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10 pages, 1139 KiB  
Article
Adaptive Evolution and Functional Differentiation of Testis-Expressed Genes in Theria
by Yukako Katsura, Shuji Shigenobu and Yoko Satta
Animals 2024, 14(16), 2316; https://doi.org/10.3390/ani14162316 - 9 Aug 2024
Viewed by 160
Abstract
Gene expression patterns differ in different tissues, and the expression pattern of genes in the mammalian testis is known to be extremely variable in different species. To clarify how the testis transcriptomic pattern has evolved in particular species, we examined the evolution of [...] Read more.
Gene expression patterns differ in different tissues, and the expression pattern of genes in the mammalian testis is known to be extremely variable in different species. To clarify how the testis transcriptomic pattern has evolved in particular species, we examined the evolution of the adult testis transcriptome in Theria using 10 species: two marsupials (opossum and Tasmanian devil), six eutherian (placental) mammals (human, chimpanzee, bonobo, gorilla, rhesus macaque, and mouse), and two outgroup species (platypus and chicken). We show that 22 testis-expressed genes are marsupial-specific, suggesting their acquisition in the stem lineage of marsupials after the divergence from eutherians. Despite the time length of the eutherian stem lineage being similar to that of the marsupial lineage, acquisition of testis-expressed genes was not found in the stem lineage of eutherians; rather, their expression patterns differed by species, suggesting rapid gene evolution in the eutherian ancestors. Fifteen testis-expressed genes are therian-specific, and for three of these genes, the evolutionary tempo is markedly faster in eutherians than in marsupials. Our phylogenetic analysis of Rho GTPase-activating protein 28 (ARHGAP28) suggests the adaptive evolution of this gene in the eutherians, probably together with the expression pattern differentiation. Full article
(This article belongs to the Section Animal Genetics and Genomics)
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Figure 1

Figure 1
<p>Acquisition/loss of testis-expressed genes in 10 species. The genes at each clade are listed in <a href="#animals-14-02316-t001" class="html-table">Table 1</a>, <a href="#animals-14-02316-t002" class="html-table">Table 2</a>, <a href="#app1-animals-14-02316" class="html-app">Tables S2 and S3</a>, and the left and right of the ‘/’ symbols mean acquisition and loss of testis-expressed genes, respectively.</p>
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<p>The phylogenetic trees, branch lengths, and dn/ds ratios for three therian-specific testis genes in 10 species. (<b>A</b>) <span class="html-italic">ARHGAP28</span> genes (1833 bp). (<b>B</b>) <span class="html-italic">SYNM</span> genes (2235 bp). (<b>C</b>) <span class="html-italic">PDZRN3</span> genes (2256 bp). The trees were constructed by the NJ method using the number of nucleotide differences. The average and standard deviation of branch lengths in six eutherians are shown above the black bold branch, and those in two marsupials are shown above the gray bold branch. The dn/ds ratio in six eutherian or two marsupial pairs is shown under the branch. ** and * mean <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.05, respectively, and are supported by Fisher’s exact test.</p>
Full article ">Figure 2 Cont.
<p>The phylogenetic trees, branch lengths, and dn/ds ratios for three therian-specific testis genes in 10 species. (<b>A</b>) <span class="html-italic">ARHGAP28</span> genes (1833 bp). (<b>B</b>) <span class="html-italic">SYNM</span> genes (2235 bp). (<b>C</b>) <span class="html-italic">PDZRN3</span> genes (2256 bp). The trees were constructed by the NJ method using the number of nucleotide differences. The average and standard deviation of branch lengths in six eutherians are shown above the black bold branch, and those in two marsupials are shown above the gray bold branch. The dn/ds ratio in six eutherian or two marsupial pairs is shown under the branch. ** and * mean <span class="html-italic">p</span> &lt; 0.01 and <span class="html-italic">p</span> &lt; 0.05, respectively, and are supported by Fisher’s exact test.</p>
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14 pages, 1013 KiB  
Review
Exploring Iguape Virus—A Lesser-Known Orthoflavivirus
by Marielena V. Saivish, Maurício L. Nogueira, Shannan L. Rossi and Nikos Vasilakis
Viruses 2024, 16(6), 960; https://doi.org/10.3390/v16060960 - 14 Jun 2024
Viewed by 1027
Abstract
Brazil has earned the moniker “arbovirus hotspot”, providing an ideal breeding ground for a multitude of arboviruses thriving in various zoonotic and urban cycles. As the planet warms and vectors expand their habitat range, a nuanced understanding of lesser-known arboviruses and the factors [...] Read more.
Brazil has earned the moniker “arbovirus hotspot”, providing an ideal breeding ground for a multitude of arboviruses thriving in various zoonotic and urban cycles. As the planet warms and vectors expand their habitat range, a nuanced understanding of lesser-known arboviruses and the factors that could drive their emergence becomes imperative. Among these viruses is the Iguape virus (IGUV), a member of the Orthoflavivirus aroaense species, which was first isolated in 1979 from a sentinel mouse in the municipality of Iguape, within the Vale do Ribeira region of São Paulo State. While evidence suggests that IGUV circulates among birds, wild rodents, marsupials, bats, and domestic birds, there is no information available on its pathogenesis in both humans and animals. The existing literature on IGUV spans decades, is outdated, and is often challenging to access. In this review, we have curated information from the known literature, clarifying its elusive nature and investigating the factors that may influence its emergence. As an orthoflavivirus, IGUV poses a potential threat, which demands our attention and vigilance, considering the serious outbreaks that the Zika virus, another neglected orthoflavivirus, has unleashed in the recent past. Full article
(This article belongs to the Special Issue Zoonotic and Vector-Borne Viral Diseases)
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<p>Geographic range and epidemiological landscape of Iguape virus. Brazilian states with evidence of IGUV circulation are named. Hosts from which IGUV and/or antibodies have been identified within a given Brazilian state are indicated by a representative graphic(s). Pie charts within a given state indicate the number of studies identifying IGUV by size and the method of their identification by color.</p>
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<p>The possible transmission cycles of IGUV.</p>
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17 pages, 2047 KiB  
Review
The Ecological Separation of Deer and Domestic, Feral and Native Mammals in Tropical Northern Australia—A Review
by Peter J. Murray and Timothy D. Nevard
Animals 2024, 14(11), 1576; https://doi.org/10.3390/ani14111576 - 26 May 2024
Viewed by 669
Abstract
We explored the ecological and historical factors that led to formation of the unique guild of native and introduced mammalian herbivores between 5 and 1000 kg in northern Australia. Following the disappearance of large native herbivores about 46 kya, and until the arrival [...] Read more.
We explored the ecological and historical factors that led to formation of the unique guild of native and introduced mammalian herbivores between 5 and 1000 kg in northern Australia. Following the disappearance of large native herbivores about 46 kya, and until the arrival of Europeans and their livestock, the only herbivorous mammals were mid-sized endemic marsupial macropods, which continued to utilise the same vegetation as their much larger former neighbours. Only one species of contemporary native herbivore has an adult bodyweight approaching 100 kg, and for the past 150–200 years, the total biomass of introduced domestic and wild vertebrate herbivores has massively exceeded that of native herbivorous species. We conclude that the current guild of native and introduced mammalian herbivores differentially utilises the landscape ecologically. However, climate- and anthropogenically related changes due to fire, drought, flooding, predation and introduced weeds are likely to have significant impacts on the trajectory of their relative ecological roles and populations. Given their differing ecological and dietary characteristics, against this backdrop, it is unclear what the potential impact of the dispersal of deer species could have in northern Australia. We hence focus on whether sufficient knowledge exists against which the potential impacts of the range expansion of three deer species can be adequately assessed and have found a dearth of supporting evidence to inform appropriate sustainable management. We identify suitable research required to fill the identified knowledge gaps. Full article
(This article belongs to the Section Ecology and Conservation)
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<p>Extent of tropical savannas in northern Australia (after [<a href="#B3-animals-14-01576" class="html-bibr">3</a>]).</p>
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<p>Distribution of macropod species &gt; 5 kg found in northern Australia [<a href="#B32-animals-14-01576" class="html-bibr">32</a>].</p>
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<p>Distribution of the introduced feral pig and herbivorous mammal species &gt; 5 kg found in northern Australia [<a href="#B32-animals-14-01576" class="html-bibr">32</a>]. Not shown are the distributions of feral cattle and horses, as both are widely distributed across northern Australia.</p>
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<p>Mean mature bodyweights of native and introduced mammals &gt; 5 kg found in northern tropical Australia; deer species shown in green [<a href="#B32-animals-14-01576" class="html-bibr">32</a>].</p>
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<p>Distribution and abundance of (<b>a</b>) chital, (<b>b</b>) rusa and (<b>c</b>) sambar deer in Australia in 2000 (herd data points not to scale). Insets show the bioclimatic (predicted) distribution of chital, rusa and sambar, respectively (M Bomford, unpublished data). The scale of predicted habitat suitability ranges from pink and dark green, indicating high habitat suitability, to light green and orange, indicating medium habitat suitability, and then to yellow and blue, indicating poor habitat suitability. Taken from Moriarty [<a href="#B34-animals-14-01576" class="html-bibr">34</a>].</p>
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30 pages, 6582 KiB  
Article
Trends in Rescue and Rehabilitation of Marsupials Surviving the Australian 2019–2020 Bushfires
by Holly R. Cope, Clare McArthur, Rachael Gray, Thomas M. Newsome, Christopher R. Dickman, Aditi Sriram, Ron Haering and Catherine A. Herbert
Animals 2024, 14(7), 1019; https://doi.org/10.3390/ani14071019 - 27 Mar 2024
Viewed by 1808
Abstract
The 2019–2020 Australian bushfire season had a devastating impact on native wildlife. It was estimated that 3 billion native animals were impacted by the fires, yet there are few estimates of the number of animals that were rescued and rehabilitated post-fire. Focusing on [...] Read more.
The 2019–2020 Australian bushfire season had a devastating impact on native wildlife. It was estimated that 3 billion native animals were impacted by the fires, yet there are few estimates of the number of animals that were rescued and rehabilitated post-fire. Focusing on the state of New South Wales (NSW) and Kangaroo Island, South Australia, we used a case study approach to determine the number of marsupials that were reported rescued due to the 2019–2020 bushfires in these areas and analysed species-specific trends in rescue and release success. In NSW, we found 889 reports of fire-affected marsupials in 2019–2020, mostly comprising kangaroos and wallabies (macropods; n = 458), koalas (n = 204), and possums (n = 162), with a smaller number of wombats (n = 43) and other marsupial species. Most reports of fire-affected marsupials occurred 6–8 weeks after fire ignition, and there was no difference in temporal frequency of rescues between marsupial groups. For the three main groups, the probability of survival and subsequent release differed, with macropods having the lowest probability of release after rescue (0.15 ± 0.04) compared to koalas (0.47 ± 0.04) and possums (0.55 ± 0.10). The type of injury was the main predictor of survival during rehabilitation for all three marsupial groups, with those malnourished/moribund or with traumatic injuries less likely to survive rehabilitation. Death or euthanasia occurred on the day of rescue for 77% of macropods, 48% of possums and 15% of koalas. Koalas most often died during rehabilitation rather than on the day of rescue, with 73% either dying or being euthanised between day 1 and 30 post-rescue, representing a potential welfare concern. On Kangaroo Island, koalas were the most frequently rescued marsupial species; most euthanasia cases and deaths occurred in a hospital, whereas other marsupials were mostly euthanised at triage. In both jurisdictions, koalas were over-represented while possums were under-represented relative to baseline population densities and wildlife rescue trends in the years before the 2019–2020 bushfires. These species differences in presentation post-fire warrant further investigation, as do the differences in triage, survival and release outcomes. It is hypothesised that the high intensity and large scale of the 2019–2020 fires impeded marsupial fire evasion tactics, as evidenced by the small number of animals found for rescue, and the differing rates of presentation relative to underlying population densities for the main marsupial groups. Based on our findings, there is a need for detailed record keeping and data sharing, development of consistent and evidence-based triage, treatment and euthanasia guidelines and deployment of trained wildlife emergency rescue teams with advanced search techniques to minimise animal suffering where safe to do so. Full article
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<p>Marsupial group as a proportion (%) of (<b>a</b>) all marsupial records and (<b>b</b>) fire-affected marsupial records for the years 2015/16–2018/19 (blue columns; <span class="html-italic">n</span> = 125,439 for all records; <span class="html-italic">n</span> = 235 for fire records) and 2019/20 (red columns; <span class="html-italic">n</span> = 37,076 for all records; <span class="html-italic">n</span> = 889 for fire records). Record counts (<span class="html-italic">n</span>) are presented above columns for each taxonomic group.</p>
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<p>Fate as a proportion (%) of (<b>a</b>) all marsupial records and (<b>b</b>) fire-affected marsupial records in 2015/16–2018/19 (blue columns) and 2019/20 (red columns). Record counts (<span class="html-italic">n</span>) are presented above columns. The fate of “In care” indicates that the animal was still in care at the time of reporting, and the fate of “Dead on arrival” indicates that the animal died between the time it was reported and when a rescuer arrived.</p>
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<p>Mean ± 95% CI probability of release (0 = euthanised/died in care, 1 = released/relocated) of fire-affected marsupials in 2019/20 for koalas, macropods, and possums and gliders, by injury type. Superscript letters indicate injury types for each marsupial group that are significantly different based on pairwise comparisons.</p>
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<p>Number of fire-affected marsupial rescue records in 2015/16–2018/19 and 2019/20 for koalas, possums and macropods by sex (<b>a</b>, <b>b</b> and <b>c</b>, respectively) and by age (<b>d</b>, <b>e</b> and <b>f</b>, respectively). The data for the period 2015/16–2018/19 are presented as yearly mean ± SD, while the data for the year 2019/20 are absolute numbers. LR chi-squared analyses are testing for an overall difference in proportions of ages (where no change occurred between years), and chi-squared analyses are testing for a change in relative proportions of sexes between years.</p>
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<p>Cumulative number of koalas, macropods, and possums rescued due to fires in 2019/20 (<span class="html-italic">n</span> = 480 with accurate dates of rescue and fire ignition, excluding those found dead) over 12 months following ignition of the fire at the relevant rescue location. Cumulative <span class="html-italic">n</span> values are presented above each line.</p>
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<p>Mean ± 95% CI length of stay (days) of fire-affected koalas, macropods, and possums and gliders that were (<b>a</b>) released or relocated (<span class="html-italic">n</span> = 183) and (<b>b</b>) died or euthanised in rehabilitation after rescue (<span class="html-italic">n</span> = 369) in 2019/20. Note the different scales on the <span class="html-italic">y</span>-axis between figure (<b>a</b>,<b>b</b>). Marsupial groups with significantly different lengths of stay are denoted with different superscript letters (a or b) at <span class="html-italic">p</span> ≤ 0.001.</p>
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<p>Mean ± 95% CI length of stay (days) relative to significant predictor variables for fire-affected marsupial groups released/relocated in 2019/20: (<b>a</b>) macropods by injury type, (<b>b</b>) koalas by fire severity, (<b>c</b>) koalas by injury type, and (<b>d</b>) possums and gliders by injury type that were. Note: The 95% CI for koala injury type malnourished/moribund was wide [−763 and 902] and is not displayed. Superscript letters denote significant differences between categorical variables as determined using pairwise comparisons.</p>
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<p>Length of stay (days) for fire-affected macropods and koalas that died or were euthanised in rehabilitation after rescue in 2019/20 relative to the significant predictor variables outlined in <a href="#animals-14-01019-t003" class="html-table">Table 3</a>. Macropod mean (±95% CI) length of stay (days) versus (<b>a</b>) injury type and (<b>b</b>) age. Superscript letters denote significant differences between categorical variables as determined using pairwise comparisons.</p>
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<p>Location of rescue due to 2019–2020 bushfires for (<b>a</b>) macropods, (<b>b</b>) possums and gliders, (<b>c</b>) koalas and (<b>d</b>) wombats.</p>
Full article ">Figure 10
<p>Number of records for possums, macropods and koalas in each of four main IBRA regions (NSW North Coast, South East Corner, South Eastern Highlands, Sydney Basin) with any cause for rescue (all records) and fire-affected animals (fire records) across the years preceding the 2019–2020 bushfires in eastern Australia (2015/16–2018/19; blue columns) and the year of the 2019–2020 bushfires (2019/20; red columns). Density estimates for possums, macropods and koalas are annotated down the centre of the chart (per ha); macropod and possum densities were extracted from van Eeden et al. [<a href="#B4-animals-14-01019" class="html-bibr">4</a>]; koala densities were calculated as mean population size from Adams-Hosking et al. [<a href="#B38-animals-14-01019" class="html-bibr">38</a>] over IBRA size (ha) from DAWE [<a href="#B41-animals-14-01019" class="html-bibr">41</a>].</p>
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<p>Wildlife rescue records in NSW between 2015/16 and 2019/20 for all species and for marsupials alone. Data sourced from NSW Wildlife Rehabilitation dashboard [<a href="#B29-animals-14-01019" class="html-bibr">29</a>].</p>
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23 pages, 6722 KiB  
Article
SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization
by Nathaly Cormier, Asha E. Worsham, Kinsey A. Rich and Daniel M. Hardy
Int. J. Mol. Sci. 2024, 25(7), 3652; https://doi.org/10.3390/ijms25073652 - 25 Mar 2024
Viewed by 838
Abstract
Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 [...] Read more.
Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4aHaus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals. Full article
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Figure 1
<p>Sequences of SMA20 composite cDNA and its predicted 126 amino acid polypeptide. (<b>A</b>) Nucleotide and deduced amino acid sequences of the 786-bp SMA20 composite cDNA. The tryptic peptide obtained by de novo sequencing is boxed in black. Numbers on the left denote positions in the nucleotide sequence, and numbers on the right denote positions in the amino acid sequence. The 5′- and 3′-UTRs (lowercase letters) flank a 378 nt coding sequence (upper case letters) with the predicted initiator methionine (boxed in gray) embedded in a good Kozak consensus (AcAGcccccg; Ref. [<a href="#B23-ijms-25-03652" class="html-bibr">23</a>]), a non-canonical ATTAAA polyadenylation signal (red underline) specifying transcript termination, and in-frame stop codons (black underlines) in both UTRs. Features of the encoded protein include, in order, a region of AP-rich sequence (boxed in green) that includes two PGAP putative SH3 domain binding sites (yellow underlines) but no contiguous hydrophobic amino acids expected for an N-terminal signal peptide [<a href="#B24-ijms-25-03652" class="html-bibr">24</a>], followed by two predicted transmembrane segments (pink underlines). (<b>B</b>) Predicted SMA20 properties. Shown are the hydrophilicity (“Kyte–Doolittle”), antigenicity (“Jameson–Wolf”), and surface probability (“Emini”) inferred from the SMA20 protein sequence. The horizontal ruler denotes the amino acid position, and the vertical axes represent arbitrary units. Peptides used for antibodies (overlined in red) spanned amino acids P<sup>21</sup>-E<sup>53</sup> (anti-P-rich) and S<sup>82</sup>-E<sup>96</sup> (anti-loop) between the two putative transmembrane segments (TMS; boxed in pink). (<b>C</b>) SMA20 structural modules. Asterisks and small ovals and rectangles mark locations of predicted sites for posttranslational modification as indicated in the alanine- and proline-rich (“A/P-rich”) region containing two predicted SH3 binding motifs (PGAP, black bars), and in the loop region flanked by predicted transmembrane segments (“TMS1/2”).</p>
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<p>Tissue specificity of SMA20 mRNA expression. (<b>A</b>) Upper panel: Detection of SMA20 mRNA on Northern blot of total RNAs (5 μg/lane) isolated from pig tissues via hybridization of a 5′-end cDNA probe (nts 1–395; 2-day exposure). All tissues except epididymis and testis came from a female pig. Lower panel: The ethidium bromide-stained gel before blot transfer, confirming quality and equal loading of the total RNA preparations. (<b>B</b>) Northern blot confirmation of the full-length SMA20 composite cDNA via hybridization of oligonucleotides (30 mer each) complementary to 5′- and 3′-end SMA20 cDNA to blots of pig testis total RNA (20 μg/lane; 4-day exposure). (<b>C</b>) Upper panel: Northern blot detection of SMA20 mRNA (5 μg total RNA per lane) in pig testis from a pre-pubertal boar (“pre”) and from three mature boars (“1–3”) via hybridization with the same SMA20 cDNA probe as for panel A. Lower panel: hybridization of the stripped blot with a probe for S16 ribosomal RNA to verify equivalent loading.</p>
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<p>Membrane and lipid raft association of SMA20 in pig spermatozoa. Shown are Western blots probed with raw heteroantisera (“As”) or affinity-purified antibodies (“Ab”) directed against the SMA20 loop or proline-rich regions (“αLoop” or “αP-rich” respectively; panels (<b>A</b>–<b>C</b>)), or with alloantisera raised against pig sperm TWM or lipid rafts (“αTWM” or “αRafts”; panel (<b>D</b>)). (<b>A</b>) TWM resolved on 8–15% linear gradient SDS-PAGE (10 μg protein/lane) and detected with αLoop or αP-rich antisera or purified antibodies. The blue arrowhead marks the location of the M<span class="html-italic">r</span> 37,000 band recognized by the αP-rich peptide antibody. (<b>B</b>) 20 μg of TWM resolved on 15% SDS-PAGE; detected with αLoop or αP-rich antisera or purified antibodies preabsorbed (+), or not (−), with their cognate peptides. Note the robust blocking of immunoreactivity by the peptides. (<b>C</b>) SMA20 in proteins extracted sequentially from 5 × 10<sup>6</sup> spermatozoa with 1% Triton X-100 (TX100) and 1% SDS [<a href="#B26-ijms-25-03652" class="html-bibr">26</a>] and then resolved and blotted as for panel (<b>A</b>). (<b>D</b>) Alloantigens in 50 μg of TWM resolved by two-dimensional gel electrophoresis and detected on blots with αTWM or αRafts alloantisera. Red arrows indicate SMA20 protein (<span class="html-italic">M</span>r 20,000 and p<span class="html-italic">I</span> 4.3).</p>
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<p>Localization of SSMP20 protein on pig spermatozoa by immunofluorescence. Shown are paired fluorescence and phase contrast images of identical microscopic fields (400× magnification). WGA = labeling with wheat germ agglutinin to detect plasma membrane. (<b>A</b>) Detection of SMA20 in MeOH-fixed (=membrane permeabilized) spermatozoa. Red fluorescence depicts SMA20 immunoreactivity detected with affinity-purified antibodies directed against the SMA20 loop (“αLoop”, 1.1 μg/mL) or proline-rich (“αP-rich”, 0.8 μg/mL) regions, respectively, and green fluorescence depicts WGA detection of the acrosome. (<b>B</b>) Detection of SMA20 on live-labeled spermatozoa.</p>
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<p>Ultrastructural localization of SMA20 by immuno-EM with αP-rich affinity-purified antibody. Shown are multiple fields of cross-sections through heads of mature spermatozoa labeled after embedding and sectioning. (<b>A</b>) Cells post-fixed with osmium tetroxide. Red arrows mark SMA20-associated gold nanoparticles localized in apposition to the inner leaflet of the plasma membrane (pm) overlying the anterior head, with its ruffled appearance characteristic of spermatozoa in TEM [<a href="#B26-ijms-25-03652" class="html-bibr">26</a>], as well as much fewer on the outer acrosomal membrane (oam). The bar graph summarizes relative quantification of gold particles’ specific association with the head plasma membrane, evident as nearly complete blocking of immunoreactivity by cognate peptide in 40 randomly chosen microscopic fields. a = acrosome; n = nucleus. (<b>B</b>) Cells without osmium post-fixation. The bar graph summarizes relative quantification of specific labeling as for panel (<b>A</b>).</p>
Full article ">Figure 5 Cont.
<p>Ultrastructural localization of SMA20 by immuno-EM with αP-rich affinity-purified antibody. Shown are multiple fields of cross-sections through heads of mature spermatozoa labeled after embedding and sectioning. (<b>A</b>) Cells post-fixed with osmium tetroxide. Red arrows mark SMA20-associated gold nanoparticles localized in apposition to the inner leaflet of the plasma membrane (pm) overlying the anterior head, with its ruffled appearance characteristic of spermatozoa in TEM [<a href="#B26-ijms-25-03652" class="html-bibr">26</a>], as well as much fewer on the outer acrosomal membrane (oam). The bar graph summarizes relative quantification of gold particles’ specific association with the head plasma membrane, evident as nearly complete blocking of immunoreactivity by cognate peptide in 40 randomly chosen microscopic fields. a = acrosome; n = nucleus. (<b>B</b>) Cells without osmium post-fixation. The bar graph summarizes relative quantification of specific labeling as for panel (<b>A</b>).</p>
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<p>SMA20 is the pig ortholog of PMIS2. Shown are pairwise dot-plot comparisons of the SMA20 cDNA sequence (vertical dimension in all panels) to the warthog coding sequence (cds) and, as indicated, warthog, pig, mouse, human, and armadillo genomic sequences between <span class="html-italic">Atp4a</span> and <span class="html-italic">Haus5</span> in their respective species (horizontal dimensions).</p>
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<p>SMA20/PMIS2 is unique to Eutheria. Shown are dotplot comparisons constructed with a concatemeric assembly of armadillo reverse-complemented <span class="html-italic">Atp4a</span>, <span class="html-italic">Pmis2</span>, and <span class="html-italic">Haus5</span> coding sequences (cds) as the vertical dimension for interrogation of armadillo and opossum genomic loci, and a comparable assembly with reverse-complemented <span class="html-italic">Atp4a</span>, <span class="html-italic">Pmis2</span>, and reverse-complemented <span class="html-italic">Etv2</span> cds for interrogation of platypus genomic loci.</p>
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<p>Multiple alignment (MUSCLE) of pig (<span class="html-italic">Sus scrofa</span>) SMA20 polypeptide sequence to PMIS2 orthologs from 69 species representing 17 of the 19 extant orders of placental mammals (Afrosoricida, Carnivora, Cetartiodactyla, Chiroptera, Cingulata, Dermoptera, Eulipotyphla, Lagomorpha, Perissodactyla, Pholidota, Pilosa, Primates, Proboscidea, Rodentia, Scandentia, Sirenia, and Tubulindentata; missing are Hyracoidea and Macroscelidea). (<b>A</b>) Alignment of N-terminal region sequences. The bottom seven species are myomorph rodents. (<b>B</b>) Alignment of TMS1 and TMS2 region sequences showing the sequence variation in segments upstream of the relatively more conserved transmembrane segments (red overlines). Colored shading denotes similar amino acid side chain chemistries: light yellow = aromatic, dark yellow = hydrophobic/aliphatic, green = hydrophilic, blue = basic, red = acidic.</p>
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<p>Multiple alignment (MUSCLE) of pig (<span class="html-italic">Sus scrofa</span>) SMA20 polypeptide sequence to PMIS2 orthologs from 69 species representing 17 of the 19 extant orders of placental mammals (Afrosoricida, Carnivora, Cetartiodactyla, Chiroptera, Cingulata, Dermoptera, Eulipotyphla, Lagomorpha, Perissodactyla, Pholidota, Pilosa, Primates, Proboscidea, Rodentia, Scandentia, Sirenia, and Tubulindentata; missing are Hyracoidea and Macroscelidea). (<b>A</b>) Alignment of N-terminal region sequences. The bottom seven species are myomorph rodents. (<b>B</b>) Alignment of TMS1 and TMS2 region sequences showing the sequence variation in segments upstream of the relatively more conserved transmembrane segments (red overlines). Colored shading denotes similar amino acid side chain chemistries: light yellow = aromatic, dark yellow = hydrophobic/aliphatic, green = hydrophilic, blue = basic, red = acidic.</p>
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<p>Genomic ontogeny of SMA20/PMIS2. Shown is the comparative synteny of regions encompassing the four upstream and four downstream genes immediately flanking <span class="html-italic">Pmis2</span> in the mouse genome. The compared <span class="html-italic">Sbsn</span>–<span class="html-italic">Cox6b1</span> regions span 140, 146, and 62 kb in the mouse, opossum, and platypus genomes, respectively (loci not drawn to scale). Blue arrows denote major changes that occurred in the evolution of Mammalia, including gene inversions associated with the divergence of Theria and Prototheria (with inverted three-gene cassette denoted by red arrows), as well as de novo genesis of <span class="html-italic">Pmis2</span> associated with the divergence of Metatheria and Eutheria.</p>
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<p>Candidate topologies (TopI-III) of SMA20 in the head of mature, ejaculated boar spermatozoa. All three topologies reflect cytoplasmic orientation of the AP-rich region in accordance with immunolocalization results, with “Outside” being extracellular or acrosomal lumen for SMA20 in the peri-acrosomal plasma membrane or outer acrosomal membrane, respectively. Squiggly lines depict the two presumably alpha helical hydrophobic segments, TMS1 and TMS2, proposed to span or embed in the lipid bilayer, as shown, and the black bars mark locations of possible SH3 binding motifs (<span class="html-italic">PGAP</span>) in the AP-rich region.</p>
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23 pages, 2938 KiB  
Article
Spontaneous Lesions of Endangered Geriatric Julia Creek Dunnarts (Sminthopsis douglasi, Archer 1979) with Emphasis in Reproductive Pathology
by Viviana Gonzalez-Astudillo, Andrea Schaffer-White, Lawrence Noble, Patricia O’Hara, Peter Murray, Tamsin S. Barnes and Rachel Allavena
Vet. Sci. 2024, 11(4), 142; https://doi.org/10.3390/vetsci11040142 - 22 Mar 2024
Viewed by 1385
Abstract
Julia Creek dunnarts are an endangered species of carnivorous marsupials and the focus of multiple conservation strategies involving significant resources such as captive breeding programs. Despite the relevance for conservation, no study to date has focused on evaluating geriatric diseases in dunnarts. This [...] Read more.
Julia Creek dunnarts are an endangered species of carnivorous marsupials and the focus of multiple conservation strategies involving significant resources such as captive breeding programs. Despite the relevance for conservation, no study to date has focused on evaluating geriatric diseases in dunnarts. This study describes the pathology findings in a group of one wild and thirty-five captive-born, mostly geriatric Julia Creek dunnarts that failed to produce offspring over multiple breeding periods. A total of 20 females and 16 males were submitted for a postmortem examination, with ages ranging from 9 to 42 and 12 to 42 months for females and males, respectively. Of these, 10 had unremarkable findings. The most common condition in females was cystic glandular hyperplasia (n = 8), typical of hormonal dysregulation profiles in senescence, particularly hyperestrogenism. Rarely, cutaneous disease represented by unidentified dermal round cell infiltrates was observed in females (n = 2). Primary reproductive hormonal dysregulation was also suspected in males diagnosed with testicular degeneration, aspermatogenesis and/or atrophy (n = 3). Cutaneous round cell infiltrates, possibly compatible with epitheliotropic lymphomas, were seen in males (n = 3), and 2/3 affected males also had concurrent testicular degeneration or atrophy, indicating male sex could be a predictor for lymphoid neoplasia in aged dunnarts, especially in individuals with concurrent testosterone-luteinizing hormone dysregulation as it occurs in gonadectomized animals. The role of an underlying viral etiology is also explored. This study is the first to describe major spontaneous diseases in endangered aged Julia Creek dunnarts, providing an important understanding of senescence and geriatric diseases within a conservation context. Full article
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<p><b>Gross female reproductive lesions in geriatric Julia Creek dunnarts.</b> (<b>a</b>) <b>Cystic glandular hyperplasia (CGH), dunnart 14.</b> A 2.5 cm diameter, pale pink to tan, firm abdominal cyst filled by a pale tan fluid expands the cavity and displaces abdominal viscera. (<b>b</b>) <b>CGH, dunnart 11.</b> The right uterus measures 3.5 cm in diameter, is diffusely dark purple, firm and is found expanding the abdominal cavity, displacing and compressing the abdominal viscera cranially as well as compressing the thoracic cavity, causing pulmonary collapse. The uterine mass is filled with approx. 5 mL of dark red, inspissated fluid. (<b>c</b>) <b>CGH and endometrial papillary adenoma, dunnart 13.</b> Rarely, a single uterus is expanded by two separate masses; the largest mass measures 2.5 cm diameter (arrowhead) and corresponds to CEH. The second mass (asterisk) is an endometrial papillary adenoma, measuring about 2 cm in diameter and filled by viscous dark brown-orange material. (<b>d</b>) <b>Ovarian cyst, Not Otherwise Specified—NOS, dunnart 6.</b> The ovary appears to be expanded by a single, unilocular mass filled by light yellow-pink fluid; bilaterally, the uteri are appreciated below the mass. Photo credit: Peter Moore. (<b>e</b>) <b>Vaginal neutrophilic infiltrates, dunnart 10.</b> Grossly, the lateral vaginas (arrowhead) are discolored off-white and moderately expanded, possibly indicating underlying pathologic inflammation. However, physiologic neutrophilic infiltrates that occur in certain small mammal species during metestrus and diestrus cannot fully be ruled out. This dunnart also had CGH and a cystic oviduct (not on image).</p>
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<p><b>Female reproductive lesions in geriatric Julia Creek dunnarts.</b> (<b>a</b>) <b>Age-related ovarian atrophy, dunnart 8.</b> Low numbers of primordial or primary follicles, few secondary follicles and 2 corpora albicans are observed. No corpora lutea are present, potentially indicating acyclic state. Hematoxylin and eosin (H&amp;E) stain, 10×. (<b>b</b>) <b>Cystic oviduct, dunnart 9.</b> The proximal end of the oviduct (ampulla or isthmus region) is expanded by a single, 5–7 mm diameter; fluid-filled cyst lined by a single layer of ciliated attenuated columnar epithelium; H&amp;E stain, 40×. (<b>c</b>) <b>Cystic glandular hyperplasia (CGH), dunnart 3.</b> Subgross view of the uterine wall with diffusely, actively proliferating, tortuous, dilated and often cystic endometrial glands. Hyperplastic glands lie back-to-back, supported by stromal bands of variable width and segmentally contain papillae covered by columnar, stratified to pseudostratified epithelium. H&amp;E stain, 4×. (<b>d</b>) <b>CGH with mucin, dunnart 7.</b> High magnification view into one of the cystic structures found expanding the endometrium, formed by anastomosing trabeculae of thin fibrous connective tissue septa lined by a single line of ciliated columnar secretory epithelium, with abundant goblet cells; H&amp;E stain, 20×. (<b>e</b>) <b>CGH with squamous metaplasia, dunnart 11.</b> The endometrium is expanded by numerous islands and anastomosing trabeculae forming cysts filled by fluid and necrotic debris lined by tall columnar cells. Foci of metaplastic, stratified, squamous, non-keratinizing epithelium are observed (asterisk). H&amp;E stain, 20×. (<b>f</b>) <b>Endometrial (glandular) polyp, dunnart 14.</b> A polypoid mass protrudes into the uterine lumen comprising numerous glands lined by predominantly hyperplastic/dysplastic columnar glandular epithelium, mimicking endometrial mucosa, and supported by a stroma comprising spindle cells with variable amounts of collagen and scattered small-caliber vasculature. Focal squamous metaplasia (asterisk) is also observed. H&amp;E stain, 4×. (<b>g</b>,<b>h</b>) <b>Endometrial adenoma, papillary, with focal squamous metaplasia, dunnart 13.</b> A neoplastic mass supported by a broad base formed by well-differentiated columnar, pseudostratified, mucus-secreting epithelial cells arranged in a papillary pattern (<b>f</b>) and mimicking endometrial epithelium is observed replacing the uterine stroma. No invasion to the endometrium or myometrium is observed. Focal squamous stratified and pseudostratified non-keratinizing epithelium is also observed forming fronds, covered by large amounts of necrotic debris. H&amp;E stain, 20×. (<b>i</b>) <b>Vaginal neutrophilic infiltrates, dunnart 10.</b> Florid neutrophilic infiltrates are observed multifocally performing exocytosis into the vagina mucosa and infiltrating the lamina propria, possibly supporting a pathologic vaginitis over those granulocytic infiltrates of physiological origin that infiltrate during metestrus or diestrus. H&amp;E stain, 10×.</p>
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<p><b>Gross male reproductive and non-reproductive lesions in geriatric Julia Creek dunnarts.</b> (<b>a</b>) <b>Testicular degeneration/atrophy, dunnart 9.</b> The scrotal sac has a markedly shortened inguinal canal, turning it into a sessile structure with no obvious palpable testes, easily exposing the urogenital sinus (arrowhead) and bulbourethral glands (asterisks). (<b>b</b>) <b>Normal testes and scrotum, control dunnart.</b> Scrotum contains palpable testes and is suspended from the abdomen by a regular stalk with the inguinal canal containing the spermatic cord, completely covering the urogenital sinus and partially the bulbourethral glands. (<b>c</b>) <b>Bilateral testicular degeneration/atrophy, dunnart 4.</b> The left testicle (arrowhead) is around 1.5 times smaller than the globally larger right testicle, indicating a unilateral testicular tubular degeneration/atrophy in this dunnart. (<b>d</b>,<b>e</b>) <b>Cutaneous epitheliotropic lymphoma, dunnart 4.</b> Extensive areas of alopecia affecting 90% of the body with diffusely markedly thickened skin that is mottled pink, red and with multiple scabs and flaking in the caudo-dorsal region. The coat is maintained in the distal cervical and proximal dorsal and flank regions. (<b>f</b>) <b>Dermatitis with alopecia, dunnart not included in study included for comparison.</b> An approx. 2 cm band of circumferential alopecia is observed in the cranial abdomen. Follicular atrophy was noticed on histopathology. Note that the skin is smooth and not crusty, ulcerated or thickened, as in the epitheliotropic lymphoma cases. (<b>g</b>) <b>Telogen alopecia, dunnart 25.</b> Lateral view. Regionally extensive areas of alopecia are observed predominantly affecting the lateral thighs and rump region in this view. The skin appears thickened and focally wrinkly. (<b>h</b>) <b>Telogen alopecia, dunnart 25.</b> Dorsal view. About 50% of the body presents a bilaterally symmetrical alopecia, particularly focused in the head (arrowhead), lateral thighs and rump region (asterisks). (<b>i</b>) <b>Telogen alopecia, dunnart 31.</b> There is bilaterally symmetrical alopecia affecting the lateral thighs, rump and craniodorsal abdominal region. <b>Inset:</b> Alopecia was occasionally observed along comedone formation. The right pinna has been removed postmortem from all dunnarts in the photographs for DNA analysis.</p>
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<p><b>Male reproductive lesions in geriatric Julia Creek dunnarts.</b> (<b>a</b>) <b>Testicular diffuse testicular tubular degeneration/atrophy with azoospermia, left testis, dunnart 4.</b> Despite autolytic artefact, which has removed the germinal epithelium and Sertolli cells from the seminiferous tubular lining, there are multiple, rounded, variably sized cells that are observed floating within the tubular lumina. Hematoxylin and eosin (H&amp;E) stain, 4×. <b>Inset:</b> Small aggregates of large, rounded multiple-germ-cell nuclei of similar maturity are observed within degenerated seminiferous tubular lumina, interpreted as multinucleated giant cells of germ cell origin. (<b>b</b>) <b>Testicular degeneration with azoospermia, right testis, dunnart 4.</b> The seminiferous tubules are devoid of early and late spermatids; no spermatogenesis is observed. Supporting basement membrane is wavy, buckled and thickened. Few granulomas are observed replacing seminiferous tubules (arrowheads), likely resulting from tubular shrinkage and collapse and/or spermiostasis. Numerous peritubular interstitial (Leydig) cell aggregates are observed forming non-compressive clusters, likely enhanced by the disappearing tubules. Increased numbers of interstitial cells are a normal finding in multiple marsupial species. H&amp;E stain, 4×. (<b>c</b>) <b>Segmental tubular degeneration/atrophy, dunnart 4.</b> Degeneration/atrophy also affected segments of seminiferous tubules, resulting in the visualization of tubules still containing germ cells in various stages of maturation (arrowhead) abutting others virtually devoid of germ cell epithelium but with a few remaining Sertolli cells (arrow). H&amp;E stain, 4×. (<b>d</b>) <b>Metastatic testicular lymphoma, dunnart 5.</b> The interstitium is unilaterally expanded by dense aggregates of lymphocytes, mainly within vasculature. H&amp;E stain, 10×. (<b>e</b>) <b>Epididymal segmental ductal atrophy, dunnart 5.</b> Segmental narrowing of the ductal lumina is observed with normal appearing epithelium, along occasionally lower epithelial height, likely due to testicular degeneration/atrophy in ipsilateral testis. H&amp;E stain, 10×. (<b>f</b>) <b>Severe bilateral testicular atrophy, dunnart 8.</b> No identifiable testicular tissue was found within the scrotum, only a portion of the epididymis. Some of these tubules contain protein; however, epithelium is attenuated and no sperm is visualized. H&amp;E stain, 20×. (<b>g</b>) <b>Prostatitis, dunnart 12.</b> The interstitium is multifocally expanded by small aggregates of lymphocytes and occasional plasma cells. H&amp;E stain, 20×. (<b>h</b>) <b>Calcified prostatic concretions, dunnart 9.</b> Multifocally within glandular lumina, there are variably sized, irregularly shaped mineral deposits. H&amp;E stain, 40×.</p>
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<p><b>Non-reproductive lesions of geriatric male Julia Creek dunnarts.</b> (<b>a</b>) <b>Primary cutaneous epitheliotropic lymphoma (mycosis fungoides), dunnart 5.</b> The dermis is infiltrated and replaced by a non-encapsulated and densely cellular neoplasm arranged in sheets. In multiple areas, the subcutis is also infiltrated. Hematoxylin and eosin (H&amp;E) stain, 4×. (<b>b</b>) <b>Primary cutaneous epitheliotropic lymphoma (mycosis fungoides), dunnart 4.</b> Dermal infiltrate predominantly comprises lymphocytes with hyperchromatic, convoluted or cerebriform nuclei admixed with occasional histiocytes and granulocytes. Multifocally, hair follicles are effaced by the infiltration of low numbers of neoplastic cells within the outer root sheath. H&amp;E, 40×. (<b>c</b>) <b>Primary cutaneous epitheliotropic lymphoma (mycosis fungoides), dunnart 5.</b> Multiple intraepidermal vesicles are filled with pleomorphic lymphoid cells (Pautrier’s microabscesses) and occasionally by solitary cells surrounded by a clear halo. H&amp;E, 40×. (<b>d</b>) <b>Peripheral diffuse cutaneous epitheliotropic lymphoma, dunnart 1.</b> Sheets of neoplastic lymphocytes are observed expanding the dermis, displacing adnexa and infiltrating and expanding the epidermis forming discrete and often coalescing Pautrier’s microabscesses. CD3 immunohistochemistry—IHC, 20×. (<b>e</b>) <b>Peripheral diffuse cutaneous epitheliotropic lymphoma, dunnart 1.</b> Neoplastic lymphocytes also reached into and isolated adipocytes. CD3 IHC, 10×.</p>
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11 pages, 1114 KiB  
Perspective
Beyond Borders: Investigating the Mysteries of Cacipacoré, a Lesser-Studied Arbovirus in Brazil
by Marielena V. Saivish, Maurício L. Nogueira, Shannan L. Rossi and Nikos Vasilakis
Viruses 2024, 16(3), 336; https://doi.org/10.3390/v16030336 - 22 Feb 2024
Cited by 1 | Viewed by 1196
Abstract
Cacipacoré virus (CPCV) was discovered in 1977 deep in the Amazon rainforest from the blood of a black-faced ant thrush (Formicarius analis). As a member of the family Flaviviridae and genus orthoflavivirus, CPCV’s intricate ecological association with vectors and hosts raises [...] Read more.
Cacipacoré virus (CPCV) was discovered in 1977 deep in the Amazon rainforest from the blood of a black-faced ant thrush (Formicarius analis). As a member of the family Flaviviridae and genus orthoflavivirus, CPCV’s intricate ecological association with vectors and hosts raises profound questions. CPCV’s transmission cycle may involve birds, rodents, equids, bovines, marsupials, non-human primates, and bats as potential vertebrate hosts, whereas Culex and Aedes spp. mosquitoes have been implicated as potential vectors of transmission. The virus’ isolation across diverse biomes, including urban settings, suggests its adaptability, as well as presents challenges for its accurate diagnosis, and thus its impact on veterinary and human health. With no specific treatment or vaccine, its prevention hinges on traditional arbovirus control measures. Here, we provide an overview of its ecology, transmission cycles, epidemiology, pathogenesis, and prevention, aiming at improving our ability to better understand this neglected arbovirus. Full article
(This article belongs to the Section Insect Viruses)
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<p>Geographic range and epidemiological landscape of Cacipacoré virus. Brazilian states with evidence of CPCV circulation are named. Hosts from which CPCV and/or antibody have been identified within a given Brazilian state are indicated by representative graphic(s). Pie charts within a given state indicate the number of studies identifying CPCV by size and the method of identification by color.</p>
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<p>Transmission cycles of CPCV (adapted from [<a href="#B72-viruses-16-00336" class="html-bibr">72</a>]).</p>
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20 pages, 10628 KiB  
Article
Astrocytes of the Anterior Commissure Regulate the Axon Guidance Pathways of Newly Generated Neocortical Neurons in the Opossum Monodelphis domestica
by Katarzyna Bartkowska, Paulina Koguc-Sobolewska, Ruzanna Djavadian and Krzysztof Turlejski
Int. J. Mol. Sci. 2024, 25(3), 1476; https://doi.org/10.3390/ijms25031476 - 25 Jan 2024
Viewed by 882
Abstract
In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the [...] Read more.
In marsupials, upper-layer cortical neurons derived from the progenitors of the subventricular zone of the lateral ventricle (SVZ) mature morphologically and send their axons to form interhemispheric connections through the anterior commissure. In contrast, eutherians have evolved a new extra callosal pathway, the corpus callosum, that interconnects both hemispheres. In this study, we aimed to examine neurogenesis during the formation of cortical upper layers, including their morphological maturation in a marsupial species, namely the opossum (Monodelphis domestica). Furthermore, we studied how the axons of upper layers neurons pass through the anterior commissure of the opossum, which connects neocortical areas. We showed that upper-layer II/III neurons were generated within at least seven days in the opossum neocortex. Surprisingly, these neurons expressed special AT-rich sequence binding protein 2 (Satb2) and neuropilin 1 interacting protein (Nrp1), which are proteins known to be essential for the formation of the corpus callosum in eutherians. This indicates that extrinsic, but not intrinsic, cues could be key players in guiding the axons of newly generated cortical neurons in the opossum. Although oligodendrocyte precursor cells were present in the neocortex and anterior commissure, newly generated upper-layer neurons sent unmyelinated axons to the anterior commissure. We also found numerous GFAP-expressing progenitor cells in both brain structures, the neocortex and the anterior commissure. However, at P12–P17 in the opossums, a small population of astrocytes was observed only in the midline area of the anterior commissure. We postulate that in the opossum, midline astrocytes allow neocortical axons to be guided to cross the midline, as this structure resembles the glial wedge required by fibers to cross the midline area of the corpus callosum in the rodent. Full article
(This article belongs to the Special Issue Advances in Research on Neurogenesis: 3rd Edition)
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<p>The distribution of BrdU+ and Satb2+ cells in the developing opossum brains. (<b>A</b>–<b>C</b>) coronal sections from the brain rostral part of P12 (<b>A</b>), P14 (<b>B</b>), and P17 (<b>C</b>), showing BrdU-labeled cells in the cingulate cortex, motor cortex, somatosensory cortex, granular insular cortex, piriform cortex, and anterior commissure. (<b>E</b>,<b>G</b>,<b>I</b>) coronal section from the brain caudal part of P12 (<b>E</b>), P14 (<b>G</b>), and P17 (<b>I</b>), demonstrating BrdU-labeled cells in the retrosplenial cortex and visual cortex. (<b>A</b>–<b>C</b>,<b>E</b>,<b>G</b>,<b>I</b>) BrdU-labeled cells were located in the neocortical upper layers of opossums that were injected at P12, P14, and P17, and sacrificed at P90. A few BrdU-immunopositive cells were observed in the anterior commissure at P12 (<b>A</b>,<b>D</b>), and their numbers gradually increased at P14 (<b>B</b>,<b>F</b>), and P17 (<b>C</b>,<b>H</b>); (<b>D</b>,<b>F</b>,<b>H</b>) Zoomed areas shown in (<b>A</b>), (<b>B</b>) and (<b>C</b>), respectively. (<b>J</b>–<b>L</b>) Immunostaining of developing cortical plate with Satb2 (red) at P12, P14, and P17 in opossum brains. The scale bar in (<b>C</b>), (<b>H</b>), and (<b>I</b>) refers to (<b>A</b>,<b>B</b>), (<b>D</b>,<b>F</b>), and (<b>E</b>,<b>G</b>), respectively. The scale bar in (<b>L</b>) refers to both images (<b>J</b>) and (<b>K</b>). AC, anterior commissure; Cg, cingulate cortex; ec, external capsule; GI, granular insular cortex; M, motor cortex; P, postnatal day; Pir, piriform cortex; RS, retrosplenial cortex; S, somatosensory cortex; V, visual cortex.</p>
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<p>Nrp1 protein in the developing cerebral cortex of opossums. (<b>A</b>–<b>C</b>) The distribution of Nrp1-immunolabeled cells in the cerebral cortex of P14, P17, and P19 opossums. (<b>D</b>) Representative Western blot of Nrp1 and loading control GAPDH protein expression in the cerebral cortex of opossums at P14, P17, P24, and P35. (<b>E</b>) The quantification of Nrp1 protein in the developing cerebral cortex. The scale bar in (<b>C</b>) refers to both (<b>A</b>,<b>B</b>).</p>
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<p>Development and cellular organization of the anterior commissure. (<b>A</b>–<b>C</b>) Double GFAP (red) and DAPI (blue)-immunolabeled brain sections from the anterior commissure at P12 (<b>A</b>), P14 (<b>B</b>) and P17 (<b>C</b>), and the cerebral cortex (<b>F</b>,<b>I</b>) at P17 (<b>F</b>) and P31 (<b>I</b>) in the opossums. (<b>D</b>,<b>E</b>,<b>G</b>,<b>H</b>) High magnification confocal images showing GFAP-labeled cells in the anterior commissure of P17 (<b>D</b>) and cerebral cortex of opossums at P17 (<b>E</b>) and P31 (<b>G</b>,<b>H</b>). (<b>L</b>) Double labeled with DAPI (<b>K</b>) and vimentin (<b>J</b>)-stained brain coronal sections at the level of the anterior commissure. The scale bar in (<b>C</b>), and (<b>L</b>) refers to (<b>A</b>,<b>B</b>), and (<b>J</b>,<b>K</b>), respectively.</p>
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<p>Development of oligodendrocytes in opossums. (<b>A</b>–<b>C</b>) Olig2 (red) and DAPI (blue) immunostaining in the anterior commissure of opossums at P17 (<b>A</b>), P21 (<b>B</b>) and P30 (<b>C</b>). (<b>D</b>–<b>F</b>) BrdU (green) and CC1 (red) immunolabeled cells in brain sections presenting the anterior commissure in opossums at P19 (<b>D</b>), P21 (<b>E</b>) and P30 (<b>F</b>). (<b>G</b>,<b>H</b>) Percentage of cells colocalizing Olig2 and DAPI (<b>G</b>) or BrdU and CC1 (<b>H</b>) expressed as mean ± SEM. * <span class="html-italic">p</span> = 0.02, ** <span class="html-italic">p</span> &lt; 0.0001. The scale bar in (<b>C</b>), and (<b>F</b>) refers to (<b>A</b>,<b>B</b>), and (<b>D</b>,<b>E</b>), respectively.</p>
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<p>Myelination of the anterior commissure in the developing opossum. (<b>A</b>–<b>C</b>) Myelin basic protein (MBP) immunostaining in the anterior commissure of opossums at P30 (<b>A</b>), P40 (<b>B</b>) and P50 (<b>C</b>). (<b>D</b>–<b>L</b>) Fiber staining of the anterior commissure using the Gallyas silver impregnation method. Individual myelinated axons were visualized in the anterior commissure in the 30-day-old opossum brain (<b>D</b>), and the intense staining of fiber tracts was seen in 50-day-old (<b>F</b>–<b>I</b>) and 90-day-old opossums (<b>J</b>–<b>L</b>). The scale bar in (<b>C</b>) refers to both (<b>A</b>) and (<b>B</b>). The scale bar in (<b>F</b>) and (<b>J)</b> refers to (<b>D</b>,<b>E</b>,<b>H</b>,<b>I</b>,<b>K</b>,<b>L</b>), and (<b>G</b>), respectively.</p>
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<p>The pattern of axon projection neocortical neurons in the developing opossum brain is defined by DiI labeling (<b>A</b>–<b>F</b>) or virus infection (<b>G</b>–<b>J</b>). The pattern of the DiI-labeled axons of neocortical neurons in 12-day-old (<b>A</b>–<b>C</b>), 14-day-old (<b>D</b>–<b>F</b>), and 30-day-old (<b>G</b>–<b>J</b>) opossum brains. (<b>B</b>,<b>E</b>) and (<b>C</b>,<b>F</b>) The zoomed area showing the external capsule (<b>B</b>,<b>E</b>) and the anterior commissure (<b>C</b>,<b>F</b>) in 12-day-old (<b>B</b>,<b>C</b>) and 14-day-old (<b>E</b>,<b>F</b>) opossums. (<b>H</b>) the zoomed area from the coronal brain section (<b>G</b>), showing just below the injection site; (<b>I</b>) the zoomed area from the anterior cortex, showing mCherry-expressing axons; and (<b>J</b>) the zoomed area from the contralateral neocortex. (<b>K</b>) Schematic representation of the coronal section presented in G, showing the injection site and fiber tract containing neocortical neuronal axons that cross the anterior commissure and reach the contralateral neocortex. The scale bar in (<b>A</b>), and (<b>F</b>) refers to (<b>D</b>), and (<b>B</b>,<b>C</b>,<b>E</b>), respectively. The scale bar in (<b>J</b>) refers to both images (<b>H</b>,<b>I</b>). 3V, third ventricle; AC, anterior commissure; CTX, cerebral cortex; HIP, hippocampus; PIR, piriform cortex; STR, striatum.</p>
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29 pages, 3557 KiB  
Article
Gut Microbiome Profiling of the Endangered Southern Greater Glider (Petauroides volans) after the 2019–2020 Australian Megafire
by Jordyn Clough, Sibylle Schwab and Katarina Mikac
Animals 2023, 13(22), 3583; https://doi.org/10.3390/ani13223583 - 20 Nov 2023
Viewed by 2367
Abstract
Studying the gut microbiome can provide valuable insights into animal health and inform the conservation management of threatened wildlife. Gut microbiota play important roles in regulating mammalian host physiology, including digestion, energy metabolism and immunity. Dysbiosis can impair such physiological processes and compromise [...] Read more.
Studying the gut microbiome can provide valuable insights into animal health and inform the conservation management of threatened wildlife. Gut microbiota play important roles in regulating mammalian host physiology, including digestion, energy metabolism and immunity. Dysbiosis can impair such physiological processes and compromise host health, so it is essential that the gut microbiome be considered in conservation planning. The southern greater glider (Petauroides volans) is an endangered arboreal marsupial that faced widespread habitat fragmentation and population declines following the 2019–2020 Australian bushfire season. This study details baseline data on the gut microbiome of this species. The V3–V4 region of the 16S rRNA gene was amplified from scats collected from individuals inhabiting burnt and unburnt sites across southeastern Australia and sequenced to determine bacterial community composition. Southern greater glider gut microbiomes were characterised by high relative abundances of Firmicutes and Bacteroidota, which is consistent with that reported for other marsupial herbivores. Significant differences in gut microbial diversity and community structure were detected among individuals from different geographic locations. Certain microbiota and functional orthologues were also found to be significantly differentially abundant between locations. The role of wildfire in shaping southern greater glider gut microbiomes was shown, with some significant differences in the diversity and abundance of microbiota detected between burnt and unburnt sites. Overall, this study details the first data on greater glider (Petauroides) gut microbiomes, laying the foundation for future studies to further explore relationships between microbial community structure, environmental stressors and host health. Full article
(This article belongs to the Section Wildlife)
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<p>Map of southern greater glider sampling locations across southeastern NSW, Australia. Locations include Seven Mile Beach National Park (NP); Meroo NP; Murramarang NP; a public reserve in Broulee; Eurobodalla NP; Monga NP; and Sharewater, a wildlife sanctuary near Braidwood. Fire severity was taken from NSW Department of Planning and Environment Fire Extent and Severity Mapping (FESM) 2019/20 overlay [<a href="#B29-animals-13-03583" class="html-bibr">29</a>]. Sampling locations are marked as burnt (red) or unburnt (green). Map generated using qGIS v3.32.1.</p>
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<p>Alpha diversity of southern greater glider gut microbiomes from southeastern NSW. Boxplots show mean alpha diversity of samples at each location, with interquartile range and outlier values indicated. (<b>a</b>) Chao1 index. (<b>b</b>) Shannon index. 7MBNP = Seven Mile Beach NP (<span class="html-italic">n</span> = 4), BROUL = Broulee (<span class="html-italic">n</span> = 4), CONGO = Eurobodalla NP (<span class="html-italic">n</span> = 2), MEROO = Meroo NP (<span class="html-italic">n</span> = 3), MONGA = Monga NP (<span class="html-italic">n</span> = 6), MURRA = Murramarang NP (<span class="html-italic">n</span> = 3), SHARE = Sharewater (<span class="html-italic">n</span> = 2).</p>
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<p>Principal coordinate analysis of beta diversity distances between southern greater glider gut microbiomes. (<b>a</b>) Bray–Curtis distances. (<b>b</b>) Jaccard distances. The proportion of variance among the samples explained by each axis is indicated in brackets. Individuals sampled from various geographic locations across southeastern NSW. 7MBNP = Seven Mile Beach NP (<span class="html-italic">n</span> = 4), BROUL = Broulee (<span class="html-italic">n</span> = 4), CONGO = Eurobodalla NP (<span class="html-italic">n</span> = 2), MEROO = Meroo NP (<span class="html-italic">n</span> = 3), MONGA = Monga NP (<span class="html-italic">n</span> = 6), MURRA = Murramarang NP (<span class="html-italic">n</span> = 3), SHARE = Sharewater (<span class="html-italic">n</span> = 2). Plots generated in R v4.3.1.</p>
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<p>Taxonomic bar plots showing relative abundances of microbial taxa in southern greater glider gut microbiomes. (<b>a</b>) Phylum-level plot. (<b>b</b>) Family-level plot. (<b>c</b>) Genus-level plot. The size of the coloured regions on each plot indicates the proportional contributions (%) of each taxon to total composition (100%). Individuals sampled from various geographic locations across southeastern NSW. 7MBNP = Seven Mile Beach NP (<span class="html-italic">n</span> = 4), BRO = Broulee (<span class="html-italic">n</span> = 4), CONGO = Eurobodalla NP (<span class="html-italic">n</span> = 2), MEROO = Meroo NP (<span class="html-italic">n</span> = 3), MONGA = Monga NP (<span class="html-italic">n</span> = 6), MUR = Murramarang NP (<span class="html-italic">n</span> = 3), SW = Sharewater (<span class="html-italic">n</span> = 2). Plots generated in R v4.3.1.</p>
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<p>Heatmap plot showing relative abundance of KEGG orthologues (KOs) coding for plant-fibre-degrading enzymes detected across southern greater glider gut microbiomes from southeastern NSW. Geographic location and burn status of the sampling site are indicated. Relative abundance (%) of KO in each sample indicated by colour gradient. White cells indicate that the KO was not detected in the sample. K01179: endoglucanase, K05350: beta-glucosidase, K18650: exo-poly-alpha-galacturonosidase, K06113: arabinan endo-1,5-alpha-L-arabinosidas, K01051: pectinesterase, K01728: pectate lyase, K01181: endo-1,4-beta-xylanase, K15924: glucuronoarabinoxylan endo-1,4-beta-xylanase, K01811: alpha-D-xyloside xylohydrolase, K01805: xylose isomerase. Plot generated in R v4.3.1.</p>
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<p>Differentially abundant KOs detected between southern greater glider gut microbiomes from across southeastern NSW. (<b>a</b>) Volcano plot from ANCOM differential abundance analysis. Only significantly differentially abundant KOs (assuming a 0.05 significance threshold) are labelled. The y-axis value (W) represents the number of times the null hypothesis (that the average abundance of a given KO in one population is equal to that in another population) was rejected. The x-axis value (clr) represents the effect size change between the compared populations. (<b>b</b>) Heatmap showing relative abundance of significantly differentially abundant KOs at each sampling location. Relative abundance (%) of KO in each sample indicated by colour gradient. White cells indicate that the KO was not detected in the sample. Plots generated in R v4.3.1.</p>
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34 pages, 4361 KiB  
Article
Anatomy of the Female Koala Reproductive Tract
by Sara Pagliarani, Chiara Palmieri, Michael McGowan, Frank Carrick, Jackson Boyd and Stephen D. Johnston
Biology 2023, 12(11), 1445; https://doi.org/10.3390/biology12111445 - 17 Nov 2023
Viewed by 3270
Abstract
The koala (Phascolarctos cinereus), while being an iconic Australian marsupial, has recently been listed as endangered. To establish an improved understanding of normal reproductive anatomy, this paper brings together unpublished research which has approached the topic from two perspectives: (1) the [...] Read more.
The koala (Phascolarctos cinereus), while being an iconic Australian marsupial, has recently been listed as endangered. To establish an improved understanding of normal reproductive anatomy, this paper brings together unpublished research which has approached the topic from two perspectives: (1) the establishment of an artificial insemination program, and (2) the definition of Chlamydia spp.-derived histopathological changes of the female koala urogenital system. Based on the presentation and histological processing of over 70 opportunistic specimens, recovered from wildlife hospitals in Southeast Queensland (Australia), we describe the gross and microanatomy of the koala ovary, oviduct, uteri, vaginal complex, and urogenital sinus during the interestrous, proliferative, and luteal phases of the reproductive cycle. Full article
(This article belongs to the Section Reproductive Biology)
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<p>In situ location of the female koala reproductive system. Insert—Ovary within the ovarian bursa. Am—Ampulla of oviduct, At—Adipose tissue in pelvic canal, Bl—Bladder, Bu—Ovarian bursa, Ce—Cervix, Is—Isthmus of oviduct, Lv—Lateral vagina, Pe—Pelvic floor bisected, Ov—Ovary, Vc—Vaginal cul-de-sac, Us—Urogenital sinus. Bar = 1 cm. Inset: Bar = 0.5 cm.</p>
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<p>Schematic drawing of an example of the female koala reproductive system in the different stages of the reproductive cycle showing the relative cross-sectional sections and changes in volume of the respective components. lv—lateral vagina.</p>
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<p>Histological features of the koala’s ovary in the interestrous phase. (<b>A</b>) Outer cortical and inner medullary region of the ovary, converging into the hilus containing numerous blood vessels (asterisk). HE. Bar = 1 mm. (<b>B</b>) Primordial follicle with an oogonium surrounded by a single layer of flat follicular cells. HE. Bar = 20 µm. (<b>C</b>) Primary follicle with a central oocyte surrounded by a single layer of cuboidal follicular cells. HE. Bar = 20 µm. (<b>D</b>) Secondary follicle with columnar follicular cells and the appearance of the zona pellucida (arrow). HE. Bar = 20 µm. (<b>E</b>) Early stage of antrum formation (asterisk) with multiple layers of follicular cells. HE. Bar = 20 µm. (<b>F</b>) Graafian follicle with theca cells (arrow) and a single cavity containing follicular fluid (asterisk). HE. Bar = 100 µm.</p>
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<p>Histological features of follicular atresia (<b>A</b>,<b>B</b>) and hemorrhagic follicles (<b>C</b>) in the koala’s ovary. (<b>A</b>) Dilated atretic follicles with detachment and irregularity of the stratum granulosum (arrow). HE. Bar = 700 µm. (<b>B</b>) Obliterative atresia with invasion of the antral space by hypertrophic granulosa and theca cells. HE. Bar = 100 µm. (<b>C</b>) Hemorrhagic follicle consisting of a large blood-filled cavity (asterisk). HE. Bar = 100 µm.</p>
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<p>Histological features of the interstitial-like tissue within the koala’s ovary. (<b>A</b>) Multiple aggregates of interstitial-like tissues occupying 80–90% of the ovary (asterisks). HE. Bar = 200 µm. (<b>B</b>) Type I cells with pales eosinophilic often vacuolated cytoplasm (arrowheads) and Type II cells with strong eosinophilic cytoplasm (arrows). HE. Bar = 50 µm. (<b>C</b>) Residual follicle with multiple layers of granulosa cells (asterisk) encircled by the interstitial-like tissue. HE. Bar = 50 µm.</p>
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<p>Oviduct (<b>A</b>), uterus (<b>B</b>), and cervix (<b>C</b>) of the female koala in the interestrous phase. (<b>A</b>) Ampulla (a) and isthmus (i) of the oviduct (inset: reduced number of the mucosal folds in the isthmus close to the uterine junction). HE. Bar = 2 mm (inset—Bar = 100 µm). (<b>B</b>). Uterine wall with mucosa/submucosa (asterisk), myometrium (arrow), and serosa/perimetrium (arrowhead). HE. Bar = 600 µm. (<b>C</b>) Protrusion of the caudal extremities of the muscular cervices (arrows) into the corresponding vaginal cul-de-sac complex (asterisks). HE. Bar = 1 mm.</p>
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<p>Vaginal cul-de-sac (<b>A</b>) and lateral vagina (<b>B</b>) of the female koala in the interestrous phase. (<b>A</b>) Medial septum (asterisk) partitioning the vaginal cul-de-sac. HE. Bar = 200 µm. (<b>B</b>) Stratum basale (arrow) and stratum spinosum (asterisk) lining the lumen of the lateral vaginae. HE. Bar = 60 µm.</p>
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<p>Urogenital sinus (<b>A</b>) and urogenital strand (<b>B</b>) of female koala in the interestrous phase. (<b>A</b>) Prominent longitudinal mucosal folds lining the lumen of the urogenital sinus. HE. Bar = 500 µm. (<b>B</b>) Urogenital strand (us) positioned between the urethra (u) and lateral vaginae (lv), and consisting of connective tissue, smooth muscle fibers, and blood vessels. HE. Bar = 500 µm.</p>
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<p>Histological features of the koala’s ovary in the proliferative phase. Large tertiary follicle (asterisk) in the ovarian cortex. HE. Bar = 2 mm.</p>
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<p>Histological features of the uterus (<b>A</b>), vaginal cul-de-sac (<b>B</b>), vagina (<b>C</b>), and urogenital sinus (<b>D</b>) of the koala’s ovary in the proliferative phase. (<b>A</b>) High number of glandular structures within the mucosa/submucosa of the uterus (inset: glandular tissue lined by columnar epithelial cells). HE. Bar = 200 µm (inset—Bar = 20 µm). (<b>B</b>) Increased folding of the epithelium lining the lumen of the vaginal cul-de-sac with cells transitioning to a columnar epithelium (inset). HE. Bar = 1 mm (inset—Bar = 5 µm). (<b>C</b>) Formation of the stratum granulosum (sg) over the stratum basale (sb) in the vaginal epithelium; note the desquamation of cornified epithelial cells in the lumen (asterisk). HE. Bar = 20 µm. (<b>D</b>) Hyperplastic and hypertrophic epithelial layer lining the lumen of the urogenital sinus (asterisk). Note the increased number of cells in the inset. HE. Bar = 1 mm (inset—Bar = 100 µm).</p>
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<p>Histological features of ovary (<b>A</b>) and uterus (<b>B</b>) from pregnant koalas. (<b>A</b>) Large corpus luteum (asterisk), consisting of numerous polygonal cells with abundant eosinophilic granular cytoplasm (inset). HE. Bar = 1 mm (inset—Bar = 50 µm). (<b>B</b>) Bi-layered appearance of the uterus, with a basal layer consisting of glands lined by epithelial cells with a pale eosinophilic cytoplasm (bl), and a superficial layer (sl) with densely packed glands lined by epithelial cells with a hypereosinophilic cytoplasm. Note the interstitial edema (asterisk) in the superficial layer. HE. Bar = 600 µm.</p>
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<p>Histological features of the ovary (<b>A</b>–<b>C</b>) and uterus (<b>D</b>) from a post-partum koala. (<b>A</b>) Corpus luteum with a central cavity (C) lined by an amorphous eosinophilic material (asterisk). HE. Bar = 1 mm. (<b>B</b>,<b>C</b>) Numerous Graafian follicles in the process of degeneration in a post-partum ovary. Higher magnification of a generated follicle in (<b>C</b>)—note the irregular detachment of the stratum granulosum. HE. Bar = 1 mm (<b>B</b>), 200 µm (<b>C</b>). (<b>D</b>) Reduced thickness and regression of the superficial (sl) and basal layer (bl). HE. Bar = 500 µm.</p>
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<p>(<b>A</b>). Transverse section through the koala urogenital strand during interoestrus (11.5×); (<b>B</b>). Transverse section of the central portion of the urogenital stand tissue of a pregnant koala showing oedema associated with the formation of the birth canal (11.5×); (<b>C</b>). Slit-like birth canal found in the urogenital strand of a recently post-partum koala (11.5×); (<b>D</b>). The birth canal of the koala showing no evidence of an epithelial lining (76×). Bc—Birth canal; Bl—Bladder; Lv—Lateral vagina; Ur—Urethra; Us—Urogenital strand; Black arrow—possible weak point in the connective tissue of the urogenital strand where the birth canal first forms; White “X”—Evidence of oedema formation; White arrow—Transverse tear in urogenital strand.</p>
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16 pages, 2755 KiB  
Review
Oral Cavity Mucocele and Different Surgical Treatment Strategies: Is Laser Excision Effective? A Scoping Review
by Andrea Scribante, Matteo Pellegrini, Federica Pulicari, Francesca De Martino, Giacomo Li Vigni, Martina Ghizzoni and Francesco Spadari
Appl. Sci. 2023, 13(22), 12327; https://doi.org/10.3390/app132212327 - 14 Nov 2023
Cited by 3 | Viewed by 2056
Abstract
Mucocele is a common lesion localized in the oral cavity; it originates from the lesion of a salivary duct and the consequent accumulation of mucin in soft tissues. It is a common lesion of young patients and is frequently associated with areas subject [...] Read more.
Mucocele is a common lesion localized in the oral cavity; it originates from the lesion of a salivary duct and the consequent accumulation of mucin in soft tissues. It is a common lesion of young patients and is frequently associated with areas subject to traumas. Surgical treatment is needed especially if it reaches considerable dimensions, as it can cause discomfort in the patient and tends to increase in size if subjected to further traumatism. This scoping review aims to investigate which treatments are used for this type of lesion, which are the most suitable to prevent recurrence, and if laser-assisted surgery is considered more efficient than other methods. An electronic search was performed within the PubMed (MEDLINE) and Scopus databases. Articles published in the years 2010 to 2023 were selected. Techniques like marsupialization and laser-assisted excision effectively treat mucoceles, with no clear prevalence between them. Laser surgery offers speed, precision, and minimal post-operation discomfort, but randomized trials are needed for conclusive comparisons. Full article
(This article belongs to the Section Applied Dentistry and Oral Sciences)
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<p>Flow chart of the review process.</p>
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68 pages, 40066 KiB  
Review
Pheromone Sensing in Mammals: A Review of the Vomeronasal System
by Mateo V. Torres, Irene Ortiz-Leal and Pablo Sanchez-Quinteiro
Anatomia 2023, 2(4), 346-413; https://doi.org/10.3390/anatomia2040031 - 9 Nov 2023
Cited by 4 | Viewed by 3654
Abstract
This review addresses the role of chemical communication in mammals, giving special attention to the vomeronasal system in pheromone-mediated interactions. The vomeronasal system influences many social and sexual behaviors, from reproduction to species recognition. Interestingly, this system shows greater evolutionary variability compared to [...] Read more.
This review addresses the role of chemical communication in mammals, giving special attention to the vomeronasal system in pheromone-mediated interactions. The vomeronasal system influences many social and sexual behaviors, from reproduction to species recognition. Interestingly, this system shows greater evolutionary variability compared to the olfactory system, emphasizing its complex nature and the need for thorough research. The discussion starts with foundational concepts of chemocommunication, progressing to a detailed exploration of olfactory systems. The neuroanatomy of the vomeronasal system stands in contrast with that of the olfactory system. Further, the sensory part of the vomeronasal system, known as the vomeronasal organ, and the integration center of this information, called the accessory olfactory bulb, receive comprehensive coverage. Secondary projections of both the olfactory and vomeronasal systems receive attention, especially in relation to the dual olfactory hypothesis. The review concludes by examining the organization of the vomeronasal system in four distinct mammalian groups: rodents, marsupials, herpestids, and bovids. The aim is to highlight the unique morphofunctional differences resulting from the adaptive changes each group experienced. Full article
(This article belongs to the Special Issue Advances in Anatomy and Its History)
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<p>Dissection of the turbinate complex in a dog. The difference between the red respiratory mucosa (RM) and the brown olfactory mucosa (OM) is observed. 1. Dorsal turbinate; 2. Ventral turbinate; 3. Endoturbinate; 4. Ectoturbinates; 5. Olfactory bulb; 6. Telencephalon frontal lobe; 7. Frontal sinus.</p>
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<p>Lateral view of the nasal septum of a dog showing the projection area of the vomeronasal organ (open arrowheads) and the vomeronasal nerves (arrows). FS, frontal sinus; 1. Olfactory bulb, 2. Olfactory peduncle; 3. Telencephalon. Scale bar: 5 cm.</p>
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<p>Grüneberg ganglion of a mouse. Transverse section of the dorsal part of the nasal vestibule (NV) immunostained with anti-OMP showing immunopositive ganglion cells (arrowheads). NVC, Cartilage of the nasal vestibule. Scale bar: 100 μm.</p>
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<p>Septal organ of a mouse. Decalcified transverse section of the nasal cavity immunostained with anti-OMP. The immunopositive olfactory epithelium (OE) lines the roof of the nasal cavity, the nasal septum, the dorsal part of the ventral turbinate (VT) and the branches of the olfactory nerve (nI). On the basal part of the nasal septum a patch of OMP-positive epithelium corresponding to the septal organ (SO) can be observed. VN, vomeronasal nerves. Scale bar: 1 mm.</p>
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<p>Septal organ of a mouse. Viewed at higher magnification of the septal organ (SO) area than is shown in the <a href="#anatomia-02-00031-f004" class="html-fig">Figure 4</a>. Decalcified transverse section of the nasal cavity immunostained with anti-OMP. Immunopositive neuroreceptor cells and the branches of the septal olfactory axons (SON) can be observed. VN, vomeronasal nerves. Scale bar: 100 μm.</p>
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<p>Vomeronasal organ of a horse. Autofluorescence transversal section of the VNO in which all components of the organ are identified. Aa, Artery; RE, Respiratory epithelium; RM, Respiratory mucosa; SE, Sensory epithelium; VC, Vomeronasal cartilage; VG, Vomeronasal glands; VN, Vomeronasal nerves; Vv, Veins. Nuclear contrast: TOPRO-3 iodide. Scale bar: 250 μm.</p>
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<p>Sensory epithelium of the dama gazelle vomeronasal organ stained with hematoxylin-eosin. 1. Basal cells; 2. Neuroreceptor cells; 3. Sustentacular cells; 4. Mucomicrovillar complex; 5. Vomeronasal nerve axons; 6. Veins; 7. Collagen fibers. Scale bar: 50 μm.</p>
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<p>Vomeronasal organ of a horse. Autofluorescence confocal microscopy of the sensory epithelium (SE) allows for the clear differentiation of the zones corresponding to the main strata: Basal (1); Neuroreceptor (2); Sustentacular (3); Mucomicrovillar (4). RE, respiratory epithelium; Red nuclear contrast: TOPRO-3 iodide. Scale bar: 50 μm.</p>
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<p>Vomeronasal gland of a cow stained with PAS-Alcian blue-hematoxylin staining. Both the glandular acini and the duct are stained. Scale bar: 150 μm.</p>
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<p>Sagittal section of the nose of a fetal mouse. The vomeronasal organ is located in the base of the nasal septum. A functional opening to the nasal cavity (NC) can be observed. The NC is lined by olfactory epithelium (OE). Hematoxylin-eosin staining. Scale bar: 500 μm.</p>
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<p>The vomeronasal organ of a rabbit. Hematoxylin-eosin staining. Transverse section of the head showing the nasal septum (1) with the VNOs located over the palatine processes of the incisive bone (2). The J-shaped cartilaginous envelope of the VNO (3) is covered by a thin bony layer (4). The ventral recess of the nasal cavity (5) is shaped by the cartilage of the incisive duct (6). (7). Ventral turbinate; (8). Palate. Scale bar: 500 μm.</p>
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<p>The vomeronasal organ of a fox. Transverse section of the base of the nasal septum (NS) showing the topographic relationships of the vomeronasal organs (arrows) with the ventral recesses of the nasal cavity (arrowheads). CT, canine tooth; SP, soft palate; Vm, vomer bone. Scale bar: 2 mm.</p>
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<p>The accessory olfactory bulb of a postnatal mouse. Confocal double immunofluorescence of a sagittal section stained with anti-MAP2 (green) and GFAP (red) showing the lamination of the AOB. 1. Vomeronasal nerve layer; 2. Glomerular layer; 3. Mitral plexiform layer; 4. Somas of mitral cells appear as unstained circular structures; 5. Lateral olfactory tract; 6. Granular cells layer. FL, Frontal lobe. Scale bar: 250 μm.</p>
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<p>The accessory olfactory bulb of a mouse. Immunohistochemical staining against MAP2. FL. Frontal lobe; GlL, glomerular layer; GrL, granular layer; MPL, mitral-plexiform layer; VNL, vomeronasal nerve layer; 2. Glomerular layer; 3. Mitral plexiform layer; 4. Somas of mitral cells appear as unstained circular structures; 5. Lateral olfactory tract; 6. Granular cells layer. FL, Frontal lobe. Scale bar: 250 μm.</p>
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<p>The accessory olfactory bulb of a cat. Nissl staining of a histological transverse section. The red box shows the topographic relationship of the olfactory bulb and the telencephalon frontal lobe. At higher magnification the yellow area containing the AOB allows to differentiate the lamination of the AOB. 1. Vomeronasal nerve layer; 2. Glomerular layer; 3. Mitral-plexiform layer; 4. Granular cell layer. Scale bar: 250 μm.</p>
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<p>The accessory olfactory bulb of a sheep. Consecutive Nissl (<b>A</b>) and Tolivia (<b>B</b>) staining of a sagittal section. The red box shows the topographic relationship of the olfactory bulb and the telencephalon frontal lobe. At higher magnification the yellow area containing the AOB allows to differentiate the lamination of the AOB. 1. Duramater; 2. Vomeronasal nerve layer; 3. Glomerular layer; 4. Mitral-plexiform layer; 5. Lateral olfactory tract; 6. Granular cell layer. Scale bar: 500 μm.</p>
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<p>The vomeronasal organ of a capybara. Transverse decalcified histological section stained with hematoxylin-eosin. RE, respiratory epithelium; SE, sensory epithelium; VC, vomeronasal cartilage; VD, vomeronasal duct; VG, vomeronasal glands; VD, vomeronasal duct; Vm, vomer bone; Vv, veins. Scale bar: 500 μm.</p>
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<p>Histological section of a capybara VNO stained with hematoxylin-eosin. A migratory stream (arrows) departing from the sensory epithelium (SE) to the vomeronasal axons (VN) can be observed. Scale bar: 100 μm.</p>
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<p>Immunohistochemical staining of a capybara AOB labelled with anti-GAP43 shows strong immunolabelling in the superficial layers: vomeronasal nerve (VNL) and glomerular (GlL) layers. GrL, Granular layer; LOT, Lateral olfactory tract; LV, lateral ventricle; MPL, Mitral-plexiform layer; WM, White matter. Scale bar: 100 μm.</p>
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<p>Hematoxylin-eosin histological staining of a wallaby vomeronasal duct. RE. Respiratory epithelium; SE. Sensory epithelium; VN. Vomeronasal nerve. Scale bar: 100 μm.</p>
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<p>PAS staining of the wallaby VNO showing a high density of PAS+ glandular acini (VG) in the dorsolateral part of the parenchyma. The vomeronasal duct contains PAS+ secretion (*). RE. Respiratory epithelium; RM. Respiratory mucosa; SE. Sensory epithelium; VN. Vomeronasal nerve; Vv. Veins. Scale bar: 200 mm.</p>
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<p>Anti-calretinin immunostaining of a wallaby SE of the VNO. Neuroreceptor cells somata (open arrowheads); Neuroreceptor cells dendritic knobs (arrowheads). Intraepithelial blood vessel (asterisk). Scale bar: 50 μm.</p>
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<p>UEA histochemical labelling of a wallaby VNO sensory epithelium. Neuroreceptor cells somata (open arrowheads); Neuroreceptor cells dendritic knobs (arrowheads). Dendritic processes of the neuroreceptor cells (arrows); Intraepithelial blood vessel (asterisk). Scale bar: 50 μm.</p>
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<p>(<b>A</b>) The vomeronasal organ of a meerkat. Transverse decalcified histological section stained with PAS. (<b>B</b>) Magnification in box A. NVN, vomeronasal nerves; RM, respiratory epithelium; V, vomer; VNC, vomeronasal cartilage; VNG, vomeronasal glands. Scale bars: A, 200 μm; B, 100 μm.</p>
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<p>Histological transverse section of the VNO of a dama gazelle showing its main components. NCd, nasal caudal nerve; RE, respiratory epithelium; RM, respiratory mucosa; SE, sensory epithelium; VD, vomeronasal duct; VG, vomeronasal glands; VN, vomeronasal nerve; Vv, veins. Scale bar: 500 μm.</p>
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<p>Hematoxilin-eosin staining of a dama gazelle VNO sensory epithelium. Neuroreceptor cells (arrowhead); Sustentacular cells (white arrowheads); Basal cells (asterisk); MMC, mucomicrovillar complex; SE, sensory epithelium; VN, vomeronasal nerve; Vv, veins. Scale bar: 250 μm.</p>
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<p>PGP9.5 immunostaining of the SE of a dama gazelle VNO showing the morphology and distribution of the neuroreceptor somata, and their dendritic knobs and processes. Scale bar: 150 μm.</p>
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16 pages, 8033 KiB  
Case Report
Giant Mandibular Ameloblastoma with Rare Hypercalcemia: A Case Report and Literature Review
by Wenyi Shen, Chenlu Xu, Pan Wang, Junpeng Chen, Dan Yu and Huiyong Zhu
Medicina 2023, 59(11), 1956; https://doi.org/10.3390/medicina59111956 - 6 Nov 2023
Cited by 1 | Viewed by 2046
Abstract
Ameloblastoma is the most common benign odontogenic tumor with local invasion and high recurrence, which generally occurs in the jaw bones. Hypercalcemia is a common paraneoplastic syndrome that is commonly observed in patients with malignancies but rarely encountered in patients with benign tumors. [...] Read more.
Ameloblastoma is the most common benign odontogenic tumor with local invasion and high recurrence, which generally occurs in the jaw bones. Hypercalcemia is a common paraneoplastic syndrome that is commonly observed in patients with malignancies but rarely encountered in patients with benign tumors. Thus far, not many cases of ameloblastoma with hypercalcemia have been reported, and the pathogenic mechanism has not been studied in depth. This paper presents a case report of a 26-year-old male diagnosed with giant ameloblastoma of the mandible, accompanied by rare hypercalcemia. Additionally, a review of the relevant literature is conducted. This patient initially underwent marsupialization, yet this treatment was not effective, which indicated that the selection of the appropriate operation is of prime importance for improving the prognosis of patients with ameloblastoma. The tumor not only failed to shrink but gradually increased in size, accompanied by multiple complications including hypercalcemia, renal dysfunction, anemia, and cachexia. Due to the contradiction between the necessity of tumor resection and the patient’s poor systemic condition, we implemented a multi-disciplinary team (MDT) meeting to better evaluate this patient’s condition and design an individualized treatment strategy. The patient subsequently received a variety of interventions to improve the general conditions until he could tolerate surgery, and finally underwent the successful resection of giant ameloblastoma and reconstruction with vascularized fibular flap. No tumor recurrence or distance metastasis was observed during 5 years of follow-up. Additionally, the absence of hypercalcemia recurrence was also noted. Full article
(This article belongs to the Section Dentistry and Oral Health)
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<p>CBCT imaging (<b>A</b>) and three-dimensional reconstruction of the skull (<b>B</b>) demonstrated extensive bony destruction and soft tissue involvement by the right mandibular tumor.</p>
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<p>Right mandibular ameloblastoma resulted in huge swelling of mandible and significant facial asymmetry (<b>A</b>,<b>B</b>). Intraoral examination showed that the right buccal vestibule and gums presented widely swollen with a cauliflower-like mass (<b>C</b>).</p>
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<p>Panoramic radiography (<b>A</b>), CT (<b>B</b>), three-dimensional reconstruction (<b>C</b>), and MRI (<b>D</b>) showed an expansive growth pattern of the tumor with bony destruction and huge soft tissue mass.</p>
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<p>The change in total serum calcium levels, serum creatinine levels, and hemoglobin levels after interventions.</p>
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<p>The computerized tomography with angiography of the neck vessels suggested that the right mandibular ameloblastoma was supplied by multiple branches of the right external carotid artery.</p>
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<p>Gross appearance of the ameloblastoma after surgical excision (<b>A</b>,<b>B</b>). Histological biopsy of the resected specimen showed the ameloblastoma was a mainly plexiform type (<b>C</b>) with a small amount of follicular structure (<b>D</b>).</p>
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<p>The patient’s facial deformity had improved greatly after tumor resection. (<b>A</b>–<b>C</b>) Clinical photos and (<b>D</b>,<b>E</b>) three-dimensional reconstruction.</p>
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<p>The patient received mandibular defect reconstruction with vascularized fibular myocutaneous flap. (<b>A</b>) Surgical photograph. (<b>B</b>) Clinical photograph. (<b>C</b>) Panoramic radiography. (<b>D</b>) Three-dimensional reconstruction.</p>
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33 pages, 2376 KiB  
Article
Three Blind Moles: Molecular Evolutionary Insights on the Tempo and Mode of Convergent Eye Degeneration in Notoryctes typhlops (Southern Marsupial Mole) and Two Chrysochlorids (Golden Moles)
by Mark S. Springer, Christopher A. Emerling and John Gatesy
Genes 2023, 14(11), 2018; https://doi.org/10.3390/genes14112018 - 28 Oct 2023
Cited by 1 | Viewed by 1821
Abstract
Golden moles (Chrysochloridae) and marsupial moles (Notoryctidae) are textbook examples of convergent evolution. Both taxa are highly adapted to subterranean lifestyles and have powerful limbs for digging through the soil/sand, ears that are adapted for low-frequency hearing, vestigial eyes that are covered by [...] Read more.
Golden moles (Chrysochloridae) and marsupial moles (Notoryctidae) are textbook examples of convergent evolution. Both taxa are highly adapted to subterranean lifestyles and have powerful limbs for digging through the soil/sand, ears that are adapted for low-frequency hearing, vestigial eyes that are covered by skin and fur, and the absence of optic nerve connections between the eyes and the brain. The eyes of marsupial moles also lack a lens as well as retinal rods and cones. Two hypotheses have been proposed to account for the greater degeneracy of the eyes of marsupial moles than golden moles. First, marsupial moles may have had more time to adapt to their underground habitat than other moles. Second, the eyes of marsupial moles may have been rapidly and recently vestigialized to (1) reduce the injurious effects of sand getting into the eyes and (2) accommodate the enlargement of lacrimal glands that keep the nasal cavity moist and prevent the entry of sand into the nasal passages during burrowing. Here, we employ molecular evolutionary methods on DNA sequences for 38 eye genes, most of which are eye-specific, to investigate the timing of relaxed selection (=neutral evolution) for different groups of eye-specific genes that serve as proxies for distinct functional components of the eye (rod phototransduction, cone phototransduction, lens/cornea). Our taxon sampling included 12 afrothere species, of which two are golden moles (Amblysomus hottentotus, Chrysochloris asiatica), and 28 marsupial species including two individuals of the southern marsupial mole (Notoryctes typhlops). Most of the sequences were mined from databases, but we also provide new genome data for A. hottentotus and one of the two N. typhlops individuals. Even though the eyes of golden moles are less degenerate than the eyes of marsupial moles, there are more inactivating mutations (e.g., frameshift indels, premature stop codons) in their cone phototransduction and lens/cornea genes than in orthologous genes of the marsupial mole. We estimate that cone phototransduction recovery genes were inactivated first in each group, followed by lens/cornea genes and then cone phototransduction activation genes. All three groups of genes were inactivated earlier in golden moles than in marsupial moles. For the latter, we estimate that lens/cornea genes were inactivated ~17.8 million years ago (MYA) when stem notoryctids were burrowing in the soft soils of Australian rainforests. Selection on phototransduction activation genes was relaxed much later (5.38 MYA), during the early stages of Australia’s aridification that produced coastal sand plains and eventually sand dunes. Unlike cone phototransduction activation genes, rod phototransduction activation genes are intact in both golden moles and one of the two individuals of N. typhlops. A second marsupial mole individual has just a single inactivating mutation in one of the rod phototransduction activation genes (PDE6B). One explanation for this result is that some rod phototransduction activation genes are pleiotropic and are expressed in extraocular tissues, possibly in conjunction with sperm thermotaxis. Full article
(This article belongs to the Section Population and Evolutionary Genetics and Genomics)
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<p>Simplified phylogeny for therian mammals that shows the placement of three mole families (Chrysochloridae, Talpidae, Notoryctidae). Divergence times are from Meredith et al. [<a href="#B11-genes-14-02018" class="html-bibr">11</a>]. Shaded boxes are as follows: blue = Laurasiatheria; green = Euarchontoglires; red = Afrotheria; orange = Xenarthra; and purple = Marsupialia. Silhouettes are for the same species or a closely related species.</p>
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<p>Summary of genes with and without inactivating mutations in <span class="html-italic">Notoryctes typhlops</span> (<b>left</b>) and two species of Chrysochloridae (<span class="html-italic">Amblysomus hottentotus</span>, <span class="html-italic">Chrysochloris asiatica</span>) (<b>right</b>). Vertical rectangles with light-gray shading indicate genes for which there are no inactivating mutations in both individuals of <span class="html-italic">N. typhlops</span> or both species of Chrysochloridae. Vertical rectangles with yellow shading indicate genes for which there is an inactivating mutation(s) in one of the two individuals of <span class="html-italic">N. typhlops</span> or one species of Chrysochloridae. Vertical rectangles with red shading indicate genes for which there is an inactivating mutation(s) in both individuals of <span class="html-italic">N. typhlops</span> or both species of Chrysochloridae.</p>
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<p>Examples of inactivating mutations in <span class="html-italic">Notoryctes typhlops</span> (<b>left</b>) and chrysochlorids (<b>right</b>). Gene names are shown above taxon names, and exon numbers are shown above alignments. Only 11 of 28 marsupial species are shown. Silhouettes are for the same species or a closely related species.</p>
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<p>Estimated times for the commencement of relaxed selection (neutral evolution) in five groups of eye genes in <span class="html-italic">Notoryctes typhlops</span>. Mean estimates are the mean of eight individual estimates for each gene group (<a href="#app1-genes-14-02018" class="html-app">Table S2</a>). Divergence times for Pseudocheiridae to <span class="html-italic">Gymnobelideus leadbeateri</span> are from Mitchell et al. [<a href="#B86-genes-14-02018" class="html-bibr">86</a>]. All other divergence dates are from Duchêne et al. [<a href="#B77-genes-14-02018" class="html-bibr">77</a>]. Silhouettes are for the same species or a closely related species.</p>
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<p>Estimated times for the commencement of relaxed selection (neutral evolution) in four groups of eye genes in Chrysochloridae. Mean estimates are the mean of eight individual estimates for each gene group (<a href="#app1-genes-14-02018" class="html-app">Table S2</a>). The divergence time for <span class="html-italic">Echinops telfairi</span> to <span class="html-italic">Microgale talazaci</span> is from Foley et al. [<a href="#B74-genes-14-02018" class="html-bibr">74</a>]. All other divergence times are from Meredith et al. [<a href="#B11-genes-14-02018" class="html-bibr">11</a>]. Silhouettes are for the same species or a closely related species.</p>
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17 pages, 4911 KiB  
Article
Development of Seven New dPCR Animal Species Assays and a Reference Material to Support Quantitative Ratio Measurements of Food and Feed Products
by Kate R. Griffiths, Jacob L. H. McLaughlin, Felicity Hall, Lina Partis, Samuel C. Hansen, Rachel Tulloch and Daniel G. Burke
Foods 2023, 12(20), 3839; https://doi.org/10.3390/foods12203839 - 20 Oct 2023
Cited by 1 | Viewed by 5401
Abstract
Laboratory testing methods to confirm the identity of meat products and eliminate food fraud regularly rely on PCR amplification of extracted DNA, with most published assays detecting mitochondrial sequences, providing sensitive presence/absence results. By targeting single-copy nuclear targets instead, relative quantification measurements are [...] Read more.
Laboratory testing methods to confirm the identity of meat products and eliminate food fraud regularly rely on PCR amplification of extracted DNA, with most published assays detecting mitochondrial sequences, providing sensitive presence/absence results. By targeting single-copy nuclear targets instead, relative quantification measurements are achievable, providing additional information on the proportions of meat species detected. In this Methods paper, new assays for horse, donkey, duck, kangaroo, camel, water buffalo and crocodile have been developed to expand the range of species that can be quantified, and a previously published reference assay targeting the myostatin gene has been modified to include marsupials and reptiles. The accuracy of this ratio measurement approach was demonstrated using dPCR with mixtures of meat DNA down to 0.1%. However, the limit of detection (LOD) of this approach is not just determined by the assay targets, but by the samples themselves, with food or feed ingredients and processing impacting the DNA yield and integrity. In routine testing settings, the myostatin assay can provide multiple quality control roles, including monitoring the yield and purity of extracted DNA, identifying the presence of additional meats not detected by the suite of species-specific assays and potentially estimating a sample-specific LOD based on measured copy numbers of the myostatin target. In addition to the myostatin positive control assay, a synthetic DNA reference material (RM) has been designed, containing PCR targets for beef, pork, sheep, chicken, goat, kangaroo, horse, water buffalo and myostatin, to be used as a positive template control. The availability of standardised measurement methods and associated RMs significantly improves the reliability, comparability and transparency of laboratory testing, leading to greater confidence in results. Full article
(This article belongs to the Special Issue Advanced Technologies in Detecting Food Fraud and Authenticity)
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<p>dPCR analysis of novel species assays. (<b>A</b>) Kangaroo assay. (<b>B</b>) Water Buffalo assay. (<b>C</b>) Camel/alpaca assay. (<b>D</b>) Crocodile/alligator assay. (<b>E</b>) Duck/goose assay. (<b>F</b>) Horse assay. (<b>G</b>) Donkey assay. (<b>H</b>) Published goat assay [<a href="#B24-foods-12-03839" class="html-bibr">24</a>]. One-dimensional scatter graphs are generated by the Bio-Rad QuantaSoft<sup>®</sup> software (version 1.7.4.0917), with each vertical column capturing data from an individual well. DNA samples analysed: cow, pig, chicken, sheep, horse, kangaroo, water buffalo, camel, goat, turkey, duck, goose, donkey, alpaca, red deer, bison, crocodile, alligator and quail. Droplets coloured blue or green demonstrate positive PCR amplification using FAM or HEX-labelled probes, respectively. Black droplets are PCR-negative. The dPCR assays were run as duplexes: water buffalo(FAM)/goat(HEX), camel(FAM)/crocodile(HEX), turkey(FAM)/duck(HEX), horse(FAM)/kangaroo(HEX), horse(FAM)/donkey(HEX). The horse results shown in F were duplexed with the donkey assay.</p>
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<p>dPCR analysis of horse and donkey assays run in simplex and duplex. One-dimensional scatter graphs showing analysis of horse DNA (left well) and donkey DNA (right well). (<b>A</b>) European horse assay (simplex). (<b>B</b>) Novel horse assay (simplex). (<b>C</b>) Novel horse assay (duplexed with donkey assay). (<b>D</b>) Novel donkey assay (simplex). (<b>E</b>) Novel donkey assay (duplexed horse with assay).</p>
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<p>Sequence line-up of the myostatin amplicon targeted by the MY77 PCR assay [<a href="#B4-foods-12-03839" class="html-bibr">4</a>]. Underlined sequences in the reference sequence indicate the binding positions for the published MY77 PCR assay and include the wobbles introduced to match the single nucleotide variants [<a href="#B4-foods-12-03839" class="html-bibr">4</a>,<a href="#B24-foods-12-03839" class="html-bibr">24</a>]. The nucleotides highlighted in bold and lowercase are those that deviate from the reference sequence.</p>
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<p>Comparison of performance between the published MY77 assay and a modified version of this assay using a novel reverse primer and probe to expand species suitability. DNA samples: chicken (wells 1–2), kangaroo (wells 3–4), crocodile (wells 5–6) and alligator (wells 7–8). For each species, the first well shows the MY77 assay published by Druml et al. [<a href="#B4-foods-12-03839" class="html-bibr">4</a>] (primer and probe sequences underlined in <a href="#foods-12-03839-f003" class="html-fig">Figure 3</a>); the second well shows the MY77 assay using a longer reverse primer (MY77R_251895) and the new probe (MY97P_256895F_YR), including the second wobble at position 12. The pink lines indicate where the boundaries between positive and negative populations were added manually.</p>
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<p>Copy number concentration ratios for each of the animal species assays relative to myostatin. Ratios were calculated as the copy number concentration from the species-specific assay divided by the copy number concentration from the MY77 assay. The error bars show two standard deviations (95% coverage) from four replicate ratio measurements.</p>
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<p>Measured versus expected DNA copy number ratios (%) for the DNA mixtures prepared, as shown in <a href="#foods-12-03839-t002" class="html-table">Table 2</a>, using seven of the animal species-specific assays. (<b>A</b>) Mixture 1. (<b>B</b>) Mixture 2. (<b>C</b>) Mixture 3. (<b>D</b>) Mixture 4. (<b>E</b>) Mixture 5. (<b>F</b>) Mixture 6. Measured (blue) and calculated (green) copy number ratios for mixtures of extracted DNA. B = beef, P = pork, S = sheep, C = chicken, H = horse, K = kangaroo, G = goat. The expected ratios for the individual species were calculated using the sum of species-specific copies as the denominator (Equation (1) shows the beef ratio calculation). The measured ratios for each of the species are the average copy number concentrations of duplicate dPCRs for each assay, as a proportion of the total dPCR copy number concentrations measured for that sample. The expected value for myostatin (100%) was calculated from the sum of species-specific copies added to the mixture, using the pre-measured DNA copy number concentrations and DNA volumes. The measured ratio for myostatin was calculated from the MY77 copy number concentrations relative to its expected copy number value.</p>
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<p>Two-dimensional scatter graphs generated by the Bio-Rad QuantaSoft<sup>®</sup> software (version 1.7.4.0917) of the synthetic DNA animal species reference material, RM1, using duplex dPCR assays. The animal species assays are analysed in pairs using four primers and two probes: (<b>A</b>) Cow/pig duplex assays. (<b>B</b>) Sheep/chicken duplex assays. (<b>C</b>) Horse/kangaroo duplex assays. (<b>D</b>) Water buffalo/goat duplex assays. The FAM-labelled probes (cow, sheep, horse and water buffalo assays) are analysed in Channel 1 and viewed on the y-axis. The HEX-labelled probes (pig, chicken, kangaroo and goat assays) are analysed in Channel 2 and viewed on the x-axis. In each scatter graph, black populations represent negative droplets that contain neither of the species targets, the blue populations contain targets for the FAM-labelled assay only, the green populations contain targets for the HEX-labelled assay only, the orange populations contain targets for both assays. When quantifying the number of positive droplets, the FAM assay is the sum of the blue and orange populations and the HEX assay is the sum of the green and orange populations.</p>
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<p>Examples of food, pet food and MBM analysed using duplex dPCR. (<b>A</b>) Kangaroo meat dog food roll. (<b>B</b>) Teriyaki beef strips. (<b>C</b>) Beef and pork MBM. (<b>D</b>) Rendered ovine MBM. (<b>E</b>) Cow’s milk camembert cheese. (<b>F</b>) Sheep’s milk feta. (<b>G</b>) Goat’s milk hard cheese. (<b>H</b>) Water buffalo blue cheese.</p>
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