Bombyx Neuropeptide GPCRs Figure 2. Neighbor-joining phylogenetic trees for neuropeptide GPCRs in Bombyx and Drosophila. The trees are rooted with DHR and BAR as outgroups for family A (rhodopsin-like) and family B (secretin receptor-like; shown in inset) GPCRs, respectively. Bootstrap support is indicated with an open circle for 50–70%, gray circle for 70–90%, and filled circle for 90–100%. Bombyx GPCRs are in bold type: AKHR, adipokinetic hormone receptor; BAR, Bombyx allatostatin (A-type) receptor; BMSR, Bommo-myosuppressin receptor; BRFaR, Bommo-FMRFamide receptor; DHR, diuretic hormone receptor; DpHR, diapause hormone receptor; PBANR, pheromone biosynthesis-activating neuropeptide receptor; SIFR, SIFamide receptor. The endogenous Drosophila ligands are given in parentheses: AKH, adipokinetic hormone; CCAP, crustacean cardioactive peptide; DH, diuretic hormone; ETH, ecdysis-triggering hormone; NPF, neuropeptide F; PDF, pigment-dispersing factor. doi:10.1371/journal.pone.0003048.g002 effect is most likely inhibitory, because sNPFs have clear allatostatic activity (Figure 5D). Thus, AT could inhibit the allatostatic effect of sNPFs produced in the CC, thereby exerting its indirect allatotropic effect by ‘‘derepression’’. Our preliminary results that the larval CA incubated alone secretes more JH than CC-CA complex in vitro (Y.K. and K.H., unpublished) further support this idea. However, this hypothesis must be carefully considered from several aspects. First, some allatoregulatory peptides are known to change their activity during development [43]. Detailed physio- logical analyses of sNPF and AT along developmental stages, as well as the improvement of a reliable in vitro CC-CA co-culture system, are thus necessary to examine the above hypothesis. Second, we cannot fully rule out the possibility that the low, but still detectable expression of the AT receptor in the CA (Figure 6A) may mediate the allatotropic activity of its ligand, though we could not detect any allatotropic activity of AT using our in vitro CA culture (data not shown). Considering the varying activity of AT during development in many insects [4,44], the temporal expression analysis of the AT receptor within the CC-CA complex, as well as the other receptors expressed in the CA, may help clarify such a possibility. Thus, both of these alternative possibilities require the introduction of the ‘‘fourth’’, temporal axis to our three-dimensional, spatial expression analysis. Although this is beyond the scope of this study, our work provides a firm foundation upon which such further analyses can be based. Considering that sNPF has been reported in most insects examined [37], it is possible that sNPF has a conserved role in the regulation of JH biosynthesis. In relation to this, it is interesting to note that several physiological events related to sNPF in other insects (ovarian growth, body size control and adult diapause) are all reminiscent of its regulatory role in JH biosynthesis [37,40,41,45]. Together with the recently reported role of sNPF in the regulation of insulin expression [46], investigation of the functional conservation of sNPF in other insect species is clearly warranted. Identification of sNPFs as CC-produced allatoregulatory peptides may also provide a direct link between JH biosynthesis and energy balance. JH acts as a signal for starvation-induced growth inhibition, which suggests a tight link between JH biosynthesis and energy balance [47]. On the other hand, CC is well known for its glucose-sensing capacity, which regulates energy homeostasis in insects [42]. Investigation of the sNPF secretion from the CC under a variety of physiological conditions will test the validity of this possibility. The high expression of the ETH receptor in the CA (Figure 6A) is another unexpected finding of this study. This result was further verified by in situ hybridization (Figure S3), suggesting an unknown physiological role of ETH in the regulation of JH biosynthesis. ETH is produced by Inka cells of epitracheal glands and released into the hemolymph during ecdysis [9,39], so the putative allatoregulatory role of ETH may explain the transient rise of JH titer at ecdysis reported in many insects [48–50]. Together with the as-yet-unknown ligand of BNGR-B3, elucidation of the physiological significance of these putative regulators of JH biosynthesis will elucidate a number of unexpected features of this important endocrine pathway. All of the above results clearly suggest that the regulatory mechanisms of JH biosynthesis are as complex as those of ecdysteroidogenesis, in which multiple factors are involved [51]. Indeed, most tissues investigated in this study express various neuropeptide GPCRs (Figure 3), indicating the necessity of a systematic approach to reveal regulatory mechanisms of their functions. The present study provides a solid basis for such an approach, which leads to our thorough understanding of the neuropeptide circuitry that extends beyond individual tissues to form a system-wide network in multicellular organisms. Materials and Methods Comprehensive identification of BNGRs BNGR genes were identified by a cloning-based approach combining homology searching and RACE, using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Moun- tain View, CA) according to the manufacturer’s instructions. Whole coding regions of all the identified BNGR genes were cloned, sequenced, and confirmed to have seven transmembrane regions and full conserved domains for GPCRs. See Results and Tables S1, S2, S3 for the detailed procedure. The primers used for RACE and cloning are listed in Table S4 and S5, respectively. Phylogenetic analysis Deduced amino acid sequences of Bombyx and Drosophila neuropeptide receptors were aligned and rooted neighbor-joining trees were generated using ClustalW. Bootstrap analyses of 1,000 replications were conducted to assess the relationships. Quantitative RT-PCR (Q-RT-PCR) analysis of the neuropeptide GPCR genes cDNAs from various tissues were synthesized and Q-RT-PCR was carried out essentially as described [52]. After PCR, the absence of unwanted byproducts was confirmed by automated melting curve analysis. Serial dilutions of pME18S plasmids containing coding sequences of the neuropeptide GPCR genes were used for standards. Rp49 was chosen as a reference gene, and serial dilutions of a pCR2.1 plasmid containing 340 bp of Rp49 cDNA were used for standards. The molar amounts of the GPCR genes and Rp49 were calculated, and the transcript levels of the GPCR genes were normalized with Rp49 levels in the same samples and presented as arbitrary units. The primers used for Q- RT-PCR are listed in Table S6. Cloning of AST-C Using the amino acid sequence of Manduca sexta allatostatin (GenBank accession no. P42559) as a query, BLAST analysis (TBLASTN) was performed against Bombyx whole-genome shotgun sequence contigs using KAIKOBLAST. Based on the putative coding sequence of the obtained contig (contig 454046, GenBank accession no. BAAB01095856), RACE primers were designed as follows: sense primer, 59-ATGGCAGATTGGTGC- PLoS ONE | www.plosone.org 6 August 2008 | Volume 3 | Issue 8 | e3048