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All data were collected through self-report questionnaires due to the study’s focus on workers’ perceptions of working conditions. Given the subjective nature of emotional dissonance, the appraisal of leadership quality, and mental distress, objective assessment methods pose challenges. To address potential issues arising from self-report, such as subjective interpretations, response set tendencies, and common method variance ( 58 ), steps were taken, including varying response anchors, separating independent and dependent variables in the survey, and ensuring participant anonymity ( 59 ). Given that this study stands as a singular contribution to documenting how high quality leadership behaviors buffers the impact of emotional dissonance on mental distress, replication in diverse populations is warranted for a more comprehensive understanding. To determine if the results of this study are replicable for mental health outcomes beyond self-reporting, future research should incorporate medically determined diagnoses for anxiety and depression.
|
39561242_p27
|
39561242
|
Strengths and limitations
| 3.580178 |
biomedical
|
Study
|
[
0.7511947154998779,
0.0008102417341433465,
0.2479950338602066
] |
[
0.9982580542564392,
0.0012594955042004585,
0.0004254753584973514,
0.00005696316293324344
] |
en
| 0.999996 |
Our nationwide study on home-care workers highlights the role of high-quality leadership in mitigating the negative impact of emotional dissonance on mental health. The study results underscore the benefits for organizations, particularly in human services where the prevalence of emotionally demanding job tasks is known to be high, of prioritizing the enhancement of leadership behaviors as a viable measure to prevent employee mental distress. Future studies should investigate whether our findings can be replicated in different occupational settings and whether they hold relevance to clinically diagnosed anxiety and depression outcomes.
|
39561242_p28
|
39561242
|
Concluding remarks
| 3.368607 |
biomedical
|
Study
|
[
0.7005765438079834,
0.0010155423078686,
0.2984079122543335
] |
[
0.9948069453239441,
0.004172676708549261,
0.0009143520728684962,
0.0001060405484167859
] |
en
| 0.999996 |
Symbiosis between a heterotrophic host and a photosynthetic partner (photosymbiosis) is considered to be the primary event which led to the acquisition and distribution of plastids in the evolution of eukaryotes . Photosymbiosis remains an essential life strategy and supports the functioning of today’s aquatic ecosystems especially in oligotrophic waters [ 3–6 ]. This partnership, considered as either mutualism or farming in the spectrum of symbioses , can provide a competitive advantage in a nutritionally challenging environment where nutrients and preys are scarce (oligotrophic waters). Therefore, partners need to establish a metabolic connection in order to exchange metabolites and nutrients. The microalgae need to be supplied by the host with all the essential macro- and micro-nutrients (e.g. iron, nitrogen) to maintain their metabolic and physiological activity. In turn, the host can benefit from photosynthetic products (photosynthates) exported from the microalgae .
|
39658219_p0
|
39658219
|
Introduction
| 4.200401 |
biomedical
|
Study
|
[
0.9988308548927307,
0.00025372864911332726,
0.0009154137806035578
] |
[
0.9812002778053284,
0.002373893279582262,
0.016285862773656845,
0.00013997257337905467
] |
en
| 0.999998 |
In the past decade, NanoSIMS (Nanoscale Secondary Ion Mass Spectrometry) studies coupled with 13 C labeling improved our knowledge on carbon transfer and allocation in different photosymbiotic systems [ 9–11 ]. Transferred photosynthates have been mostly investigated in benthic multicellular photosymbioses such as reef-dwelling invertebrates (e.g. anemones, jellyfish, giant clams, corals) living with Symbiodiniaceae microalgae. Sugars, which are the main photosynthetic products, and lipids are considered to be the main photosynthates exported from the symbiotic microalgae [ 12–14 ]. Glucose has been shown to be a major transferred metabolite in some photosymbioses , as well as inositol or galactose . Glycerol has also been suggested as a putative transferred metabolite since it is significantly released by free-living Symbiodiniaceae in culture [ 19–21 ]. However, the exact nature of translocated carbohydrates is still uncertain since experimental evidence is difficult to obtain on such photosymbiotic systems and on these very rapid metabolic processes.
|
39658219_p1
|
39658219
|
Introduction
| 4.15189 |
biomedical
|
Study
|
[
0.99895179271698,
0.00030393421184271574,
0.0007442182395607233
] |
[
0.9876044988632202,
0.00027873367071151733,
0.012019890360534191,
0.00009677858906798065
] |
en
| 0.999998 |
In symbiosis, both partners need to reprogram their transportome (defined as “membrane proteins responsible of the translocation of any kind of solutes across the lipid layer” ) in order to establish metabolic connectivity. Most metabolites including sugars require a complete set of transporters to traverse algal and host membranes. For example, in symbioses between plants and fungi, changes in expression of the transporter genes are essential to connect and integrate different metabolisms . In marine photosymbioses, some transporters have been highlighted in genomic and transcriptomic studies . A glucose transporter ( GLUT8 ) and an aquaporin ( GflP ) that could putatively transport glycerol, were described in anemones and the jellyfish Cassiopea . Even though most studies have focused on host transporters, less is known about the ones of the symbiotic microalgae that can export energy-bearing metabolites derived from photosynthesis. So far, a Sugars Will Eventually be Exported Transporter ( SWEET ) has been described as a glucose transporter located in the cell membrane of the microalga Breviolum (Symbiodiniaceae), the symbiont of the anemone Exaiptasia diaphana .
|
39658219_p2
|
39658219
|
Introduction
| 4.543318 |
biomedical
|
Study
|
[
0.999255359172821,
0.0003558018943294883,
0.0003888403589371592
] |
[
0.9831228852272034,
0.0008331254939548671,
0.01583201065659523,
0.00021201089839451015
] |
en
| 0.999995 |
Relatively less studied than reef ecosystems, a wide diversity of photosymbioses are also found in marine and freshwater plankton. For instance, radiolarians and foraminiferans that are widespread in the sunlit layer of the ocean can host diverse microalgae . Among radiolarians, some species of acantharia live in symbiosis with different species of Phaeocystis (Haptophyta) depending on the oceanic regions . For instance, P. cordata , P. globosa, and P. antarctica are known to be abundant in their free-living phase, and are found in symbiosis when acantharia are present (e.g. P. antarctica is the symbiont in the Southern Ocean . Symbiotic acantharia significantly contribute to primary production (up to 20% in surface oligotrophic oceans) and carbon fluxes to deep layers of the ocean . Previous studies using 3D electron microscopy have shown that the microalga Phaeocystis undergoes drastic morphophysiological changes in symbiosis: cell and plastid volume, as well as plastid number, greatly increased compared to free-living cells in culture . Whereas cell division is very likely arrested in symbiosis, photosynthesis and carbon fixation are enhanced, corroborated by an upregulation of many genes of the Calvin–Benson cycle . Symbiotic microalgae with their expanded photosynthetic apparatus therefore produce a substantial amount of organic carbon but the identity of these compounds and the mechanisms by which they are transferred to the host remain largely unknown. Investigating the composition and expression of the algal transportome can reveal how symbiotic microalgae metabolically connect to their host and can provide insights on the putative exchanged metabolites.
|
39658219_p3
|
39658219
|
Introduction
| 4.477295 |
biomedical
|
Study
|
[
0.9987683892250061,
0.00047879331395961344,
0.0007528821006417274
] |
[
0.9981088638305664,
0.0003810939670074731,
0.001414988306351006,
0.00009506996138952672
] |
en
| 0.999996 |
Here, we conducted genomic and transcriptomic analyses on an uncultivable planktonic photosymbiosis between the microalga Phaeocystis and acantharia hosts in order to shed light on the molecular mechanisms of their metabolic connectivity. More specifically, we investigated whether the algal transportome is remodeled in symbiosis. We first compared sugar transporter genes in haptophyte genomes and studied their expression in free-living and symbiotic stages of Phaeocystis using a combination of single-holobiont transcriptomics and in situ environmental metatranscriptomics. We evaluated the transcriptional dynamics of these sugar transporters in symbiosis at different periods of the day. This study reveals that the transportome of the microalga is significantly remodeled in symbiosis within a host and pinpoints putative key sugar transporters with different transcriptional patterns during the day. This work significantly improves our understanding of the metabolic connectivity between a host and microalgae, and so provides fundamental knowledge of the ecological success of this widespread symbiosis in the ocean.
|
39658219_p4
|
39658219
|
Introduction
| 4.354623 |
biomedical
|
Study
|
[
0.9992653727531433,
0.0004148231237195432,
0.00031988078262656927
] |
[
0.9983997941017151,
0.00023407961998600513,
0.0012523591285571456,
0.00011385251127649099
] |
en
| 0.999997 |
Genomic identification of transporter genes was obtained from a re-annotation of the 186 115 protein sequences of six haptophyte species ( Diacronema lutheri , Emiliania huxleyi , Chrysochromulina tobinii , Phaeocystis antarctica , Phaeocystis globosa from Phycocosm and Phaeocystis cordata from ). Sequences were re-annotated with multiple tools in order to have a complete description of each transporter useful for downstream analysis: InterProScan 5.60 with best scores for P- values < .000001 , Transporter Classification Database , blastp of the protein sequences with diamond , to the Uniprot release 2022 02, Hmmscan with Pfam-A.hmm 2021-11-15 , EggNog mapper . To identify transporters, we first searched in the merged files of annotations for the terms: “carrier|transport|channel|permease|symporter|exchanger|antiporter|periplasmic|facilitator". We searched sugar transporters using terms such as: “disaccahride|carbohydrate|sugar|ose|saccharide|glucose|polysaccharide”.
|
39658219_p5
|
39658219
|
Dataset of genomic sequences of haptophyte transporters
| 4.214759 |
biomedical
|
Study
|
[
0.9994938373565674,
0.00027205224614590406,
0.00023407010303344578
] |
[
0.999268114566803,
0.00027000776026397943,
0.00039485874003730714,
0.00006696443597320467
] |
en
| 0.999997 |
We used Phobius and tmhmm 2.0 to predict the number of transmembrane domains. We selected proteins that presented at least two transmembrane domains and less than three differences in terms of transmembrane domain number between Phobius and tmhmm. For PF00083 and PF07690 families, we used protein models (from InterPro database, https://www.ebi.ac.uk/interpro/ ) of the subfamilies domains described in Table S1 , in order to build hmm profiles and use hmmsearch (best value, P- value E-23) to carefully identify these transporters. Subcellular localization of P. cordata transporters was evaluated through five different tools: Deeploc 2.0 , TargetP 2.0 , Hectar 1.3 , WoLF PSORT , and MuLocDeep 1 . We used two thresholds: (i) a Deeploc score > 0.5 (as used in ) and (ii) the consistency of prediction should be the same for at least two tools, to select the most accurate putative predictions of transporter localization.
|
39658219_p6
|
39658219
|
Dataset of genomic sequences of haptophyte transporters
| 4.127511 |
biomedical
|
Study
|
[
0.999477207660675,
0.0002575560938566923,
0.00026517538935877383
] |
[
0.9994055032730103,
0.00016701477579772472,
0.0003715301281772554,
0.00005604627222055569
] |
en
| 0.999997 |
For the free-living stage, a total of nine replicates of P. cordata cells maintained in K2 medium at 50–60 μmol photons m −2 s −1 and 20°C were harvested at 7 p.m. at both late and stationary growth stages. Symbiotic acantharians (holobionts) with intra-cellular P. cordata were collected with a 150 μm plankton net in the Mediterranean Sea at Villefranche-sur-Mer, France. Individual holobionts were manually isolated with a micropipette under a binocular microscope, rapidly transferred into filtered seawater (0.2 μm), and maintained in an incubator (50–75 μmol photons m −2 s −1 , 20°C, 12 h/12 h). Free-living and symbiotic samples were frozen in the same conditions, in liquid nitrogen in a 0.2 μl polymerase chain reaction (PCR) tube containing 4.4 μl of Smart-Seq2 buffer (Triton X-100 0.4% / RNase inhibitor (ratio 19/1), dNTPs 10 mM, oligo dT 5 uM, ). Each sample was sequenced at 75 million reads, 2 × 150 paired-end with an Illumina NextSeq 500 instrument. A total of 1.9 billion reads were produced for this study.
|
39658219_p7
|
39658219
|
Single-holobiont transcriptomics: sampling and analysis
| 4.145491 |
biomedical
|
Study
|
[
0.9993688464164734,
0.00030629217508248985,
0.00032489447039552033
] |
[
0.9995465874671936,
0.0002049043687293306,
0.00019085950043518096,
0.00005763180161011405
] |
en
| 0.999997 |
In order to identify the symbiotic microalgae, we retrieved 18S ribosomal ribonucleic acid (rRNA) sequences in the assemblies of each samples using Barrnap (v0.9, Seemann T., Booth T. https://github.com/tseemann/barrnap ) and taxonomically identified them using vsearch on the Pr2 database v2 ( Table S7 ).
|
39658219_p8
|
39658219
|
Single-holobiont transcriptomics: sampling and analysis
| 4.047044 |
biomedical
|
Study
|
[
0.9992524981498718,
0.00023316062288358808,
0.0005143421003594995
] |
[
0.9994256496429443,
0.000347430061083287,
0.00017583457520231605,
0.00005112555663799867
] |
en
| 0.999997 |
Reads were first trimmed using trimmomatic (version 0.39, option PE -phred33; ILLUMINACLIP: contams_forward_rev.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 ) and bacterial, virus, human, and fungal sequence contaminants removed using kraken2 (2.1.2) and the k2_standard_202310 database . In order to maximize the mapping rates of the reads, we built a new reference transcriptome of P. cordata from the reads obtained with the sequencing of our culture plus the reads from the PRJNA603434 BioProject deposited at NCBI GenBank from the same Phaeocystis strain. Briefly, reads were assembled using rnaSPAdes v3.15.5 and peptides were predicted using TransDecoder v5.7.1 and Transdecoder.Predict using the Pfam. A database and UniProt database to identify accurate protein coding sequences. The peptides were annotated with the same method as for the genomic proteome. Conserved orthologous scores were calculated with BUSCO v5.4.4 and re-alignment rates with Bowtie2 . We reached a 68.87% re-mapping rate of the reads ; the two transcriptome references (this study and ) presented the same completeness .
|
39658219_p9
|
39658219
|
Single-holobiont transcriptomics: sampling and analysis
| 4.223901 |
biomedical
|
Study
|
[
0.9994805455207825,
0.00026813705335371196,
0.0002514141087885946
] |
[
0.999325156211853,
0.0002992335066664964,
0.00030485380557365716,
0.00007077218469930813
] |
en
| 0.999997 |
To verify if the decontamination of the reference transcriptome step using kraken2 was sufficient, we applied two blastp searches of the predicted peptides (as queries): (i) against the P. cordata protein sequences from the Joint Genome Institute’s genome portal; and (ii) against the NCBI nr database. For the 330 transcripts annotated as sugar transporter in the reference transcriptome, 96% of them were found in the protein sequences of P. cordata genome. The contigs presenting <50% identity (4 contigs) have a NCBI blast with Oryza sativa , Arabidopsis thaliana , and 2 bacteria but with a percentage of identity very close to the one found with the blastp against the P. cordata genome-derived protein models. For the 14 proteins not found in the P. cordata genome, only 4 have an NCBI match with a bacterial assignment but again with a < 50% identity. Thus, in total, six sugar transporter genes putatively presented a bacterial homolog but with ~35% identity on average ( Table S1 , “Blastp refTrans JGIgenome”).
|
39658219_p10
|
39658219
|
Single-holobiont transcriptomics: sampling and analysis
| 4.211823 |
biomedical
|
Study
|
[
0.9993926286697388,
0.00030510302167385817,
0.00030226135277189314
] |
[
0.9994970560073853,
0.0002186635829275474,
0.00022167705174069852,
0.00006267905700951815
] |
en
| 0.999997 |
For the Differential Expression (DE) analysis, read counts were obtained using Kallisto (0.48.0, ) on the reference transcriptome coding sequences. Prior to the DE analysis, a supplementary step of normalization was conducted using preprocesCore R package that enables a quantile normalization . The differential gene expression analysis between free-living vs symbiotic stages was conducted using the DESeq2 R package (1.36.0, ). We used the threshold of normalized read counts >10 among all replicates to qualify a gene as expressed in a given condition .
|
39658219_p11
|
39658219
|
Single-holobiont transcriptomics: sampling and analysis
| 4.128131 |
biomedical
|
Study
|
[
0.9995786547660828,
0.00018652122525963932,
0.00023486392456106842
] |
[
0.9992151260375977,
0.00042042852146551013,
0.0003113770217169076,
0.00005302642603055574
] |
en
| 0.999996 |
Reads of the Mediterranean stations 11, 9, 22, 23, 25, and 30 of the Tara Oceans expedition were obtained from the published dataset . To compare the expression of sugar transporter genes between two size fractions, we transformed their TPM values into ratios between the expression of genes of interest and housekeeping genes that we identified in a similar way to the approach used by quantitative PCR ( Table S4 “HousekeepingGenes”). Briefly, the community expression patterns are compositional data, not absolute counts, and this type of data is constrained to an arbitrary fixed total defined by the sequencing depth, creating potentially spurious correlations through changes in abundance of other organisms in the system (see for a review on the topic). A solution to this problem is to analyze ratios of gene expression instead of the proportions of the total. In this approach, choosing an appropriate denominator (housekeeping genes) to calculate ratios is critical . We established a robust denominator via 4 criteria: (i) analyzing only samples in which at least 20% of the Phaeocystis transcriptome was expressed and retained only genes expressed in all samples showing at least 20% Phaeocystis expression in every analyzed sample (this procedure eliminated 7 of the top 19 genes with the most reads mapped, likely because their high expression was the result of non-specific mapping in samples in which <20% of the Phaeocystis transcriptome was expressed), (ii) selecting genes whose expression across all analyzed samples has a coefficient of variation below 200 (mean CV all genes = 374.8) and a fold change from the mean below 2, (iii) checking that the genes present a functional annotation corresponding to typical housekeeping genes, and (iv) checking that the genes correlate enough between them and through a k-means clustering, selecting the cluster with the highest amount of genes presenting r > 0.5 (adapting ). We then used the sugar transporters’ TPM as a numerator and the geometric mean of the housekeeping selected genes as a denominator to calculate our statistics. See also supplemental file Figs S6 and S7 and Table S6 where additional information on the method for this analysis.
|
39658219_p12
|
39658219
|
Tara oceans metatranscriptomic data analysis
| 4.165644 |
biomedical
|
Study
|
[
0.9991838335990906,
0.00029896872001700103,
0.0005171330994926393
] |
[
0.9995313882827759,
0.00014510026085190475,
0.00027275478350929916,
0.000050800164899555966
] |
en
| 0.999996 |
To evaluate the expression of the sugar transportome during the day, we collected more symbiotic acantharia in surface waters of the Mediterranean Sea (Villefranche-sur-Mer, France). For “Morning” samples, we sampled and isolated acantharia, maintained them in an incubator overnight (50–75 μmol PAR m −2 s −1 , 20°C, 12 h/12 h), and harvested them the day after at 9 a.m. in the morning (one hour of light exposure). For “evening” samples, symbiotic acantharia were collected, and isolated in filtered seawater, and frozen the same day ~7 p.m. in the evening after 10 hours of light in the incubator. For “dark-evening” samples, holobionts were collected and maintained in the incubator with light until 8 p.m., and then transferred into a black box until 7 p.m. the following day. In order to compare gene expression across time/light conditions (morning, evening, and dark-evening), we built a matrix of read counts for the three conditions using kallisto on the reference transcriptome of P. cordata, as explained above, and normalized these counts using DESeq2 R package without “reference condition” for the dds object creation.
|
39658219_p13
|
39658219
|
Temporal transcriptional dynamics of sugar transporters of the microalga Phaeocystis cordata
| 4.105772 |
biomedical
|
Study
|
[
0.9993823766708374,
0.00028910418041050434,
0.00032848710543476045
] |
[
0.999559223651886,
0.00018266162078361958,
0.00020470422168727964,
0.000053370506066130474
] |
en
| 0.999996 |
To identify the molecular toolbox underlying metabolic fluxes and investigate the putative genomic footprint of symbiosis, we unveiled the transportome of Phaeocystis (three species: P. antarctica , P. cordata , P. globosa ) at the genomic level, and we compared this transportome with three non-symbiotic haptophyte species . We also predicted subcellular localization of transporters using a combination of different in silico tools. We particularly focused on sugar transporters, since soluble sugars may be the main exported currency to the host. Using the same method of protein annotation for each species, we identified transporters based on the presence of transmembrane domains and protein domain annotations using the InterPro/Pfam classification. In total, we found 270 unique Pfam domains for the transporter genes in haptophyte genomes. The three different Phaeocystis species analyzed here contained an average of 965 transporter genes corresponding to 3% of all genes (1244 genes or 4% if we include those lacking a predicted transmembrane domain, see Methods, Fig. 1A , Table S1 : “General values1” and “General values2 (TMD)”).
|
39658219_p14
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.208583 |
biomedical
|
Study
|
[
0.9994126558303833,
0.0003521295147947967,
0.00023520816466771066
] |
[
0.9993940591812134,
0.00016340560978278518,
0.00036391173489391804,
0.00007860425830585882
] |
en
| 0.999999 |
Across Phaeocystis genomes, sugar transporter genes represented on average 18% (175 out of 965) of all transporter genes and were classified into eight different core Pfams (i.e. shared among all haptophyte species, Fig. 1A , Table S1 : “General values2 (TMD)”). The largest Pfam family of sugar transporters is the Triose Phosphate Transporters with 64.7 genes on average across Phaeocystis species, similar to E. huxleyi , but higher than in diatom genomes (between 13 and 22 TPTs for Phaeodactylum tricornutum ). In plants, TPTs are transporters located in the plastid envelope and export photosynthetically-derived sugars to the cytosol . For P. cordata , in silico subcellular localization analyses predicted only three out of 60 TPTs associated to the plastid membranes whereas the majority were predicted to be located in the endomembrane system . Similar to diatoms, the localization of TPTs in the four membranes of the secondary red plastid of Phaeocystis remains ambiguous and TPTs could also be localized elsewhere in the cell [ 65–67 ].
|
39658219_p15
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.325712 |
biomedical
|
Study
|
[
0.9993639588356018,
0.00033427774906158447,
0.0003017250564880669
] |
[
0.9992843270301819,
0.0002559643762651831,
0.00037405427428893745,
0.00008569457713747397
] |
en
| 0.999996 |
The second largest sugar transporter family was the “Sugar (and Other) transporters” Pfam: PF00083, containing 31 genes in Phaeocystis on average . This Pfam is composed of different transporters with various substrates, such as glucose, galactose, mannose, polyol, and inositol sugars . For instance, using a complementary hidden Markov model (HMM) search of InterPro domains in P. cordata PF00083 proteins, we found 5 GLUT transporter , 6 sugar transporter ERD6/Tret1-like , and 12 Sugar transport protein STP/Polyol transporter PLT domains ( Table S1 : “GLUTcharacterization”). In silico subcellular localization successfully assigned a prediction for 21 of the 33 transporter genes from PF00083 of P. cordata in the cell membrane, three in vacuoles, and one in the plastid membrane . These results suggest that some of these transporters might be involved in sugar flux at the cell surface. Nucleotide-sugar transporters play an important role in the biosynthesis of glycoproteins, glycolipids, and non-cellulosic polysaccharides translocating nucleotide-sugars to the Golgi apparatus . In Phaeocystis species, we found 34 NSTs genes on average (31 in E. huxleyi ). In P. cordata , 50% of the NSTs were predicted to be localized to the Golgi apparatus, in accordance with their known biological function .
|
39658219_p16
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.346591 |
biomedical
|
Study
|
[
0.9993725419044495,
0.0002990337379742414,
0.0003284048871137202
] |
[
0.9992642998695374,
0.00031381542794406414,
0.000341114035109058,
0.00008073725621215999
] |
en
| 0.999997 |
SWEET transporters are bidirectional transporters of small sugars following the concentration gradient . SWEETs have been highlighted in terrestrial and aquatic symbioses (Fabaceae- Rhizobium , cnidarians-Symbiodiniaceae), particularly in sugar efflux from the photosynthetic to the heterotrophic partner [ 28 , 73–75 ]. Between two and four SWEET genes were found across Phaeocystis genomes. Among the four SWEET of P. cordata , one was predicted in the lysosome/vacuole, one in the endomembrane system, and one in the cell membrane. For the Pfam PF13347 (MFS/sugar transport protein) , we found four genes in Phaeocystis genomes.
|
39658219_p17
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.232915 |
biomedical
|
Study
|
[
0.9993696808815002,
0.00020199529535602778,
0.0004283403104636818
] |
[
0.9993389248847961,
0.0003617845068220049,
0.0002435001515550539,
0.00005577636693487875
] |
en
| 0.999997 |
We investigated the presence of putative sugar transporters from the Major Facilitator Superfamily PF07690 (MFS). Through an HMM search , we detected 43 putative sugar transporters for P. cordata and 25 and 20 for P. globosa and P. antarctica , respectively. Among them, many genes corresponded to glucose-6-phosphate transporter . In Phaeocystis , most of these PF07690 transporters were predicted to be either at the cell membrane or ER ( Table S1 , “Subcellular Localization P.cord”). Finally, we investigated the presence of genes belonging to PF00230 that correspond to a specific type of aquaporin, found to be involved in different reef photosymbioses for putative glycerol transport (anemones, jellyfish, and giant clams ). In Phaeocystis genomes, seven ( P. cordata ) to nine ( P. antarctica ) homologs of these aquaporin genes were found.
|
39658219_p18
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.18678 |
biomedical
|
Study
|
[
0.9993340373039246,
0.0002766709076240659,
0.00038936713826842606
] |
[
0.9995286464691162,
0.00018076025298796594,
0.00023565578158013523,
0.00005497724123415537
] |
en
| 0.999998 |
This inventory of sugar transporters in haptophyte genomes unveiled different categories that could be involved in the influx and efflux of sugars in the microalga Phaeocystis . Overall, 25 sugar transporters were predicted to be localized at the cell membrane , and the vast majority presented a putative localization in the endomembrane system (including ER or Golgi). We also hypothesize that sugar transporters can be located on vesicles from the endomembrane system that could fuse to the cell membrane . These predictions provide some localization patterns for the different categories of sugar transporters in Phaeocystis but require experimental validation in order to compare with plants and other algae. We did not find any evidence of large copy number variations of sugar transporter genes in Phaeocystis genomes compared to other haptophytes, which could have been a first indication of a genomic footprint to explain the predominance of this genus in symbiosis. This hypothesis has already been explored and validated in symbiotic microalgae: Symbiodiniaceae clades presented enriched functions related to transmembrane transport in their genomes compared to other non-symbiotic dinoflagellates, especially for the major facilitator superfamily . This genomic characterization of the Phaeocystis transportome generates fundamental knowledge on this key marine phytoplankton taxon and is an essential step for unveiling the expression dynamics of sugar transporter genes in symbiosis.
|
39658219_p19
|
39658219
|
Genomic inventory of sugar transporters in Phaeocystis species
| 4.550986 |
biomedical
|
Study
|
[
0.999259889125824,
0.0004575348284561187,
0.00028253140044398606
] |
[
0.9983606934547424,
0.0004751959349960089,
0.0010063140653073788,
0.00015788232849445194
] |
en
| 0.999997 |
To reveal which sugar transporters might play a role in symbiosis, we assessed their gene expression based on single-holobiont transcriptomic analyses. More specifically, we compared the expression of transporter genes of the microalga P. cordata between free-living (four and five culture replicates in exponential and stationary growth phases, respectively) and symbiotic conditions through a Differential Expression (DE) analysis . Each sample was frozen at the same period of the day (late evening, 7 p.m.). About 75 million of reads were obtained per holobiont sample, producing a total of 1950 billion of reads in this study. A de novo reference transcriptome from total RNA sequences of cultured P. cordata was built and used to quantify gene expression.
|
39658219_p20
|
39658219
|
Algal transportome is significantly remodeled in symbiosis
| 4.120991 |
biomedical
|
Study
|
[
0.9992963075637817,
0.0003129958058707416,
0.0003907881327904761
] |
[
0.99960857629776,
0.0001369319943478331,
0.00020443960966076702,
0.00005007524669053964
] |
en
| 0.999997 |
Before comparing the expression of the transportome between the free-living and symbiotic stages, we assessed whether the transportome of free-living Phaeocystis cells maintained in culture varies with respect to the growth phase (i.e. exponential vs stationary phase). Only 3.3% of the transporters were found to be differentially expressed between exponential and stationary phase, indicating that the expression of the transportome is not drastically modified ( Table S2 ). We therefore considered hereafter both growth phases as free-living replicates (nine in total) to compare with the symbiotic stage.
|
39658219_p21
|
39658219
|
Algal transportome is significantly remodeled in symbiosis
| 4.073814 |
biomedical
|
Study
|
[
0.9993607401847839,
0.00029792406712658703,
0.0003413221857044846
] |
[
0.9995779395103455,
0.00016117982158903033,
0.00021260381618048996,
0.00004826663644053042
] |
en
| 0.999997 |
From the 2806 transporters genes considered as expressed in this analysis (sum of normalized read counts >10 in all replicates), 26% (724 genes) were found to be exclusively expressed in free-living and 10% (267 genes) exclusively expressed in symbiosis ( Table S3 : “Exp FL Symb”). A principal component analysis (PCA) of the DESeq2 dataset showing the gene expression variance revealed: (i) a clear separation between symbiosis and free-living samples; (ii) a high variance in expression of all genes across symbiotic replicates, and (iii) clustering of expression of transporter genes in symbiotic samples . We verified this clustering pattern by using different subsets of expressed genes with similar numbers of genes as controls . These results show the existence of two distinct transportomes of the microalga Phaeocystis expressed in the symbiotic and free-living stages.
|
39658219_p22
|
39658219
|
Algal transportome is significantly remodeled in symbiosis
| 4.150278 |
biomedical
|
Study
|
[
0.9994645714759827,
0.0002541672729421407,
0.0002812814782373607
] |
[
0.999528169631958,
0.00017582443251740187,
0.0002443402772769332,
0.000051664690545294434
] |
en
| 0.999998 |
Compared to the free-living stage, 42% of the transportome was significantly remodeled in symbiosis with 982 differentially expressed genes . Among those, 33% (768 genes) were downregulated and 9% (214 genes) were upregulated in symbiosis ( Table S3 : “UP-DOWN global”). These numbers are higher than the ones found in a symbiotic dinoflagellate ( Breviolum ) , and with an opposite trend : 213 upregulated and 167 downregulated . We explain this remodeling of Phaeocystis transportome in symbiosis by a significant global decrease of the transporter gene expression and high positive fold changes for some transporter genes . Note that 58% of the algal transportome remained expressed in symbiosis but without differential expression. Therefore, these results suggest that many algal transporters could be less required in symbiosis likely due to the transition from the ocean to the host microhabitat, but some transporters could be specifically induced in symbiosis with high transcriptional activity in response to metabolic changes within a host and potentially enable the metabolic connectivity between the two partners.
|
39658219_p23
|
39658219
|
Algal transportome is significantly remodeled in symbiosis
| 4.268065 |
biomedical
|
Study
|
[
0.9994198083877563,
0.00029878620989620686,
0.00028139230562373996
] |
[
0.9993382096290588,
0.00021416270465124398,
0.0003802525461651385,
0.00006738543015671894
] |
en
| 0.999998 |
We focused on the expression of sugar transporter genes of the microalga in symbiosis. From our dataset, 261 genes representing eight Pfams were found to be expressed (out of the 330 sugar transporter genes in the reference transcriptome; Table 1 and Table S3 “UP-DOWN global” and “ P.cordata SugarTR Anno”). At the Pfam level, we observed that five sugar transporter families presented a global downregulation of gene expression . Yet, at the gene level, some transporters presented a high upregulation within each Pfam. Among the 36% of sugar transporters remodeled in symbiosis, 8% (19 genes) were found as upregulated and 28% (74 genes) downregulated . Note that 64% (167 genes out of the 259) of the sugar transporter genes were still expressed in symbiosis (named “neutral” in Table 1 ). Among the 19 up-regulated genes in symbiosis, we found nine TPTs , two aquaporins , two monosaccharide transporters , two MFS/sugar transport protein , three Nucleotide sugar transporters , and one gene from PF07690 . Note that these Pfams are the same that were found upregulated in the study of Maor-Landaw et al. 2020 , translating Uniprot IDs in Pfam accession numbers. These transporters therefore contribute to the significant remodeling and specialization of the algal sugar transportome in symbiosis, and potentially play a key role in the flux and exchange of sugars.
|
39658219_p24
|
39658219
|
Expression of the algal sugar transporters in symbiosis
| 4.248987 |
biomedical
|
Study
|
[
0.9992951154708862,
0.00035182194551452994,
0.0003530434041749686
] |
[
0.9994403719902039,
0.00016376140411011875,
0.00033029328915290534,
0.00006556165317306295
] |
en
| 0.999997 |
Concerning the TPTs, only nine out of 96 genes (9.4%) were upregulated and 66% were still expressed in symbiosis. It is possible that the observed upregulation of some TPTs can be linked to the multiplication of plastids in symbiosis (from two when free-living to up to 60 plastids when symbiosis ) and would ensure enhanced sugar export into the cytosol. The most upregulated transporter genes correspond to two aquaporins . This type of aquaporin, also known as glycerol facilitator ( GlpF ), was found to be involved in two benthic photosymbioses and suggested to be involved in glycerol transport . The three upregulated transporter genes with glycerol as putative substrate raise the hypothesis that this metabolite can be important for the carbon metabolism of the holobiont and possibly transported to the host. Two other upregulated genes corresponded to putative transporters of monosaccharides , with assignment to GLUT proteins . One of these two GLUT proteins was predicted to be localized at the cell membrane ( Table S3 , “SubcellLoc SugarTR_UP”). Four SWEET transporter genes were also expressed in symbiosis (log2FC ranging between −6.9 to 0.68), but not upregulated. The two upregulated MFS/sugar transport proteins could also play a role in sugar flux as shown in plants . This transcriptomic analysis from freshly collected holobionts allowed us to reveal the sugar transportome expressed in the symbiotic microalga and identify candidate genes for future functional characterizations. To have an alternative line of evidence, we investigated the expression of this algal transportome in situ, exploiting metatranscriptomic data collected in the Mediterranean Sea.
|
39658219_p25
|
39658219
|
Expression of the algal sugar transporters in symbiosis
| 4.348474 |
biomedical
|
Study
|
[
0.99919193983078,
0.00043013758840970695,
0.0003779635881073773
] |
[
0.9993719458580017,
0.00019912597781512886,
0.00033765361877158284,
0.00009136521839536726
] |
en
| 0.999999 |
Using the Tara metatranscriptomic dataset from the Mediterranean Sea, we evaluated the expression of sugar transporter genes of the microalga Phaeocystis in two size fractions collected in surface waters of six stations from the Tara Oceans expedition . Phaeocystis reads were specifically recruited in the metatranscriptome dataset based on stringent criteria . The small size fraction mainly corresponds to the free-living stage of P. cordata (4 μm in size ) whereas its symbiotic stage within acantharians is mainly detected in the large size fraction (note that the Mediterranean species, P. cordata, does not form colonies ). From this metatranscriptomic dataset, we identified 270 sugar transporters with a difference between the large (123 genes) and small (266 genes) size fraction . This can be partially explained by the lower abundance of Phaeocystis transcripts in the 180–2000 μm fraction that might have been diluted and so less sequenced due to the high abundance of transcripts from large multicellular organisms (zooplankton).
|
39658219_p26
|
39658219
|
In situ sugar transportome expression of the microalga Phaeocystis in the Mediterranean Sea
| 4.17494 |
biomedical
|
Study
|
[
0.9992116689682007,
0.000318075530230999,
0.00047027261462062597
] |
[
0.9996030926704407,
0.00015904386236798018,
0.0001874337758636102,
0.00005043817509431392
] |
en
| 0.999997 |
In order to normalize and compare gene expression between the two size fractions, we calculated the ratio between the expression (TPM) of the 270 sugar transporter genes and a selected set of 149 housekeeping genes (e.g. ribosomal proteins, tubulin, ATP synthase…) whose expression levels were expected to correspond to basic cellular activity (see Materials and methods and Table S4 : “sugarTR counts”). Based on this normalization method, we found 24 sugar transporter genes with a higher mean expression value in the large size fraction compared to the small one: one SWEET gene, twelve TPTs, six NSTs, three aquaporins, one GLUT , and one member for the MFS/sugar transporter protein PF13347 . Eighteen out of the nineteen sugar transporter genes found as upregulated in symbiosis were also detected in both size fractions of the metatranscriptomic dataset. Of these, six of them were found to present a higher TPM value in the large size fraction and correspond to two aquaporin PF00230 genes, the transporter from PF07690, two TPTs, and one PF04142 . Note that the two aquaporin genes identified here correspond to the genes that presented the highest positive fold change values in symbiosis in the differential expression analysis from isolated holobionts . This in situ metatranscriptomic analysis provides an ecological context for our experimental study, and further confirms some sugar transporter candidates as key players in symbiosis, such as TPTs and aquaporin.
|
39658219_p27
|
39658219
|
In situ sugar transportome expression of the microalga Phaeocystis in the Mediterranean Sea
| 4.264165 |
biomedical
|
Study
|
[
0.9994518160820007,
0.00030090095242485404,
0.0002472798805683851
] |
[
0.9993379712104797,
0.00020516544464044273,
0.00037968700053170323,
0.00007715078390901908
] |
en
| 0.999997 |
It is well established that photosynthesis and central carbon metabolism depend on the circadian rhythm and light conditions [ 83–85 ]. Therefore, in order to further understand the metabolic connectivity, we investigated the transcriptional dynamics of the sugar transportome of symbiotic Phaeocystis by harvesting acantharia hosts at three different times of day and light exposure periods: (i) morning (9 a.m., after 1 h light exposure), (ii) evening (7 p.m., after 10 h light exposure), and (iii) dark-evening (7 p.m., after an incubation in darkness for 24 hours). The dark-evening condition corresponds to a situation where holobionts, and thus microalgae experience a non-photosynthetic day (absence of light). In total, we found 209 sugar transporter genes expressed in these three conditions, representing 96% (214/223) of the sugar transportome . Overall, 28% (60 genes) of sugar transporters were found to be expressed in the morning, 43% (91 genes) expressed in the evening and 29% (63 genes) in the dark-evening ( Table S5 ). Among them, some were exclusively expressed in the morning (18), evening (43) or dark-evening . These results demonstrate that many sugar transporter genes of the symbiotic microalga tend to be induced during the day. From the comparison of holobionts collected in the evening and submitted or not to darkness (dark-evening), we found 53% (113/214) of genes downregulated when incubated in the dark (FC < −2, Table S5 ). 34% (72/214) were still expressed in the holobionts exposed to darkness and thus, their expression does not seem to be linked to the presence of light. These results show that the majority of sugar transporter expression of the symbiotic microalga is modulated by light conditions.
|
39658219_p28
|
39658219
|
Dynamic expression of the algal sugar transportome in symbiosis during the day
| 4.293817 |
biomedical
|
Study
|
[
0.9993218183517456,
0.00039612920954823494,
0.0002821398957166821
] |
[
0.9993828535079956,
0.00019309700292069465,
0.00033572723623365164,
0.00008826034172670916
] |
en
| 0.999997 |
At the Pfam level, we found specific expression patterns modulated by either light or period of the day . For instance, the expression of TPTs and Glycerol 3-Phosphate transporter tended to be modulated only by light since they were significantly more expressed in evening vs dark-evening conditions and did not show significantly higher expression between morning and evening . On the contrary, transcription of sugar hexose transporters seemed to be more regulated by the period of the day as shown by a significant higher expression in the evening compared to morning but not differentially expressed between evening and dark-evening conditions . NSTs seemed to be regulated by both parameters (light and period of the day) as they were found to be significantly more expressed in the evening vs morning, and dark-evening vs evening . MFS/sugar transport protein genes tended to present a higher expression in the dark . SWEET genes were not differentially expressed between the three conditions, yet two pairs of genes seemed to present opposite patterns (more expressed in the morning or in the dark). Generally, aquaporin genes exhibited a lower expression in the darkness and two of them were only expressed in the evening, after the normal daylight exposure. These results show specific transcriptional patterns of sugar transporters in response to light , to the period of the day or both parameters .
|
39658219_p29
|
39658219
|
Dynamic expression of the algal sugar transportome in symbiosis during the day
| 4.294769 |
biomedical
|
Study
|
[
0.9993512034416199,
0.00028788793133571744,
0.0003608127881307155
] |
[
0.9991329312324524,
0.0002325256500625983,
0.000564646499697119,
0.00006989306712057441
] |
en
| 0.999997 |
We also paid attention to the dynamics of the sugar transporter genes found to be upregulated in symbiosis from our holobiont transcriptomes. The nine upregulated TPTs in symbiosis exhibited several transcriptional patterns: two genes exclusively expressed in the morning and two genes exclusively in the evening; in addition, two genes had a higher expression in the evening compared to morning, and six genes had a higher expression in light (evening) compared to dark-evening . This suggests that different TPT genes might have specific roles at different periods of the day and this could depend on their subcellular localization. GLUT and aquaporin genes upregulated in symbiosis showed a higher expression in morning vs evening, or dark-evening vs evening, raising two hypotheses: (i) the transcription is activated in the morning to produce transporters during the day or (ii) transcription mainly takes place in the dark for sugar excretion at night. Further studies should increase the temporal resolution during a day-night cycle to fully reveal the dynamics of the transportome expression of the symbiotic microalga.
|
39658219_p30
|
39658219
|
Dynamic expression of the algal sugar transportome in symbiosis during the day
| 4.185338 |
biomedical
|
Study
|
[
0.9993199110031128,
0.00030705571407452226,
0.00037294960930012167
] |
[
0.9994285702705383,
0.00018278430798090994,
0.00033199565950781107,
0.00005655712084262632
] |
en
| 0.999996 |
This study improves our understanding of the molecular players that are potentially involved in the carbon metabolism, and metabolic connectivity between the symbiotic microalga Phaeocystis and its acantharian host. We found that Phaeocystis species share a conserved sugar transportome among haptophytes at the Pfam level with few differences in gene copy number. Therefore, this genomic analysis did not reveal a specific sugar transportome linked to the symbiotic life stage of the microalga Phaeocystis , compared to non-symbiotic haptophytes. This can be explained by the fact that Phaeocystis symbionts are not vertically transmitted across host generations, do not depend on symbiosis for survival, and genome evolution would rather occur in the extensive free-living population . Our study shows that the capacity of the microalga Phaeocystis to be in symbiosis may be rather due to the large plasticity of the transportome expression with 42% of transporter genes of metabolites and nutrients being differentially expressed. This suggests a drastic change in the flux and homeostasis of metabolites and nutrients in the symbiotic microalgae. More specifically, downregulation of most transporter genes along with high expression of a few ones suggests (i) lower trafficking of metabolites linked to the intracellular life stage (perhaps due to the arrested cell division), and (ii) specialization toward some metabolite fluxes putatively beneficial for the host. The transcriptional plasticity of the algal sugar transportome not only takes place during the free-living-to-symbiosis transition but also throughout the day. This reveals the complex dynamics of the carbon homeostasis and fluxes in the holobiont system.
|
39658219_p31
|
39658219
|
Discussion
| 4.446276 |
biomedical
|
Study
|
[
0.9991940855979919,
0.0005186551716178656,
0.0002873473276849836
] |
[
0.9987282156944275,
0.00030813319608569145,
0.0008257710724137723,
0.00013787824718747288
] |
en
| 0.999998 |
Among the 19 sugar transporters of the microalga Phaeocystis upregulated in symbiosis, we found two GLUT and two aquaporin genes, which were also found to be more expressed in the large size fraction of environmental metatranscriptomes. This study provides further evidence that GLUT and aquaporin transporters, which are also upregulated in other photosymbiotic hosts , play a key role in symbiosis. Similarly, the higher expression of a SWEET gene in this large size fraction suggests that this transporter may also be involved in metabolic connectivity in this planktonic symbiosis, as shown for anemone/dinoflagellate symbiosis .
|
39658219_p32
|
39658219
|
Discussion
| 4.184401 |
biomedical
|
Study
|
[
0.99935382604599,
0.0002791113802231848,
0.00036707433173432946
] |
[
0.9995421171188354,
0.00018168542010243982,
0.000224086397793144,
0.00005211797542870045
] |
en
| 0.999997 |
The diversity of transporters and their expression patterns raise the hypothesis that several algal carbohydrates (glucose, glycerol) might be transferred to the host at different temporal windows. Future functional characterization (e.g. expression in heterologous systems) of the candidate sugar transporters revealed here will be essential to fully understand the role of these transporters in symbiosis. Overall, this study expands the list of holobionts using similar transporter genes and raises the hypothesis of a convergence for carbon exchange mechanisms in photosymbiosis.
|
39658219_p33
|
39658219
|
Discussion
| 4.127755 |
biomedical
|
Study
|
[
0.999187171459198,
0.00030988120124675333,
0.0005029518506489694
] |
[
0.9991369843482971,
0.00020151537319179624,
0.0006015409016981721,
0.00006002455484122038
] |
en
| 0.999995 |
The rumen is a digestive organ unique to ruminants. It has a distinctive microbial fermentation system that can efficiently convert macromolecular substances from the diet, such as lignin, cellulose, and non-protein nitrogen, into nutrients that are easier for the host to utilize . Early research focused on the structure of the complex microbial community in the rumen and its impact on nutrient usage from feeds of different compositions. These microorganisms metabolize dietary compounds and produce volatile fatty acids (VFAs), amino acids and other essential nutrients, thereby providing energy for the host body and maintaining the optimal functioning of the rumen .
|
PMC11697150_p0
|
PMC11697150
|
1 Introduction
| 3.979796 |
biomedical
|
Study
|
[
0.9994112253189087,
0.0001622626732569188,
0.000426548212999478
] |
[
0.9247739911079407,
0.01515361201018095,
0.05975286662578583,
0.00031960869091562927
] |
en
| 0.999994 |
The hindgut of ruminants was previously considered the endpoint of digestion; however, in-depth research on the fungal microbiota of ruminants has shown that the fungi in the rectum produce a large number of lignin-degrading enzymes that can ferment unused lignin. These enzymes effectively decomposed the structural polymers of plant cell walls, thereby improving the absorption and utilization rate of their nutrients .
|
PMC11697150_p1
|
PMC11697150
|
1 Introduction
| 3.853888 |
biomedical
|
Study
|
[
0.9989588260650635,
0.00010897024912992492,
0.000932157738134265
] |
[
0.9796561002731323,
0.016281187534332275,
0.003886644495651126,
0.00017600944556761533
] |
en
| 0.999996 |
Compared with the traditional measurement of feed conversion ratio (FCR), residual feed intake (RFI) is considered to be a more accurate and flexible assessment method. RFI refers to the difference between the actual feed intake of an individual animal and the expected feed intake based on its body size and production performance. Low RFI indicates that individual animals consume less feed than predicted and therefore have a lower environmental pollution capacity, without affecting individual body weight, daily weight gain, or body shape.
|
PMC11697150_p2
|
PMC11697150
|
1 Introduction
| 3.771678 |
biomedical
|
Study
|
[
0.9808206558227539,
0.0003538894816301763,
0.01882542483508587
] |
[
0.7022623419761658,
0.2699156403541565,
0.027477577328681946,
0.00034444063203409314
] |
en
| 0.999998 |
In studies on the microbiota of ruminants with different RFIs, Herd and Arthur and Paz et al. reported that rumen fermentation patterns and the microbial composition of the ruminant gut accounted for 19%-20% of the variation in RFI. Ellison et al. found that six types of rumen microorganisms were highly correlated with actual feed efficiency in ewes. Liu et al. showed that the abundance of Firmicutes and Bacteroidetes in the intestines of Angus cattle with low RFI was significantly higher than that of cattle with high RFI. Elolimy et al. found that the abundance of Bacteroidota in rectal feces of Holstein cattle was higher in low-RFI cattle than in high-RFI cattle. Both Firmicutes and Bacteroidetes are the most abundant bacterial groups in the digestive tract of ruminants and play a major role in fiber fermentation in the diet.
|
PMC11697150_p3
|
PMC11697150
|
1 Introduction
| 4.027049 |
biomedical
|
Study
|
[
0.999177873134613,
0.0002736902970355004,
0.0005483662243932486
] |
[
0.9166159629821777,
0.000605061068199575,
0.0825795978307724,
0.0001993309415411204
] |
en
| 0.999998 |
Previous studies have shown that the gastrointestinal microbiota of ruminants affects RFI expression, which is one of the important indicators of feed efficiency type. Digestion and absorption in ruminants are processes that involve a number of interconnected steps in the gastrointestinal tract (GIT). For our experiments, we chose Dexin fine-wool meat sheep of the same age and under the same feeding conditions. To systematically examine the influence of RFI on the digestive tract as a whole, the type and abundance of microorganisms in the rumen, ileum and rectum were determined to represent the overall GIT of the sheep under different feed efficiencies.
|
PMC11697150_p4
|
PMC11697150
|
1 Introduction
| 4.0412 |
biomedical
|
Study
|
[
0.9986226558685303,
0.0002317480102647096,
0.0011455221101641655
] |
[
0.9995469450950623,
0.0002305820380570367,
0.00018539524171501398,
0.00003704582559294067
] |
en
| 0.999996 |
All animal procedures were approved by the Agreement Management and Review Committee of the Feed Research Institute of the Xinjiang Academy of Animal Sciences .
|
PMC11697150_p5
|
PMC11697150
|
2 Materials and methods
| 0.845133 |
biomedical
|
Other
|
[
0.669766366481781,
0.0044355737045407295,
0.32579806447029114
] |
[
0.00858251377940178,
0.9893583059310913,
0.0011947478633373976,
0.0008644174668006599
] |
en
| 0.999997 |
The test was conducted at the Baicheng County Breeding Sheep Farm in Xinjiang, China . Fifty 70-day-old Dexin fine-wool meat sheep were purchased from the Baicheng County Breeding Sheep Farm. Deworming was performed twice, and the sheep were weighed before the experiment started (30.77 ± 3.17 kg). The trial period lasted 100 days, including a 14-day pre-feeding period. The animals were weighed every 20 days during the trial period and slaughtered on the 100 th day of the trial period.
|
PMC11697150_p6
|
PMC11697150
|
2.1 Animals and experimental period
| 2.598253 |
biomedical
|
Study
|
[
0.9936476349830627,
0.0013881133636459708,
0.004964236170053482
] |
[
0.9926226139068604,
0.0067482879385352135,
0.00027454717201180756,
0.000354556628735736
] |
en
| 0.999998 |
The feed formula selected was the 678S-2 experimental pelleted feed produced by Tiankang Feed Technology Co., Ltd. The nutrient levels in the feed are shown in Table 1 .
|
PMC11697150_p7
|
PMC11697150
|
2.2 Feed formulation and processing
| 1.783435 |
biomedical
|
Study
|
[
0.9550319314002991,
0.0011739471228793263,
0.04379413649439812
] |
[
0.8049553632736206,
0.1918824464082718,
0.0019116236362606287,
0.0012506421189755201
] |
en
| 0.999996 |
Before the test, the pen was thoroughly disinfected and the sheep entering the pen were marked with numbered ear tags. Before the start of the pilot test period, the experimental animals were dewormed using a combination of intramuscular injection and feeding of anthelminthics. Sheep in individual cages were fed twice a day at 10:00 and 18:00, with free access to food and water, ensuring that the amount of leftover feed for each sheep was >15% per day. During the experiment, three test sheep were eliminated due to disease, and five test rams were selected for breeding.
|
PMC11697150_p8
|
PMC11697150
|
2.3 Experiment management
| 3.144807 |
biomedical
|
Study
|
[
0.9954326152801514,
0.001556257251650095,
0.0030110888183116913
] |
[
0.9900499582290649,
0.009428163059055805,
0.0002496959059499204,
0.0002721966302487999
] |
en
| 0.999997 |
Using SPSS26.0 for analysis, the R 2 values of dry matter feed intake, daily weight gain and average mid-term metabolic weight of the test sheep met the conditions of the regression equation. A linear model was constructed to calculate the RFI of the test sheep as follows:
|
PMC11697150_p9
|
PMC11697150
|
2.4.1 Calculation and grouping of residual feed intake
| 2.930028 |
biomedical
|
Study
|
[
0.9967505931854248,
0.00038885639514774084,
0.0028604846447706223
] |
[
0.9945846199989319,
0.004975536372512579,
0.00029058798099868,
0.0001492706360295415
] |
en
| 0.999999 |
The average daily weight gain, ADGi = (FBWi – IBWi)/N was also calculated. FBWi (final body weight) is the weight of individual i at the end of the trial, IBWi (initial body weight) is the initial weight of individual i , and N is the number of trial days. The formula for average metabolic body weight is MBWi = [1/2 × (FBWi + IBWi)] 0.75, where β0 is the regression intercept, β1 and β2 are fixed values, and ei is the RFI of sheep i .
|
PMC11697150_p10
|
PMC11697150
|
2.4.1 Calculation and grouping of residual feed intake
| 4.069062 |
biomedical
|
Study
|
[
0.9992715716362,
0.000197890869458206,
0.0005306056118570268
] |
[
0.9978877902030945,
0.0017407065024599433,
0.00030995201086625457,
0.0000615075186942704
] |
en
| 0.999996 |
Based on the mean and standard deviation of the RFI, the test sheep were divided into a high RFI (H-RFI) group (RFI > mean + 0.5SD) with 11 sheep, a medium RFI (M-RFI) group (mean + 0.5SD < RFI < mean – 0.5SD) with 18 sheep, and a low RFI (L-RFI) group (RFI < mean – 0.5SD) with 13 sheep .
|
PMC11697150_p11
|
PMC11697150
|
2.4.1 Calculation and grouping of residual feed intake
| 3.407807 |
biomedical
|
Study
|
[
0.9989107847213745,
0.00027701404178515077,
0.0008122670697048306
] |
[
0.9963854551315308,
0.0032856143079698086,
0.00023579593107569963,
0.00009313294140156358
] |
en
| 0.999998 |
On the 90 th day of the experimental period, six lambs from each group were selected to collect feces, twice a day for 3 days. The samples were placed in plastic bags and stored at −20°C until used. On the 100 th day of the experimental period, the animals were humanely slaughtered before feeding. Six Dexin male lambs were randomly selected from each group and 10 ml of solid digesta from the rumen and the ileum solid digesta were taken, transferred to cryopreservation tubes, and stored in liquid nitrogen. During the collection process, there was no chyme or feces in the rectum of some slaughtered sheep, so only the rectal feces of three lambs from each group were collected and stored in liquid nitrogen. The rumen chyme, ileum chyme and rectal feces were sent to the Xinjiang Morgan Biotechnology Co., Ltd. for sequencing analysis of the bacterial and fungal microbiota.
|
PMC11697150_p12
|
PMC11697150
|
2.4.2 Sample collection
| 4.06428 |
biomedical
|
Study
|
[
0.9993361830711365,
0.0003595612070057541,
0.00030424376018345356
] |
[
0.9994162321090698,
0.00035455074976198375,
0.00016537644842173904,
0.00006386650784406811
] |
en
| 0.999995 |
Apparent digestibility was determined according to a published method using hydrochloric acid-insoluble ash content of feces and feeds as a readout :
|
PMC11697150_p13
|
PMC11697150
|
2.4.3 Measurement of GIT indices and data analysis
| 3.430014 |
biomedical
|
Study
|
[
0.9980317950248718,
0.00035861379001289606,
0.001609577564522624
] |
[
0.9883660674095154,
0.010279649868607521,
0.0011487328447401524,
0.0002054579381365329
] |
en
| 0.999999 |
The nutrient digestibility of dry matter, crude protein and neutral detergent fiber was also calculated.
|
PMC11697150_p14
|
PMC11697150
|
2.4.3 Measurement of GIT indices and data analysis
| 2.482938 |
biomedical
|
Study
|
[
0.9909489750862122,
0.0016433049459010363,
0.0074077569879591465
] |
[
0.9733083844184875,
0.022406885400414467,
0.0033122284803539515,
0.0009725051932036877
] |
en
| 0.999997 |
Ammoniacal nitrogen was determined using indophenol blue colorimetry according to a published method . Concentrations of volatile fatty acids were determined by gas chromatography. Briefly, VFAs were separated on a 2 m glass column (3 mm i.d.) using a Fisons HRGC MEGA 2 Series model 8560 chromatograph (Fison Instruments, Glasgow, UK) equipped with a flame ionization detector (Fison Instruments, Glasgow, UK). The chromatographic column was 10% SP-1000 + 1% H3PO4, the chromatographic column was 100/120 ChromosorbWAW (Tehnokroma Analitica SA, Sant Cugat del Valles, Spain), and the carrier gas was nitrogen. The injector and detector temperatures were 200°C, and the column temperature was 155°C. The internal standard used was 2-ethylbutyric acid (Sigma Aldrich, Taufkirchen, Germany). GC was used to determine the concentration of acetic acid, propionic acid, butyric acid, and valeric acid in the rumen.
|
PMC11697150_p15
|
PMC11697150
|
2.4.3 Measurement of GIT indices and data analysis
| 4.120026 |
biomedical
|
Study
|
[
0.9995866417884827,
0.00020021657110191882,
0.00021323657711036503
] |
[
0.9990938901901245,
0.0004933283198624849,
0.0003508212976157665,
0.00006191906140884385
] |
en
| 0.999995 |
For determining the genus and species of microbiota in the rumen, genomic DNA rumen fluid sample was extracted by the cetyltrimethylammonium bromide (CTAB) method. DNA concentration and purity was determined by 0.8% agarose gel electrophoresis, and the DNA was diluted to 1 ng/μL for use. PCR amplification was performed using the V3 region-specific primers of 16S rDNA, the extracted DNA, a fungal ITS1 primer pair (F - 5′CTTGGTCATTTAGAGGAGTAA3 and R - 5′GCTGGTTTCTTTCATATCGATTGCB), and the appropriate combination of PCR reagents and polymerase for amplification.
|
PMC11697150_p16
|
PMC11697150
|
2.4.3 Measurement of GIT indices and data analysis
| 4.104827 |
biomedical
|
Study
|
[
0.9996055960655212,
0.0002157340495614335,
0.00017878007201943547
] |
[
0.9990289211273193,
0.000596972240600735,
0.00030648455140180886,
0.00006767526792827994
] |
en
| 0.999997 |
The PCR products were run on an agarose gel and isolated with an OmegaDNA purification kit (Omega Corporation, USA). The purified PCR products were collected and sequenced on an IlluminaNovaSeq. PCR amplification, paired-end sequencing on the 6,000 platform, Illumina HiSeq sequencing, and analysis of the results were performed by the Beijing NuoHe Bioinformation Technology Co., Ltd. Amplicon qiime2 cloud platform .
|
PMC11697150_p17
|
PMC11697150
|
2.4.3 Measurement of GIT indices and data analysis
| 3.751296 |
biomedical
|
Study
|
[
0.9992664456367493,
0.00026879439246840775,
0.0004648052854463458
] |
[
0.9635015726089478,
0.035556916147470474,
0.0005596227711066604,
0.0003818904224317521
] |
en
| 0.999997 |
As shown in Table 2 , the daily feed intake and RFI in the H-RFI group were significantly higher than those in the L-RFI and the M-RFI groups ( P < 0.01). The F/G was significantly higher than that of the L-RFI and M-RFI groups ( P < 0.05), and there was no significant difference in the daily weight gain and mid-term metabolic weight of the sheep ( P < 0.05).
|
PMC11697150_p18
|
PMC11697150
|
3.1 Differences in growth performance of male Dexin lambs with different RFI
| 4.028683 |
biomedical
|
Study
|
[
0.998852014541626,
0.0003710429591592401,
0.000776908709667623
] |
[
0.9994943141937256,
0.00022149998403619975,
0.0002380269579589367,
0.00004614424688043073
] |
en
| 0.999998 |
As shown in Figure 2A , the apparent digestibility of dry matter in the L-RFI group was extremely significantly higher than the H-RFI group ( P < 0.01) and significantly higher than the M-RFI group ( P < 0.05), the M-RFI group was extremely significantly higher than the H-RFI group( P < 0.01), and the apparent digestibility of crude protein in the L-RFI group was significantly higher than M-RFI group ( P < 0.05), and the apparent digestibility of neutral detergent fiber the L-RFI group was significantly higher than the M-RFI group and the H-RFI group ( P < 0.05), the M-RFI group was extremely significantly higher than the H-RFI group( P < 0.01). There was no significant difference in ammonia nitrogen and volatile fatty acids between lambs with different RFI ( P > 0.05) . RFI and DMI were significantly negatively correlated with dry matter digestibility (r = −0.765, −0.546) and significantly positively correlated with propionic acid (r = 0.518, 0.500). ADG was significantly positively correlated with isobutyric acid (r = 0.578) .
|
PMC11697150_p19
|
PMC11697150
|
3.2 Differences in apparent digestibility and rumen fermentation parameters of male Dexin lambs with different RFIs
| 4.141829 |
biomedical
|
Study
|
[
0.9986984729766846,
0.0003220341168344021,
0.0009794770739972591
] |
[
0.9995594620704651,
0.000145685306051746,
0.00025740047567524016,
0.00003741582986549474
] |
en
| 0.999997 |
As shown in Supplementary Figure S1A , in the analysis of rumen chyme samples of Dexin lambs with different RFIs, 4,160 bacterial OTUs were found in the L-RFI group, 1,359 OTUs in the M-RFI, and 3,872 OTUs in the H-RFI. Among them, there were 845 OTUs in common in the three groups, 1,078 OTUs in common in L-RFI and M-RFI, 1,698 OTUs in common in L-RFI and H-RFI, and 1,081 OTUs in common in H-RFI and M-RFI. With respect to the fungi, 775 OTUs were found in L-RFI, 934 OTUs were found in M-RFI, and 747 OTUs were found in H-RFI . Among them, L-RFI and H-RFI had 97 OTUs in common, L-RFI and M-RFI had 115, M-RFI and H-RFI had 102, and among all three groups there were 63 OTUs in common.
|
PMC11697150_p20
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 4.128356 |
biomedical
|
Study
|
[
0.9993799924850464,
0.00025775021640583873,
0.0003621692885644734
] |
[
0.999553382396698,
0.00020365542150102556,
0.00019360554870218039,
0.00004926605470245704
] |
en
| 0.999999 |
As shown in Supplementary Tables S1 , S2 , there were no significant differences in Chao1, Shannon, and Simpson indices of bacteria and fungi in rumen digesta among male Dexin lambs with different RFIs ( P > 0.05).
|
PMC11697150_p21
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 3.805156 |
biomedical
|
Study
|
[
0.9989575147628784,
0.000308844493702054,
0.0007337088463827968
] |
[
0.9993871450424194,
0.0003686108102556318,
0.00019201803661417216,
0.000052127885282970965
] |
en
| 0.999997 |
As shown in Supplementary Figures S2A , B , the PCoA plots of rumen digesta of male Dexin lambs with different RFIs partially overlapped without obvious separation, indicating that the differences in microbial communities between and within rumen fluid sample groups were small.
|
PMC11697150_p22
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 3.552698 |
biomedical
|
Study
|
[
0.998979389667511,
0.00031510289409197867,
0.0007054454181343317
] |
[
0.9989086389541626,
0.0007517579942941666,
0.00026124235591851175,
0.0000783639625296928
] |
en
| 0.999997 |
As shown in Figure 3A and Table 3 , at the phylum level, Firmicutes, Bacteroidia , and Proteobacteria were the dominant bacterial groups in rumen fluid, The Bacteroidota bacteria in the L-RFI group were significantly lower than those in the M-RFI group( P < 0.05), while there were no significant differences in the other bacteria ( P > 0.05). At the genus level, Escherichia-Shigella, Prevotella _7 and Methanobrevibacter were the dominant bacterial genera in rumen fluid, and there was no significant difference among the top ten bacterial genera ( P > 0.05) . At the fungal phylum level, Ascomycota, Basidiomycota , and Mortierellomycota were the dominant phyla, and there was no significant difference among the top ten fungal phyla groups ( P > 0.05) . At the fungal genus level, Cladosporium, Fusarium , and Debaryomyces were the dominant genera, and there was no significant difference among the top ten fungal genera ( P > 0.05) .
|
PMC11697150_p23
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 4.136199 |
biomedical
|
Study
|
[
0.9993487000465393,
0.0003133341670036316,
0.00033800097298808396
] |
[
0.9995050430297852,
0.00014408060815185308,
0.00030030461493879557,
0.00005061367482994683
] |
en
| 0.999995 |
As shown in Figure 4 , there were seven species with LDA difference >4.0. The results show that the genera with the greatest impact on RFI on community structure are p__Proteobacteria and g__Roseburia in H-RFI , and o__Bacteroidales, c__Bacteroidia, o__Oscillospirales, p__Bacteroidota , and f__Eubacterium__coprostanoligenes_group in M-RFI. No differences were detected in fungi and L-RFI group.
|
PMC11697150_p24
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 3.962487 |
biomedical
|
Study
|
[
0.9989637136459351,
0.00026886959676630795,
0.0007673394284211099
] |
[
0.9995160102844238,
0.0002774717577267438,
0.00016359098663087934,
0.000042955529352184385
] |
en
| 0.999997 |
It can be seen from Figures 5A , C that the bacteria identified in the different RFI groups are mainly involved in membrane transport, gene translation, carbohydrate metabolism, energy production, and amino acid metabolism. The main differences in fungi in rumen digesta occurred in the L-RFI group and included wood-digesting saprophytes, soil saprophytes, plant pathogens and endophytic plant pathogens. The H-RFI group contained unclassified saprophytes, while the M-RFI samples mainly included animal pathogens, unclassified, and undefined saprophytes .
|
PMC11697150_p25
|
PMC11697150
|
3.3 Differences in rumen microorganisms of Dexin male lambs with different RFIs
| 4.02861 |
biomedical
|
Study
|
[
0.9992222785949707,
0.0002762271324172616,
0.0005015249480493367
] |
[
0.9994285702705383,
0.00025382169405929744,
0.0002722906647250056,
0.00004534379331744276
] |
en
| 0.999996 |
As shown in Supplementary Figure S3A , 1,781 OTUs were found in L-RFI, 1,195 in M-RFI, and 1,454 in H-RFI among the bacteria identified in ileum digesta samples of male Dexin lambs with different RFIs. Among them, there were 401 OTUs in common among all three groups, 578 OTUs in common between L-RFI and M-RFI, 652 OTUs in common between L-RFI and H-RFI, and 533 OTUs in common between H-RFI and M-RFI. With respect to the fungi, 496 OTUs were found in L-RFI, 354 OTUs were found in M-RFI, and 448 OTUs were found in H-RFI . Among them, L-RFI and H-RFI had 107 OTUs in common, L-RFI and M-RFI had 102, M-RFI and H-RFI had 103, and among all three groups there were 80 OTUs in common.
|
PMC11697150_p26
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 4.124346 |
biomedical
|
Study
|
[
0.9994274377822876,
0.0002374016330577433,
0.0003351951309014112
] |
[
0.999575674533844,
0.0001893985172500834,
0.00018911280494648963,
0.000045778437197441235
] |
en
| 0.999998 |
As shown in Supplementary Table S3 , in the ileal digesta samples of male Dexin lambs with different RFIs, the bacterial there were no significant differences in Chao1, Simpson, and Simpson indices between any of the groups ( P > 0.05). There were no significant differences in the Chao1, Shannon, and Simpson indices among the fungal groups ( P > 0.05) ( Supplementary Table S4 ).
|
PMC11697150_p27
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 4.025928 |
biomedical
|
Study
|
[
0.9991642236709595,
0.00028341481811366975,
0.0005524566513486207
] |
[
0.9995608925819397,
0.00019332360534463078,
0.000198383757378906,
0.00004742295277537778
] |
en
| 0.999997 |
As shown in Supplementary Figures S4A , B , the PCA plots of ileal chyme partially overlapped without obvious separation, indicating that the microbial communities in the ileal chyme samples were less diverse between and within groups.
|
PMC11697150_p28
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 3.554961 |
biomedical
|
Study
|
[
0.9989469647407532,
0.00033703382359817624,
0.0007160783861763775
] |
[
0.9989017248153687,
0.0007396096480078995,
0.0002833536418620497,
0.0000752409323467873
] |
en
| 0.999997 |
At the phylum level, Firmicutes, Bacteroidota , and Proteobacteria were the dominant phyla in ileal chyme, The Campylobacterota bacteria in the L-RFI group were significantly higher than those in the H-RFI group ( P < 0.05), while there were no significant differences in the other bacteria ( P > 0.05) . At the genus level, Escherichia-Shigella, Bacteroides , and Erysipelatoclostridium were the main dominant genera in the ileal chyme . For the Methanobrevibacter genus, L-RFI was significantly higher than M-RFI and H-RFI ( P < 0.05), and there were no significant differences in the remaining genera ( P > 0.05). At the fungal phylum level, the dominant phyla in ileal chyme samples were Ascomycota, Basidiomycota and Mortierellomycota , and there was no significant difference among the top ten dominant phyla ( P > 0.05) . At the fungal genus level, Geotrichum, Penicillium , and Cladosporium were the dominant genera, and there was no significant difference among the top ten dominant genera ( P > 0.05) .
|
PMC11697150_p29
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 4.162072 |
biomedical
|
Study
|
[
0.9993863105773926,
0.00028968878905288875,
0.00032394350273534656
] |
[
0.9994844198226929,
0.000160666459123604,
0.0003059953742194921,
0.00004896424798062071
] |
en
| 0.999999 |
As shown in Figures 7A , B , there are three species with LDA values >4, indicating that the bacterial genera that have a greater impact on ileal chyme due to residual feed intake are f-Family-Xi in M-RFI , f_Anaerovoracaceae and g_Christensenellaceae_R_7_group in L-RFI. No differences were detected in fungi and H-RFI group.
|
PMC11697150_p30
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 4.037497 |
biomedical
|
Study
|
[
0.9992660880088806,
0.00025334733072668314,
0.000480562710436061
] |
[
0.9995044469833374,
0.0002607046044431627,
0.00018721753440331668,
0.000047672201617388055
] |
en
| 0.999996 |
The differentially expressed functions of ileal digesta bacteria from different RFI groups were mainly membrane transport, carbohydrate metabolism, and amino acid metabolism . Among the ileal chyme fungi, the functional annotations of L-RFI and H-RFI fungi were quite different, mainly concentrated in plant pathogens, undefined animal pathogens, endophytic plant pathogens and undefined saprophytes .
|
PMC11697150_p31
|
PMC11697150
|
3.4 Relative bacterial populations in ileal digesta of male Dexin lambs with different RFIs
| 4.025049 |
biomedical
|
Study
|
[
0.9995558857917786,
0.00018544180784374475,
0.00025865159113891423
] |
[
0.9989866614341736,
0.000514960556756705,
0.0004395844880491495,
0.000058752819313667715
] |
en
| 0.999997 |
As shown in Supplementary Figure S5A , 979 OTUs were found in the samples of rectal feces from L-RFI lambs, 870 from M-RFI, and 1,454 from H-RFI. There were 293 OTUs in common among the three groups, 404 OTUs in common between L-RFI and M-RFI, 442 OTUs in common between L-RFI and H-RFI, and 425 OTUs in common between H-RFI and M-RFI. A total of 500 OTUs were found in the fungi of the rectal fecal samples from L-RFI, 1,170 OTUs from M-RFI, and 1,246 OTUs from H-RFI, of which 31 OTUs were found in common in all three groups, 101 OTUs in common in L-RFI and M-RFI, 99 OTUs in common in L-RFI and H-RFI, and 72 OTUs in common in H-RFI and M-RFI .
|
PMC11697150_p32
|
PMC11697150
|
3.5 Differences in bacterial microbiota from rectal feces of male Dexin lambs with different RFIs
| 4.104232 |
biomedical
|
Study
|
[
0.999366819858551,
0.00023908077855594456,
0.0003940370224881917
] |
[
0.9995561242103577,
0.00021455615933518857,
0.00018558940791990608,
0.00004366945358924568
] |
en
| 0.999996 |
As shown in Supplementary Tables S5 , S6 , there were no significant differences in the Chao1, Shannon, and Simpson indices observed in any of the groups of rectal fecal samples ( P > 0.05).
|
PMC11697150_p33
|
PMC11697150
|
3.5 Differences in bacterial microbiota from rectal feces of male Dexin lambs with different RFIs
| 3.618346 |
biomedical
|
Study
|
[
0.9989963173866272,
0.0003940062306355685,
0.0006097639561630785
] |
[
0.9991907477378845,
0.0004720189026556909,
0.00027118317666463554,
0.00006605563248740509
] |
en
| 0.999995 |
As shown in Supplementary Figure S6 , there is a clear separation between L-RFI and H-RFI in the rectal feces' samples of male Dexin lambs with different RFI, indicating that the microbiota in the two groups are quite different.
|
PMC11697150_p34
|
PMC11697150
|
3.5 Differences in bacterial microbiota from rectal feces of male Dexin lambs with different RFIs
| 2.873598 |
biomedical
|
Study
|
[
0.996842622756958,
0.0003340598486829549,
0.002823383780196309
] |
[
0.9926294088363647,
0.006726392544806004,
0.0004794334527105093,
0.00016467987734358758
] |
en
| 0.999996 |
As shown in Figure 9A and Table 11 , at the phylum level, Euryarchaeota, Bacteroidia , and Firmicutes were the main bacterial groups in rectal feces. The Uryarchaeota phylum in L-RFI was extremely significantly higher than that in M-RFI and H-RFI ( P < 0.01), the Actinobacteriota phylum in L-RFI and H-RFI was significantly higher than that in M-RFI ( P < 0.05), the Patescibacteria phylum in H-RFI was significantly higher than that in M-RFI and L-RFI ( P < 0.05), but there were no significant differences in other phyla ( P > 0.05). At the genus level, Achromobacter, Chryseobacterium , and Methanobrevibacter were the dominant bacterial genera in rectal feces . The abundance of Methanobrevibacter in L-RFI was significantly higher than in M-RFI and H-RFI ( P < 0.05), and there were no significant differences in the other genera ( P > 0.05). The dominant rectal fecal fungi at the phylum level were Ascomycota, Basidiomycota and Mortierellomycota , and there were no significant differences among the three groups in the top ten dominant phyla . At the genus level for rectal fecal fungi, Penicillium, Acaulium , and Scopulariopsis were the dominant genera . The levels of Aspergillus and Ustilago genera in the L-RFI group were significantly higher than those in the other two groups ( P < 0.05), but those in the “other” group were significantly lower than those in the two RFI groups ( P < 0.05).
|
PMC11697150_p35
|
PMC11697150
|
3.5 Differences in bacterial microbiota from rectal feces of male Dexin lambs with different RFIs
| 4.185194 |
biomedical
|
Study
|
[
0.9993810653686523,
0.0003115850267931819,
0.00030740388319827616
] |
[
0.9994834661483765,
0.0001740186708047986,
0.0002861238026525825,
0.00005640428571496159
] |
en
| 0.999997 |
As shown in Figures 10A , B , the expression differences in the microbial functions of rectal fecal bacteria are mainly concentrated in membrane transport, energy production, carbohydrate and amino acid metabolism.
|
PMC11697150_p36
|
PMC11697150
|
3.5 Differences in bacterial microbiota from rectal feces of male Dexin lambs with different RFIs
| 3.648723 |
biomedical
|
Study
|
[
0.9993282556533813,
0.00019396476272959262,
0.00047786301001906395
] |
[
0.9981977343559265,
0.001233765040524304,
0.00048173501272685826,
0.00008686007640790194
] |
en
| 0.999995 |
The rumen is the main site for ruminants to receive feed, water and saliva. It is a humid environment with a favorable temperature of 36–40°C, which makes it an excellent place for microbial growth and reproduction . A large number of studies have shown that the various microbial communities in the rumen work synergistically to convert substances such as cellulose and hemicellulose into volatile fatty acids, and convert the nitrogen produced by dietary degradation into microbial proteins that are absorbed and utilized by the host . 1 The apparent digestibility of dry matter, crude protein, neutral detergent fiber and other insdicators can accurately mirror a sheep's ability to digest and absorb nutrients . In a study on the effect of RFI on apparent digestibility, Bonilha et al. found that the digestibility of neutral detergent fiber and total digestible nutrients in Neruda cattle in the H-RFI group was significantly lower than in the L-RFI group. The research results of Arce-Recinos et al. on growing beef cattle showed that the digestibility of dry matter, crude protein, neutral detergent fiber and acid detergent fiber of L-RFI cattle was 4%−5% higher than that of H-RFI cattle, similar to the results in this study. The apparent digestibility of dry matter, crude protein, neutral detergent fiber and acid detergent fiber of L-RFI male Dexin lambs was higher than in the H-RFI group, indicating that apparent digestibility may be one of the reasons why some sheep show higher feed efficiency. In this experimental study, no difference in rumen volatile fatty acids was found among the three groups of sheep, which is similar to the findings of two studies by Arce-Recinos et al. and Zhang et al. . This may be due to the fact that the feed substrates were exactly the same, making it difficult to establish a difference in the volatile fatty acid content.
|
PMC11697150_p37
|
PMC11697150
|
4 Discussion
| 4.158604 |
biomedical
|
Study
|
[
0.9992519021034241,
0.0002797474735416472,
0.00046836156980134547
] |
[
0.9990567564964294,
0.00014350336277857423,
0.000744096003472805,
0.00005564019738812931
] |
en
| 0.999996 |
In the OTU results of this experiment, the fungal and bacterial microbiota detected in the L-RFI group were greater than those in the M-RFI and H-RFI groups, suggesting that an enrichment of beneficial microbes could enable individuals to have higher environmental adaptability and stress resistance. There were no significant differences in the Chao1, Shannon, and Simpson indices of bacteria and fungi in the rumen digesta, ileum digesta, and rectal feces samples among the three groups. The PCoA plots did not show significant differences in the microorganisms from the three groups of sheep, which is consistent with the results of Pinnell et al. on the rumen of Holstein cows. This may be because the RFI only affects individual microbial types and not the overall microbial community.
|
PMC11697150_p38
|
PMC11697150
|
4 Discussion
| 4.072669 |
biomedical
|
Study
|
[
0.9994223117828369,
0.0002471096522640437,
0.0003305670979898423
] |
[
0.9995511174201965,
0.0001502418308518827,
0.00025455173454247415,
0.0000441449046775233
] |
en
| 0.999997 |
Ruminants rely on the abundant microbial communities in their digestive tract to digest feed and convert it into nutrients that are easily absorbed. The most abundant gut bacteria are from the Firmicutes and Bacteroidota phyla . Recent progress in microbial research indicates that Bacteroidota can produce large amount of glycoside hydrolases, which can effectively degrade nutrients such as cellulose, pectin and starch, and plant polysaccharides in the rumen . Firmicutes are mainly polysaccharide-degrading microorganisms in the rumen, but recent studies indicate that Firmicutes can produce biotin, playing a major role in cellulose degradation, VFA production, and metabolism . In this study, at the bacterial phylum level, the dominant phyla of bacteria in rumen digesta, ileum digesta and rectal feces were Firmicutes, Bacteroidota , and Proteobacteria . The rumen bacteria results were consistent with the results of Liu et al. on the rumen microbiota of Hu sheep with different RFIs. The rumen of healthy ruminants is characterized by the dominance of anaerobic bacteria of Firmicutes and Bacteroidota . The results of ileal digesta were consistent with those of Elolimy et al. , with Firmicutes and Bacteroidota as the main bacterial communities, and the abundance of Firmicutes and Bacteroidota in the L-RFI group was higher than that in the H-RFI group. The rectal feces results were similar to those of Elolimy , with the abundance of Bacteroidota in cattle with L-RFI being higher than that in cattle with H-RFI. At the bacterial genus level, the genera with greatest abundance in rumen chyme were Bacteroides, Rikenellaceae_RC9_gut_group , and Prevotella _7. Among them, the abundance of Bacteroides in the L-RFI group was higher than that in the H-RFI group, and the abundance of Rikenellaceae_RC9_gut_group was lower than that in the H-RFI group. The Bacteroides genus has been shown to effectively degrade plant cell wall polysaccharides and improve fiber utilization . The specific role of the Rikenellaceae _RC9_gut_group genus is still unclear, and it has so far only been shown to be related to butyrate and propionate metabolism . In the ileal digesta, Escherichia-Shigella, Bacteroides , and Turicibacter were the main genera. The abundance of Bacteroides in L-RFI lambs was higher than that in the H-RFI group, while the abundance of Escherichia-Shigella was lower than that in the H-RFI group. Escherichia-Shigella is a harmful bacterium that may cause bacterial dysentery . Methanobrevibacter is a methanogen. Although it was not the main genus in terms of abundance in the ileal digesta, the data showed that its numbers in the L-RFI group were significantly higher than in the H-RFI group. This was contrary to the results of Mia , which showed that the ileal digesta of L-RFI male Dexin lambs contained more methanogens. In our results, the abundance of methanogens in the three parts of the L-RFI group was higher than that in the H-RFI group. This may be a result of the sheep production model and feed type. However, recent microbiological research indicates that methanogens are of great significance to the early intestinal microbial colonization of ruminants. They can effectively reduce the H 2 produced by the fermentation and decomposition of plant fibers in the digestive tract, reduce the hydrogen partial pressure, and improve the body's hydrogen nutrition pathway . However, the mechanistic details and regulatory pathways need to be verified by subsequent studies. In rectal feces, Achromobacter, Chryseobacterium , and Methanobrevibacter were the main genera. The abundance of Methanobrevibacter in the L-RFI group was significantly higher than that in the H-RFI group, while the abundance of Achromobacter and Chryseobacterium was lower than in the H-RFI group. Achromobacter is a conditionally pathogenic bacterium that can cause urinary tract infections under certain conditions . Chryseobacterium has a strong ability to digest collagen and can cause disease in the body . Through the abundance analysis of bacterial microbiota under the three RFI conditions, we can conclude that L-RFI sheep achieve higher digestion efficiency of feed nutrients by having a greater abundance of Firmicutes and Bacteroidetes in the digestive tract. This echoes the results of apparent digestibility in this study, indicating that L-RFI sheep have a higher efficiency of decomposition and absorption of feed nutrients through an enriched population of microorganisms. The increased abundance of pathogenic bacteria detected in the H-RFI group may be a result of differences in their digestive tract microbiota, which makes them less adaptable and resistant than the L-RFI sheep, which is consistent with our OTU results.
|
PMC11697150_p39
|
PMC11697150
|
4 Discussion
| 4.292289 |
biomedical
|
Study
|
[
0.9992464780807495,
0.00036315873148851097,
0.00039037346141412854
] |
[
0.999224066734314,
0.00017535104416310787,
0.0005297759198583663,
0.00007079492934281006
] |
en
| 0.999997 |
There are a large number of anaerobic fungi in the GIT of ruminants. Previous studies have generally concluded that these fungi exist in the animal body in the form of zoospores, which produce highly active, fiber-degrading enzymes, and play a major role in the digestion of fibrous plant material . It is increasingly recognized that fungi can optimize rumen fermentation, enhance nutrient availability, and promote intestinal health. Anaerobic fungi degrade plant cell walls through both enzymatic reactions and physical means targeting fibers that are difficult for bacteria to degrade . In addition to fiber degradation in the rumen, 5% to 10% of carbohydrate degradation occurs in the hindgut, indicating that fiber degradation by anaerobic fungi can occur over the entire digestive tract . It has been proven that Basidiomycota and Ascomycota can produce mycelial hyphae, which can penetrate the silica cuticle produced on the surface of forage through stomata and damaged parts of the dermis, thereby promoting more efficient digestion of plant fiber . Basidiomycota and Ascomycota are aerobic fungi with the highest relative abundance, based on sampling of rumen chyme. Within 2 h of eating, the oxygen concentration in the rumen is sufficient for the survival of Ascomycota. As aerobic fungi multiply, the oxygen content gradually decreases. This process causes the rumen to turn into an anaerobic environment, and anaerobic fungi and bacteria begin to multiply in large numbers . Mortierellomycota is a type of saprophytic fungus that is widely distributed in the digestive tract of ruminants and has the ability to efficiently decompose lignin .
|
PMC11697150_p40
|
PMC11697150
|
4 Discussion
| 4.516819 |
biomedical
|
Study
|
[
0.9992038607597351,
0.0002914602227974683,
0.0005047426675446332
] |
[
0.9821042418479919,
0.0007605222053825855,
0.016964253038167953,
0.0001709001953713596
] |
en
| 0.999997 |
In this experiment, at the phylum level, the main fungal phyla in rumen and ileum digesta were Basidiomycota, Ascomycota and Mortierellomycota, among which Basidiomycota and Mortierellomycota were more abundant in L-RFI sheep, and Ascomycota was more abundant in the H-RFI group. At the genus level, Cladosporium, Fusarium , and Debaryomyces were the dominant genera in rumen digesta. Current microbiological studies have shown that Cladosporium fungi can produce lipases, proteases, urease, and chitinase, which can help the host digest corn-rich diets . Fusarium can produce biomass that is broken down and transformed by the animal rumen; however, in vivo research on the effects of Fusarium on nutrient digestibility and rumen function is lacking and the details of the specific mechanism of action are still unclear . Debaryomyces has emerged as a potentially valuable probiotic. Its cell wall and the polyamines it produces have been shown to stimulate immunity, regulate the microbiome, and improve digestive function . However, the abundance of Debaryomyces in the H-RFI group was higher than that in the L-RFI group.
|
PMC11697150_p41
|
PMC11697150
|
4 Discussion
| 4.165673 |
biomedical
|
Study
|
[
0.9994357228279114,
0.00028826971538364887,
0.0002759167109616101
] |
[
0.9992092251777649,
0.0001898798655020073,
0.0005407651769928634,
0.000060173617384862155
] |
en
| 1 |
At the genus level in the ileal digesta, the dominant fungal genera were Geotrichum, Penicillium , and Cladosporium . Geotrichum are believed to have the potential to be useful probiotics, which can increase the production of acetic acid and propionic acid and the ratio of propionic acid to acetic acid in ruminants; their effectiveness is higher than that of traditional brewer's yeast . The cellulase produced by Penicillium can effectively increase the content of oleic acid, linoleic acid and linolenic acid in milk, and can also affect the fat yield and unsaturated fatty acid content of milk . In the results of this study, although there was no significant difference in the abundance of Geotrichum in the ileal digesta among the three groups, the abundance in the L-RFI group was 21% higher than that in the H-RFI group, which may be related to the improvement in feed efficiency. Aspergillus can secrete cellulase and protease to improve the digestibility of the feed . Ustilago plays a role in lignin degradation and participates in the fermentation of feed . In our experiments, Aspergillus and Ustilago were significantly more abundant in the L-RFI group than in the other two groups, suggesting that Aspergillus may play a major role in improving feed efficiency.
|
PMC11697150_p42
|
PMC11697150
|
4 Discussion
| 4.14451 |
biomedical
|
Study
|
[
0.9993878602981567,
0.0002737652976065874,
0.00033835365320555866
] |
[
0.9994615912437439,
0.00018993417324963957,
0.0002977277326863259,
0.000050736682169372216
] |
en
| 0.999998 |
LefSE is a high-dimensional biomarker mining tool based on the LDA algorithm, which is used to identify significantly characterized microorganisms . In the results of this experiment, when LDS > 4.0, the characteristic bacteria were only found in the rumen and ileum digesta. Among them, the g__ Roseburia genus found in the H-RFI group in rumen chyme is believed to affect the core populations and produce ketones that affect host development . P_Proteobacteria may parasitize the phylum Proteobacteria in rumen fluid and rumen epithelium, and Proteobacteria may oxidize ammonia and methane on the surface of the rumen epithelium . In the ileal chyme, the f_Anaerovoracaceae bacteria found in the L-RFI group can utilize a variety of types of organic matter as carbon sources and participate in the fermentation of plant polysaccharides in the GIT . The g_Christensenellaceae_R_7_group is currently thought to be a new type of probiotic that can effectively improve the growth performance and meat quality of ruminants. It plays an important role in the degradation of carbohydrates and amino acids into acetate and ammonia, respectively . The LEfSE results suggest that there are differences in some probiotics in the digestive tracts of sheep in the L- and H-RFI groups, which may be the main reason for the differences in feed utilization efficiency. The Proteobacteria found in the rumen chyme will compete for H ions with methanogens in the digestive tract, which may be one of the reasons for the differences in the abundance of methanogens among the three groups.
|
PMC11697150_p43
|
PMC11697150
|
4 Discussion
| 4.222295 |
biomedical
|
Study
|
[
0.9994761347770691,
0.0002493038191460073,
0.0002746141981333494
] |
[
0.9993742108345032,
0.00023230646911542863,
0.0003348934988025576,
0.00005867346771992743
] |
en
| 0.999999 |
The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis is a software module that builds a manually curated pathway graph representing the current knowledge on biological networks under defined conditions in a specific organism. The pathway diagrams are graphical representations of the networks of interacting molecules responsible for specific cellular functions . In our study, the rumen chyme KEGG diagram showed that the differential metabolic pathways between L-RFI and H-RFI were mainly concentrated in Cellular Process (L-RFI), Metabolism (L-RFI), and Environmental Information Processing (H-RFI). Among these, the Cellular Process pathway is mainly concentrated in the Transport and Catabolism pathway, the Metabolism pathway is mainly related to carbohydrate metabolism, amino acid metabolism and energy production pathways, and the Environmental Information Processing metabolic pathway was mainly concentrated in the Membrane Transport pathway. This is similar to the research results of Zhang et al. and Zhou et al. . The ileal chyme KEGG map shows that the pathway differences are mainly Cellular Process (L-RFI), Metabolism (L-RFI), Genetic Information Processing (L-RFI), and Environmental Information Processing (H-RFI). Among these, the Cellular-Process pathway was mainly concentrated in cell motility, the Genetic Information Processing pathway was mainly concentrated in replication and repair, the Metabolism pathway was mainly concentrated in carbohydrate metabolism, amino acid metabolism and energy production, and the Environmental Information Processing metabolic pathway was mainly concentrated in membrane transport. In the rectal fecal KEGG map, only the Environmental Information Processing pathway and the Human Diseases pathway were overexpressed in the H-RFI group. The Environmental Information Processing pathway was mainly concentrated in the membrane transport pathway, and the Human Diseases pathway was mainly concentrated in the endocrine system pathway. This is quite different from the research results of Elolimy et al. on Holstein cattle, which may be related to the breed, production mode and gender.
|
PMC11697150_p44
|
PMC11697150
|
4 Discussion
| 4.250752 |
biomedical
|
Study
|
[
0.9994093179702759,
0.00026785419322550297,
0.0003228360728826374
] |
[
0.9992241859436035,
0.00020115968072786927,
0.0005113305523991585,
0.000063326682720799
] |
en
| 0.999997 |
Overall, the KEGG analysis indicated that the overexpression in the L-RFI group was mainly concentrated in carbohydrate metabolism, amino acid metabolism and energy production, which may be related to the high abundance of Bacteroidetes and Firmicutes in the microbiota of L-RFI sheep, which could effectively improve the conversion and absorption of nutrients such as cellulose and amino acids. In the LEfSE results, some differentially expressed microbial metabolites had the function of promoting carbohydrate metabolism and amino acid decomposition and conversion, which may be related to the differences in KEGG metabolic pathways.
|
PMC11697150_p45
|
PMC11697150
|
4 Discussion
| 4.126337 |
biomedical
|
Study
|
[
0.9994962215423584,
0.0002398018550593406,
0.00026390739367343485
] |
[
0.9994434714317322,
0.00018599779286887497,
0.0003114368882961571,
0.00005911524567636661
] |
en
| 0.999998 |
In the KEGG diagram of fungi from the rumen and ileum digesta, the main differences observed in the fungi in rumen fluid were wood saprophytes (L-RFI), soil saprophytes (L-RFI), plant pathogens and endophytic plant pathogens (L-RFI), and unclassified saprophytes (H-RFI). The functions of ileal chyme fungi are mainly concentrated in undefined (H-RFI), endophytic plant pathogens (H-RFI) and animal pathogens (H-RFI). Given the current limitations in the KEGG functional annotation of fungi, the specific metabolic pathways should be further explored.
|
PMC11697150_p46
|
PMC11697150
|
4 Discussion
| 4.106399 |
biomedical
|
Study
|
[
0.9995096921920776,
0.00016717243124730885,
0.0003231028968002647
] |
[
0.9987170696258545,
0.0007362343021668494,
0.0004859234031755477,
0.00006088167356210761
] |
en
| 0.999994 |
According to the results of this study, different RFI did not significantly affect the overall digestive tract microbial community of Dexin male lambs. Its main mechanism of action may be to improve feed efficiency by changing the abundance of certain beneficial bacteria.
|
PMC11697150_p47
|
PMC11697150
|
5 Conclusions
| 2.889673 |
biomedical
|
Study
|
[
0.9975113868713379,
0.0004738833522424102,
0.0020147839095443487
] |
[
0.997204065322876,
0.002251095836982131,
0.00039067212492227554,
0.0001542496174806729
] |
en
| 0.999996 |
Living in a microbe-rich environment, insects, such as Dipteran species, interact with a broad range of symbionts, opportunists, and pathogens. This diversity of cohabitants has likely shaped the sophisticated insect immune system, which must be capable to combat pathogens while promoting the growth of beneficial microbes. Studies mostly done in Drosophila melanogaster have shown the multiple roles of the gut microbiota on insect health by promoting host development under poor nutrient conditions, providing vitamins, or preventing infection [ 1–3 ]. Over-growth of one or more normal species making up the microflora causes intestinal dysfunction and diseases, affecting gut homeostasis and shortens lifespan . Accumulating evidence has shown that microbiota–host interactions are complex that cannot be simplified by good or bad.
|
39530356_p0
|
39530356
|
Introduction
| 4.151671 |
biomedical
|
Study
|
[
0.9993911981582642,
0.00025879128952510655,
0.0003499778686091304
] |
[
0.8488200306892395,
0.001485785935074091,
0.14939621090888977,
0.0002979489217977971
] |
en
| 0.999996 |
Whereas many gut-dwelling bacteria generally benefit the host’s physiology, they can also be detrimental and lead to host pathogenesis in some cases. These bacteria are called pathobiont or pathogenic potential . In D. melanogaster , the over-growth of a gut-dwelling bacteria, Gluconobacter morbifer G707T, induces intestinal dysbiosis, which causes gut cell apoptosis . These studies suggested gut microbiota must be tightly controlled by the host immune system, which is complex, encompassing various constitutive and inducible defenses. First, physical barriers protect the gut epithelium from direct contact with bacteria. These include the cuticle that lines the foregut and hindgut, and a chitinous layer called the peritrophic membrane (PM) that lines the midgut . The PM forms a permeable porous matrix to allow the digestive process while separating bacteria and harmful particles from the gut epithelium . An acidic region in the middle part of the midgut also contributes to eliminating ingested bacteria . Second, the mechanical flushing of pathogens out of the intestine by peristalsis is thought to be a vital host defense mechanism. Peristalsis involves a wave-like longitudinal and circular muscle contraction that moves food and bacteria through the digestive tract . The waves can be short, regional reflexes or long, continuous contractions that travel the whole gut. In insects, as in mammals, gastrointestinal motility is controlled by the secretory products of enteroendocrine cells as well as enteric and central nervous systems . Ingestion of bacteria promotes strong midgut visceral contractions, a process involving reactive oxygen species (ROS) production by the NADPH Duox by enterocytes .
|
39530356_p1
|
39530356
|
Introduction
| 4.680328 |
biomedical
|
Study
|
[
0.999350368976593,
0.00042317877523601055,
0.00022652768529951572
] |
[
0.9924939870834351,
0.0011214582482352853,
0.006108880043029785,
0.0002755489549599588
] |
en
| 0.999997 |
Ingestion of bacteria triggers the production of hypochlorous acid (HOCl) by Duox. Its microbicidal role is thought to be crucial for insects to survive oral bacteria infection . Production of ROS by Duox is achieved through recognizing pathogen and pathobiont-released uracil, but not the symbiotic bacteria via the Gαq-PLCβ-Ca 2 + pathway and activation of MEKK1-MKK3-p38 MAPK pathways . In addition, lipid catabolism also upregulated transcription of the Duox gene 1 . Although initial studies have shown that Duox-derived HOCl has a direct bactericidal effect, other studies revealed another role for Duox in gut homeostasis. As described above, Duox is also involved in peristalsis but also contributes to the increased stem cell proliferation observed upon bacterial infection .
|
39530356_p2
|
39530356
|
Introduction
| 4.44909 |
biomedical
|
Study
|
[
0.9995853304862976,
0.00021419013501144946,
0.00020038746879436076
] |
[
0.9976921081542969,
0.0007023775251582265,
0.001468693488277495,
0.0001367682998534292
] |
en
| 0.999996 |
We have recently shown that Malpighian tubules are required for gut homeostasis post oral infection . Malpighian tubules form the insect kidney, regulating osmolarity/water homeostasis . Malpighian tubules are connected at the posterior end of the midgut, where they generate primary urine . Studies done in several insect species, particularly locusts, have revealed the existence of counterflows that can flush Malpighian tubule liquid toward the anterior parts of the gut . The existence of retrograde fluid flow in the insect gut is possibly due to the peritrophic matrix that transversally compartmentalizes the midgut. According to this model, food and bacteria transit forward along the gut in the endoperitrophic space formed by the PM, whereas the countercurrent flow initiated by Malpighian tubules takes place in the ectoperitrophic space between the PM and the epithelium. Our previous study reveals that this countercurrent flow increases upon bacterial oral infection and brings the tubules-produced Jak–STAT pathway ligand Unpaired 3 (Upd3) to the anterior part of the midgut to promote an increased epithelium renewal. The observation that this counterflow is increased upon bacterial infection suggested that it could play an important role in host defense.
|
39530356_p3
|
39530356
|
Introduction
| 4.420753 |
biomedical
|
Study
|
[
0.9993367791175842,
0.0003929564554709941,
0.0002702344791032374
] |
[
0.9989705085754395,
0.00042159701115451753,
0.0004921744111925364,
0.00011571207869565114
] |
en
| 0.999997 |
Here, we used the Tephritidae fruit fly Bactrocera dorsalis , an invasive dipteran Bactrocera pest that has continued to pose a significant threat to the fruit and vegetable industry, causing enormous economic losses worldwide . B. dorsalis larva lives in rotten fruits and owns a complex gut microbiota, making it a good model for gut microbiota study . Here, we show that pathobiont and other symbiotic bacteria-derived tyramine induce the formation of Malpighian tubules–gut countercurrent flow, which requires aquaporin Prip. This flow ensures the accumulation of ROS produced by Duox upon recognition of pathobiont. Our results show that this countercurrent flow is required for increased gut peristalsis and gut microbiota homeostasis. Collectively, our data highlights the roles of pathobiont and other symbiotic bacteria in the early stage gut immune response. Our study also reveals the importance of Malpighian tubules–gut countercurrent flow in gut microbiota maintenance.
|
39530356_p4
|
39530356
|
Introduction
| 4.280302 |
biomedical
|
Study
|
[
0.999394416809082,
0.00032811969867907465,
0.00027751369634643197
] |
[
0.9993333220481873,
0.00022583855025004596,
0.00035715335980057716,
0.00008371892181457952
] |
en
| 0.999995 |
The B. dorsalis were raised at the Institute of Horticultural and Urban Entomology, Huazhong Agricultural University (Wuhan, China), under conditions of 28 ± 1°C and 70%–80% relative humidity, with a 14 h light/10 h dark cycle. Larvae were raised on an artificial diet constituting of 300 g banana, 300 g corn meal, 60 g yeast, 60 g sucrose, 1.2 g sodium paraben methyl ester, 60 g tissue paper, 2.4 ml hydrochloric acid, 600 ml water. Upon emergence, the adult flies were transferred to cages measuring 30 × 30 × 30 cm and nourished with a synthetic food source. The artificial diet made of 10% sucrose, 3.4% yeast, 1% agar, 1.7% honey, and supplemented with 1.6 g/L sodium paraben methyl ester. Female flies that emergence for 5 to 8 days were used for all experiments.
|
39530356_p5
|
39530356
|
Insect rearing
| 4.063841 |
biomedical
|
Study
|
[
0.9991413354873657,
0.00015999109018594027,
0.0006986424559727311
] |
[
0.9986214637756348,
0.0011475086212158203,
0.00018636714958120137,
0.00004459046249394305
] |
en
| 0.999998 |
Providencia rettgeri , Citrobacter koseri , and Enterobacter hormaechei were cultured in Luria–Bertani (LB) medium for 14 h at 37°C overnight, 220 rpm from single colony. Bacteria culture were harvested by centrifuge . By combining an equal volume of an overnight-grown culture of P. rettgeri and commensal bacteria (having an optical density of OD600 = 200) with a 5% sucrose solution in a 1:1 ratio, the infection solution was prepared, which was then applied to cover the surface of the artificial diet. Adult female B. dorsalis , aged between five and eight days, were subjected to a 2 h starvation period in a plastic box maintained at 28°C, then supplied with the infection solution. For all experiments in this paper, the flies were subjected to a 2 h infection and then switched to a clean fly food for the following sampling. The time points mentioned in the paper indicate the time post the 2 h infection process. For the bacteria persistence experiment, the flies were firstly washed twice in 75% ethanol for 30 s, then washed using phosphate-buffered saline (PBS) for 30 s. The flies were then homogenized in a 2 ml freezing tube containing 200 μl of LB solution and 3 steel beads. For feces samples collection, flies were moved to a 50 mL falcon tube after infection and then feces were washed and collected using LB at desired time point. The samples were serially diluted and dilutions were then plated (2 μl) onto the LB plates. The plates were cultivated overnight at 37°C ( P. rettgeri ). In the vitamin C feeding experiment (Coolaber, China), the infection solution was supplemented with 50 mg/ml of vitamin C. Similarly, for the Tyramine feeding experiment (MACKLIN, China), the infection solution was enriched with 70 mM of Tyramine.
|
39530356_p6
|
39530356
|
Oral infection and bacteria persistence
| 4.132527 |
biomedical
|
Study
|
[
0.9995355606079102,
0.00027167960070073605,
0.00019276216335128993
] |
[
0.9992615580558777,
0.0002795760228764266,
0.0003937900182791054,
0.00006502752512460575
] |
en
| 0.999994 |
Two small wells were dug next to each other in a polydimethylsiloxane (PDMS) plate. A mixture containing an equal proportion of Schneider’s insect medium (Pricella, China) and insect saline buffer was dispensed into the two wells. Brilliant Blue solution (Sigma, Germany) was added into one of the wells to make the final concentration of 0.5 g/L. The gut and Malpighian tubules of adult female B. dorsalis were dissected. The Malpighian tubules were placed in the well containing Brilliant Blue dye, while the gut was put into the other well. The PDMS plate was then sealed with mineral oil (Beyotime, China) to prevent tissue evaporation. The gut was imaged under the microscope (Olympus SZX7, Japan) with a camera system MZX81 (MshOt, China) after 2 h. This method typically produces very clear results—either a “Yes” or “No” outcome. In simpler terms, the signal will either be present or absent in a specific region of the gut. The presence of the signal in the anterior and posterior midgut, or its absence, was recorded accordingly. In vivo measurement of countercurrent flow was performed as previously described . However, due to the gut’s absorption of the dye in B. dorsalis , the dye accumulation anterior midgut might be a combined result of gut absorption and countercurrent flow pushback.
|
39530356_p7
|
39530356
|
Countercurrent flow measurement
| 4.151594 |
biomedical
|
Study
|
[
0.9994919300079346,
0.00020271110406611115,
0.000305323104839772
] |
[
0.998847246170044,
0.000802268332336098,
0.0002878352242987603,
0.0000626523542450741
] |
en
| 0.999997 |
Adult female B. dorsalis , aged between five and eight days, underwent a 2-h starvation period in a plastic container maintained at 28°C. The flies were then infected with infection solution supplied with FITC dextran (Sigma, Germany). The flies were placed on a sticky tape with their backs fixed to the tape with their abdomen facing upside. One minute gut peristalsis videos were recorded using the microscope (Olympus SZX7, Japan) with a camera system MZX81 (MshOt, China) 2 h after infection. The gut peristalsis times were counted from the videos.
|
39530356_p8
|
39530356
|
Monitor of intestinal peristalsis frequency
| 4.050351 |
biomedical
|
Study
|
[
0.999484658241272,
0.00018553879635874182,
0.0003297596122138202
] |
[
0.9991036057472229,
0.0005643198965117335,
0.00028139344067312777,
0.000050618873501662165
] |
en
| 0.999998 |
To quantify ROS using dihydroethidium (DHE), flies were chilled on ice to induce anesthesia, followed by dissection of their guts in Schneider’s insect medium. The dissected guts were then immediately submerged in a 30 mM solution of DHE (Life Technologies) and incubated for 7 min at ambient temperature. Afterward, the guts were rinsed twice with Schneider’s insect medium and promptly visualized using a SP8 LIGHTNING confocal microscope manufactured by Leica (Germany). The signal intensity was quantified using FIJI.
|
39530356_p9
|
39530356
|
Gut reactive oxygen species staining and quantification
| 4.08927 |
biomedical
|
Study
|
[
0.9996138215065002,
0.00017436979396734387,
0.0002119276177836582
] |
[
0.9960789084434509,
0.003360528964549303,
0.00044364103814586997,
0.00011698593152686954
] |
en
| 0.999996 |
Rhodamine 6G (R6G) HOCI sensor was purchased from Heliosense (Xiamen, China). To facilitate microscope examination of the R6G fluorescence signal, both the infection solution and the sucrose control solution were supplemented with 50 μM of R6G. The flies were permitted to consume these solutions for a period of 2 h. Following a 2 h interval post infection, the flies were gathered and dissected in Schneider’s insect medium. The dissected guts were then mounted in an antifade medium sourced from Beyotime (China), and promptly inspected using a SP8 LIGHTNING confocal microscope.
|
39530356_p10
|
39530356
|
Gut reactive oxygen species staining and quantification
| 3.966376 |
biomedical
|
Study
|
[
0.9994993209838867,
0.00013795349514111876,
0.000362708029570058
] |
[
0.9928526282310486,
0.006655980832874775,
0.0003693058679345995,
0.00012207288818899542
] |
en
| 0.999997 |
For detecting H 2 O 2 in the gut, we used hydrogen peroxide assay kit (Beyotime, China) following the manufactural protocol. Briefly, flies were anesthetized on the ice and the guts were dissected in PBS at desired time points. The guts were collected and homogenized with glass beads. The kit is based on the production of trivalent iron ions by oxidizing divalent iron ions with hydrogen peroxide, which then forms a purple product with xylenol orange. The absorbance was measured at 560 nm. H 2 O 2 concentration was adjusted to the protein quantity using bicinchoninic acid assay Protein Assay Kit (Beyotime, China).
|
39530356_p11
|
39530356
|
Gut reactive oxygen species staining and quantification
| 4.089341 |
biomedical
|
Study
|
[
0.9996351003646851,
0.00013644585851579905,
0.00022848292428534478
] |
[
0.9977750182151794,
0.0017539423424750566,
0.00038725807098671794,
0.00008379556675208732
] |
en
| 0.999995 |
Primers bearing the T7 ribonucleic acid (RNA) polymerase promoter sequence (5′- GGATCCTAATACGACTCACTATAGG-3′) at their 5′ ends were utilized for cloning the desired sequence fragments via polymerase chain reaction (PCR). The primer sequences are detailed in Supplementary Table 1 . A quantity of 1 μg of the PCR product served as the template for in vitro synthesis of double-stranded RNA (dsRNA) using the T7 Ribomax Express RNAi System from Promega (Madison, WI, USA). The concentration of the synthesized dsRNA was measured at 280 nm using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc.). The integrity and quality of the dsRNA were assessed through agarose gel electrophoresis. Needles for dsRNA injection were fabricated using a puller (PC 10, Narishige, Tokyo, Japan) set at a heat level of 60.8. The Nanoinject III system from Drummond Scientific (USA) was employed for microinjecting the dsRNA. For the RNAi experiments, 1 μl of a 2 μg per microliter dsRNA solution was injected into the thorax of flies aged 5 to 8 days. The experiments were conducted three days post injection. As a control, flies injected with ds-egfp were used.
|
39530356_p12
|
39530356
|
Ribonucleic acid interference
| 4.173564 |
biomedical
|
Study
|
[
0.9995359182357788,
0.0002790860889945179,
0.00018510168592911214
] |
[
0.9992251396179199,
0.00037575591704808176,
0.00032488370197825134,
0.00007429647666867822
] |
en
| 0.999996 |
To analyze gene expression, RNA was extracted from either 8 whole flies or 20 guts. Subsequently, 500 nanograms of total RNA or equivalent tissue samples were reverse transcribed using PrimeScript RT enzyme from TAKARA, in conjunction with a blend of oligo-dT and random hexamer primers. Quantitative PCR (qPCR) reactions were carried out on complementary deoxyribonucleic acid (cDNA) samples in a 20 μl volume, comprising 10 μl of SYBR Green Mix (Vazyme, China), 400 nM of each primer, and 2 μl of cDNA (diluted 1:10). These reactions were conducted on an FQD-96X instrument (Bioer, China), utilizing 96 well plates. The PCR protocol involved an initial denaturation step at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Relative gene expression levels were determined using the 2 -ΔΔCt method. A minimum of three independent biological replicates were performed for each experiment. RpL32 served as the reference gene. The primers utilized in the qPCR analysis are listed in Supplementary Table 1 .
|
39530356_p13
|
39530356
|
Ribonucleic acid extraction and quantitative polymerase chain reaction
| 4.137981 |
biomedical
|
Study
|
[
0.9995416402816772,
0.0002629751688800752,
0.00019541371148079634
] |
[
0.9992464780807495,
0.0003997049934696406,
0.0002849222219083458,
0.00006896578270243481
] |
en
| 0.999996 |
For collecting hemolymph, eight individuals were placed in a bottom opened 0.6 ml tube covered with glass beads. The tube was transferred into a 1.5 ml tube and spun in a centrifuge for 10 min at 4°C with a rotational speed of 1500 rpm. To clear hemolymph samples, an additional centrifugation step was carried out for 5 min at 13 000 rpm. For gut and Malpighian tubules tyramine detection, the desired tissues were dissected in PBS. The whole flies, gut, Malpighian tubules and hemolymph samples were homogenized in PBS. For bacteria tyramine detection, overnight culture (OD600 = 100) was used. Tyramine detection was performed using insect tyramine kit (MEMIAN, China) according to the manufactural protocol with 10 μl infection solution. All data were normalized to a single fly. To normalize the tyramine content of ingested bacteria, we measured bacterial consumption by individual flies. Twenty flies were placed in a plastic box containing 500 μl of infection solution, and the remaining volume was measured at 0 h . The tyramine content consumed per fly was then calculated.
|
39530356_p14
|
39530356
|
Hemolymph extraction and tyramine detection
| 4.10662 |
biomedical
|
Study
|
[
0.9995394945144653,
0.00023988679458852857,
0.0002206564968219027
] |
[
0.9993213415145874,
0.0003538312448654324,
0.0002658449811860919,
0.00005899156167288311
] |
en
| 0.999998 |
Bacteria DNA extraction and 16S ribosomal RNA (rRNA) amplicon sequencing were performed using the established method in the lab. DNA was extracted from the guts of 40 flies aged 5–8 days after emergence for each biological replicate utilizing the E.Z.N.A. Soil DNA kit provided by Omega (Norcross, GA, USA), following the manufacturer’s guidelines. Two or three biological replicates were carried out for consistency. To amplify the 16S rRNA gene’s variable regions V3 and V4, the broad-spectrum forward primer 341F (CCTAYGGGRBGCASCAG) and reverse primer 806R (GGACTACNNGGGTATCTAAT) were employed in conjunction with Phusion High Fidelity PCR Master Mix from New England Biolabs (Beverley, MA). The PCR amplification protocol involved an initial heating at 95°C for 5 min, followed by 35 cycles of annealing at 56°C for 45 s, extension at 72°C for 1 min, and denaturation at 94°C for 45 s, culminating in a final extension at 72°C for 10 min. The PCR products were then submitted to Novogene’s Experimental Department for sequencing. Upon receiving the raw sequencing data, the single end reads were assigned to their respective samples based on unique barcodes and truncated by removing the barcode and primer sequences. Quality filtering was applied to the raw reads under specified conditions to generate high quality clean reads, utilizing the Cutadapt (version b1.9.1, accessible at http://cutadapt.readthedocs.io/en/stable/ ) quality control process as described by Martin in 2011. The reads were then compared against the reference database (Silva database, accessible at https://www.arb-silva.de/ ) using the UCHIME algorithm (available at http://www.drive5.com/usearch/manual/uchime_algo.html ) to detect and subsequently remove chimeric sequences, yielding the final set of clean reads. Alpha diversity (Simpson index) was calculated using qime and visualized with R software.
|
39530356_p15
|
39530356
|
Bacteria deoxyribonucleic acid extraction and 16S ribosomal ribonucleic acid amplicon sequencing
| 4.240093 |
biomedical
|
Study
|
[
0.9995115995407104,
0.00030963821336627007,
0.00017878423386719078
] |
[
0.9990713596343994,
0.0003539413446560502,
0.00047852835268713534,
0.00009613225847715512
] |
en
| 0.999997 |
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