Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei , China b

Transcription

1 General and Comparative Endocrinology 141 (2005) Developmental expression of FXPRLamide neuropeptides in peptidergic neurosecretory cells of diapause- and nondiapausedestined individuals of the cotton bollworm, Helicoverpa armigera Jiu-Song Sun a,1, Qi-Rui Zhang a,1, Tian-Yi Zhang a, Zhong-Liang Zhu a, Hong-Min Zhang a, Mai-Kun Teng a, Li-Wen Niu a, Wei-Hua Xu a,b, a Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, Hefei , China b State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Science, Beijing , China Received 13 May 2004; revised 22 November 2004; accepted 29 November 2004 Available online 18 January 2005 Abstract The diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) gene encodes Wve neuropeptides, DH, PBAN, α-sgnp, β-sgnp, and γ-sgnp (subesophageal ganglion neuropeptide). All share the C-terminal pentapeptide FXPRLamide sequence and are produced in the subesophageal ganglion (SG). Expression of the DH PBAN gene in the central nervous system of embryonic, larval, pupal, and adult Helicoverpa armigera (Har) was studied using in situ hybridization, whole-mount immunocytochemistry, and competitive ELISA. Both Har-DH PBAN mrna and protein are localized in the mandibular, maxillary, and labial cell clusters of the SG and a pair of ventral midline neurons of each thoracic ganglion. The FXPRLamide titers in hemolymph are signiwcantly higher in diapause-destined larvae during the Wfth and sixth instar than in similar nondiapause-destined individuals. In contrast, the FXPRLamide titers in diapause-destined pupae are signiwcantly lower than in nondiapause-destined pupae. The results from immunocytochemistry and in situ hybridization are consistent with changes of FXPRLamide titers as measured by ELISA. These data suggest that the expression of DH PBAN might be correlated with diapause induction at the larval stage of diapause-destined individuals and continuous development at pupal stage of nondiapause-destined individuals. Thus, the DH PBAN gene may play an important regulatory role in aspects of insect development besides diapause termination and pheromone biosynthesis. The transport pathways of FXPRLamide neuropeptides suggest that humoral route is involved in their regulation of development Elsevier Inc. All rights reserved. Keywords: Diapause hormone; FXPRLamide neuropeptide; Neurosecretory cell; Development; Helicoverpa armigera 1. Introduction Diapause is an adaptive strategy for animals to survive unfavorable environmental conditions. Many insects arrest their development and initiate diapause at a speciwc stage in their life cycles. A period during which Corresponding author. Fax: address: whxu@ustc.edu.cn (W.-H. Xu). 1 These authors contributed equally to this work. growth or development is suspended and physiological activity is diminished, as in certain insects in response to adverse environmental conditions. In Bombyx mori (Bom), the neuropeptide diapause hormone (DH) has been shown to be a speciwc factor for inducing embryonic diapause (Yamashita, 1996). From the structural analysis of DH cdna, the pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid neurohormone that controls sex pheromone biosynthesis in females of Lepidopteran species, was found to be /$ - see front matter 2004 Elsevier Inc. All rights reserved. doi: /j.ygcen

2 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) encoded in Bom-DH cdna (Raina and Klun, 1984; Raina et al., 1989; Sato et al., 1993). Thus, the cdna and gene were designated DH PBAN cdna and DH PBAN gene, respectively (Xu et al., 1995a). Shortly thereafter, a DH-like peptide in the PBAN cdna from Helicoverpa zea (Hez), a species that undergoes pupal diapause, was Wrst reported by Ma et al. (1994), and later DH-like peptides were also found in several Lepidopteran species (Choi et al., 1998; Duportets et al., 1999; Iglesias et al., 2002; Jacquin-Joly et al., 1998). However, in pupal diapause species, the function of the DH-like peptide remains unknown. Recently, three DH PBAN cdnas were cloned from Helicoverpa assulta (Has) (Choi et al., 1998), Heliothis virescens (Hvi) (Xu and Denlinger, 2003), and Helicoverpa armigera (Har) (Zhang et al., 2004a), and showed that a single mrna encodes a common polyprotein precursor, from which DH-like peptide, PBAN, and three other peptides that all share an FXPR/KL sequence at the C-terminus. Interestingly, Has-, Hvi-, and Har-DH-like peptides encoded by DH PBAN gene were found to break pupal diapause evectively in H. assulta (Zhao et al., 2004), H. virescens (Xu and Denlinger, 2003), and H. armigera (Zhang et al., 2004a). Expression analysis during pupal development showed that Has-, Har-, or Hvi-DH PBAN mrnas are signiwcantly higher in nondiapause subesophageal ganglion (SG) than in diapause-destined SG (Xu and Denlinger, 2003; Zhang et al., 2004a,b; Zhao et al., 2004), whereas Bom- DH PBAN mrna levels are threefold lower in nondiapause-type pupae than in diapause-type pupae (Xu et al., 1995b). Ma et al. (1998) reported that Hez-PBAN gene expression was related to pheromone synthesis in H. zea, but they did not examine its relationship to pupal diapause. Using whole-mount immunocytochemistry and in situ hybridization, Kingan et al. (1992), Ma et al. (1998), and Sato et al. (1994, 1998) localized DH PBAN gene products in the mandibular, maxillary, and labial cell clusters of the SG in H. zea and B. mori, respectively. In H. armigera, Sun et al. (2003) and Zhang et al. (2004b) documented the locations of Har-DH PBAN mrna and peptides in the central nervous system (CNS) at the pupal stage using the same techniques, and showed high expression of Har-DH PBAN gene in nondiapause-destined pupae and low expression in diapause-destined pupae. Zhang et al. (2004b) demonstrated that the diversity of DH PBAN gene expression in the SG of nondiapause- and diapause-type pupae was Wrst found in day 2 pupae. To better understand the possible functions of FXPRLamide peptides in H. armigera, we systematically investigated the developmental expression of the Har- DH PBAN gene at embryonic and postembryonic stages, focusing on the similarities and diverences in the DH PBAN gene expression between diapause- and nondiapause-destined larvae and pupae using cytochemical methods and statistical analysis. The FXPRLamide titers in the hemolymph of larval and pupal stages were also measured by competitive ELISA. 2. Materials and methods 2.1. Insects Larvae of the bollworm, H. armigera were reared according to the method of Sun et al. (2003). Each larva was fed an artiwcial diet at 22 C under a photoperiod of 14 h light:10 h dark (nondiapause type), and 10 h light:14 h dark (diapause type). Developmental stage was selected at the Wfth larval ecdysis and pupation Preparation of anti-dh serum and dot-immunoblotting assay The Har-DH (NDVKDGAASGAHSDRLGLWFG PRL-amide) was synthesized by Genemed Synthesis (South San Francisco, CA, USA). The preparation of anti-dh antibody was the same as that described in Sun et al. (2003). The method of dot-immunoblotting assay was adopted from Sato et al. (1998), except that PVDF membrane (Hybond-P, Amersham) was used instead of the nitrocellulose membrane, and the immunological signals were detected using color development reaction instead of chemiluminescence. All procedures were performed at room temperature. Peptides to be tested were dissolved in distilled water. One microliters of the peptide solution was dotted on methanol-treated PVDF membrane and the membrane was air-dried. The membrane was then blocked with PBST 0.05 (PBS D 20 mm Na-phosphate, 130 mm NaCl, ph 7.2, containing 0.05% Tween 20) containing 5% skim-milk (SM-PBST 0.05 ), and incubated for 1.5 h with serum diluted at 1/1000 with SM-PBST After PBST 0.05 wash, the secondary antibody (HRP-conjugated goat anti-rabbit IgG, Promega), diluted to 1/2500 with PBST 0.05, was added to the membrane and incubated for 1.5 h. It was then washed again with PBST 0.05 and used for detection. Immunological signals were visualized by DAB Stock Stain Kit (Sino- American Biotechnology) Whole-mount immunocytochemistry The distribution of FXPRLamide immunoreactivity in the CNS of H. armigera throughout all developmental stages except for embryonic stage was investigated using whole-mount immunocytochemistry as described previously (Sun et al., 2003). Embryos were dissected in 0.75% NaCl from chorion. An incision was made in the dorsal epidermis and the gut was removed to expose the CNS. Embryo Wxation and processing were carried out as described in Sun et al. (2003). Finally, after color

3 50 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) development, the CNS, including brain, SG, thoracic ganglion (TG), and abdominal ganglion (AG) were dissected from the tissues and directly mounted with glycerol for observation with a light microscope (Olympus, BX60). For statistical analysis, the area of FXPRLamideimmunoreactive somata in SG was determined at larval and pupal stages in diapause-destined and nondiapausedestined individuals using the digital camera SPOT, and its corresponding software SPOT-Advanced (version 3.0) according to the manufacturer s instructions (Diagnostic Instruments, USA). More than 20 specimens were counted for each group and statistical signiwcance was calculated using Student s t test. P < 0.01 was considered very signiwcant (**) Whole-mount in situ hybridization Using in vitro transcription with T7 or T3 RNA polymerase, sense or antisense RNA hybridization probe for Har DH PBAN cdna labeled with digoxigenin-utp (Boehringer Mannheim) was generated. The procedure for larval and pupal in situ hybridization was described previously in Sun et al. (2003), except that the stained tissues were dehydrated through increasing concentrations of ethanol, cleared in xylene, and mounted with neutral balsam. The procedure for embryonic dissection was identical to that used for in the whole-mount immunocytochemistry. All the remaining procedures were as described above and the specimens were mounted directly with glycerol for observation after staining. The controls were performed according to the same procedure, except that the antisense crna probe was replaced by sense crna probe. The principles and procedures for the measurement of stained neurosecretory cells have been described before (Hu et al., 2002; Liu et al., 2000). The optical density of staining neurons was measured automatically using the MetaMorph image acquisition and processing software (Universal Imaging, USA). The data reported are means SE. Statistical signiwcance was calculated using Student s t test as described above Competitive ELISA The titers of FXPRLamide in hemolymph from diapause and non-diapause larvae were measured by competitive ELISA according to the procedures described in Sun et al. (2003). 3. Results 3.1. Characterization of the antibody The speciwcity of the anti-dh serum was determined by dot-immunoblotting assay using various synthetic peptides including the FXPRLamide family of peptides and other unrelated peptides (Fig. 1). With 1/1000 dilution of the serum, 0.69 pmol of Har-DH was detectable. Cross-reactivity with other FXPRLamide peptides was also detected. PBAN showed the highest reactivity after DH, followed by β-sgnp, γ-sgnp, and α-sgnp. Two FXPRLamide peptides, Drosophila melanogaster DHlike peptide (GenBank No. AF203878) and Manduca sexta DH-like peptide (Xu and Denlinger, 2004) showed high cross-reactivity, but not prothoracicotropic hormone (PTTH, corresponding to a 20-residue peptide from N-terminus of Har-PTTH, GenBank No. AY286543), which does not have FXPRLamide at its C- terminal. These results indicate that the C-terminal pentapeptide, FXPRLamide might contain the major epitope and the antiserum could recognize FXPRLamide family of peptides evectively Expression of DH PBAN gene at embryonic stage By whole-mount in situ hybridization, the expression of DH PBAN gene at embryonic stage was detected in SG and TGs (Fig. 2A). The DH PBAN mrna were present mainly in the SG (Fig. 2A(a)) and were restricted to the mandibular (Md), maxillary (Mx), and labial (Lb) cell clusters, as reported at pupal or adult stage B. mori, H. zea, and H. armigera (Ma et al., 1998; Sato et al., 1994, 1998; Sun et al., 2003). The hybridization signals were also localized in a pair of ventral midline neurons in all TGs (Figs. 2A(b d)), but not in the rest of the nervous system. Whole-mount immunocytochemistry revealed that the FXPRLamide immunoreactive cells were present in SG (Fig. 2B(a)) and TGs (Figs. 2B(b d)), as was shown by wholemount in situ hybridization, but the stained cell bodies in the lateral regions were also detected in each TG. Two projections from the three cell clusters in the SG extended to the corpus cardiacum corpus allatum (CC CA) complex (Fig. 2B(a)) DiVerential expression of DH PBAN in diapauseand nondiapause-destined individuals at postembryonic stage The intensity of hybridization signals and FXPRLamide immunoreactivity in SGs of last larval instar of diapause- and nondiapause-destined individuals (Fig. 3) were compared. Similar to Wndings in the embryonic stage, the cell bodies show hybridization signals and FXPRLamide immunoreactivity in three cell clusters of larval SG. In whole-mount immunocytochemistry, no signiwcant diverence is observed between nondiapauseand diapause-destined larvae (Figs. 3A D). The hybridization signals of the mrna are similar in Md and Mx cell clusters of diapause-destined larvae (Fig. 3F) and nondiapause-destined larvae (Fig. 3E), but much

4 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) Fig. 1. Immunological speciwcity of the anti-dh serum. (A) Synthetic peptides used for dot-immunoblotting assay. The conserved FXPRLamide sequences are highlighted with white letters on black. (B) Dot-immunoblotting assays. The synthetic peptides were dotted on PVDF membrane. The membrane was probed with the antiserum and immunological signals were visualized by DAB Stock Stain Kit. DH, PBAN, α-sgnp, β-sgnp, γ- SGNP, and N-PTTH are from H. armigera. N-PTTH is corresponding to a 20-residue peptide from N-terminus of PTTH (GenBank No. AY286543). Drm-DH, Drosophila melanogaster DH-like peptide (GenBank No. AF203878). Mas-DH, Manduca sexta DH-like peptide (Xu and Denlinger, 2004). stronger in the Lb cluster of diapause-destined larvae (Fig. 3F). The diapause-destined pupae incubated at 22 C entered diapause within 4 5 days after pupation in our preliminary experiments. We thus focused on the developmental expression of Har-DH PBAN gene in the early middle stage pupae (days 0, 3, and 5 pupae). For 0- day-old pupae, there was no observable diverence in the intensity of DH PBAN positive cells in SGs of nondiapause- and diapause-destined individuals using either whole-mount immunocytochemistry or in situ hybridization (Fig. 4). Obvious diverences in size, shape, and axonal projections of immunoreactive neurosecretory cells were observed in SGs between diapause- and nondiapause-destined pupae of 3-day-old (Fig. 5) and 5-dayold (Fig. 6) pupae using whole-mount immunocytochemistry. In nondiapause pupae, the FXPRLamide immunoreactive cells were larger (Figs. 5A and B, 6A and B) and the axonal projections of Lb cluster were much clearer (Figs. 5B and 6B) than those of diapausetype pupae (Figs. 5C and D, 6C and D). The hybridization signals in SGs of 3-day-old and 5-day-old nondiapause pupae could be clearly detected (Figs. 5E and 6E), whereas signals from diapause-destined pupae were very weak (Figs. 5F and 6F). To clarify the diverence in expression between diapause- and nondiapause-destined individuals, we measured the area of immunoreactive cells and the optical density of hybridization signal based on the results of immunocytochemistry and in situ hybridization. For the mid-stage of sixth larval instars and 0-day-old pupa, there was no signiwcant diverence in the area of immunoreactive cells between diapause- and nondiapause-destined individuals, but signiwcant diverence was detected between in 3-day-old and 5-day-old pupae (Fig. 7). The optical density of hybridization signal in diapause-destined larvae was signiwcantly higher than that in nondiapause-destined individuals (Fig. 8). At day 0 after pupation, the optical density of DH PBAN positive cells was similar between diapause- and nondiapause-destined individuals, but signiwcantly higher in nondiapause-destined pupae at days 3 and 5. The expression of the Har-DH PBAN gene in 0-dayold adults was also measured using whole-mount immunocytochemistry and in situ hybridization. These

5 52 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) Fig. 2. Expression of DH PBAN gene at embryonic stage of H. armigera. Anterior is at the top of in all photomicrographs. (A) Localization of Har- DH PBAN mrna in CNS by in situ hybridization. Hybridization signal was not detected in control tissues where the hybridization probe was sense-strand RNA (data not shown). Frontal view of SG (a) showing hybridization signal in the presumptive mandibular (Md), maxillary (Mx), and labial (Lb) cell clusters. Frontal view of TG1-3 (b,c,d) showing two ventral midline neurons (arrowheads) expressing the Har-DH PBAN transcript. (B) FXPRLamide peptides immunoreactivity in the CNS by whole-mount immunocytochemistry. Pre-adsorption of the antiserum with Har-DH abolished all immunoassaying of nervous tissues (data not shown). Frontal view of SG (a) and TG1-3 (b,c,d) showing three cell clusters and neuritis projections (thin arrow) in SG expressing FXPRLamide peptides immunoreactivity and the antiserum immunoassaying of a pair of ventral midline (arrowhead) and lateral neurons (arrow) in TGs. Scale bars equal 15 μm. Fig. 3. Expression of DH PBAN gene in SG of last larval instar. Pre-adsorption of the antiserum with Har-DH abolished all immunoassaying of SGs (data not shown) and hybridization signal was also not detected in control tissues, where the hybridization probe was sense-strand RNA (data not shown). Anterior is at the top of all photomicrographs. (A,B) Nondiapause-destined and (C,D) diapause-destined are the SGs of last larval instar showing FXPRLamide peptides immunoreactivity in the Md, Mx, and Lb cell clusters. (E) Nondiapause-destined and (F) diapause-destined are the SGs last larval instar showing by in situ hybridization results. Cellular localization of Har-DH PBAN gene expression is restricted to three cell clusters. Scale bars equal 30 μm. neurosecretory cells of FXPRLamide immunoreactivity were localized in three cell clusters of SG, similar to Wndings in pupae (Figs. 9A and B). The hybridization signals were present only in SG (Fig. 9C) and are absent from other components of the nervous system, such as brain, TGs, and AGs (data not shown).

6 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) Fig. 4. Expression of DH PBAN gene in SG of 0-day-old pupae. Anterior is on the top in all the photomicrographs. (A,B) Nondiapause-destined and (C,D) diapause-destined are the pupal SGs of day 0 showing the Md, Mx, and Lb cell clusters immunoassayed with the antiserum. Both (E) Nondiapause-destined and (F) diapause-destined are the SGs, showing three cell clusters expressing the Har-DH PBAN transcript by in situ hybridization. Scale bars equal 30 μm. Fig. 5. Expression of DH PBAN gene in SG of 3-day-old pupae. (A,B) Nondiapause-destined and (C,D) diapause-destined are frontal views of SGs showing FXPRLamide peptides immunoreactivity in the Md, Mx, and Lb cell clusters. Both (E) nondiapause-destined and (F) diapause-destined are frontal views of SGs, showing hybridization signals in three cell clusters by in situ hybridization. Scale bars equal 30 μm Changes of FXPRLamide titers in hemolymph of diapause- and nondiapause-destined larvae To elucidate the relationship between the synthesis and release of DH PBAN gene products, we measured the titer of FXPRLamide neuropeptides in hemolymph of diapause- and nondiapause-destined individuals using competitive ELISA (Fig. 10). During Wfth and sixth larval instars, the FXPRLamide titers in hemolymph of diapause-type larvae were signiwcantly higher than those in nondiapause-type larvae. In the middle of the sixth larval instar, the titer was 4-fold higher in diapause-destined larvae. On the contrary, at pupal middle stage, the FXPRLamide titers in diapause-type were signiwcantly lower than in nondiapause-type, as described in Sun et al. (2003) Release site of DH PBAN gene products Earlier studies in B. mori and H. zea have shown that the corpus cardiacum corpus allatum (CC CA) complex and terminal abdominal ganglion (TAG) are the release sites of DH PBAN gene products at pupal and adult stages (Ma et al., 1996, 1998; Sato et al., 1998). We are interested in conwrming the releasing route of FXPRLamide neuropeptides in H. armigera at embryo and Wrst larval instar. The immunoreactivity of TAG and the retrocerebral complex (CC CA and associated nerves) was assessed. The CC and the associated nerves were stained heavily, but no positive immunoreactivity was detected in CA (Figs. 11A and C). Two projections were clearly stained in all TGs (Figs. 2B(c d)) and all AGs (data not shown), and terminated in the last abdominal ganglion (Figs. 11B and D).

7 54 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) Fig. 6. Expression of Har-DH PBAN gene in SG of 5-day-old pupae. (A,B) Nondiapause-destined and (C,D) diapause-destined are frontal views of SGs showing the Md, Mx, and Lb cell clusters, as immunoassayed by the antibody. (E) Nondiapause-destined and (F) diapause-destined are frontal views of SGs. Cellular localization of Har-DH PBAN gene expression is restricted to three cell clusters by in situ hybridization. Scale bars equal 30 μm. Fig. 7. Quantitative analysis of stained area of FXPRLamide immunoreactive somata in SGs of diapause-destined and nondiapause-destined individuals (n 7 20). L, middle stage of sixth instar larvae; P0, P3, and P5: 0-day-, 3-day-, and 5-day-old pupae. The SE is shown by a vertical bar. **P < Fig. 8. Quantitative analysis of the optical density of DH PBAN positive mrna somata in SGs of diapause- and nondiapause-destined individuals (n 7 20). L, middle stage of sixth instar larvae; P0, P3, and P5: 0-day-, 3-day-, and 5-day-old pupae. The SE is shown by a vertical bar. **P < Discussion In the present study, the expression of DH PBAN gene in the CNS of H. armigera through embryonic and postembryonic development was investigated using in situ hybridization and immunocytochemistry. We compared the diverences in mrna and peptide levels between diapause- and nondiapause-destined larvae and pupae. In addition, we also showed the localization and possible pathway for release of FXPRLamide neuropeptides in embryos. The speciwcity of Har-DH antiserum was characterized by the dot-immunoblotting method and the results showed that the polyclonal antibodies could evectively recognize other members of the FXPRLamide family. Hence, we named the immunoreactivity in the ELISA and immunocytochemistry results as FXPRLamide immunoreactivity.

8 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) Fig. 9. Expression of Har-DH PBAN gene in SG of 0-day-old adult H. armigera. (A,B) Frontal view of SG showing FXPRLamide peptides immunoreactivity in the Md, Mx, and Lb cell clusters. (C) Frontal view of SG showing hybridization signal in three cell clusters by whole-mount in situ hybridization. Scale bars equal 30 μm. Fig. 10. Changes of FXPRLamide peptides titers in hemolymph. The hemolymph of 10 individuals from diapause-destined and nondiapause-destined was mixed, respectively, as a sample for competitive ELISA, and each point represents the mean values from Wve samples. L5 mid: the middle stage of the Wfth larval instar; L6 0-d and L6 mid: 0-day-old and the middle stage of the sixth larval instar; W: wandering; P0, P3, and P5: 0-day-, 3-day-, and 5-day-old pupae. The SE is shown by a vertical bar. **P <0.01. Recently, it has been reported that both Drosophila ETH1 and Manduca ETH antisera could cross-react with other neuropeptides containing the conserved C- terminal PRXamide motif, which is shared by ETHs (Drm-ETH1, Drm-ETH2, and Mas-ETH), the cardioactive peptide CAP2b, PBANs, and DHs in moths, and the Drosophila neuropeptides CG15520 and CG6371 (Park et al., 2002). Therefore, we also tested the cross-reactivity of anti-dh serum with other PRXamide peptides, Drm- ETH1 (PRLamide), -ETH2 (PRIamide), and -CAP2b (PRVamide) and found that the antiserum we used here could only detect Drm-ETH1 at relatively high concentration (more than 56 pmol), but not Drm-ETH2 and - CAP2b even though 500 pmol peptides were used (data not shown). The result suggests that the last amino acid lysine in the FXPRLamide motif is an important part of Fig. 11. Localization of FXPRLamide peptides immunoreactivity in the CC CA and TAG. Dorsal views of brain retrocerebral complex (Br CC CA) of embryo (A) and Wrst larval instar (C) showing FXPRLamide peptides immunoreactivity (anterior at the bottom). Arrowheads, CA; arrow, CC; Br, brain. (B) Embryo and (D) Wrst larval instar showing the frontal views of TAG with neuritis projections (arrow) immunoassayed by the antibody. Scale bars equal 10 μm. the FXPRLamide epitope and the PRXamide peptides as ETH or CAP2b could not interfere in the related immunological assays. At the embryonic stage, DH PBAN gene expression was detected in SG and TG. Although the cell bodies cannot be clearly discerned in SG, the hybridization signal is clear. In H. armigera, the DH regulates the development

9 56 J.-S. Sun et al. / General and Comparative Endocrinology 141 (2005) of pupae by stimulating the synthesis and release of ecdysone (Zhang et al., 2004b), and PBAN/pyrokinin can accelerate pupariation in the Xesh Xy, Sarcophaga bullata (Nachman et al., 1997; Zdarek et al., 1998). Thus, the FXPRLamide peptides may be related to embryonic development. However, the function of these FXPRL peptides remains to be further investigated. The transport pathway of FXPRLamide neuropeptides in embryonic and larval stages was also revealed by immunocytochemistry. In the retrocerebral complex, CC was stained intensely, but there was no immunoreactivity in CA. The results are consistent with the observations reported in B. mori and H. zea (Golubeva and Raina, 1997; Ma et al., 1996; Sato et al., 1998). By comparing our results with those from previous studies, it can be concluded that the CC is a neurohemal organ releasing FXPRLamide neuropeptides through circulation of hemolymph, which in turn transports them to their target tissue through circulation. Furthermore, the FXPRLamide neuropeptides have been detected in the hemolymph of larvae and pupae of H. armigera by competitive ELISA. Our results strongly support the humoral-route hypothesis put forward by Raina (1993) in which PBAN functions as a neurohormone. The high FXPRLamide titer present in the hemolymph of diapause-destined larvae may be closely correlated to sensitive stage for diapause induction in H. armigera. It is well known that diapause-destined larvae need to eat more food than nondiapause-destined larvae to store energy for pupal diapause; this feeding pattern prolongs the larval stage by about two days. A very similar phenomenon is noted in B. mori, which shows high expression of Bom-DH PBAN gene during the last two larval stages in diapause-destined individuals and thus was related to the regulation of larval growth through prolonged feeding periods (Xu et al., 1995b). We injected Wve synthetic FXPRLamide neuropeptides from DH PBAN gene into nondiapause-destined larvae and found that these peptides could signiwcantly delay development in H. armigera (data not shown). Therefore, most probably, the high expression of the Har-DH PBAN gene in diapause-destined larvae regulates larval growth and contributes to physiological preparations for pupal diapause, as reported in B. mori (Xu et al., 1995b). The expression of the DH PBAN gene during the pupal stage has drawn considerable attention. Using Northern blotting or quantitative RT-PCR, Has-, Har-, and Hvi-DH PBAN mrna remained at a relatively low levels in diapause-type pupae, while they increased steadily with the development of nondiapause-type pupae (Xu and Denlinger, 2003; Zhao et al., 2004; Zhang et al., 2004a,b). Based on our immunocytochemistry results, in the SG of nondiapause pupae, the neurosecretory cells of DH PBAN were enlarged and the axonal projections from the neurosecretory cells stained heavily, especially in the Lb cell cluster. This indicates vigorous synthesis of the FXPRLamide neuropeptides, as reported in Sun et al. (2003) and Zhang et al. (2004a,b). However, in diapause pupae, no signiwcant diverence was noticed in the neurosecretory cells of DH PBAN from day 0 to day 5. These results indicate that the neurosecretory cells could enter into the arrested or developmental status decided by diapause- or nondiapausedevelopmental programs. The high level of FXPRLamide peptides (Figs. 5 7 and 10) may contribute to continuous development in nondiapause pupae, because DH, a member of the FXPRLamide peptides family, could stimulate prothoracic glands to synthesize ecdysone (Zhang et al., 2004b). In Lepidopteran species, the expression of the DH PBAN gene has been detected in both males and females throughout the embryonic and postembryonic stages (Choi et al., 1998; Duportets et al., 1999; Iglesias et al., 2002; Jacquin-Joly et al., 1998; Ma et al., 1994; Sato et al., 1994; Xu et al., 1995b; Xu and Denlinger, 2003, 2004; Zhang et al., 2004a). It is well known that PBAN regulates sex pheromone biosynthesis in Lepidopteran females and that DH induces egg diapause in the developing ovaries of B. mori. Thus, the FXPRL peptides expressed in males should have other biological functions besides the stimulation of pheromone synthesis and diapause induction. 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