Drosophila melanogaster flatline encodes a myotropin orthologue to Manduca sexta allatostatin

doi:10.1016/S0196-9781(01)00649-0

Peptides

Volume 23, Issue 4, April 2002, Pages 787-794

https://doi.org/10.1016/S0196-9781(01)00649-0 Get rights and content

Abstract

We identified a Drosophila melanogaster gene encoding a peptide that dramatically decreases spontaneous muscle contractions and, correspondingly, named the peptide flatline (FLT). This gene consisted of 4 exons and was cytologically localized to 32D2–3. Processing of a predicted 122 amino acid precursor would release pEVRYRQCYFNPISCF that differs from Manduca sexta allatostatin (Mas-AST) by one amino acid, Y4→F4. FLT does not act as an allatostatin. In situ tissue hybridization further suggests FLT is a novel brain-gut peptide and specifically, the measured activity indicates that it is a potent myotropin. Despite its profound myotropic effect, pupae injected with FLT eclosed.

Introduction

Juvenile hormones (JHs) are sesquiterpenoids that play critical roles in the control of insect development and reproduction. The rate of JH biosynthesis in different insect species appears to be regulated by stimulatory and inhibitory peptides, the allatotropins and allatostatins (ASTs), respectively. These factors originate principally in cells of the brain and are transported to the corpora allata (CA), the site of JH biosynthesis and release.

Three unrelated neuropeptide primary sequences that have allatostatic activity have been identified in different insect species. The first type of AST, identified and purified from cockroach brain, is defined by a core C-terminal structure Y/FXFGL/I-NH₂ [19], [26]. Neuropeptides with C-terminal homology to the cockroach ASTs comprise a large interphyletic family and have been purified from numerous insect Orders, as well as molluscs and crustaceans [3]. This peptide family appears to have adapted numerous roles that include interneuronal functions, neuromodulatory roles, myotropic and myoendocrine roles and direct action on biosynthetic pathways. The myomodulatory function appears to be conserved through evolutionary time whereas the JH inhibitory activity appears to be confined specifically to certain insect groups such as cockroaches and crickets.

In addition to the “cockroach-type” of AST, the cricket, Gryllus bimaculatus, also expresses a second type of peptide inhibitor of JH biosynthesis [13]. The cricket inhibitor is a family of C-terminal amidated nonapeptides that share sequence identity with M. sexta myoinhibitory peptide [5], Locusta migratoria locustamyoinhibiting peptide [21] and five peptides (drostatin-B) from D. melanogaster [24].

A third type of AST (Manse-AST) is a single non-amidated 15-residue peptide inhibitor of JH biosynthesis isolated from M. sexta. Mas-AST inhibits JH biosynthesis in vitro by CA from adult M. sexta 100% (0.1 μM) and Helicoverpa zea 77% (0.5 μM) [12]. In other moth species, Pseudaletia unipuncta [11] and Lacanobia oleracea [2], Mas-AST is a relatively poor inhibitor of JH biosynthesis in vitro and there is no correlation between Mas-AST expression and measured fluctuations in JH titers.

In D. melanogaster the major biosynthetic product of the CA is JH bisepoxide (JHB₃) [20]. The rate of JHB₃ biosynthesis appears to be regulated in vitro by AST(s) as brain-ring gland complexes are far less active than isolated ring glands [20]. An octapeptide sharing the C-terminal core structure of the “cockroach-type” of AST along with its corresponding receptor has been identified in D. melanogaster [4]. Antibodies raised against cockroach AST are immunoreactive with numerous cells of the D. melanogaster CNS, interneurons and endocrine cells, yet not with nerve fibers that terminate in the CA [27]. Cockroach-type ASTs purified from the blowfly, Calliphora vomitoria, also do not have an allatostatic effect on the CA of D. melanogaster but do inhibit JH production in cockroach CA [7]. Together, these observations suggest that cockroach-type ASTs do not function as regulators of the CA in flies.

In this paper we describe the molecular characterization of D. melanogaster flatline that encodes the flatline peptide, a structural orthologue of Mas-AST with myoinhibitory effects [12]. This peptide has recently been shown to be a potent prolonged inhibitor of muscle contraction in D. melanogaster rather than an inhibitor of JH biosynthesis and, thus, has been named flatline (FLT) to reflect this activity [18], [15]. FLT potently reduces the number of spontaneous contractions of D. melanogaster adult crop and pupal and young adult heart (Nichols et al. unpublished). Microinjection of FLT in white pupae slows heart contraction dramatically for several minutes. Even at nine minutes post-FLT injection, for concentrations of 10⁻⁶ M and greater, the heart rate does not recover greater than forty percent of the basal rate. In spite of the duration and profundity of the effect, these pupae eclosed and produced apparently healthy adults. Thus, to date, a peptide that functions as an AST with inhibitory activity on JH biosynthesis in D. melanogaster has yet to be uncovered.

Section snippets

Animals

D. melanogaster Oregon R strain flies were maintained on cornmeal molasses media at room temperature under a 12 h light/dark cycle.

Molecular analysis

A ³²P-end labeled oligonucleotide with the sequence: 5′-GAAGCAIGAGATIGGGTTAAGTAGCACTGICGGAAIC GCACCTG-3′ was used in initial screening of a λ charon-4A genomic library [14]. This antisense probe corresponds to the entire Manse-AST peptide sequence. Restriction fragments from the genomic clone were used to screen a head specific cDNA library constructed in λEXLX [9]

Molecular characterization of D. melanogaster FLT

To isolate a D. melanogaster Flt gene and cDNA, a λCharon 4A D. melanogaster genomic library and two head-specific cDNA libraries were screened. Seven cDNA clones were isolated and sequences represented a cDNA of 1214 nucleotides in length (Fig 1A). This size is in close correspondence with the transcript size found on Northern blots (data not shown). The cDNA contained a 5′ non-coding region of 260 nucleotides, an open reading frame of 366 nucleotides corresponding to 122 amino acid residues

Discussion

In this paper we describe the molecular characterization of D. melanogaster FLT, a structural orthologue of Mas-AST [12]. A gene encoding a FLT precursor was identified in D. melanogaster using a synthetic oligonucleotide designed against the coding sequence of Mas-AST. This gene was recently identified through the Drosophila genome project and then later shown to be expressed [25]. The gene covers a region of approximately 10 kb at cytological position 32D2–3 on the left arm of the second

Acknowledgments

This work was supported by Natural Sciences and Engineering Research Council of Canada grants (OPG0036481, WGB and A9407, SST), and a National Science Foundation grant (IBN 0076615, RN).

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^☆: The research in this manuscript was presented at the Winter Neuropeptide symposium 1999, and the Invertebrate Neuropeptide conferences 2000 and 2001.

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Drosophila melanogaster flatline encodes a myotropin orthologue to Manduca sexta allatostatin☆

Abstract

Introduction

Section snippets

Animals

Molecular analysis

Molecular characterization of D. melanogaster FLT

Discussion

Acknowledgments

Regul Pept

Insect Biochem Mol Biol

Regul Peptides

Cell

Regul Pept

Biochem Biophys Res Commun

Biochem Biophys Res Commun

Dev Biol

Peptides

Promoter elements in Drosophila melanogaster revealed by sequence analysis

Genetics

The significance of Manduca sexta allatostatin in the tomato moth Lacanobia oleracea

Ann NY Acad Sci

Allatostatinsa growing family of neuropeptides with structural, and functional diversity

Ann NY Acad Sci

Reverse physiology in Drosophilaidentification of a novel allatostatin-like neuropeptide and its cognate receptor structurally related to the mammalian somatostatin/galanin/opioid receptor family

EMBO J

A yeast artificial chromosome clone map of the Drosophila genome

Genetics