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Molecular phylogenetic and in silico analysis of glyceraldeyde-3-phosphate dehydrogenase (GAPDH) gene from northern bobwhite quail (Colinus virginianus)


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Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon–intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.
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Molecular Biology Reports
Molecular phylogenetic andin silico analysis
ofglyceraldeyde‑3‑phosphate dehydrogenase (GAPDH) gene
fromnorthern bobwhite quail (Colinus virginianus)
AravindanKalyanasundaram1· BrettJ.Henry1· CassandraHenry1· RonaldJ.Kendall1
Received: 15 October 2020 / Accepted: 28 January 2021
© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021
Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi
and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have
attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper
reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic
enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite
GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified
in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR prod-
ucts. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame
(ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon–intron
pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes.
Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like
the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR
primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this pre-
liminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first
to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there
is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary
qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.
Keywords Bobwhite· Exon· GAPDH· Gene expression· Intron· qPCR· Reference gene
The northern bobwhite (Colinus virginianus; hereafter
bobwhite) is an economically important game bird in North
America and is very popular among hunters [1, 2]. Although
declining bobwhite populations have been noted for over
a century [3] nationwide conservation efforts for bobwhite
began only a couple decades ago as a result of the articles
published by Brennan and co-workers [4, 5]. However, bob-
white have exhibited a relatively steady decline of 3.5% per
year over the last 50years despite this effort and is currently
declining at a faster rate that 94% of all other declining
birds [6]. This decline has been attributed to many factors
including habitat loss, fragmentation, agricultural practices,
extreme weather events, and more recently parasitic infec-
tion [4, 5, 7]. Early hypotheses stated that parasitic infection
had minimal impacts on bobwhite populations [8] but as
populations continued to decline, more studies have been
conducted in an effort to assess the potential impact of para-
sitic infections in bobwhite [911].
Two helminths, eyeworm Oxyspirura petrowi and caecal
worm Aulonocephalus pennula, have been demonstrated to
have high prevalence of 66% and 91%, respectively, in wild
bobwhite in the Rolling Plains ecoregion of Texas [12]. A
number of studies have been published in relation to preva-
lence and diagnostic techniques of these parasitic infections
* Ronald J. Kendall
1 The Wildlife Toxicology Laboratory, Texas Tech University,
Lubbock, TX79409-3290, USA
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Molecular Biology Reports
1 3
in wild bobwhite [1317]. However, the available knowledge
on host parasite interaction in this species is limited due to
very few studies having utilized histological or proteomic
approaches to assess the host response to these parasites [12,
18]. A pilot study on host immune response demonstrated
the ability to quantify immune response through quantita-
tive PCR (qPCR) analysis of cytokine and toll-like receptor
(TLR) gene expression in bobwhite challenged intramuscu-
larly with crude glycoproteins from both O. petrowi and A.
pennula [18]. Aside from the aforementioned study, little
has been done to unravel how the bobwhite immune system
responds to infection from these two helminth parasites.
Therefore, more research must be conducted on the host
parasite interaction of bobwhite to understand the implica-
tions of these infections.
Furthermore, very few functional genes for bobwhite have
been completely or partially characterized with sequences
available within the GenBank database. This difficulty is due
to this lack of information for many genes and can constrain
our ability to conduct research on host parasite interactions
for not only bobwhite, but also the majority of avian species
aside from chicken (Gallus gallus). Consequently, it is dif-
ficult to study the gene expression profile or immunological
aspects of bobwhite with insufficient genomic information.
However, for species that have a more complete understand-
ing of expressed genes and their corresponding sequences,
such as chicken, assessing gene expression by qPCR is a
widely used and reliable technique for studying host parasite
interactions [1921].
In qPCR, controlling or avoiding error is critical in gen-
erating reliable data and is of particular importance for
gene expression analyses [22]. One widely used approach
to achieve this is using housekeeping or reference genes to
normalize the variation [23]. Reference genes should have a
constant gene expression in various organs and under vari-
ous physiological conditions [24]. Some genes alter their
expression in different physiological conditions based on
the experimental study and therefore would not be good
candidates as a reference gene [25]. As such, choosing an
appropriate reference gene is important before conduct-
ing a study on gene expression analysis. Currently several
genes are often used as reference or reference genes in qPCR
analysis, and GAPDH (glyceraldeyde-3-phosphate dehydro-
genase) is one of the most widely used reference gene in
experimental studies for many organisms due to its constant
expression [24, 26, 27].
GAPDH, is a ubiquitous glycolytic enzyme with a key
role in energy production by conversion of glyceralde-
hyde-3-phosphate (G3P) to 1,3-bisphosphoglyceric acid
(BPGA) in the presence of nicotinamide adenine dinucleo-
tide (NAD+) and inorganic phosphate [28, 29]. GAPDH is
mainly located in the cytosol of cells, which also found in
nucleus, mitochondria, and cytoskeleton [30]. Apart from
glycolytic pathways, GAPDH plays a variety of roles in
other cellular processes such as cell death, DNA repair, oxi-
dative stress repair, and is a critical regulator of cancer cells
[3133]. Its ubiquity and multiple functions are what results
in its constant expression, and therefore make it a quality
candidate for use as a reference gene.
In this study, we obtained the full-length gene sequence
of bobwhite GAPDH. Using this complete GAPDH gene
sequence, we predict the evolutionary relationship, exon
intron pattern, proteomic structural analysis, and suitability
of its use as a reference gene. Results of this study provide
not only genetic information and structural similarities to
other species’ GAPDH, but also describes the potential of
GAPDH to be a reference gene in bobwhite gene expression
Materials andmethods
Study area andsample collection
The experimental study area of the present manuscript is
consistent with the study area described in Dunham etal.
[13]. The broader range of application (e.g., Rolling Plains)
was described by Rollins [7]. Wild bobwhite were collected
in July of 2019 from the same study area, in the same man-
ner, and using the same techniques previously described by
Dunham etal. [13].
RNA extraction
For primer optimization, 100mg of quail breast tissue was
collected from wild bobwhite and stored in RNAlater™ sta-
bilization solution (Invitrogen, USA) with 1:10 ratio (1g of
tissue in 10ml of RNAlater™). RNeasy Mini Kit (Qiagen,
USA) was used to extract total RNA from the breast tissue
according to manufacturer’s instructions. Following elu-
tion, quantity and quality of total RNA was estimated by
absorbance at 260–280nm using Qubit 3.0 and then stored
at –80°C for further use. cDNA was synthesized from total
RNA using the QuantiTect Reverse Transcription kit (Qia-
gen, USA) according to manufacturer’s instructions, with
quantity and quality of the cDNA estimated using the Qubit
3.0 as described previously. Synthesized cDNA was stored at
–40°C prior to primer optimization and full-length GAPDH
gene amplification.
Primer design
A gene specific set of PCR primers (cvGAPDH F 5ʹ-ATG
CTC CTT GGA TGC CAT GTG-3ʹ) for the complete bobwhite
GAPDH gene were designed using IDT Oligo Analyzerand
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Molecular Biology Reports
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utilizing sequences retrieved from northern bobwhite whole
genome project (PRJNA188411) [34, 35]. This sequence
was confirmed by comparison to GAPDH sequences from
other avian species through multiple sequence alignment
using ClustalW2. A set of qPCR primers (qGAPDH F
5ʹ-GGT GCA CGA CGC ATT GCT GAC-3ʹ) was also designed
spanning an exon-exon junction in the GAPDH gene to use
as a reference gene in bobwhite gene expression analyses.
PCR amplification ofbobwhite GAPDH
Newly synthesized GAPDH primers were optimized using
total RNA template extracted from bobwhite breast tis-
sue. Gradient PCR reactions were performed with 5μL of
MyTaq™ Red Mix (Bioline, USA), 1μL of 10μM forward,
1μL of 10μM reverse primer, 1μL of cDNA template, and
2μL of nuclease free water for 10µL reactions. PCR run
conditions were as follows: 95°C for 3min; 30 cycles of
95°C for 40s, 50–60°C for 1min, and 72°C for 30s; and a
final extension step of 72°C for 5min. Gene amplification
was confirmed by 1.5% agarose gel electrophoresis.
Sequence analysis
The GAPDH PCR product was purified from the agarose
gel and sequenced in both directions using their respec-
tive forward and reverse primers. Raw sequences were
trimmed to remove poor reads using BioEdit 7.2 and these
trimmed sequences were used for BLAST analysis. The
processed forward and reverse sequences were overlapped
to obtain full-length sequence using EMBOSS Merger. The
intron–exon distribution was predicted using HMMgene tool
( ces/HMMge ne/). The genomic
region of bobwhite and chicken GAPDH were retrieved from
NCBI [34, 36] and Splign (
sutil s/splig n/splig n.cgi) was then used to predict exon–intron
patterns of the GAPDH gene by using cDNA and genomic
regions. We estimated the intron similarity between mul-
tiple avian species such as chicken (Gallus gallus), new-
world quail (northern bobwhite, scaled quail and marbled
wood quail) and old-world quail (Japanese quail) by multiple
alignment search using the genomic region of GAPDH. We
predicted microsatellites in intron one using microsatel-
lite repeats finder (http://insil /micro
satel lites /info). The complete open reading frame (ORF) of
GAPDH sequence obtained in this study was submitted in
DDBJ (LC569866).
Phylogenetic analysis
The evolutionary relationship of bobwhite GAPDH was
inferred by MEGA X using the maximum likelihood (ML)
method and Tamura–Nei model [37]. Full gene sequences
of GAPDH from 76 other avian species were retrieved from
the NCBI GenBank database. Multiple alignments of the
bobwhite GAPDH sequences were completed with these
76 sequences using ClustalW2. Sequences were taken from
order Passeriformes, Accipitriformes, Columbiformes,
Gruiformes, Galliformes, and Anseriformes for construct-
ing a phylogenetic tree for avian GAPDH. Short sequences
(< 500bp) were removed to ensure high quality results from
the phylogenetic tree. GAPDH from burflower Neolamarckia
cadamba (KY922898) and benthi Nicotiana benthamiana
(AB937979) from kingdom Plantae were specified as out-
groups in this analysis. Initial tree(s) for the heuristic search
were obtained automatically by applying Neighbor-Join and
BioNJ algorithms to a matrix of pairwise distances esti-
mated using the Maximum Composite Likelihood (MCL)
approach, and then selecting the topology with superior log
likelihood value. The tree is drawn to scale, with branch
lengths measured in the number of substitutions per site.
There were a total of 1926 positions in the final dataset. The
bootstrap value was set at 1000 to represent strong evolution-
ary relationships between bobwhite and other avian species
of the phylum Chordata.
Three‑dimensional structure
Homology modeling was used to predict the three-dimen-
sional (3D) structure of bobwhite GAPDH. This was con-
ducted following steps such as template selection, sequence
alignment, backbone model building, loop modeling, side
chain refinement, model refinement using energy function,
and model evaluation. A total of 2179 templates were gen-
erated, and 50 templates were then found to match with the
bobwhite GAPDH sequence. These templates were screened
and the template with the highest quality was then selected
for building structure models. A crystal structure of GAPDH
from wild pig Sus scrofa (5tso.1) showed 86.49 identity to
bobwhite GAPDH. Three-dimensional structure of the bob-
white GAPDH was built based on the X-ray crystal structure
of GAPDH from wild pig Sus scrofa (5tso.1) available in the
protein data bank (https :// The model for the
bobwhite GAPDH protein was built with SWISS-MODEL
(https ://swiss model .expas, and WHATIF (https ://
swift f/WIF1_4.html). The predicted
model reliability was assessed by What-Check and PRO-
CHECK. The predicted model was then visualized using
PyMol (https ://pymol .org/2/). The functional motif or active
site of GAPDH was predicted using Motif finder.
GAPDH primer set forqPCR
Quantitative PCR was performed using an API StepOne-
Plus Real-Time PCR System in a 96-well microplate with
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Molecular Biology Reports
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a final reaction volume of 10µL. Amplification of GAPDH
(143bp) was done using blood samples collected from
experimental bobwhites. Total RNA was extracted using
QIAamp RNA Blood Mini Kit as per the manufacturer’s
instructions. Quantitative PCR was carried out as described
by Kalyanasundaram etal. [18] using the qGAPDH F and
qGAPDH R primers. GADPH expression was analyzed by
qPCR in two groups of ten bobwhite. The treated group
contained bobwhite that were experimentally infected with
O. petrowi as described in Kalyansundaram etal. [38] and
the control group were uninfected. Statistical analyses were
conducted using the Real Statistics Resource Pack software
(Release 7.2). Data normality was assessed for normality
and outliers using the Shapiro–Wilk test and Grubb’s test,
respectively. A two-factor ANOVA was used to evaluate
whether sex and/or treatment had a significant effect on the
GAPDH qPCR Cq values.
The PCR amplification product of bobwhite GAPDH was
obtained at an annealing temperature of 60°C. A single
amplification band was observed in the gel analysis. The
sequence results of this product revealed a 1002bp size
bobwhite GAPDH gene (Fig.1a, b). BLAST results on
the gene sequence showed high sequence identity to Pha-
sianus colchicus (ring-necked pheasant) (97.6%), Numida
Meleagris (helmeted guineafowl) (97.6%), and Gallus gallus
(chicken) (97.5%) at the nucleotide level.
The GADPH gene in bobwhite and chicken have a similar
number of exons and have comparable intron length, except
for the intron located between exon one and two (Fig.2).
In chicken, the length of this intron is 623bp [36] but this
intron is 2001bp in bobwhite. Utilizing mRNA from this
study and the GAPDH genomic region from bio-project
(PRJNA188411), we predicted eleven exons and ten introns
in the bobwhite GAPDH (Fig.2). In total, 13 microsatel-
lites were found within intron one of the GAPDH gene
(Table1). These microsatellite regions were widely distrib-
uted throughout the intron. All microsatellites were dinu-
cleotide repeats except the 12th microsatellite which is a
tri-nucleotide repeat. The role of these microsatellites was
not determined as this was beyond scope of this study. We
predicted the sequence identity with an average of 90–96%
of the bases at the same location within the chicken and new
world quail. However, surprisingly we found considerable
sequence variation between new and old-world quail within
the introns. The highest identity (84.7%) was found with
intron four and the least identity (24.9%) found in intron one.
Fig. 1 DNA agarose gel of the
PCR products. a PCR amplifica-
tion of complete GAPDH gene
(1002bp). b PCR amplification
of GAPDH gene product using
qGAPDH primers (143bp)
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A phylogenetic tree (Fig.3) was constructed based on
the full-length gene sequence of bobwhite GAPDH from
this study along with GAPDH genetic information for
other avian species acquired from the GenBank database.
Results of the evolutionary tree shows that the GAPDH gene
sequences for Numida Meleagris (helmeted guineafowl),
Coturnix japonica (Japanese quail), Phasianus colchicus
(common pheasant), Meleagris gallopavo (wild turkey), and
Gallus gallus (chicken) were closely related to the bobwhite
sequence. This is an expected result as these species are
closely related as they all belong to the order Galliformes.
Based upon the 1002 bp sequence, the bobwhite
GAPDH gene encodes for a 334 amino acid protein.
The molecular weight of this protein is estimated to be
35.95kDa with a pI of 9.06. The active site of the GAPDH
enzyme (ASCTTNCL) was determined to be at the 148
to 155 amino acid position and predicted to function as a
nucleophile. Based on the 3D model of GAPDH predicted
using Swiss-PDB model, a crystal structure of GAPDH
from wild pig Sus scrofa (5tso.1) showed 86.49% iden-
tity to bobwhite GAPDH in target–template alignment.
The model was then visualized using the PyMol (Fig.4a,
b). Homology modeling of GAPDH predicts a homo-
tetrameric structure with four ligands that each interact
with an NAD+ molecule. The Qualitative Model Energy
Analysis (QMEAN) local quality score estimates each
residue of the model (x-axis) and the expected similar-
ity to the native structure (y-axis). Usually, a score below
0.6 indicates a low quality model. The GAPDH model
residues all score above 0.6 which implies that this model
is of acceptable quality (Fig.4c). The QMEAN Z score
estimates the model quality through assessment of the
agreement between the predicted model and experimental
structures of similar size. Scores below −4.0 are consid-
ered indicative of a low quality models. In this predicted
model, the QMEAN Z score (−0.41) is above the −4.0
threshold. (Fig.4d).
To assess the difference in bobwhite GAPDH gene
expression, a preliminary qPCR was conducted on samples
from 20 bobwhite, with 10 from bobwhite infected with
O. petrowi and 10 uninfected control bobwhite. The mean
Cq values were in agreement between the infected group
(28.62 ± 3.53) and the control group (29.128 ± 2.67). The
increased standard deviation within the infected group is
likely due to the presence of a single outlier within the
dataset, which was confirmed through the Grubb’s test.
However even with that outlier included in the data set, the
ANOVA analysis of bobwhite GAPDH expression showed
no significant difference in Cq values between sexes or
infection status with the associated p-values being 0.35
and 0.73, respectively.
Fig. 2 Comparison of intron–exon pattern between bobwhite and
chicken GAPDH genomic region. The figure shows exons as yellow
boxes and introns as yellow lines. Exon 1 has been highlighted as a
blue box in both bobwhite and chicken. The length of the intron 1
of bobwhite and chicken GAPDH showed with black arrow. (Color
figure online)
Table 1 Prediction of microsatellites from intron one
Position in Intron1
Cycle Repeats Sequence
65 2 4 GGG GGG GG
82 2 10 GGG GGG GGG
134 2 3 CCC CCC
142 2 4 CCC CCC CC
313 2 3 CCC CCC
483 2 3 CCC CCC
653 2 3 CCC CCC
823 2 3 CCC CCC
993 2 3 CCC CCC
1163 2 3 CCC CCC
1333 2 3 CCC CCC
1503 2 3 CCC CCC
1709 2 3 GTG TGT
1968 3 3 CTC CTC CTC
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GAPDH is an enzyme present in most organisms and its
primary function is to catalyze a step in the breakdown of
glucose through the glycolysis pathway. Through this role,
it maintains relatively constant levels of expression which
has made it one of the most utilized reference genes for gene
expression studies in many organisms including in avian spe-
cies such as chicken and Japanese quail [39, 40]. While the
bobwhite is from the same order, Galliformes, as both the
chicken and Japanese quail, much less is known specifically
about GAPDH and its expression levels in bobwhite. As the
interest in studying the bobwhite immune system is increas-
ing, it is essential to identify genes that can be utilized as
reference genes for gene expression studies in bobwhite.
In this study, we are developing the understanding of
GAPDH in bobwhite, and to do this, we first obtained the
full length ORF of the GAPDH sequence for bobwhite. We
then used this sequence to determine the evolutionary rela-
tionship of bobwhite GAPDH. The phylogenetic analysis
Fig. 3 Phylogenetic analysis of bobwhite GAPDH gene using Maxi-
mum Likelihood methods. The evolutionary history was inferred
using the ML method based on the Tamura–Nei model. Bootstrap
values above 50 are shown in the tree. All positions containing gaps
and missing data were eliminated. Species name and their nucleotide
accession numbers were included in the tree. There were 1926 posi-
tions for the final dataset. Evolutionary analyses were conducted in
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revealed high similarity (90–96%) of bobwhite GAPDH to
other avian GAPDH particularly to the order Galliformes.
Subsequently, the intron–exon analysis of bobwhite GAPDH
showed high similarity to chicken GAPDH aside from the
increased length of the intron one (2001bp in bobwhite ver-
sus 623bp in chicken) between exon one and two at the 5
This configuration of a gene, with intron one being signif-
icantly longer than the following introns, has been reported
in many plant and animal species [41]. Lengthened introns
at the 5 ends have been reported to have a significant role
directly or indirectly in gene transportation, regulation, and
alternative splicing [42, 43]. Identifying the reasons for this
increased intron length was beyond the scope of this study
as it must be done experimentally due to the lack of in silico
methods for predicting intron function [44]. However, the
increased length of intron one and the microsatellites found
within could have some important role in GAPDH regulation
in the bobwhite. This could be critical to understand, as stud-
ies have suggested that microsatellites can alter the expres-
sion profile between reference and tissue-specific genes [45].
Additionally, we used the ORF sequence to generate the
theoretically encoded protein, which predicted a 35.95kDa
protein. Utilizing this information and homology modeling,
we predicted that bobwhite GAPDH exists primarily as a
homo-tetramer. This is in agreement with what one would
expect for GAPDH as it is typically reported to be ~ 37kDa
and its primary active form is that of a homo-tetramer [29].
Additionally, the active site is predicted to have four ligands
each capable of binding a single NAD+ for catalytic pro-
cesses, which further solidifies the similarities of bobwhite
GAPDH to other avian GAPDH [36].
The high similarity displayed in these analyses add to the
potential of GAPDH as a reference gene for gene expres-
sion studies in bobwhite. To that end, a preliminary study
assessing Cq value variation of bobwhite GAPDH in qPCR
Fig. 4 Molecular model of bobwhite GAPDH. a The homomeric
structure of the bobwhite GAPDH predicted by homology modeling.
b Bobwhite GAPDH monomer with arrows indicating the α-helix,
β-pleated sheet, and the active site. c and d QMEANZ and QMEAN
score of the predicted model
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analyses was conducted. This demonstrated no significant
difference in the generated Cq values between two groups
of bobwhite. This is a necessary characteristic for a gene
expression reference gene. However, further studies would
need to be conducted to thoroughly validate GAPDH’s
potential as a reference gene. These would include verifying
the stability of GAPDH gene expression between male and
female bobwhite, in which tissues the expression is stable,
and if the expression is stable under a range of treatments.
This study is the first to elucidate information about bob-
white GAPDH. Through experimentation and modeling, we
have confirmed that the bobwhite GAPDH gene has high
sequence identity to other avian GAPDH, including chicken,
and the encoded protein is of similar size and structure as
well. As a result, we performed a preliminary qPCR as a
test of GAPDH’s potential for use as a reference gene in
bobwhite gene expression studies. This preliminary experi-
ment showed no statistical significance in the variation of Cq
values between the uninfected and infected groups. However,
further studies would need to be conducted to thoroughly
validate GAPDH’s potential as a reference gene.
Funding This research received funding and support form Park Cit-
ies Quail Coalition (Grant No. 24A125) and the Rolling Plains Quail
Research Foundation (Grant No. 23A751).
Data availability All data generated or analyzed during this study are
included in this manuscript. Sequencing data obtained from this study
has been submitted in DNA Data Bank of Japan (DDBJ) (Acc No.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
Ethical approval This experiment was approved by Texas Tech Uni-
versity Animal Care and Use Committee under protocol 18044-05 and
16071-08. All bobwhites were trapped and handled according to Texas
Parks and Wildlife permit SRP-0715-095.
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Background: Oxyspirura petrowi (Spirurida: Thelaziidae), a heteroxenous nematode of birds across the USA, may play a role in the decline of the northern bobwhite (Colinus virginianus) in the Rolling Plains Ecoregion of West Texas. Previous molecular studies suggest that crickets, grasshoppers and cockroaches serve as potential intermediate hosts of O. petrowi, although a complete study on the life-cycle of this nematode has not been conducted thus far. Conse-quently, this study aims to improve our understanding of the O. petrowi life-cycle by experimentally infecting house crickets (Acheta domesticus) with O. petrowi eggs, feeding infected crickets to bobwhite and assessing the life-cycle of this nematode in both the definitive and intermediate hosts.Methods: Oxyspirura petrowi eggs were collected from gravid worms recovered from wild bobwhite and fed to house crickets. The development of O. petrowi within crickets was monitored by dissection of crickets at specified intervals. When infective larvae were found inside crickets, parasite-free pen-raised bobwhite were fed four infected crickets each. The maturation of O. petrowi in bobwhite was monitored through fecal floats and bobwhite necropsies at specified intervals.Results: In this study, we were able to infect both crickets (n = 45) and bobwhite (n = 25) with O. petrowi at a rate of 96%. We successfully replicated and monitored the complete O. petrowi life-cycle in vivo, recovering embryonated O. petrowi eggs from the feces of bobwhite 51 days after consumption of infected crickets. All life-cycle stages of O. petrowi were confirmed in both the house cricket and the bobwhite using morphological and molecular techniques.Conclusions: This study provides a better understanding of the infection mechanism and life-cycle of O. petrowi by tracking the developmental progress within both the intermediate and definitive host. To our knowledge, this study is the first to fully monitor the complete life-cycle of O. petrowi and may allow for better estimates into the potential for future epizootics of O. petrowi in bobwhite. Finally, this study provides a model for experimental infection that may be used in research examining the effects of O. petrowi infection in bobwhite. (PDF) Life-cycle of Oxyspirura petrowi (Spirurida: Thelaziidae), an eyeworm of the northern bobwhite quail (Colinus virginianus). Available from: [accessed Nov 22 2019].
Full-text available
Helminth parasites have been a popular research topic due to their global prevalence and adverse effects on livestock and game species. The Northern bobwhite (Colinus virginianus), a popular game bird in the USA, is one species subject to helminth infection and has been experiencing a decline of > 4% annually over recent decades. In the Rolling Plains Ecoregion of Texas, the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) helminths are found to be highly prevalent in bobwhite. While there have been increasing studies on the prevalence, pathology, and phylogeny of the eyeworm and caecal worm, there is still a need to investigate the bobwhite immune response to infection. This study utilizes previously sequenced bobwhite cytokines and toll-like receptors to develop and optimize qPCR primers and measure gene expression in bobwhite intramuscularly challenged with eyeworm and caecal worm glycoproteins. For the challenge experiments, separate treatments of eyeworm and caecal worm glycoproteins were administered to bobwhite on day 1 and day 21. Measurements of primary and secondary immune responses were taken at day 7 and day 28, respectively. Using the successfully optimized qPCR primers for TLR7, IL1β, IL6, IFNα, IFNγ, IL10, and β-actin, the gene expression analysis from the challenge experiments revealed that there was a measurable immune reaction in bobwhite in response to the intramuscular challenge of eyeworm and caecal worm glycoproteins.
Full-text available
Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein–protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.
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RT-qPCR is the gold standard for candidate gene expression analysis. However, the interpretation of RT-qPCR results depends on the proper use of internal controls, i.e., reference genes. Japanese quail is an agronomic species also used as a laboratory model, but little is known about RT-qPCR reference genes for this species. Thus, we investigated 10 putative reference genes (ACTB, GAPDH, PGK1, RPS7, RPS8, RPL19, RPL32, SDHA, TBP and YWHAZ) in three different female and male quail tissues (liver, brain and pectoral muscle). Gene expression stability was evaluated with three different algorithms: geNorm, NormFinder and BestKeeper. For each tissue, a suitable set of reference genes was defined and validated by a differential analysis of gene expression between females and males (CCNH in brain and RPL19 in pectoral muscle). Collectively, our study led to the identification of suitable reference genes in liver, brain and pectoral muscle for Japanese quail, along with recommendations for the identification of reference gene sets for this species.
Full-text available
A picture is beginning to emerge from a variety of organisms that for a subset of genes, the most important sequences that regulate expression are situated not in the promoter but rather are located within introns in the first kilobase of transcribed sequences. The actual sequences involved are difficult to identify either by sequence comparisons or by deletion analysis because they are dispersed, additive, and poorly conserved. However, expression-controlling introns can be identified computationally in species with relatively small introns, based on genome-wide differences in oligomer composition between promoter-proximal and distal introns. The genes regulated by introns are often expressed in most tissues and are among the most highly expressed in the genome. The ability of some introns to strongly stimulate mRNA accumulation from several hundred nucleotides downstream of the transcription start site, even when the promoter has been deleted, reveals that our understanding of gene expression remains incomplete. It is unlikely that any diseases are caused by point mutations or small deletions that reduce the expression of an intron-regulated gene unless splicing is also affected. However, introns may be particularly useful in practical applications such as gene therapy because they strongly activate expression but only affect the transcription unit in which they are located.
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The northern bobwhite quail (Colinus virginianus) is a popular gamebird in the Rolling Plains Ecoregion of West Texas. However, there has been a population decline in this area over recent decades. Consistent reports indicate a high prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula), which may be of major influence on the bobwhite population. While research has suggested pathological consequences and genetic relatedness to other pathologically significant parasites, little is known about the influence of climate on these parasites. In this study, we examined whether seasonal temperature and precipitation influences the intensity of these parasites in bobwhite. We also analyzed quantitative PCR results for bobwhite feces and cloacal swabs against temperature and precipitation to identify climatic impacts on parasite reproduction in this region. Multiple linear regression analyses were used for parasite intensity investigation while binary logistic regression analyses were used for parasite reproduction studies. Our analyses suggest that caecal worm intensity, caecal worm reproduction, and eyeworm reproduction are influenced by temperature and precipitation. Temperature data was collected 15, 30, and 60 days prior to the date of collection of individual bobwhite and compared to qPCR results to generate a temperature range that may influence future eyeworm reproduction. This is the first preliminary study investigating climatic influences with predictive statistics on eyeworm and caecal worm infection of northern bobwhite in the Rolling Plains.
Full-text available
Physaloptera spp. are common nematodes found in the stomach and muscles of mammals, reptiles, amphibians, and birds. Physaloptera spp. have a complicated life cycle with multiple definitive hosts, arthropod intermediate hosts, aberrant infections, and possible second intermediate hosts or paratenic hosts. For example, Physaloptera sp. larvae have been found within the tissues of wild northern bobwhite quail (Colinus virginianus), and it is suspected that quail may serve as paratenic or secondary hosts of these parasites. However, because it is not known what role quail play in the life cycle of Physaloptera spp. and descriptions of Physaloptera spp. larvae are limited, molecular tools may be beneficial when identifying these helminths. In this study, we generated primers using universal nematode primers and obtained a partial mitochondrial cytochrome oxidase 1 (COX 1) sequence. Morphological identification of Physaloptera sp. in bobwhite was confirmed via polymerase chain reaction (PCR), and a phylogenetic tree was constructed using the maximum likelihood method. BLAST analysis revealed a strong identity to other Physaloptera spp. and the phylogenetic tree placed all Physaloptera spp. in the same cluster. We also documented a marked increase in Physaloptera infections in bobwhite from 2017 to 2018, and the similarity of these parasites to Onchocerca volvulus and Wuchereria bancrofti may give insight into the increased prevalence we observed. This study demonstrates the usefulness of molecular techniques to confirm the identity of species that may lack adequate descriptions and provides new insight for the diagnosis and potentially overlooked significance of Physaloptera sp. infections of bobwhite in the Rolling Plains ecoregion of Texas.
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The molecular evolutionary genetics analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from free of charge.
Full-text available
We have been monitoring wild Northern bobwhite quail (Colinus virginianus) on a research transect in Mitchell County, Texas. We captured a total of 51 bobwhites in March-May of 2016 and 2017 and examined them for eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) infections. In March 2017, bobwhites averaged 15 ± 10 eyeworms and 269 ± 90 caecal worms, and by mid-April averages had increased to 18 ± 13 eyeworms and 372 ± 144 caecal worms. These averages were much higher than those observed in March 2016 (11 ± 13 eyeworms and 160 ± 57 caecal worms) and April 2016 (12 ± 12 and 216 ± 56, respectively). We observed a precipitous decline in quail numbers by late April 2017, and average infection had dropped to 7 ± 2 eyeworms and 252 ± 109 caecal worms. The number of trapping sessions needed to capture one bobwhite also increased from 14.26 in 2016 to 36.46 in 2017. These observations warrant further investigation into the effects these helminth parasites may have on bobwhites and their populations within the Rolling Plains.
One hundred and sixty-one northern bobwhites ( Colinus virginianus ; hereafter ‘bobwhite’) were examined from the Rolling Plains ecoregion of Texas and western Oklahoma from 2011 to 2013. Complete necropsies yielded 13 species, of which two are new host ( Gongylonema phasianella ) and region ( Eucoleus contortus ) records and three ( Dispharynx nasuta , Tetrameres pattersoni and Oxyspirura petrowi ) are known to cause morbidity and mortality. Of the species found, Aulonocephalus pennula commonly occurred, Oxyspirura petrowi was intermediate in prevalence, and the remaining species were rare. Species richness was similar compared to studies from the southeastern U.S., but higher than studies from the same region. In addition, 12 of the 13 species were heteroxenous helminths, supporting the theory that heteroxenous helminths in semi-arid regions are more successful than monoxenous helminths. Prevalence and abundance of A. pennula and O. petrowi were higher in adult bobwhites than in juveniles. Abundance of A. pennula and O. petrowi was higher at southern locations compared to northern locations in the study area. Our study is the first to provide a current assessment of the bobwhite helminth community across the Rolling Plains ecoregion of the U.S.