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Preliminary Findings of Structure and Expression of Opioid Receptor Genes in a Peregrine Falcon (Falco pevegrinus), a Snowy Owl (Bubo scandiacus), and a Blue-fronted Amazon Parrot (Amazona aestiva).

Abstract: To further knowledge of the physiology of opioid receptors in birds, the structure and expression of the [mu]-, [delta]-, and [kappa]-opioid receptor genes were studied in a peregrine falcon (Falco peregrinus), a snowy owl (Bubo scandiacus), and a blue-fronted Amazon parrot (Amazona aestiva). Tissue samples were obtained from birds that had been euthanatized for poor release prognosis or medical reasons. Samples were taken from the brain (telencephalon, thalamus, pituitary gland, cerebellum, pons, medulla oblongata, mesencephalon), the spinal cord and dorsal root ganglions, and plantar foot skin. Messenger RNA was recovered, and reverse transcription polymerase chain reaction (RT-PCR) was performed to generate complementary DNA (cDNA) sequences. Gene structures were documented by directly comparing cDNA sequences with recently published genomic sequences for the peregrine falcon and the blue-fronted Amazon parrot or by comparisons with genomic sequences of related species for the snowy owl. Structurally, the avian [mu]-opioid receptor messenger RNA (mRNA) species were complex, displaying differential splicing, alternative stop codons, and multiple polyadenylation signals. In comparison, the structure of the avian [kappa]-receptor mRNA was relatively simple. In contrast to what is seen in humans, the avian [delta]-receptor mRNA structure was found to be complex, demonstrating novel 3prime coding and noncoding exons not identified in mammals. The role of the [delta]-opioid receptor merits further investigation in avian species.

Key words: opioid, receptor, analgesia, avian, peregrine falcon. Falco peregrinus, snowy owl. Bubo scandiacus, blue-fronted Amazon parrot, Amazon aestiva

Introduction

The variability of opioid clinical effects observed for different molecules and between different species has been regularly hypothesized in the avian analgesia literature to be at least partially explained by differences in receptor distribution, function, or density. (1-4) Unfortunately, information about the distribution and function of opioid receptors in birds is scant. Spinal distribution of the [mu]- and [delta]-opioid receptors has been examined using immunohistochemistry in the chicken. (5) The supraspinal distribution of [mu]-, [delta]-, and [kappa]-opioid receptors has been described in the pigeon (Columba livia) and the chicken by using specific radiomarked ligands and autoradiographic receptor binding techniques. (6-11) In the pigeon forebrain, the [kappa] receptor was the most abundant of the 3 classical opioid receptors; however, no relative predominance was noticed in the midbrain. (6) The [mu] receptor was predominant in both the chicken forebrain and midbrain. (8) Similar studies have been performed in a passerine songbird (Junco hyemalis) to describe the autoradiographic distribution of [mu]-, [kappa]-, and [delta]-opioid receptors in the hypothalamus and vocal control regions. (12-15) A more recent study with reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), and immunohistochemical localization described the expression of [mu]- and [delta]-opioid receptors in song control regions of the brain of the zebra finch (Taenopygia guttata). (16)

In mammals, opioids produce analgesia by binding to opioid receptors in spinal and supraspinal tissues. (17-19) Moreover, opioid receptors are expressed on mammalian peripheral sensory endings and cutaneous cells, and local application of opioids has analgesic effects on inflammatory pain. (20) To our knowledge, the presence of opioid receptors in the peripheral tissues of birds has not been documented.

Mammalian opioid receptor genes display splicing patterns leading to several receptor variants that are functionally important in opioid-induced analgesia. (21) However, the presence and the function of opioid receptor gene splicing patterns has not been studied in birds.

To our knowledge, no reports exist on the distribution and function of opioid receptors from avian species commonly seen in zoological medicine practice, such as raptors or psittacine birds. The objective of the studies reported here was to survey the structure and expression of the ([mu], [delta]-, and [kappa]-opioid receptor genes in spinal, supraspinal, and peripheral tissues of a peregrine falcon (Falco peregrinus), a snowy owl (Bubo scandiacus), and a blue-fronted Amazon parrot (Amazona aestiva). We hypothesized that the [kappa]-opioid receptor mRNA species would be the most complex in term of structure and differential splicing.

Materials and Methods

Clinical aspects

A wild male adult peregrine falcon and a wild female adult snowy owl were obtained from the Clinique des oiseaux de proie of the Faculte de medecine veterinaire, Universite de Montreal (Saint-Hyacinthe, Quebec, Canada). Both birds were euthanatized in the raptor rehabilitation program because of poor release prognosis.

The peregrine falcon presented with left shoulder osteoarthritis secondary to a left shoulder luxation. The falcon was euthanatized by administration of euthanasia solution (0.3 mL/kg IV; T-61 Euthanasia Solution, Intervet-Schering-Plough Animal Health, Kirkland, QC, Canada) in the right jugular vein after general anesthesia was induced with isoflurane. Organs were dissected and frozen on dry ice in anticipation of RNA extraction. Whole body postmortem examination was performed.

The snowy owl presented with an open comminuted fracture of the proximal left radius and ulna. The bird was euthanatized, and tissue samples were obtained as previously described for the falcon.

A 1.5-year-old male blue-fronted Amazon parrot was obtained from a veterinary clinic. The bird presented with a 2-day history of lethargy, anorexia, voice change, and sneezing. Diagnostic and treatment plans were declined, and euthanasia was elected by the owners. Anesthesia was induced via face mask by use of isoflurane delivered in oxygen. The parrot was then euthanatized by administration of euthanasia solution (1 mL/kg IV; Dolethal, Lure, France) in the right jugular vein. Tissue samples were obtained as previously described.

Postmortem dissection and examination

Dissection of the peregrine falcon, snowy owl, and blue-fronted Amazon parrot was done immediately after death. Biological samples were rapidly dissected from the brain (telencephalon, thalamus, cerebellum, pons, medulla oblongata, mesencephalon), spinal cord (cranial, middle, and caudal parts), dorsal root ganglions (emerging from the cranial, middle, and caudal parts of spinal cord), and plantar foot skin. Samples were immediately frozen at -78.5[degrees]C on dry ice for the peregrine falcon and the snowy owl. For the blue-fronted Amazon parrot, samples were placed immediately in an RNA stabilization reagent (RNAlater, Qiagen GmbH, Hilden, Germany), frozen at -78.5[degrees]C, and mailed with dry ice to the Faculte de medecine veterinaire, Universite de Montreal. Immediately after all biological samples were obtained, a complete whole body postmortem examination was performed on each bird. For the peregrine falcon and the snowy owl, no additional lesions were revealed. For the blue-fronted Amazon parrot, postmortem examination revealed severe suppurative pericarditis, pneumonia, and airsacculitis.

RT-PCR amplification, genomic amplification, and amplicon sequencing

For routine total RNA extraction, about 30 mg of frozen tissue was processed for each organ and each bird by Qiagen RNeasy Mini Kit. The DNA from samples was digested using the Qiagen RNase-Free DNase Set, and RNA was protected with 0.5 [micro]L of RNase inhibitor (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed with the Qiagen QuantiTect Reverse Transcription Kit, using the random hexamer primers supplied with the kit. The PCR reactions were performed by Qiagen HotStarTaq Plus DNA polymerase on an Applied Biosystems GeneAmp PCR 9700 machine (Applied Biosystems, Singapore). For snowy owl genomic sequences, the Qiagen Long-Range PCR kit was used with an elongation time of 6 minutes. Primers used for PCR amplifications are given in Tables 1, 2, and 3 for the peregrine falcon, the snowy owl, and the blue-fronted Amazon parrot, respectively, and include the generic polyA primers used to identify the site of 3-prime polyadenylation. All primers were purchased from ThermoFisher oligo services (ThermoFisher Scientific, Waltham, MA, USA). In general, PCR amplifications included 45 cycles of denaturation at 95[degrees]C for 30 seconds, annealing at between 60[degrees]C and 66[degrees]C for 30 seconds, and elongation at 72[degrees]C for 2 minutes. For genomic amplifications of snowy owl sequences, an elongation step of 6 minutes was used. Wherever possible, PCR amplifications were performed spanning anticipated intron sequences. Sequencing reactions of PCR amplicons were performed with Big Dye Terminator V3.1 Cycle Sequencing procedure (Applied Biosystems, Austin, TX, USA). Sequences were read using an Applied Biosystems 3500 Genetic Analyzer (Applied Biosystems, Tokyo, Japan).

cDNA assembly and gene structure deduction

Sequences were read and cDNA sequences were assembled with 4Peaks (V.1.6.1) (Nucleobytes, Amsterdam, The Netherlands) and Serial Cloner (V.2.6.1) (serialbasics.free.fr) software, respectively. For the peregrine falcon and the blue-fronted Amazon parrot, whole genome shotgun contig libraries were available as an aid for homologous primer design and cDNA sequence assembly (NCBI BLAST, peregrine falcon taxid:8954; blue-fronted Amazon parrot taxid: 12930). Sequence homology studies were performed using the NCBI BLAST function.

Results

Partial structures of [mu]-, [kappa]-, and 5-opioid receptor genes for the 3 bird species studied are presented in Figures 1, 2, and 3, respectively. In each case, a summary of the corresponding human gene structure is given for comparison. Because of the use of the polyadenylation signal in mRNA for verification of transcripts during cloning, the 3' ends of the bird transcripts were favored, whereas the 5' ends are lacking. Reverse transcription was performed by the standard procedure of using generic poly-dT primers to take advantage of the 3' polyadenylation signal of mRNA transcripts. This was followed by PCR amplification with the use of 5' primers specific for the targeted bird opioid receptor transcripts and, again, the generic poly-dT primers. This strategy favored the cloning of the 3' end of the targeted bird opioid receptor transcripts, which was the object of the current work. In the case of the peregrine falcon and blue-fronted Amazon parrot transcripts, cloning was facilitated by having access to genomic sequences for these species. For the snowy owl, heterologous coding sequences were used initially to generate homologous snowy owl sequences, which were then used to design homologous primers for continued cloning and sequencing.

Detection of opioid receptor mRNA by RT-PCR

All 3 opioid receptor genes were expressed within the tissues examined for the 3 bird species studied.

The analysis of opioid receptor transcripts by RT-PCR revealed the presence of several splice variants derived from [mu]-, [delta]-, and [kappa]-opioid receptor mRNA. The structure of these variants is presented in Figures 1, 2, and 3 for the peregrine falcon, the snowy owl, and the blue-fronted Amazon parrot.

Two [mu]-opioid, 1 [kappa]-opioid, and 3 [kappa]-opioid receptor splice variants were found in the peregrine falcon. Three [mu]-opioid, 1 [kappa]-opioid, and 3 [kappa]-opioid receptor splice variants were found in the snowy owl. Three [mu]-opioid, 1 [kappa]-opioid, and 2 [kappa]-opioid receptor splice variants were found in the blue-fronted Amazon parrot.

The [mu]- and [kappa]-opioid receptor genes were composed of 4 exons, and the [kappa]-opioid receptor gene was composed of 3 exons in the 3 birds studied.

Exon homologies via BLAST analysis

Nucleotide sequences of exon 3 (or exons 3 and 4 for the [mu]-opioid receptor) were compared between birds and with human sequences by NCBI BLAST analysis, and homologies are presented in Table 4.

Discussion

In the study reported here, the gene structures of [mu]-, [delta]-, and [kappa]-opioid receptors for a peregrine falcon, a snowy owl, and a blue-fronted Amazon parrot are partially described. Mammalian opioid receptors are classical G protein-coupled, cell membrane-anchored proteins exhibiting 7 linked transmembrane spanning domains, an extracellular N-terminus, and an intracellular C-terminal tail. (19) Opioid receptor gene structures are well conserved across mammalian species. (7) The basic opioid receptor gene is composed of 3 exons: the first exon encodes the N-terminus and the first transmembrane domain, whereas the second and third exons each encode an additional 3 transmembrane domains, yielding the 7-transmembrane structure of traditional G protein-coupled receptors. The human p-opioid receptor gene can have a fourth exon that is unique and responsible for coding 12 amino acids at the end of the intracellular C-terminus. (21) The human 8-opioid receptor can also have a fourth exon, but this is not unique because it is identical to the terminal coding sequences of exon 3. (21) Homologies of exon 3 sequences (exon 3 and 4 sequences for the [mu]-receptor) between the birds studied and human and zebra finch sequences revealed high nucleotide homologies between the birds examined in the study presented here (95%-96% identity), high homology between birds of the study presented here and the zebra finch (92%-96% identity) and lower homologies between bird and human sequences (77%-82% identities), in accordance with their evolutionary distances. (22)

In the current study on birds, we found that all 3 opioid receptor genes for the 3 birds studied undergo alternative splicing, as is seen for the mouse, (23) rat, (24) and human. (25)

In addition to gene structure, general mRNA splicing patterns for opioid receptor genes are conserved across mammalian species. (21) The [mu]-opioid receptor gene displays the most complex splicing pattern. In the mouse, rat, and human, there are 3 major classes of [mu]-opioid receptor splice variants. The first class includes full-length variants; the second class lacks exon 1 and, thus, the first transmembrane domain, and the third class lacks exon 2 (or exon 2 and 3) to generate a single transmembrane protein encoded by exon 1. All 3 classes are functionally relevant and are important for opioid-induced analgesia. (21)

Studies performed on [mu]-opioid receptor single-transmembrane variants suggest that they play an important role in enhancing morphine analgesia, presumably through stabilization of the receptor and modulation of receptor expression levels. (21,26) Moreover, activation of a truncated, 6-membrane variant of the [mu]-opioid receptor produced analgesia without the traditional side effects of opioids. (21,27) In our study, only a few [mu]-opioid receptor splice variants (2 in the peregrine falcon, 3 in the snowy owl and blue-fronted Amazon parrot) were found, compared with the 23 variants described in human and 31 variants described in the mouse (Ensembl: Human genome version GRCh37.p13; mouse genome version GRCm38.p4). This result is understandable because our study was not exhaustive, but rather of a preliminary nature, wherein we only performed 2 to 3 rounds of RT-PCR cloning and sequencing cycles for the birds studied, generally using tissues that showed comparatively high levels of RNA content based on optical density measurements. For the [kappa]-opioid receptor, only a single cDNA species was found for the birds in our study, compared with 6 transcripts described in human and 3 transcripts described in the mouse (GRCh37.p13; GRCm38.p4). Interestingly, the 5-opioid receptor splice variants were found to be more complex in the birds studied compared with those described in mammals. Three splice variants were found in both the peregrine falcon and snowy owl, incorporating novel coding and noncoding exon 4. Two splice variants were found in the blue-fronted Amazon parrot, and again a novel coding exon 4 was demonstrated. In comparison, only 2 splice variants for the [delta]-opioid receptor have been described in humans and 1 variant described in the mouse (GRCh37.p13; GRCm38.p4). Further studies targeting the avian [delta]-opioid receptor, including expression studies in other species and pharmacological trials, are warranted to further our knowledge on its function in birds.

Mutations in the [delta]-opioid receptor have been found in mammals and were associated with higher demands for alfentanil or morphine for pain relief and decreases in the potency of morphine for analgesia. (28) We currently do not know whether polymorphisms in opioid receptor genes exist in birds; further studies, including several individuals from the same species, will be necessary to ascertain whether polymorphisms in opioid receptor genes are present in birds.

In conclusion, the current studies present a preliminary characterization of the [mu]-, [delta]-, and [kappa]-opioid receptor gene structure and expression in a subset of birds seen in clinical avian practice. The role of the [delta]-opioid receptor merits further investigation in avian species. Further studies, including relative tissue expression of opioid receptor genes in several healthy individuals from the same species, as well as studies in additional species, are warranted.

Alexis Duhamelle, DVM, IPSAV, Diana L. Raiwet, BSc, Isabelle Langlois, DYM, Dipl ABVP (Avian), Guy Fitzgerald, DVM, MSc, and David W. Silversides, DVM, PhD

From the Department of Clinical Sciences (Duhamelle, Langlois, Fitzgerald) and Department of Biomedicine (Raiwet, Silversides), Faculte de medecine veterinaire. Universite de Montreal. Saint-Hyacinthe. QC. J2S2M2, Canada. Present address (Duhamelle): Clinique Veterinaire Alliance. 8 Boulevard Godard. 33300, Bordeaux, France.

References

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(15.) Deviche P, Cotter P, Gulledge CC. Identification, partial characterization, and hypothalamic distribution of [kappa], [mu], and [delta] opioid receptors in a passerine songbird (Junco hyemalis). Brain Res. 1993:614(1-2):220-226.

(16.) Khurshid N, Agarwal V, Iyengar S. Expression of [mu]- and [delta]-opioid receptors in song control regions of adult male zebra finches (Taenopygia guttata). J Chem Neuroanat. 2009:37(3): 158-169.

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Caption: Figure 1. Organization of peregrine falcon, snowy owl, and blue-fronted Amazon parrot transcripts for [mu]-opioid receptor genes. The organization of representative human transcripts is presented for comparison. The number of transcripts currently described for a human is presented (Ensembl: GRCh37.p13; GRCm38.p4).

Caption: Figure 2. Organization of peregrine falcon, snowy owl, and blue-fronted Amazon parrot transcripts for [kappa]-opioid receptor genes. The organization of representative human transcripts is presented for comparison. The number of transcripts currently described for a human is presented (Ensembl: GRCh37.p13; GRCm38.p4).

Caption: Figure 3. Organization of peregrine falcon, snowy owl, and blue-fronted Amazon parrot transcripts for [delta]-opioid receptor genes. The organization of representative human transcripts is presented for comparison. The number of transcripts currently described for a human is presented (Ensembl: GRCh37.p13;
Table 1. Primers used for amplification of opioid receptors in a
peregrine falcon.

Kappa 1 opioid receptor primers
  Primers over intron 2-3 (460-bp amplicon):
    PF2-GATCGATACATTGCCGTGTGTCACCC
    PF5-GCAGCAGTACTGTGGGACACATCC
    Primers over stop (415-bp amplicon):

  PF4-GCGTACGCCTTCTCTCTGGTTCTCG
    PF6-GAGGACAGTAAGATGACATTTCCACG
  Primers at 5' end over intron 1-2 (540-bp amplicon):
    PF1-GTCCCTATGG ATGCCCCGGTAC AG
    PF22-CTTCGCCTTGAGAGGTGTACGGAA
  PolyA primers over intron 2-3 (340-bp amplicon):
    First PCR:
      PF1-GTCCCTATGGATGCCCCGGTACAG
      DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG(T)17
    Second PCR:
      PF2-GATCGATACATTGCCGTGTGTCACCC
      DWS190-GG ATCC A AGCTTG A ATTCTA AT ACG
  PolyA primers over intron 2-3 (364-bp amplicon):
    1[degrees] PCR:
      PF1-GTCCCTATGG ATGCCCCGGTAC AG
      DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG(T),7
    2[degrees] PCR:
      PF2-GATCGATACATTGCCGTGTGTCACCC
      DWS190-GG ATCC A AGCTTG A ATTCTA AT ACG
Mul opioid receptor primers
  Primers over introns 2-3 and 3-4 including stop (640-, 515-bp
  amplicons by alternative splicing)
    PF14-GGCCTGCCAGTTATGTTCATGGC
    PF 1[degrees]-GCTTTGAGAGCTATCGGCAGGAGAC
  Primers at 5' end over intron 2-3 (510-bp amplicon)
    PF 13-GCCCTAGCAACAAGTACTCTGCC
    PF17-CAAGAACCATCCTTGTGATTCTCCG
  PolyA primers not over an intron:
    1[degrees] PCR:
       PF36-TGGAACTTCAAGAAGCTGAAACT
       DWS 190-GGATCCAAGCTTGAATTCTAATACG
    2[degrees] PCR:
       PF37-TGGAACTTCAAGAAGCTGAAACT
       DWS191-GAATTCTAATACGACTCACTATAGGG
  PolyA primers for polyA #C over intron 2-3 and partial intron 3-4
  (1248 bp):
    1[degrees] PCR:
       PF14-GGCCTGCCAGTTATGTTCATGGC
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       PF70-GCCAGTTATGTTCATGGCAAC
       DWS 190-GGATCCAAGCTTG A ATTCTA ATACG
  PolyA primers for polyA #A, #D, #E over intron 3-4 (1487-, 1514-,
  2414-bp amplicons, respectively)
    1[degrees] PCR:
       PF 15-GGTGTTTCAG AGAGTTCTGCATCC
       DWS 135[delta]-GG ATCCA AGCTTG A ATTCTA ATACGACTCACTATAGGG(T), 7
    2[degrees] PCR:
       PF 16-CCATTTCCTCA ACCATTG AGCAGC
       DWS 190-GG ATCCA AGCTTG A ATTCTAATACG
Deltal opioid receptor primers
  PolyA primers for polyA #1 not over an intron (871-bp amplicon):
    PF9-GTCATCGTCTGGACGCTGGTGGAC
    PF76-GTCGTGGCTCCCGGATGCTGCTG
  PolyA primers for polyA #2, 3 not over an intron (479-, 1096-bp
  amplicons, respectively):
    1[degrees] PCR:
       PF9-GTCATCGTCTGGACGCTGGTGGAC
       PF78-CGTTCCTATACAGAAATGCCATC
    2[degrees] PCR:
       PF 10-CCTACGTGGTGGCCAGCCTGC AC
       PF79-GCTGGAGGTACCAGCTGAAGCTC
  PolyA primers for polyA #2, 3 not over an intron (479-, 1096-bp
  amplicons, respectively):
    1[degrees] PCR:
       PF9-GTCATCGTCTGGACGCTGGTGGAC
       PF78-CGTTCCTATACAGAAATGCCATC
    2[degrees] PCR:
       PF10-CCTACGTGGTGGCCAGCCTGCAC
       PF79-GCTGGAGGTACCAGCTGAAGCTC
  PolyA primers for polyA #4 not over an intron (353-bp amplicon):
    1[degrees] PCR:
       PF67-CTCTGGACCACATTACCTGCTG
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG(T)17
    2[degrees] PCR:
       PF68-CTAAACGATGACTGATGGGTCC
       DWS191-GA ATTCTAATACG ACTC ACTATAGGG
  Primers for 5' end over intron 1-2 (457-bp amplicon):
    PF25-GCCATCGTCATCACCGCGCTCTA
    PF27-AGGGCCTTGACTGGGTGGCACAC
  Genomic primers to amplify intron 2-3 and stop (1475-bp amplicon):
    PF26-TGACTACTACAACATGTTCACCAGT
    PF 12-GGCAGCCCCGCTGGAAGCTGCCCAC
Real-time PCR primers for amplifying peregrine falcon opioid receptor
    cDNA
  Kappa 1 primers over intron 1-2 (161-bp amplicon):
    PF68-CACAACCACACCAGCATCTC
    PF69-AGCCAGATTGAAAATGTAGATGT
  Mul primers over intron 2-3 (200-bp amplicon):
    PF70-GCCAGTTATGTTCATGGCAAC
    PF71-G AGCCAGATA ACATGCG A ACA
  Deitai primers over intron 1-2 (185-bp amplicon):
    PF72-ATCGTCATCACCGCGCTCTA
    PF73-CCAGGTCTCCATCAGGTACT
  Beta-actin primers (600-bp amplicon):
    PF 19-CTTCCC ATCCATCGTGGGTCGCC
    PF21-CCATCTCCTGCTC A A A ATCC AGGGC
  Beta-actin primers (350-bp amplicon):
    PF74-TCCCATCCATCGTGGGTCG
    PF75-CATACAGGGACAACACAGCC
  GAPDH primers (185-bp amplicon):
    PF64-GTTTCCTGTGACTTCAATGGTG
    PF66-GGCTGTGTGCTCGGCTCACTC
  GAPDH primers (160-bp amplicon):
    PF65-GCCATTCCTCCACCTTTGATG
    PF66-GGCTGTGTGCTCGGCTCA

Abbreviations: bp indicates base pair; PCR, polymerase chain
reaction; Io, primary; 2[degrees], secondary: GAPDH, glyceraldehyde
3-phosphate dehydrogenase.

Table 2. Primers used for amplification of opioid receptors in a snowy
owl.

Kappa 1 opioid receptor primers
  Heterologous primers (950-bp amplicon):
    HN15-GAAGACAGCAACCAACATCTACAT
    HN18-AGTTCAAACTGCAGTGGTAATCTG
  PolyA primers for polyA #2, 3 not over an intron (479-, 1096-bp
  amplicons, respectively):
    1[degrees] PCR:
       PF9-GTCATCGTCTGGACGCTGGTGGAC
       PF78-CGTTCCTATACAGAAATGCCATC
    2[degrees] PCR:
       PF10-CCTACGTGGTGGCCAGCCTGCAC
       PF79-GCTGGAGGTACCAGCTGAAGCTC
  PolyA primers for polyA #2, 3 not over an intron (479-, 1096-bp
  amplicons, respectively):
    1[degrees] PCR:
       PF9-GTCATCGTCTGGACGCTGGTGGAC
       PF78-CGTTCCTATACAGAAATGCCATC
    2[degrees] PCR:
       PF10-CCTACGTGGTGGCCAGCCTGCAC
       PF79-GCTGGAGGTACCAGCTGAAGCTC
  PolyA primers for polyA #4 not over an intron (353-bp amplicon):
    1[degrees] PCR:
       PF67-CTCTGGACCACATTACCTGCTG
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       PF68-CTAAACGATGACTGATGGGTCC
       DWS191-GAATTCTAAT ACGACTC ACTATAGGG
  Primers for 5' end over intron 1-2 (457-bp amplicon):
    PF25-GCCATCGTCATCACCGCGCTCTA
    PF27-AGGGCCTTGACTGGGTGGCACAC
  Genomic primers to amplify intron 2-3 and stop (1475-bp amplicon):
    PF26-TGACTACTACAACATGTTCACCAGT
    PF12-GGCAGCCCCGCTGG A AGCTGCCC AC
Real-time PCR primers for amplifying peregrine falcon opioid receptor
cDNA
  Kappa 1 primers over intron 1-2 (161-bp amplicon):
    PF68-CACAACCACACCAGCATCTC
    PF69-AGCCAGATTGAAAATGTAGATGT
  Mul primers over intron 2-3 (200-bp amplicon):
    PF70-GCCAGTTATGTTCATGGCAAC
    PF71-GAGCCAGATAACATGCGAACA
  Delta 1 primers over intron 1-2 (185-bp amplicon):
    PF72-ATCGTCATCACCGCGCTCTA
    PF73-CCAGGTCTCCATCAGGTACT
  Beta-actin primers (600-bp amplicon):
    PF19-CTTCCCATCCATCGTGGGTCGCC
    PF21-CCATCTCCTGCTCAAAATCCAGGGC
  Beta-actin primers (350-bp amplicon):
    PF74-TCCCATCCATCGTGGGTCG
    PF75-CATACAGGGACAACACAGCC
  GAPDH primers (185-bp amplicon):
    PF64-GTTTCCTGTGACTTCAATGGTG
    PF66-GGCTGTGTGCTCGGCTCACTC
  GAPDH primers (160-bp amplicon):
    PF65-GCCATTCCTCCACCTTTGATG
    PF66-GGCTGTGTGCTCGGCTCA

Abbreviations: bp indicates base pair: PCR, polymerase chain
reaction: 1[degrees], primary: 2[degrees], secondary: GAPDH.
glyceraldehyde 3-phosphate dehydrogenase.

Table 3. Primers used for amplification of opioid receptors in a
blue-fronted Amazon parrot.

Kappa I opioid receptor primers
  Homologous primers over intron 2-3 (1-kbp amplicon):
    HN15-GAAGACAGCAACCAACATCTACAT
    HN18-AGTTCAA ACTGCAGTGGTAATCTG
  PolyA primers over intron 2-3 (1.7-kbp amplicon):
    1[degrees] PCR:
       AB9-CCCTGTGAAGGCTCTGGACTTC
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       AB11-CACCTCTCAAGGCAAAGATAATC
       DWS191-GAATTCTAATACGACTCACTATAGGG
Mul opioid receptor primers
  Heterologous primers across intron 2-3 (850-bp amplicon):
    HN1-CACGAA AATGA AGACTGCCACCAA
    HN5-TGGTTAGTCCTGTCCACAGTGTTGG
  PolyA primers over intron 2-3 and intron 3-4:
    1[degrees]TPCR:
       AB13-TAAGGCCCTTG ATTTCCGTACCC
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       AB15-ATGCCAAAATTGTCAATGTCTGCA
       DWS191-GAATTCTAATACGACTCACTATAGGG
Delta 1 opioid receptor primers
  Heterologous primers over intron 2-3 (700-bp amplicon):
    HN7-CGTCCGGTACACCAAGATGGAAGAC
    HN11-TTTGAAGTTTTCAGCCAGGAAAGC
  PolyA #1 primers over intron 2-3 (2591-bp amplicon):
    1[degrees]PCR:
       AB3-CACCCGGTCAAGGCCTTGGATT
       DWS1358-GGATCCAAGCTTGAATTCTAATACG ACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       AB5-AGCCAAGGCCAAGATCATCAATGT
       DWS191-GAATTCTAATACG ACTC ACTATAGGG
  PolyA #2 primers over intron 3-4 (379-bp amplicon):
    1[degrees] PCR:
       AB7-GTCATTGTCTGGACGCTGGTGG
       DWS1358-GGATCCAAGCTTGAATTCTAATACGACTCACTATAGGG[(T).sub.17]
    2[degrees] PCR:
       AB8-AAGAATCCCTACGTGGTGGC
       DWS191-GAATTCTAATACGACTCACTATAGGG
Real-time PCR primers for amplifying blue fronted Amazon parrot
receptor cDNA
  Kappa 1 primers over intron 2-3 (175-bp amplicon):
    AB10-CACCTCTCAAGGCAAAGATAA
    AB12-ACAGATTTTCATGAAGATGTCCC
  Mul primers over intron 2-3 (190-bp amplicon):
    AB14-ATGCCAAAATTGTCAATGTCTG
    AB16-GAGTACTGGCATGATGAAGGC
  Delta 1 primers over intron 2-3 (190-bp amplicon):
    AB4-AGCCAAGGCCAAGATCATCAAT
    AB6-GGACCATGAAGGCAAAGATGAA
  Beta-actin primers (200-bp amplicon):
    HN35-CAACTGGGATGACATGGAGA
    AB1-GCACAGCCTGGATGGCCAC
  GAPDH primers (177-bp amplicon):
    PF65-GCCATTCCTCCACCTTTGATG
    AB2-GCTGTGTGTTCGGCTCACTC

Abbreviations: kbp indicates kilobase pair; PCR, polymerase chain
reaction; 1[degrees], primary; 2[degrees], secondary: bp, base
pair: GAPDH. glyceraldehyde 3-phosphate dehydrogenase.

Table 4. Nucleotide identities of opioid receptor sequences
(exon 3 or exon 3 plus exon 4) for the peregrine falcon,
snowy owl, and blue-fronted Amazon parrot compared with
human and zebra finch sequences by NCBI BLAST analysis.

                  Human   Finch   Falcon   Owl

OPRM1 (receptor mu) exons 3,4 identities
  Falcon           82      96
  Owl              81      95       95
  Amazon           84      95       96     96%
OPRK1 (receptor kappa) exon 3 identities
  Falcon           77      93
  Owl              77      93       95
  Amazon           77      93       95     96%
OPRD1 (receptor delta) exon 3 identities
  Falcon           77      92
  Owl              77      93       95
  Amazon           79      92       94     95%
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Title Annotation:Original Study
Author:Duhamelle, Alexis; Raiwet, Diana L.; Langlois, Isabelle; Fitzgerald, Guy; Silversides, David W.
Publication:Journal of Avian Medicine and Surgery
Date:Sep 1, 2018
Words:4577
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