-encoding gene neuropeptides (PGN)-23 (Asn24– Leu47) and PGN-18 (Ser106–Leu123) of prepro-HezPBAN, respectively (Ma et al., 1994). Based on evidence from Northern blot analyses, the Hez-PBAN gene is transcribed in the SEG of adult H. zea (Ma et al., 1994). This finding corroborates previous studies in H. zea and other lepidopteran species, which showed that the majority of the PBAN-like immunoreactivity was present in the brain–SEG complex (Kingan et al., 1990; Rafaeli et al., 1991; Blackburn et al., 1992; Kingan et al., 1992; Davis et al., 1993, 1996; Ichikawa et al., 1995, 1996; Golubeva et al., 1997). With the exception of B. mori, these studies also revealed the occurrence of PBAN-like immunoreactivity throughout the entire ventral nervous system. In adult H. zea, we found that an antiserum, which was raised against a truncated-Hez-PBAN sequence, localized PBAN-like immunoreactivity in the ventral nervous system to paired ventral midline neurons in the thoracic ganglia (TG) and abdominal ganglia (AG) (Ma et al., 1996). However, there is a possibility that PBAN-immunostained neurons in the TG and AG could result from cross-reactivity of the PBAN-antiserum with other unidentified myotropic peptides (Nachman and Holman, 1991; Schoofs et al., 1993). Further studies are necessary to correlate localization of Hez-PBAN mRNA with that of PBAN-like immunoreactivity in the central nervous system of H. zea. Understanding the expression of the Hez-PBAN gene at different developmental stages could provide further insights on the physiological roles of the peptides that are encoded in prepro-Hez-PBAN. In the present study, developmental expression of the Hez-PBAN gene was studied in H. zea by in situ hybridization histochemistry, Northern blot analyses, RT-PCR and immunocytochemistry. Our results show that the Hez-PBAN gene is expressed primarily in the mandibular, maxillary and labial cell clusters in the SEG of adults, pupae and day 0–1 final instar larvae. In addition, low level expression of the Hez-PBAN gene also occurs in other parts of the CNS.

2. Materials and methods 2.1. Insects Insects were reared according to Jurenka et al. (1991). Unless otherwise indicated, all experiments were performed on female insects. In most cases, insects were sexed soon after pupation and transferred to 30 × 30 × 30 cm plexiglas cages until adult eclosion. Adults were fed a solution of 5% sucrose. Larvae were sexed at their penultimate instar using the methods described by Lavenseau (1982) and kept individually in molded poly-

propylene cup trays. Only 5th instar larvae (12–24 h after the last larval ecdysis) that were actively feeding were used in the present study. Insects at this developmental stage are referred to as day 0–1 final instar larvae. Pupae used for most experiments were 6-day-old pupae, in which no signs of adult cuticle were evident. In some experiments, insects that were within 24 h after the formation of the pupal cuticle were used. Insects at this stage are referred to as 0–1-day-old pupae in this paper. All insects were reared at 25 ± 1°C under a photoperiod of 14:10 (light:dark). Under these conditions, the larval instars lasted approximately 16 days, and half of this time the larvae were in their last instar. Ten to 14 days were required for pupal ecdysis to adult eclosion. 2.2. RNA isolation Total RNA was prepared from various parts of H. zea CNS. The entire CNS was removed from the insect under a modified Weevers’ insect saline (Carrow et al., 1981) and subdivided into four morphologically distinct portions: (1) brain + retrocerebral complex (Br); (2) SEG; (3) TG, which includes the prothoracic, mesothoracic, and metathoracic ganglia; and (4) AG. As a result of the fusion of the mesothoracic, metathoracic, and the first two abdominal ganglia in pupal and adult stages, it was difficult, if not impossible, to separate these ganglia during dissection. Therefore, TG in the pupal and adult stages refers to the prothoracic ganglion and the fused mesothoracic/metathoracic–abdominal 1 + 2 ganglionic complex, whereas AG refers to the 3rd, 4th, 5th and the terminal abdominal ganglia (TAG). Dissected tissues were frozen on dry ice and stored at ⫺ 80°C until the time of RNA extraction. Total RNA was extracted from dissected tissues using a guanidinium thiocyanate solution according to Chomczynski and Sacchi (1987). RNA quantification was based on UV absorbance at A260. 2.3. Northern blot analyses Total RNA was run on 1.2% agarose gels containing 0.22 M formaldehyde, and transferred onto a positively charged nylon membrane (Boehringer Mannheim, IN) using 20X SSC (1X SSC = 0.15 M NaCl, 0.015 M Na3C6H5O7, pH 7.0). The membranes were probed with a segment of the Hez-PBAN cDNA (nt ⫺ 29 to 450) (Ma et al., 1994) labeled with digoxygenin by the random primer method using a commercial kit (Boehringer Mannheim, IN). Hybridization procedures described by Engler-Blum et al. (1993) were followed, and hybridization signals were visualized by chemiluminescence. 2.4. RT-PCR One microgram total RNA was incubated with an antisense oligonucleotide primer 3⬘NCAS (5⬘-

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ATCGCGTTTTGTTTGTACTCCTGAC-3⬘) in a 20-␮l reaction mixture prepared with a Promega AMV reverse transcription system kit (Promega, WI). At the end of the reverse transcription reaction, two units of RNase H were added to digest the RNA in the reaction mixture. One microliter of the reaction mixture was added to 50 ␮l of PCR reaction buffer consisting of 10 mM TrisHCl (pH 9.0), 0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl2, 200 ␮M dNTP, 0.25 ␮M of the upstream primer EX1 (5⬘-CTCGCTGTATTCACTACGAGCAGT-3⬘) and downstream antisense primer EXA5 (5⬘-GGGGAGAAGTACTTTGTCCTGCTGTC-3⬘), and 2.5 units of Taq polymerase (Promega, WI). The reaction mixture was denatured at 95°C for 10 min and cooled to 72°C for 2 min prior to the addition of the enzyme. The reaction mixture was subjected to 33 thermal cycles that consist of 95°C/40 s, 56°C/60 s and 72°C/90 s. Fifteen microliters of the final PCR reaction mixture was analyzed on a 3% GTG agarose gel and nucleic acids were stained with ethidium bromide.

tation in a hybridization buffer consisting of prehybridization buffer components plus 100 ng/ml random-primed digoxygenin-labeled DNA probe prepared as described in the previous section. Tissues were washed (15 min each) at 45°C in post-hybridization buffers (consisting of pre-hybridization buffer components minus denatured herring sperm and tRNA) followed by washings in 2:1, 1:1 and 1:2 posthybridization buffer: PBS-Tw, 5X SSC, 2X SSC and 0.2 X SSC. After an overnight incubation in 1:2000 anti-digoxygenin antiserum (Boehringer Mannheim, IN) in PBS-Tw, hybridizing signals in the tissues were visualized by incubating tissues for 10 min at room temperature in a substrate solution containing 0.3 mg/ml nitroblue tetrazolium salt and 0.2 mg/ml 5bromo-4-chloro-3 indolyl phosphate toluidinium salt in a detection buffer (100 mM NaCl, 50 mM MgCl2, 1 mM Levamisol, 0.1% Tween-20 in 100 mM Tris pH 9.5). Signal development was terminated by repeated washing in PBS-Tw. Tissues were dehydrated in a graded series of ethanol, cleared in xylenes and mounted in Euporal.

2.5. Southern blot analyses

2.7. Whole-mount immunocytochemistry

After electrophoresis, nucleic acids were denatured and transferred to positively charged nylon membrane (Boehringer Mannheim, IN) according to standard procedures (Sambrook et al., 1989). Hybridization with the digoxygenin-labeled Hez-PBAN cDNA probe and signal detection procedures were essentially similar to those described for the Northern blot analyses.

Distribution of PBAN-like immunoreactivity in the CNS of H. zea adults, pupae and day 0–1 final instar larvae was studied using whole-mount immunocytochemistry according to the procedures described in Ma and Roelofs (1995). The primary antiserum used in the present study, I-301, was a polyclonal antiserum raised against a truncated-PBAN (Ma and Roelofs, 1995). Specificity of antiserum I-301 was studied using a competitive ELISA (Ma et al., 1996) and an antiserumblocking experiment according to previously described procedures (Ma and Roelofs, 1995). In the present study, the exact antiserum-blocking experiment was performed on H. zea adult, pupal and larval nervous tissues using antiserum I-301 pre-incubated with Hez-PBAN.

2.6. In situ hybridization histochemistry The CNS was dissected and desheathed under a modified Weevers’ saline (Carrow et al., 1981). Tissues were fixed for 3 h at room temperature in a solution containing 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4). All subsequent incubations were performed at room temperature with gentle agitation on a home-made rotator unless otherwise indicated. After a few washes in PBSTw (phosphate buffered saline: 0.89% NaCl and 0.04% KCl in 0.01 M phosphate buffer, pH 7.2 containing 0.05% tween-20), tissues were dehydrated through a graded series of methanol and rehydrated to PBS-Tw. To allow for better probe penetration, tissues were treated with 10 ␮g/ml proteinase K (Amresco, OH) in PBS-Tw. Enzyme digestion was stopped by brief washing in PBS-Tw containing 2 mg/ml glycine. Tissues were then fixed in 4% formaldehyde in 0.1 M phosphate buffer for 20 min, washed in PBS-Tw and passed (10 min each) successively through 2:1, 1:1 and 1:2 PBS-Tw:prehybridization buffer (50% formamide; 5X SSC; 10 ␮g/ml denatured herring sperm DNA; 50 ␮g heparin; 100 ␮g/ml tRNA and 0.1% Tween-20). After incubation in pre-hybridization buffer at 45°C for 2 h, tissues were incubated overnight at 45°C without agi-

3. Results 3.1. Northern analyses Expression of the Hez-PBAN gene in H. zea CNS was studied by Northern hybridization. A hybridizing transcript of ca 0.8 kb was detected in SEG of 0–1- and 1– 2-day-old adult females, pupae and day 0–1 final instar larvae (Fig. 1(a, b)). A hybridizing transcript of similar size was also detected in the SEG of 1–3-day-old adult males (Fig. 1(b)). In contrast, hybridization signals were not detected in Br, TG and AG in all of the developmental stages analyzed (Fig. 1(a, b)). 3.2. RT-PCR Tissue distribution of Hez-PBAN mRNA in H. zea CNS was examined further using RT-PCR. Total RNA

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Fig. 1. Northern hybridization analyses of Hez-PBAN mRNA levels in the central nervous system of (a) 0–1-day-old adult females, pupae and day 0–1 final instar larvae, and (b) 1–2-day-old female and 1–3-day-old male adult H. zea. Upper panels show the results of the hybridization experiment, whereas the lower panels show the relative amount of RNA loading as revealed by methylene blue staining of the ribosomal bands on the blot before hybridization. The amount of total RNA loaded was 20 ␮g/lane, except for the subesophageal ganglion, which was loaded at 5 ␮g per lane. All lanes are labeled according to the sources of RNA. Br = brain + retrocerebral complex, SEG = subesophageal ganglion, TG = thoracic ganglia and AG = abdominal ganglia (see ‘Materials and methods’ for an explanation of the sources of nervous tissues used in the experiment).

isolated from various parts of the CNS was reverse-transcribed and amplified using EX1 and EXA5 oligonucleotides as primers. High levels of Hez-PBAN mRNA, as reflected by the robust ethidium bromide staining of the predicted 440 bp amplification product, were detected in SEG from day 0–1 final instar larvae, pupae, and adults (Fig. 2(a, b)). Similar levels of Hez-PBAN mRNA were detected in the SEG of 1–3-day-old adult males (Fig. 2b). Interestingly, moderate to low levels of Hez-PBAN mRNA were also detected in other parts of the CNS besides the SEG. Hez-

PBAN mRNA was detected in Br, TG of male and female adults, pupae and day 0–1 final instar larvae (Fig. 2(a, b)). Southern hybridization experiments showed that the 440 bp amplification product from various nervous tissues hybridizes to the probe whose sequence corresponds to nucleotide ⫺ 29 to 450 of the Hez-PBAN cDNA (Fig. 2(a, b), lower panels). In AG of adult and day 0–1 final instar larvae, the 440 bp amplification product was undetectable on ethidium bromide stained gels. However, weak but detectable hybridization signals of the 440 bp amplification can be seen on the Southern

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Fig. 2. RT-PCR analyses of Hez-PBAN mRNA levels in the central nervous system of (a) 0–1-day-old female adults, pupae and day 0–1 final instar larvae, and (b) 1–2-day-old female and 1–3-day-old male adults of H. zea. DNA fragments derived from Rsa I digested pEMBL 9 + plasmid were used as a size marker. The upper panels show ethidium bromide staining of the 440 bp amplification product. The lower panels show the results of Southern hybridization of the corresponding amplification product to a digoxygenin-labeled cDNA probe. All lanes are labeled according to the sources of RNA. Br = brain + retrocerebral complex, SEG = subesophageal ganglion, TG = thoracic ganglia and AG = abdominal ganglia (see ‘Materials and methods’ for an explanation of the sources of nervous tissues used in the experiment).

blot (Fig. 2(a, b), lower panels). A similar pattern of localization of Hez-PBAN mRNA was observed in the CNS of 2–3-day-old and 4–5-day-old adult females (data not shown). The entire experiment was repeated twice using cDNA templates reverse-transcribed from different batches of tissues, and a similar pattern of Hez-PBAN mRNA localization as that described above was observed in all three experiments. Relatively similar levels of Hez-PBAN mRNA were detected in the SEG of day 0–1 final instar larvae, pupae

and adults of different ages. On the contrary, Hez-PBAN mRNA levels in other nervous tissues appear to be different in various developmental stages. This is evident especially in the Br where higher levels of Hez-PBAN mRNA were detected in day 0–1 final instar larvae and relatively low levels of Hez-PBAN mRNA were detected in pupae and 0–1-day-old adult females (Fig. 2(a)). In addition, a high level of Hez-PBAN mRNA was also observed in the Br of H. zea 1–3-day-old adult males (Fig. 2(b)). In the TG of H. zea adults, a low level

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of Hez-PBAN mRNA was detected in 0–1-day-old adult females, whereas the same transcript was not detected in 1–2-day-old adult females (Fig. 2(a, b)). 3.3. In situ hybridization histochemistry Whole-mount in situ hybridization histochemistry was used to investigate the spatial and temporal expression of the Hez-PBAN gene. Spatial distribution of neurons expressing the Hez-PBAN mRNA at different developmental stages is shown in Fig. 3. In pupae and adults of both sexes, hybridization signals were evident in the SEG and were not seen in the rest of the nervous system. The location of the Hez-PBAN mRNA hybridization signals in the SEG were restricted to the mandibular, maxillary and labial cell clusters (Homberg et al., 1990). In adults, four cells in the mandibular cluster and 8–10 cells in the maxillary clusters were stained (Fig. 4(a)). Hybridization signals also occurred in two prominent cells in the labial cluster (Fig. 4(b)). All three SEG cell clusters were stained throughout the entire adult stage (up to 4–5-day-old adult females). A similar pattern of hybridization signals was observed in pupal SEG (Fig. 4(c)). In day 0–1 final instar larvae, the pattern of the HezPBAN mRNA hybridization signals was more widespread than in pupae and adults. In addition to the prominent signals in the three SEG cell clusters of larval SEG (Fig. 4(d)), Hez-PBAN mRNA hybridization signals were detected consistently in TG. In larval TG, HezPBAN hybridization signals were localized in a pair of ventral neurons at the midline of each of TG (Fig. 4(e)). These signals were much lower than those detected in the SEG. Relatively weak Hez-PBAN mRNA hybridization signals were detected in some of the AG. When evident, hybridizing signals were localized to a pair of ventral neurons located at the midline of the ganglion (Fig. 4(f)). Among all the larval preparations examined ( ⬎ 10), Hez-PBAN hybridizing signals were not observed in the 1st AG or in any of the AG posterior to the 5th AG. 3.4. Immunocytochemistry Distribution of PBAN-like immunoreactivity in H. zea CNS at various developmental stages was studied by whole-mount immunocytochemistry (Ma and Roelofs, 1995). Spatial distribution of cell bodies expressing PBAN-like immunoreactivity in day 0–1 final instar larvae, early and 6-day-old pupae, and adults of H. zea is summarized in Fig. 3. In day 0–1 final instar larvae, PBAN-like immunoreactivity was found throughout the entire CNS. In the Br, a total of about 12 cell bodies were stained with antiserum I-301. They are localized as three distinct clusters in each of the protocerebral hemispheres (Fig. 3). The first group contains a single,

darkly-stained neuron occupying the anterior aspect of each hemisphere (Fig. 5(a)). Almost directly opposite these two neurons is the second group of neurons containing two pairs of somata at the posterior aspect of each protocerebral hemisphere (Fig. 5(b)). A third group of six to eight weakly stained cell bodies can be detected in the pars intercerebralis (Fig. 5(c)). However, the staining of these cells was not abolished by pre-adsorption of antiserum I-301 with Hez-PBAN, and the staining intensity did not vary with different dilutions of the antiserum. Therefore, we consider the staining of these cells non-specific. In the SEG of day 0–1 final instar larvae, PBAN-like immunoreactivity was localized mainly in three cell clusters located along the midline of the ganglion. The locations of these cell clusters correspond to the cell clusters in the mandibular, maxillary and labial neuromeres described in Manduca sexta (Homberg et al., 1990). The mandibular cluster contain four somata and is the most anterior location in the SEG (Fig. 5(d)). Immediately posterior and ventral to the mandibular cluster lies the maxillary cluster, which consists of 8–10 cells (Fig. 5(e)). Near the posterior end of the SEG lie the two distinct somata of the labial cell cluster (Fig. 5(e)). In some cases, two smaller cell bodies closely associated with the two larger somata in the labial cluster were faintly stained (data not shown). Along the ventral midline of each larval TG lies a pair of intensely stained neurons (Fig. 5(f)). In the AG, PBAN-like immunoreactivity is typically localized in a pair of ventral neurons located along the midline of the ganglia (Fig. 5(g)). Generally, these cells were detected in the 2nd, 3rd, 4th and 5th AG, and the staining of these cells in the 5th AG was always very faint. Among all the preparations examined ( ⬎ 20), no cells were stained by antiserum I-301 in the 1st and 6th AG, or in the TAG (Fig. 5h). In 0–1-day-old pupae, the circumesophageal connectives shorten and the Br fuses with the SEG. Except for slight differences in the spatial distribution, the number of cells showing PBAN-like immunoreactivity in the fused brain–subesophageal ganglion remained unchanged as compared to those observed in day 0–1 final instar larvae. In the SEG, however, the mandibular and maxillary cell clusters were located closer together and the mandibular cluster appeared almost directly anterior to the maxillary cluster (Fig. 6(a, b)). Later in the pupal stage (6 days following pupal ecdysis), significant fusion occurs in the ventral nerve cord. The mesothoracic and metathoracic ganglia and the 1st and 2nd AG fuse to form one ganglionic mass, the pterothoracic ganglion. The 6th AG fuses with the rest of the TAG. Morphologically, the central nervous system appears essentially the same as that seen in the adults. Staining of PBAN-like immunoreactive neurons in the Br of pupae was weak. In the SEG, the number of som-

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Fig. 3. Schematic representation of cellular localization of Hez-PBAN mRNA (filled circles) and PBAN-like immunoreactivity (filled and open circles) in the central nervous system of H. zea day 0–1 final instar larvae, 0–1-day-old pupae, 6-day-old pupae and adults. Dorsal aspect of the brain in day 0–1 final instar larvae, 0–1-day- and 6-day-old pupae are shown. The rest of the illustrations are shown from their ventral aspect. Br = brain, SEG = subesophageal ganglion, TG = thoracic ganglia and AG = abdominal ganglia (see ‘Materials and methods’ for an explanation of the terminology of various parts of the central nervous system).

ata showing PBAN-like immunoreactivity remained unchanged. In the prothoracic ganglion, two pairs of small neurons located along the ventral midline of the ganglion were stained (Fig. 6(c)). Three pairs of neurons

located along the ventral midline of the pterothoracic ganglion were stained by antiserum I-301 (Fig. 6(d)). In addition, a pair of weakly-staining neurons was detected at the lateral regions of the ganglionic complex (Fig. 6(c,

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Fig. 4. Localization of Hez-PBAN mRNA in H. zea central nervous system by in situ hybridization histochemistry. Hybridization signal was not detected in control tissues where the random-primed hybridization probe was omitted in the hybridization buffer (data not shown). Except for (a, b), anterior is to the left in the photomicrographs. Scale bars equal 35 ␮m in (b) whereas those of (a) and (c–f) equal 18 ␮m. (a, b) Frontal view of 0–1-day-old adult female subesophageal ganglion showing hybridization signal in the presumptive mandibular (Md), maxillary (Mx) and labial (Lb) cell cluster. (c) Lateral view of 6-day-old pupal subesophageal ganglion. Cellular localization of Hez-PBAN gene expression is restricted to the presumptive mandibular (Md), maxillary (Mx) and labial (Lb) cell cluster. (d) Ventral view of larval subesophageal ganglion showing hybridization signal in the presumptive mandibular (Md), maxillary (Mx) and labial (Lb) cell cluster. Note that the maxillary cell cluster is slightly out of focus in this view. (e) Ventral view of prothoracic ganglion in day 0–1 final instar larvae showing two ventral midline neurons (arrowhead) expressing the Hez-PBAN transcript. (f) Ventral view of 2nd abdominal ganglion in day 0–1 final instar larvae showing hybridizing signal in two ventral midline neurons (arrowheads).

d)). A pair of ventral neurons was stained along the midline of the 3rd, 4th, and 5th AGs and in the neuromere6 of the TAG (Fig. 3). PBAN-like immunoreactive neurons were not observed in the Br of 0–1-day- through 2–3-day-old adults. Spatial distribution of the cell bodies expressing PBAN-like immunoreactivity in the SEG and the rest of

the ventral nervous system was similar to that observed in pupae (Fig. 7(a–f)). However, the staining of the lateral neurons in the pupal TG (Fig. 7(c, d)) was no longer detectable following adult eclosion.

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Fig. 5. PBAN-like immunoreactivity in the central nervous system of day 0–1 final instar larvae. Pre-adsorption of I-301 with Hez-PBAN abolished all immunostaining of larval nervous tissues (data not shown). Unless otherwise indicated, anterior is towards the left in the photomicrographs. Scale bars equal 35 ␮m in (a, b) and (d–h) whereas that of (c) equals 18 ␮m. (a) Frontal view of cerebral ganglion showing a pair of neurons immunostained with antiserum I-301 (arrowheads). (b) Antiserum I-301 staining of two pairs of neurons at the posterior aspect of the protocerebral ganglion (arrowheads). (c) Two clusters of six neurons at the pars intercerebralis are stained by antiserum I-301. (d) Dorsal view of subesophageal ganglion showing the mandibular (Md) cell clusters expressing PBAN-like immunoreactivity. (e) Ventral view of subesophageal ganglion showing the maxillary (Mx) and the labial (Lb) cell cluster. (f) Ventral view of prothoracic ganglion showing I-301 immunostaining of a pair of ventral midline neurons (arrowhead). (g) Ventral view of 2nd abdominal ganglion showing I-301 immunostaining of a pair of ventral midline neurons (arrowheads). (h) Dorsal view of terminal abdominal ganglion. Only dendritic aborizations were observed in this ganglion.

4. Discussion In our previous study, we used Northern hybridization to show that a Hez-PBAN transcript of ca 0.8 kb in size is present in the SEG of 2–3-day-old female adults and hybridizing signal was not detected in other parts of the CNS (Ma et al., 1994). In the present study, results of Northern hybridization showed that the 0.8-kb HezPBAN transcript is also present in the SEG of H. zea pupae and day 0–1 final instar larvae. This 0.8-kb transcript was not detected in other parts of the CNS in any of the developmental stages studied. However, low levels of Hez-PBAN mRNA may occur in other parts of the CNS, although they were not detected by the Northern

hybridization experiments. Therefore, we used the more sensitive RT-PCR technique to study the localization of Hez-PBAN mRNA at a steady state in the CNS. With RT-PCR, a 440-bp DNA fragment was amplified not only from cDNA templates prepared from SEG total RNA, but also from cDNA templates prepared from total RNA of Br and TG. In the AG of adults and day 0–1 final instar larvae, a weak but detectable hybridization signal corresponding to the 440-bp amplification product can be seen on the Southern blot. Since the size of the amplified fragment (440 bp) corresponds to that predicted from the Hez-PBAN cDNA sequence, these 440bp DNA fragments could not be derived from any alternatively spliced transcripts of the Hez-PBAN gene. Fur-

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Fig. 6. PBAN-like immunoreactivity in central nervous system of H. zea pupae. Pre-adsorption of antiserum I-301 with Hez-PBAN abolished all immunostaining on pupal nervous tissues (data not shown). Anterior is toward the left in the photomicrographs. Scale bars equal 35 ␮m. (a) The presumptive mandibular (Md) and labial (Lb) cell clusters immunostained with antiserum I-301 seen at the ventral aspect of the subesophageal ganglion. (b) Ventral view of the subesophageal ganglion showing PBAN-like immunoreactivity in the presumptive maxillary (Mx) cell cluster. (c) Ventral view of the prothoracic ganglion showing two median pairs of neurons (arrow) expressing PBAN-like immunoreactivity. Note that a pair of weakly stained neurons is located at the lateral regions of the ganglion (arrowheads). (d) Ventral view of mesothoracic/metathoracic– abdominal 1 + 2 ganglion complex showing two pairs of ventral median neurons immunostained with antiserum I-301 (close arrows). Two pairs of weakly stained neurons (open arrows) can be seen at the lateral region. Note also the pair of ventral midline neurons at the posterior region of the ganglion (arrowhead and slightly out of focus) that presumably was derived from the fused 2nd abdominal ganglion.

thermore, these 440-bp DNA fragments hybridized to a Hez-PBAN cDNA probe. These results show that in addition to the SEG, a Hez-PBAN transcript similar to, if not the same as, that in the SEG is present at relatively low levels in other parts of H. zea CNS. These low levels of Hez-PBAN mRNA in nervous tissues other than SEG are not stage dependent since it was detected in adults, pupae and day 0–1 final instar larvae. Different levels of Hez-PBAN mRNA were observed in the Br and TG at various developmental stages. However, the RT-PCR procedures used in the present study were not rigorous enough for quantative comparisons between tissues and developmental stages. Whether these differences are associated with specific physiological functions in the moths await further experiments that better quantitate the mRNA levels. In addition, more sensitive procedures, such as in situ PCR, are needed to precisely locate the cellular distribution of these mRNA. This information then could be used to generate hypoth-

eses regarding the physiological significance of the different levels of Hez-PBAN mRNA. In H. zea day 0–1 final instar larvae, in situ hybridization experiments showed that low levels of Hez-PBANtranscript were localized to two ventral midline neurons in each of the TG and some of the AG. The hybridization signals observed in AG were very weak. Since a random-primed cDNA probe was used in the in situ hybridization experiments, a possibility remains that these weak hybridization signals could result from hybridization of the labeled probe with non-specific targets in the tissue. Further experiments employing an antisense cRNA probe with a sense RNA probe as a control could resolve this uncertainty. Cellular localization of Hez-PBAN mRNA in cells other than those of the SEG of adults and pupae, as revealed by the RT-PCR experiments, remains unknown. These Hez-PBAN transcripts could occur in the ventral midline cells in adult and pupal TG and AG that were shown to possess

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Fig. 7. PBAN-like immunoreactivity in the central nervous system of 2–3-day-old adults of H. zea. Pre-adsorption of antiserum I-301 with HezPBAN abolished all immunostaining on adult nervous tissues (data not shown). Anterior is to the left in the photomicrographs in (a, c, d), and to the top in the photomicrographs in (e, f). Scale bar in (a) equals 18 ␮m and the scale bars in (b–f) equal 35 ␮m. (a) Ventral view of subesophageal ganglion showing PBAN-like immunoreactivity in the presumptive mandibular (Md) and maxillary (Mx) cell cluster. (b) Frontal view of subesophageal ganglion showing the presumptive labial (Lb) cell clusters expressing PBAN-like immunoreactivity. (c) Ventral view of prothoracic ganglion showing two pairs of ventral midline neurons expressing PBAN-like immunoreactivity (arrowhead). (d) Ventral view of mesothoracic/metathoracic– abdominal 1 + 2 ganglion complex showing two clusters of ventral midline neurons immunostaining with antiserum I-301 (arrowheads). (e) Ventral view of third abdominal ganglion showing PBAN-like immunoreactivity in a pair of ventral midline neurons (arrowhead). (f) Ventral view of the terminal abdominal ganglion showing PBAN-like immunoreactivity in a pair of ventral midline neurons near the anterior aspect of the ganglion (arrowheads).

PBAN-like immunoreactivity. Absence of hybridization signals in these ventral midline cells could indicate extremely low levels, or rapid degradation of Hez-PBAN mRNA in these cells. Alternatively, low levels of HezPBAN mRNA could be present in the processes projecting from the mandibular, maxillary and labial cell clusters in the SEG. Recently, there is accumulating evidence in various eukaryotic systems that indicates that mRNAs are actively transported from cell somata into their axons to localized regions within the CNS (Ding and Lipshitz, 1993; Dirks et al., 1993; Wilhelm and Vale, 1993; Van Minnen, 1994). If active transport of mRNA occurred in the axons of the three SEG cell clusters, this could contribute to some of the signals we detected in non-SEG nervous tissues using the RTPCR technique. In B. mori, Sato et al. (1994) used Northern hybridization and RT-PCR to demonstrate that Bom-DH (BomPBAN) gene expression in adult, pupal and larval stages occurred only in the SEG. In H. zea, results of the RT-

PCR experiment showed that Hez-PBAN transcripts also occurred at low levels in Br, TG and AG. The reason for the differences observed in the expression pattern of this highly homologous gene in these two species of moths currently is unknown. One possibility could be that the projection patterns of the axons from the mandibular, maxillary and labial cell clusters in the SEG are slightly different in these insects. Ichikawa et al. (1995) showed that neurons in the maxillary clusters in B. mori do not project their axons into the ventral nervous system. By constrast, in H. zea processes derived from the mandibular, maxillary and labial cell clusters project into the ventral nerve cord. These processes were shown to contain PBAN-like immunoreactivity (Kingan et al., 1990, 1992; Blackburn et al., 1992; Ma et al., 1996). As discussed above, low levels of Hez-PBAN mRNA could be present in these processes. Furthermore, a pair of ventral midline cells in TG and some of the AG in adult, pupal and larval H. zea were stained with antiserum I301. None of these cells was seen in B. mori using anti-

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sera that were raised against Bom-DH and Bom-PBAN (Ichikawa et al., 1995). In this case, it is possible that the Bom-DH(Bom-PBAN) gene was not expressed in the homologous cells in the TG and AG of B. mori. In a previous study, HPLC-ELISA profiling in conjunction to pheromonotropic assays was used to show that Hez-PBAN and four other Hez-PBAN gene neuropeptides are present in the SEG (Ma et al., 1996). The results of that study suggested that posttranslational cleavage of pro-Hez-PBAN followed by amidation of the released peptides occurrs in the SEG. Because the study was designed to target only five biologically active peptide sequences encoded in prepro-Hez-PBAN, other peptide fragments derived from posttranslational processing of pro-Hez-PBAN that could be present in the SEG and other central nervous tissues were ignored in the experiment. In an immunoprecipitation assay, we found that antiserum I-301 also recognizes in vitro-translated prepro-Hez-PBAN and pro-Hez-PBAN sequences (Ma and Roelofs, unpublished data). Presumably, antiserum I-301 can recognize any peptide fragment that is derived from posttranslational processing of pro-HezPBAN and contains the Asp144–Leu159 amide sequence. Therefore, immunostaining of any given neuron in the CNS using antiserum I-301 could result from the presence of a large array of PBAN-related peptide fragments present in that particular neuron. Obviously, Hez-PBAN and four other Hez-PBAN encoding genederived peptides did not necessarily account for all the PBAN-like immunoreactivity in the CNS demonstrated by immunocytochemical staining using antiserum I-301. Antiserum I-301 revealed two sets of PBAN-like immunoreactive neurons in each protocerebral hemisphere of H. zea day 0–1 final instar larvae. The frontal set of neurons stained by antiserum I-301 in the Br of H. zea day 0–1 final instar larvae is comparable to a set of PBAN-like immunoreactive neurons found in M. sexta larvae (Davis et al., 1996). Because these neurons in M. sexta were stained by only one out of a panel of four anti-Hez-PBAN antisera, these authors suggested that the staining of these cells was due to the presence of other FXPRLamide family peptides. Hez-PBAN was the first neuropeptide shown to be the factor that regulates sex pheromone biosynthesis (Raina et al., 1989). The isolation of the Hez-PBAN gene sequence and subsequent experiments to show the presence of four other Hez-PBAN related peptides in the SEG, indicate that Hez-PBAN may not act alone to stimulate pheromone biosynthesis. The presence of HezPBAN mRNA and PBAN-like immunoreactivity in pupal and larval CNS further suggest that peptides encoded in the Hez-PBAN gene could be involved in other physiological functions that are developmental-stage specific. To this end, Bom-PBAN has been shown to stimulate larval cuticle melanization in P. separata (Matsumoto et al., 1990). Pss-MRCH, a peptide showing

high homology to PGN18 (Ser106–leu123) of preproHez-PBAN (Ma et al., 1996), was isolated from P. separata (Matsumoto et al., 1992a). In H. zea, a preliminary study showed that injection of antiserum I-301 into 4th instar H. zea larvae prevented expression of three longitudinal pigmented stripes in 5th instar larvae (Ma and Roelofs, unpublished observations). As in the case of regulation of pheromone biosynthesis, more than one neuropeptide could be involved in larval cuticle melanization. Future understanding of how these physiological processes are regulated by the Hez-PBAN and other PGNs requires thorough knowledge of the peptides that are released at the time of stimulation. In addition, more rigorous studies are needed to define the profile of the peptides that are secreted by individual cell clusters in the SEG.

Acknowledgements The authors thank Marion O’Connor and Kathy Poole for rearing the insects used in this study. Thanks are also extended to Marlene Campbell for assisting in tissue dissection. This research was supported in part by NRI Grant No. 94-37302-0461 to W.L.R.

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