© Friedman-Einat and Seroussi; licensee BioMed Central Ltd. 2014
Received: 26 August 2013
Accepted: 26 June 2014
Published: 3 July 2014
A LEP transcript up-regulated in lungs of ducks (Anas platyrhynchos) infected by avian influenza A virus was recently described in the Nature Genetics manuscript that reported the duck genome. In vertebrates, LEP gene symbol is reserved for leptin, the key regulator of energy balance in mammals.
Launching an extensive search for this gene in the genome data that was submitted to the public databases along with duck genome manuscript and extending this search to all avian genomes in the whole-genome shotgun-sequencing database, we were able to report the first identification of coding sequences capable of encoding the full leptin protein precursor in wild birds. Gene structure, synteny and sequence-similarity (up to 54% identity and 68% similarity) to reptilian leptin evident in falcons (Falco peregrinus and cherrug), tits (Pseudopodoces humilis), finches (Taeniopygia guttata) and doves (Columba livia) confirmed that the bird leptin was a true ortholog of its mammalian form. Nevertheless, in duck, like other domestic fowls the LEP gene was not identifiable.
Lack of the LEP gene in poultry suggests that birds that have lost it are particularly suited to domestication. Identification of an intact avian gene for leptin in wild birds might explain in part the evolutionary conservation of its receptor in leptin-less fowls.
The duck (Anas platyrhynchos) genome and transcriptome were recently reported in Nature Genetics as part of an investigation of immune-related genes implicated in the response to infection by avian influenza virus A. Using deep sequencing, the authors compared the lung transcriptomes of control and H5N1-infected ducks and used the gene symbol LEP to describe a transcript that was upregulated in the infected ducks. In vertebrates, this gene symbol is reserved for leptin, the key regulator of energy balance in mammals; however, the avian ortholog has never been established.
Leptin in poultry research
An entry in the Gene database [Gene ID: 373955] is set aside for the chicken (Gallus gallus) leptin gene. The lack of a nucleotide sequence for this entry reflects its complex history, having been cloned and its sequence then retracted [2–5]. After removing the Bos taurus sequences that contaminated the first submission of the chicken genome project and the EST database , it was finally established that no close ortholog of mammalian leptin is present in this genome. However, the obvious importance of identifying a master gene that controls appetite and fattening in poultry promoted cloning of mammalian-like leptins in turkey (Meleagris gallopavo, [GenBank: AAC32381], 95% identity to mouse leptin) and duck ([GenBank: AAT38807], 99% identity to mouse leptin). In the turkey genome housed in the ENSMBL database, there are neither annotations for LEP nor murine-like leptin sequences in its build; hence, like chickens, turkeys lack leptin.
Results and discussion
Synteny confirms leptin in birds
Leptin remains unidentifiable in domestic fowls
Examination of the recently submitted duck genome annotations revealed no gene with LEP as its symbol and no gene annotated as leptin. Moreover, BLASTN search of the WGS database using “duck leptin” [GenBank: AAT38807] or any of the novel leptin-like bird proteins described here indicated no significant similarity to leptin in this genome submission. Thus, we conclude that this gene may be also missing in duck. It is expected of the of the editorial process of a high ranking journal to ensure that when seeking a fast impact, genome publications would not turn into lists of unverified gene symbols that no one actually reads. It is further recommended that authors who deposited erroneous sequences of murine-like leptins for birds in sequence databases [GenBank: AAC32380, AAC60368, AAL35557, AAT38807, O42164, O93416] caution users of the possibility of sequence contamination. It should be also noted that 11 GenBank mRNA submissions of fish leptins with >98% identity to the mouse transcript should be similarly annotated [GenBank: DQ784814-6, AY497007, AY547279, AY547322, AY551335-9].
Moreover, a large volume of misinformation may have been generated as these murine-like leptins were the basis for studies without prior knowledge of leptin’s activity in the targeted species, including reports of the expression of the erroneous leptin gene product at the mRNA and protein levels (e.g. [9–16]). These leptins were reported to attenuate appetite, or affect other parameters related to the control of energy balance when administered to chickens [17–20], chick embryos [21–23], ovarian  and hepatoma  cells in culture or skeletal bones in an ex vivo model system .
Further BLASTN and TBLASTN searches of the WGS database using the novel avian leptin sequences revealed indications for existence of leptin in additional bird species. These include woodpecker, eagle and quails (Figure 2). Protein motifs typical of leptin were identified and annotated including leader peptide, 4-helix bundle structure and cysteine knot (Figure 2). While the leptin gene of woodpecker was apparent on an unplaced genomic scaffold [GenBank: JJRU01076739] the gene of golden eagle was much obscure. The eagle’s first coding exon (exon 2) was intact in a WGS contig [GenBank: JDSB01143511]. However, de-novo assembly of genomic raw deep-sequencing data [BioProject: PRJNA222866] was unable to extend the sequence of the last-exon-like structure [GenBank: JDSB01163119]. Yet, all the putative motifs encoded by the highly (89% identity and 91% similarity) orthologous falcon leptin gene were assembled to form disordered palindromic and repetitive contigs containing also the leptin’s syntenic gene RBM28. Such structures were also typical for the duck (data not shown). Bobwhite quail was the first galliforme with a partial exon 3 like sequence observable in a contig assembly of the WGS effort of this quail (Figure 2, [GenBank: AWGU01372785]). We used this sequence as a template for a BLAST search of the deep-sequencing data deposited for the Japanese quail in the SRA database and the related WGS assembly. The leptin gene was not identifiable in the latter, however we were able to download and assemble the matching SRA sequence reads (Figure 2), which correspond to an intact exon 3 structure. We repeated the sequence searches against the chicken genome and confirmed that even this galliforme LEP-like sequence is not detectable in Gallus gallus, in agreement with the observation that administration of a leptin antagonist had no effect on appetite and body growth of layer chickens . We could not associate any ESTs or RNA-seq reads to the quails’ leptin-like genes and moreover the role of the leptin signaling pathway may differ in galliformes. This hypothesis may also be related to the finding that the hunger hormone ghrelin , which is predominantly synthesized in the gastrointestinal tract in chickens and mammals, has been reported to have an opposite effect on appetite in chickens compared to mammals [33, 34]. Hence, galliformes provide a unique model system to decipher an alternative control mechanism of energy homeostasis and we intend to further study this in the Japanese quail.
The absence of a leptin gene in genomes related to domestic fowls seems incompatible with the presence of the leptin receptor gene, which has been cloned in chicken , turkey  and duck . Herein we report the first identification of coding sequences capable of encoding the full leptin protein precursor in birds. Identification of an intact avian gene for leptin might explain in part the evolutionary conservation of its receptor in Aves. The loss of leptin in the lineage of domestic fowls suggests that relaxing the control of appetite made these birds particularly suited to domestication.
Comparative sequence analysis
For the characterization of leptin genes not yet annotated in the avian genomes assemblies, sequence homology searches were carried out in different, publicly available database (NCBI: NR, WGS, SRA; and Ensembl) using the BLAST family of programs. Relevant sequence entries were downloaded with their quality information (FASTQ format), and reassembled using the GAP5 software . The amino acid sequences were aligned using CLUSTALW (http://www.genome.jp/tools/clustalw/) with the default parameters and the GONNET matrix; and colored using the BOXSHADE program (http://www.ch.embnet.org/software/BOX_form.html).
Sequence data accessions
The annotated sequences are available in GenBank under accessions HG425120-3.
Contribution from the Agricultural Research Organization, Institute of Animal Science, Bet Dagan, Israel, no. 678/13 is acknowledged. This research was supported by grants from the Chief Scientist of the Israeli Ministry of Agriculture & Rural Development #362-0322. We thank Andrey Shirak for performing the PCR amplification of the duck’s LEP-like sequence.
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