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Changes in Neuropeptide Prohormone Genes among Cetartiodactyla Livestock and Wild Species Associated with Evolution and Domestication

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The impact of evolution and domestication processes on the sequences of neuropeptide prohormone genes that participate in cell–cell signaling influences multiple biological process that involve neuropeptide signaling. This information is important to understand the physiological differences between Cetartiodactyla domesticated species such as cow, pig, and llama and wild species such as hippopotamus, giraffes, and whales. Systematic analysis of changes associated with evolutionary and domestication forces in neuropeptide prohormone protein sequences that are processed into neuropeptides was undertaken. The genomes from 118 Cetartiodactyla genomes representing 22 families were mined for 98 neuropeptide prohormone genes. Compared to other Cetartiodactyla suborders, Ruminantia preserved PYY2 and lost RLN1. Changes in GNRH2, IAPP, INSL6, POMC, PRLH, and TAC4 protein sequences could result in the loss of some bioactive neuropeptides in some families. An evolutionary model suggested that most neuropeptide prohormone genes disfavor sequence changes that incorporate large and hydrophobic amino acids. A compelling finding was that differences between domestic and wild species are associated with the molecular system underlying ‘fight or flight’ responses. Overall, the results demonstrate the importance of simultaneously comparing the neuropeptide prohormone gene complement from close and distant-related species. These findings broaden the foundation for empirical studies about the function of the neuropeptidome associated with health, behavior, and food production.
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Citation: Southey, B.R.;
Rodriguez-Zas, S.L. Changes in
Neuropeptide Prohormone Genes
among Cetartiodactyla Livestock and
Wild Species Associated with
Evolution and Domestication. Vet.
Sci. 2022,9, 247. https://doi.org/
10.3390/vetsci9050247
Academic Editors: Lucianna
Maruccio, Carla Lucini and Neil
Evans
Received: 3 March 2022
Accepted: 19 May 2022
Published: 23 May 2022
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4.0/).
veterinary
sciences
Article
Changes in Neuropeptide Prohormone Genes among
Cetartiodactyla Livestock and Wild Species Associated with
Evolution and Domestication
Bruce R. Southey 1, * and Sandra L. Rodriguez-Zas 1,2
1Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA;
rodrgzzs@illinois.edu
2Department of Statistics, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
*Correspondence: southey@illlinois.edu
Abstract:
The impact of evolution and domestication processes on the sequences of neuropeptide
prohormone genes that participate in cell–cell signaling influences multiple biological process that
involve neuropeptide signaling. This information is important to understand the physiological differ-
ences between Cetartiodactyla domesticated species such as cow, pig, and llama and wild species such
as hippopotamus, giraffes, and whales. Systematic analysis of changes associated with evolutionary
and domestication forces in neuropeptide prohormone protein sequences that are processed into neu-
ropeptides was undertaken. The genomes from 118 Cetartiodactyla genomes representing 22 families
were mined for 98 neuropeptide prohormone genes. Compared to other Cetartiodactyla suborders,
Ruminantia preserved PYY2 and lost RLN1. Changes in GNRH2, IAPP, INSL6, POMC, PRLH, and
TAC4 protein sequences could result in the loss of some bioactive neuropeptides in some families. An
evolutionary model suggested that most neuropeptide prohormone genes disfavor sequence changes
that incorporate large and hydrophobic amino acids. A compelling finding was that differences
between domestic and wild species are associated with the molecular system underlying ‘fight or
flight’ responses. Overall, the results demonstrate the importance of simultaneously comparing the
neuropeptide prohormone gene complement from close and distant-related species. These findings
broaden the foundation for empirical studies about the function of the neuropeptidome associated
with health, behavior, and food production.
Keywords: neuropeptide; prohormone; domestication; Cetartiodactyla; evolution
1. Introduction
Domestication typically encompasses the separation of a species from its natural
habitat and physiological and behavioral modifications through artificial selection and
environments [
1
]. This interplay is uniquely demonstrated in the mammalian superorder
Cetartiodactyla
that includes domesticated livestock species as well as species adapted to
diverse environmental conditions including alpine, desert, and marine environments, and
management practices including precision management [
2
4
]. Out of the four
Cetartiodactyla
suborders, three suborders have at least one domesticated species: Ruminantia (sheep, cattle,
goats, and deer), Suina (pigs and peccaries), and Tylopoda (camels, llamas, and alpaca).
These domesticated terrestrial Cetartiodactyla species are managed for a wide variety of
purposes including food, fiber, companion, show, and draught purposes. Whippomorpha
(hippopotamuses and whales), the fourth Cetartiodactyla suborder, encompasses species
abundant in the wild, and also present in zoos and aquariums participating in conservation
and scientific studies [5].
Livestock domestication started approximately 11,000 years ago with domestic cattle
(Bos taurus), domestic goats (Capra hircus), domestic sheep (Ovis aries), and domestic pigs
(Sus scrofa) [
2
4
]. Tylopoda were domesticated 3000 to 7000 years ago [
6
] and reindeer
Vet. Sci. 2022,9, 247. https://doi.org/10.3390/vetsci9050247 https://www.mdpi.com/journal/vetsci
Vet. Sci. 2022,9, 247 2 of 13
(Rangifer tarandus) are partly domesticated with both wild and domesticated herds [
7
].
Domestication involves the modification of biological processes associated with the regula-
tion of behaviors such as aggression, fear, and flight. Domestication also elicits changes
in biological processes associated with fertility, metabolism, development and growth,
feeding and reproductive behavior, and health through artificial selection, breeding, and
management [14].
Artificial selection has led to population genetic changes that, in turn, elicited be-
havioral and physiological changes in the domesticated populations. Underlying these
biological changes are processes associated with cell-to-cell signaling that is characterized
by small neuropeptides conveying chemical messages between neurons and other cells
when docking on prohormone receptors. Aggression, bonding, and stress response are
modulated by the neuropeptides adrenocorticotropic hormone (produced from proopi-
omelanocortin; POMC), oxytocin (produced from oxytocin/neurophysin I prepropeptide;
OXT), arginine vasopressin (produced from arginine vasopressin; AVP), and corticoliberin
(produced from corticotropin releasing hormone; CRH) [
8
]. Appetite and feeding behaviors
are modulated by neuropeptide insulinlike growth factor 2 (IGF2), neuropeptide Y (NPY),
and ghrelin (produced from ghrelin and obestatin prepropeptide; GHRL).
The evolutionary processes of natural selection mutation, gene flow, and genetic drift
have also resulted in changes in neuropeptide sequences and associated biological processes.
Sequence comparison of the genes coding for the neuropeptides’ glucagon (GCG) and
glucose-dependent insulinotropic polypeptide (GIP) across species offers insights into the
impact of evolution on associated processes [
9
]. The mutation of the melanocortin recept
or 1
(MC1R), a receptor for POMC neuropeptides, is an important component of coat color
diversity in wild and domestic species [
10
]. Moreover, while artificial and natural selection
processes can coexist, different selection pressures between wild and domestic livestock
species have been reported [
11
]. Analysis of the melanocortin system showed that MC1R
had the largest coevolutionary influence, while the POMC and agouti-related neuropeptide
(AGRP) had the lowest and third lowest coevolutionary influence, respectively [12].
The impact of domestication on neuropeptides cannot be predicted directly from
the species genome or transcriptome because neuropeptides are the result of complex
processing of a larger protein sequence. These proteins are encoded by neuropeptide
prohormone genes that contain a signal peptide which directs the protein for secretion.
After removal of the signal peptide [
13
], the resulting protein sequence undergoes cleavage
at sites compatible with the furin cleavage motif [
14
]. This motif consists of the combination
of an arginine or lysine at the cleavage site with an adjacent or near-adjacent position
containing arginine or lysine. Subsequently, cleaved peptides may undergo additional
post-translation modifications to form bioactive neuropeptides.
A bioinformatic pipeline that works with protein sequences and accommodates the
complex processing of neuropeptide prohormone gene products has been developed [
15
,
16
].
This bioinformatics strategy succeeded in the identification and characterization of neu-
ropeptide prohormone genes that have been lost or undergone the duplication part of
the evolutionary process [
17
19
]. The objective of the present study was to characterize
the changes in neuropeptide prohormone genes associated with domestication and evolu-
tionary processes. To accomplish this objective, a rich database of wild and domesticated
Cetartiodactyla species was assembled. The resulting database offered a unique opportunity
to identify neuropeptide prohormone gene gain and loss, and hybridization events that
occurred along the processes of domestication and evolution [2022].
2. Materials and Methods
The complete genomic assemblies of 118 Cetartiodactyla species distributed across all
4 suborders and encompassing 22 families were downloaded from the National Center
for Biotechnology Information (NCBI) repository [
23
]. Table 1summarizes the species
studied and Table S1 includes lists of all species and their corresponding identifiers. The
Ruminantia suborder was represented by 82 ruminant species including cattle, deer, goats,
Vet. Sci. 2022,9, 247 3 of 13
and sheep distributed across 6 families. The Suina suborder was represented by 2 species
including pigs and peccaries. The Tylopoda suborder was represented by 7 species from
2 tri
bes including camels and llamas. The Whippomorpha suborder was represented by
27 species including whales and hippopotamus.
Table 1. Summary of domestic and wild Cetartiodactyla species used by taxonomic level.
Suborder and
IF or Fam 1
N2
Parvorder/Subfamily/Tribe/Genus 3
D W
Ruminantia
Bovidae 7 50
Bovinae: 4,12; Caprinae: 2,12; Alcelaphinae: 0,4;
Reduncinae: 0,3; Cephalophinae:0,3; Hippotraginae: 0,4;
Antilopinae: 0,12; Aepycerotinae: 0,1
Cervidae 1 15 Muntiacinae: 0,3; Cervinae: 0,5; Hydropotinae: 0,1;
Odocoileinae: 1,6
Other 0 9 Moschidae: 0,3; Tragulidae: 0,2; Giraffidae: 0,3;
Antilocapridae: 0,1
Suina
1 o Sus: 1,0; Catagonus: 0,1
Tylopoda
Camelidae 4 3 Camelini: 2,1; Lamini: 2,2
Whippomorpha
0 27 Hippopotamidae: 0,1; Odontoceti: 0,19; Mysticeti: 0,7
Total 13 105
1
Cetartiodactyla suborder and infraorder (IF) or family (Fam),
2
number of domesticated (D) and wild (W) species
within infraorder or family,
3
the number of domesticated species followed by the number of wild species within
each parvorder, subfamily, or tribe.
Different groupings of the Cetartiodactyla species were identified based on domes-
tication status, suborder, family, and subfamily. Individual species were classified as
domesticated (12 species) or wild (106 species). The wild Bactrian camel, Camelus ferus, was
excluded from the previous classification due to uncertainty on the domestication status of
the sequenced individuals [
24
]. The wild species from the suborders Ruminantia,Suina, and
Tylopoda were grouped as terrestrial (nonCetacea) species (91 species), while Cetacea species
(Whippomorpha species excluding the hippopotamus, Hippopotamus amphibious) are aquatic.
Ruminantia species were also divided into domestic (8 species) and wild (74 species).
A catalog of neuropeptide gene products (prohormone proteins) was assembled and
the predicted protein sequences for each species were obtained following the bioinformatic
pipeline [
15
,
16
]. Since Cetartiodactyla species were unannotated, additional steps within
the pipeline were taken to identify the neuropeptide prohormone genes. The longest
prohormone protein isoforms were compiled from a curated list of 98 mammalian prohor-
mone protein sequences identified in Cetartiodactyla [
15
,
17
,
18
,
25
27
]. The most probable
location for each neuropeptide prohormone gene was identified using TBLASTN [
28
] using
default settings and with the low-complexity filtering disabled. An approximate 10,000 bp
region surrounding the most probable gene location was extracted and the corresponding
protein sequence was predicted from the extracted region using the gene prediction tool Ge-
newise [
29
] with default settings. Individual sequence inspection ensured that all predicted
sequences corresponded to the same longest known protein isoform. Variations in assembly
depth and quality across the genomes could result in incomplete sequences or apparent
sequence duplication. Incomplete, inconsistent, or potentially duplicate predictions were
evaluated and additional searches using alternative locations, spanning intronic regions to
accommodate genes that have short exons, and relaxing search criteria with and without
composition-based statistics [30] were used to improve the prediction.
Vet. Sci. 2022,9, 247 4 of 13
Following the compilation and validation of the neuropeptide gene protein sequences,
the similarity between species was studied. The alignment of protein sequences across
species by prohormone was implemented using the sequence aligner MAFFT [
31
]. Align-
ment accuracy was optimized using the L-INS-I parameterization that enables the alignment
of sequences containing sequences flanking around one alignable domain [
31
]. Phyloge-
netic gene trees of protein sequences were computed for each neuropeptide prohormone
gene using the software PhyML [
32
] with default parameters, and tree visualization used
the software ASTRAL with default parameters [
33
36
]. The genetic distances between
species trees and individual prohormone trees were estimated using Pearson correlation
coefficients. The protein distances within prohormones and across the different groupings
were estimated using the mean protein evolutionary distance approach [37].
The estimation of neuropeptide prohormone gene protein distances was ensued by
the identification of the evolutionary/domestication paradigm best supported by the
118 sp
ecies studied. An evolutionary model [
38
] accounting for information on amino
acid aromaticity (A), composition (C), polarity (P), and side chain volume (V) [
39
41
] was
fitted using the Jones-Taylor-Thornton (JTT) substitution model [
42
]. The final model best
supported by the sequences and species studied was selected using Akaike information
criterion [43].
3. Results
3.1. Prohormone Identification across Species
All 98 prohormone protein sequences were identified across all 118 Cetartiodactyla
species but varied across taxonomic groups. A notable finding from the comparison
across species was that relaxin 1 (RLN1) has been lost in Ruminantia but is present in all
other Cetartiodactyla suborders. Furthermore, prohormone peptide YY2 (PYY2) was only
reliably detected across the Ruminantia suborder including complete predictions in the
Giraffidae and Bovinae subfamilies and a partial PYY2 prediction in the Cervidae subfamily.
Protein sequences from AVP, secretin (SCT), tachykinin precursor 4 (TAC4), and VGF nerve
growth factor inducible (VGF) were completely or partially identified in at least 20% of
the Cetartiodactyla species. Apelin (APLN), CART pre-propeptide (CARTPT), NPY, and
somatostatin (SST) protein sequences were found to be highly conserved.
Within species, the genome assemblies of argali (Ovis ammon), Alpine ibex (Ammotragus
lervia), and blue whale (Balaenoptera musculus) enabled the prediction of 56%, 53%, and 79%
of neuropeptide prohormone genes, respectively. The hartebeest (Alcelaphus buselaphus)
genome supported the complete or partial prediction of 69% of the neuropeptide prohor-
mone genes. When compared to closely related species, the challenge in predicting the
totality of the prohormone protein sequence completely was primarily due to depth and
quality variations along the individual assemblies rather than species differences. Limita-
tions in sequence coverage, contig assembly including frame orientation, and splice site
prediction prevented an accurate census of neuropeptide prohormone gene gains and losses
despite the multiple specifications of the predictive algorithms used in the present study.
3.2. Species Tree Derived from Prohormone Sequences
3.2.1. Species Tree
The species tree followed the expected species relationships between and within the
four Cetartiodactyla suborders (Figure 1). The species tree excluded the RLN1 and PYY2 gene
trees because these genes were suborder-specific. Among the Whippomorpha species, the
hippopotamus was more distant to the rest of the species in the suborder that correspond
to the infraorder Cetacea which in turn is distributed into the parvorders Mysticeti (baleen
whales) and Odontoceti (toothed whales). The tree also demonstrates that the comparative
analysis of neuropeptide protein sequences resulted in the expected organization of species
into known families and subfamilies within the Ruminantia suborder.
Vet. Sci. 2022,9, 247 5 of 13
Vet. Sci. 2022, 9, x FOR PEER REVIEW 5 of 13
(baleen whales) and Odontoceti (toothed whales). The tree also demonstrates that the com-
parative analysis of neuropeptide protein sequences resulted in the expected organization
of species into known families and subfamilies within the Ruminantia suborder.
Figure 1. Species tree derived from individual prohomone gene trees.
3.2.2. Correlation of InterSpecies Distances Based on Individual and All Neuropeptide
Prohormone Genes
To understand the variation in species distances across neuropeptide prohormone
genes, the distances estimated within the neuropeptide prohormone gene tree were corre-
lated to the distances from the species gene tree. The correlation of interspecies distances
between individual and the species gene trees averaged 0.77 and ranged from 0.25 to 0.92.
The species distance for most individual neuropeptide prohormone genes (69 genes) was
highly correlated (between 0.8 and 0.9) with the distance estimate from the species gene tree.
Neuropeptide prohormone genes that are highly conserved tended to provide distance es-
timates less correlated with the estimates from the species gene tree, relatively with less
conserved neuropeptide prohormone genes. This was reflected by the number of unique
sequences and the length of the predicted protein sequence. The interspecies distance esti-
mated from chromogranin A (CHGA), prodynorphin (PDYN), and the thyrotropin releas-
ing hormone (TRH) were highly correlated (correlation > 0.9) with the distance from the
species gene tree. On the other hand, the interspecies distance estimated from AVP, insulin
(INS), natriuretic peptide C (NPPC), prokineticin 2 (PROK2), parathyroid hormone (PTH),
and SST had lower correlations (correlation < 0.50) with the distance from the species gene
tree resulting from extremely highly conversed protein sequences across species.
The distance between Ruminantia species computed from individual neuropeptide
prohormone genes had higher correlations with the distance computed from the species
gene tree relative to other species. This trend may reflect the higher proportion of rumi-
nant species among all species used. A higher average distance between species was esti-
mated in the second most represented suborder, Whippomorpha. The hippopotamus,
sperm whale (Physeter catodon), and baiji (Lipotes vexillifer) species were more distant from
other Whippomorpha species.
3.3. Evolutionary Model
The evolutionary model use accounts for information on amino acid aromaticity (A),
composition (C), polarity (P), and side chain volume (V) to understand the impact of evo-
lutionary and domestication changes within and across suborders. Table 2 summarizes
Figure 1. Species tree derived from individual prohomone gene trees.
3.2.2. Correlation of InterSpecies Distances Based on Individual and All Neuropeptide
Prohormone Genes
To understand the variation in species distances across neuropeptide prohormone
genes, the distances estimated within the neuropeptide prohormone gene tree were corre-
lated to the distances from the species gene tree. The correlation of interspecies distances
between individual and the species gene trees averaged 0.77 and ranged from 0.25 to 0.92.
The species distance for most individual neuropeptide prohormone genes (69 genes) was
highly correlated (between 0.8 and 0.9) with the distance estimate from the species gene
tree. Neuropeptide prohormone genes that are highly conserved tended to provide distance
estimates less correlated with the estimates from the species gene tree, relatively with less
conserved neuropeptide prohormone genes. This was reflected by the number of unique
sequences and the length of the predicted protein sequence. The interspecies distance
estimated from chromogranin A (CHGA), prodynorphin (PDYN), and the thyrotropin
releasing hormone (TRH) were highly correlated (correlation >0.9) with the distance from
the species gene tree. On the other hand, the interspecies distance estimated from AVP,
insulin (INS), natriuretic peptide C (NPPC), prokineticin 2 (PROK2), parathyroid hormone
(PTH), and SST had lower correlations (correlation <0.50) with the distance from the species
gene tree resulting from extremely highly conversed protein sequences across species.
The distance between Ruminantia species computed from individual neuropeptide
prohormone genes had higher correlations with the distance computed from the species
gene tree relative to other species. This trend may reflect the higher proportion of ru-
minant species among all species used. A higher average distance between species was
estimated in the second most represented suborder, Whippomorpha. The hippopotamus,
sperm whale (Physeter catodon), and baiji (Lipotes vexillifer) species were more distant from
other Whippomorpha species.
Vet. Sci. 2022,9, 247 6 of 13
3.3. Evolutionary Model
The evolutionary model use accounts for information on amino acid aromaticity (A),
composition (C), polarity (P), and side chain volume (V) to understand the impact of
evolutionary and domestication changes within and across suborders. Table 2summarizes
the number of neuropeptide prohormone genes by evolutionary model specification. Most
neuropeptide prohormone genes exhibited a nonzero estimate for at least one parameter;
parameters A and V commonly had negative estimates, while parameter C typically had
positive estimates, and P tended to have positive estimates (Table 2). This relationship
between parameters is consistent with the correlations of individual amino acid coefficients
for each parameter in the evolutionary model. Both the A and V parameters are positively
correlated (~0.7) and negatively correlated with the C and P parameters (
0.35 to
0.47).
The C and P parameters are positively correlated (0.4) which increased to 0.81 when cysteine
is excluded.
Table 2.
Number of neuropeptide prohormone genes by parameter specification in the evolutionary
model.
Taxonomic Group 1mS 2
Parameter 3
A C P V
++++
Overall
All 81.5 52 5 4 64 17 19 51 3
Domestic 10 31 5 2 32 11 13 26 5
Wild 75.5 50 6 3 62 17 18 49 6
Wild terrestrial 56 51 3 1 56 11 23 48 3
Ruminantia
All 55 51 5 3 50 13 24 51 5
Domestic 6 23 5 3 27 5 12 18 4
Wild 52 43 5 0 54 11 22 47 4
Bovidae 38 35 5 6 44 10 17 38 5
Bovidae Antilopinae 11 18 8 2 24 7 4 24 3
Bovidae Bovinae 10 22 1 6 20 10 6 15 6
Bovidae Caprinae 7 13 7 4 22 8 14 25 2
Cervidae 11 11 2 6 2 7 1 4 0
Tylopoda
All 4 20 1 13 10 9 7 10 1
Whippomorpha
All 20 32 3 6 23 13 11 39 0
Cetacea Mysticeti 5 16 6 4 16 18 8 13 2
Cetacea Odontoceti 14 30 3 7 21 11 10 31 3
1
Taxonomic group: All = All species overall or within each grouping,
2
Median number of species that have
prohormone sequences across neuropeptide prohormone genes,
3
Number of neuropeptide prohormone genes
with a negative (
) or positive (+) estimate in aromaticity (A), composition (C), polarity (P), or volume (V)
parameter. Neuropeptide prohormone genes may have more than 1 modified parameter.
The domestic group of species had a higher proportion of neuropeptide prohormone
genes (40%) than the wild grouping with nonzero parameter estimates especially involving
the C parameter. A smaller proportion of parameter changes occurred between domes-
ticated and wild Ruminantia species. There were seven neuropeptide prohormone genes
where the modified parameters only occurred in domestic (1) or wild terrestrial (6) groups.
These neuropeptide prohormone genes included apelin (APLN), adenylate cyclase activat-
ing polypeptide 1 (ADCYAP1), arginine vasopressin (AVP), cholecystokinin (CCK), growth
hormone releasing hormone (GHRH), torsin family 2 member A (salusin-containing iso-
form, TOR2X), and VGF nerve growth factor inducible (VGF), and the averages of the
parameter changes for these neuropeptide prohormone genes are summarized in Table 3.
The positive A parameter estimate for APLN implies a preference for aromatic amino acids
with domestication. In the wild groupings, the A and V parameters were negative estimates
Vet. Sci. 2022,9, 247 7 of 13
and the C parameter had positive estimates, implying a preference against aromatic amino
acids with domestication.
Table 3.
Parameter estimates for neuropeptide prohormone genes that presented changes in domestic
or wild species.
Parameter 1
A C P V
Symbol 2All Rum All Rum All Rum All Rum
Domestic
APLN 3.62 9.80 0.00 0.00 0.00 0.00 0.00 0.00
Wild terrestrial
ADCYAP1 1.04 1.27 0.00 0.00 0.00 0.95 0.00 0.00
AVP 0.00 0.00 0.74 1.22 0.00 0.00 0.00 0.00
CCK 0.00 1.70 2.03 2.18 0.00 0.00 0.00 0.90
GHRH 1.35 1.26 0.00 0.00 0.00 0.00 0.00 0.00
TOR2X 0.00 0.00 0.00 0.00 0.00 0.00 1.32 1.57
VGF 0.00 0.00 0.00 0.00 0.00 0.00 1.23 1.39
1
Estimated parameter change in individual aromaticity (A), composition (C), polarity (P), and volume (V)
parameters within all domestic or wild species (All) and within domestic or wild Ruminantia species (Rum).
2Neur
opeptide prohormone gene symbol. APLN: apelin; ADCYAP1: adenylate cyclase activating polypeptide 1;
AVP: arginine vasopressin; CCK: cholecystokinin; GHRH: growth hormone releasing hormone; TOR2X: torsin
family 2 member A (salusin-containing isoform); VGF: VGF nerve growth factor inducible.
4. Discussion
4.1. Prohormone Complement
The integration of bioinformatics prohormone prediction, compilation, characteriza-
tion, and analysis offered insights into changes of neuropeptide genes associated with
evolutionary and domestication processes across Cetartiodactyla suborders and species.
As expected of orders that encompass a wide range of species, the Cetartiodactyla assem-
blies differed in depth and quality across the genome. The Cetartiodactyla species tree
was virtually identical to the expected tree [
6
,
44
,
45
]. The strategy of using evolutionary
proximal species for gene prediction in weaker assemblies [
46
] enabled the recovery of
sequences and the results indicated overall sequence consistency across taxonomic groups.
The evolutionary proximal strategy minimized the identification of differences between
species that could be a result of assembly limitations.
A remarkable finding was that Suina species contained the AUG initiation codon
but all other Cetartiodactyla species in this study contained a nonAUG initiation codon of
neuropeptide W (NPW) present in other mammalian species [
47
]. The absence of the AUG
initiation codon can result in incorrect prediction of the start of the actual coding region of
NPW. The unique feature of the neuropeptide NPW is particularly important because pigs
are regularly used as biomedical models due to their higher genome similarity to humans
than rodents and several health and behavioral processes are modulated by NPW.
Another finding from the bioinformatics pipeline centered on sequence variations in
cortistatin (CORT). A 15-nucleotide insertion in signal peptide of CORT was detected in the
domestic goat and does not impact the cortistatin neuropeptides. The potential evolutionary
difference was detected in the assemblies of four domestic goat breeds currently available
and this insertion was not present in other Capra or Caprinae species.
Comparisons of the predicted prohormone sequences highlighted and refined existing
knowledge of the neuropeptide genes RLN1, PYY2, islet amyloid polypeptide (IAPP),
and galaninlike peptide (GALP). Our predictions of RLN1 extended the loss of bovine
RLN1 [
17
] to all Ruminantia species after the split from Whippomorpha. Oppositely, PYY2
was detected solely in Ruminantia species. Species from other suborders either lacked a
complete prediction or had indeterminate matches (Whippomorpha) supporting that PYY2
is a pseudogene outside Ruminantia [
48
]. While GALP was detected in most species, all
Cetartiodactyla suborders had different terminal regions and some Bovidae species lacked the
initial initiation region. Tylopoda and Cetacean species presented IAPP sequences encompass-
Vet. Sci. 2022,9, 247 8 of 13
ing discrepancies consistent with pseudogenes including stop codons and lack of a signal
peptide. Chacoan peccary (Catagonus wagneri) and all the camelid species studied, along
with previous reports on the pig IAPP [
18
], lack the sequence corresponding to traditional
cleavage sites, although analysis of RNA-seq data indicated that IAPP is expressed in
pigs [49].
The comparative analysis of neuropeptide genes between Cetartiodactyla species identi-
fied sequence differences that could impact the levels of bioactive neuropeptides including
TAC4, insulinlike 6 (INSL6), gonadotropin releasing hormone 2 (GNRH2), and prolactin
releasing hormone (PRLH). Noticeably, TAC4 exhibited sequence differences among the
Cetartiodactyla species studied, yet no sequence encompassed the cleavage motif reported
in human and mouse [
50
]. Similarly, the start and terminal regions of INSL6 in pigs and
camelids were different from the other Cetartiodactyla species that, in turn, were similar to
the corresponding human and rodent sequences [
51
]. While the N-terminal cleavage site
of the INSL6 A chain was conserved across species, the C-terminal cleavages sites of the
INSL6 A and B chains remains uncharacterized among Cetartiodactyla species. Likewise,
while the signal peptide for GNRH2 was six amino acids longer in all the species of the
Bovinae subfamily, the expected cleavage motif for the gonadoliberin-2 peptide was missing
from all the Whippomorpha species. Similarly prominent, all Cetacea species had an 8-amino
acid deletion within the PRLH signal peptide that was not present in the Hippopotamus
amphibius or other Cetartiodactyla species and this sequence difference resulted in a shorter
predicted prolactin-releasing peptide 31 (PrRP31). On the other hand, a 10 bp-insert in all
Ruminantia species results in a longer PRLH terminal region, albeit this region is not part of
a known bioactive peptide.
The variation in POMC protein sequences across the Cetartiodactyla species could
impact multiple neuropeptides. While the complete POMC sequence was predicted in
Tylopoda species, the sequence lacked a cleavage site within the N-terminal peptide (NPP)
necessary to produce the pro-
γ
-MSH, and in turn, the small amidated
γ1
-MSH. While no
peptides associated in pro-
γ
-MSH region were detected by mass spectrometry [
27
],
γ1
-MSH
has been characterized in human [
52
] and cattle [
53
]. The N-terminal cleavage site was
detected in all the other Cetartiodactyla species studied. A similar phenomenon is observed
among rodent species [
54
] with the cleavage site missing in Muridae yet observed in other
Rodentia families. Remarkably, both Tylopoda and Muridae species include the sequence for
the larger
γ3
-MSH peptide. The role of
γ
-MSH peptides in health and behavior processes
encompasses sodium metabolism and blood pressure regulation [
55
57
] and the injection
of γ1-MSH in the left ventral tegmental area of rats induced grooming [58].
Neuropeptides from calcitonin (CALC) genes have many different roles including the
regulation of calcium, vasodilation, inflammation, migraine, pain, and hypothermia [
59
62
].
Related with domestication behaviors, an SNP in the calcitonin receptor-stimulating peptide
(CRSP) gene cluster differentiated pure-breed (mating determined by human breeding
practices) from free-breeding (mating is not artificially restricted) dogs [
63
]. Moreover, the
calcitonin receptorlike receptor (CALCRL) was differentially expressed in the pituitary of
tame and aggressive foxes [
64
]. In the present study, the identification of four calcitonin
or CRSP genes across all Cetartiodactyla taxa was consistent with other mammals except
primates and rodents [
65
]. While calcitonin-related genes were detected, the comparison
of cleavage site locations indicated that the cleavage sites necessary to form all known
neuropeptides such as a calcitonin gene-related peptide 1 are absent from both CRSP2
and CRSP3.
4.2. Evolutionary Model
The differences in the evolutionary model amino acid aromaticity (A), composition (C),
polarity (P), and side chain volume (V) parameters enabled the identification of patterns
of amino acid property changes in the prohormone sequences and thus neuropeptides
across Cetartiodactyla taxa. The similarity in the number of changes between all species
and all Ruminantia species was due to the high proportion of Ruminantia species and
Vet. Sci. 2022,9, 247 9 of 13
lower similarity was observed with family or subfamily groupings. The relatively high
proportion of changes within Tylopoda was due to the limited number of species and the
close relationship between Tylopoda species. Within taxonomic groupings, Whippomorpha
exhibited a high proportion of neuropeptide prohormone genes changing in the A, C, and
V parameters that was similarly represented in Mysticeti and Odontoceti families.
The majority of neuropeptide prohormone genes exhibited changes between
Cetartiodactyla
suborders, while 18% of neuropeptide prohormone genes including promelanin concentrating
hormone (PMCH), growth hormone releasing hormone (GHRH), PROK2, AVP, platelet derived
growth factor subunit B (PDGFB), and pancreatic polypeptide (PPY) exhibited no substantial
change in parameters. The conservation of the previous prohormone sequences supported
amino acid substitution rates that are consistent with the JTT substitution model.
Among the neuropeptide prohormone genes that presented parameter changes across
species, most changes occurred in one (31%) or two parameters (34%). These changes were
primarily negative estimates for parameters A and V and positive estimates for parameter
C. For some neuropeptide prohormone genes, including hepcidin antimicrobial peptide
(HAMP), insulinlike 3 (INSL3), motilin (MLB), and spexin hormone (SPX), the amino acid
parameter changes were observed in most taxonomic groups.
A notable finding stemming from the parameter coefficient estimates implies that
changes in prohormone sequence lower the likelihood of large and hydrophobic amino
acids (i.e., phenylalanine, tryptophan and tyrosine). These amino acids may compromise
the neuropeptide function and therefore the evolutionary forces favor other types of amino
acids. Moreover, our results indicate that the cation-
π
interaction provided by the aromatic
amino acids, a strong noncovalent binding interaction that is important in protein secondary
structure and interactions with drugs and neurotransmitters such as serotonin [
66
], is
generally undesirable in prohormone sequences.
4.3. Domestication
The study of changes in neuropeptide gene protein sequences associated with do-
mestication accounted for factors such as the distribution of domestic and wild species.
Inference was partitioned orthogonally from taxonomy to remove all confounding groups
while addressing differences in the number of species and limited gene flow. The evolution
model indicated differences with wild terrestrial and domestic groupings. The domesti-
cated Ruminantia had a slightly higher proportion of changes than wild Ruminantia with
60% of the neuropeptide prohormone genes being equal or had at least one parameter in
common between domesticated and wild Ruminantia. This finding suggests that part of
the differences between parameter values can be attributed to taxonomic differences rather
than domestication.
Further challenges to the assessment of the association between domestication and
neuropeptide gene changes stem from the domestication classification of some species and
the genome assembly quality of other species. With respect to domestication assignments,
for example, the gayl (Bos frontalis) is often considered the domestic form of the wild gaur
(Bos gaurus) [
67
], however the degree of domestication is highly variable among the for-
mer group. With regards to sequence quality, the virtually identical nucleotide or protein
sequences predicted from all camelid genomes available in this study invalidates the conclu-
sion of adaptive introgression of endothelin 3 (EDN3) in South American camelids [
6
]. The
association between domestication and prohormone sequence changes is further obscured
by the counteraction of ancestral hybridization and artificial selec
tion [3,4,6,68].
Consid-
ering the distribution of species across taxa and domestication groups and adjusting for
variable assembly quality within and across domesticated groups, the present study identi-
fied notable and consistent changes among neuropeptide prohormone genes associated
with docile and herdlike behaviors.
The neuropeptide prohormone genes that presented differences in amino acid param-
eters between domestic and wild species produce neuropeptides that participate in a vast
array of functions. Among the neuropeptides from neuropeptide prohormone genes that
Vet. Sci. 2022,9, 247 10 of 13
have distinct sequence across species, APLN, AVP, TOR2X (salusin-containing torsin family
2 member A isoform), and VGF modulate angiogenesis, vasodilation, and vasoconstriction.
Likewise, neuropeptides from ADCYAP1, CCK, GHRH, and VGF are associated with feed-
ing and energy homeostasis. VGF is also associated with circadian rhythm, pain, memory,
and learning [
69
,
70
]. In the context of domestication, a commonality is that the previous
functions are associated with the sympatho-adrenomedullary system that involves the
‘fight or flight’ response where blood pressure and glucose levels increase in response to
stress [71].
5. Conclusions
A study of the association between evolutionary and domestication processes and
changes in neuropeptide gene sequences was undertaken. A bioinformatics pipeline was de-
veloped to identify the prohormone complement of 98 sequences across 11
8Cetartiodactyla
wild and domesticated species distributed across 4 suborders. An exhaustive survey of
prohormone sequences was compiled, the sequences were compared, and evolutionary
models were used to assess the change in amino acid properties including aromaticity,
composition, polarity, and volume parameters. Remarkable findings include sequence
differences among Cetartiodactyla that disrupt cleavage motifs in particular neuropeptide
prohormone genes (e.g., INSL6, GNRH2, PRLH, POMC, CALC), potentially compromis-
ing the ability of the prohormone to generate bioactive neuropeptides. Similarly notable,
the parameter coefficient estimates suggest that the evolutionary process tends to disfa-
vor large and hydrophobic amino acids in neuropeptide prohormone genes. Evolution-
ary modeling indicated that some neuropeptide prohormone genes associated with the
sympatho-adrenomedullary system and the ‘fight or flight’ response may be impacted by
domestication. The prohormone complement provides the foundation for neuropeptidomic
studies of medically and economically important characteristics and Cetartiodactyla species.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/
10.3390/vetsci9050247/s1, Table S1: Taxonomic names and National Center for Biotechnology
Information identifiers for genomes of Cetartiodactyla species. Additionally, the protein sequences
with limited multiple sequence alignments and phylogenetic tree data presented in this study are
openly available in the Illinois Data Bank, Available online: https://doi.org/10.13012/B2IDB-2071
917_V1 (accessed on 18 May 2022).
Author Contributions:
Conceptualization, B.R.S. and S.L.R.-Z.; methodology, B.R.S. and S.L.R.-Z.;
formal analysis, B.R.S.; investigation, B.R.S. and S.L.R.-Z.; writing—original draft preparation, review
and editing, B.R.S. and S.L.R.-Z.; funding acquisition, S.L.R.-Z. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by the National Institutes of Health, grant number P30 DA018310.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Publicly available datasets were analyzed in this study. This data can
be obtained from the National Center for Biotechnology Information.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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