Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the  Vibrio cholerae being the first identified . They are common features of bacterial genomes from the Vibrio genus and are generally distributed in several genomes of β- and γ-proteobacteria  widely contribute to the evolution of bacterial genomes, notably by conveying adaptive traits such as the ability to resist to antibiotic treatments . This caentified . This subacteria ,5. Thesebacteria  and the bacteria . Indeed,d by MIs ,8.attI recombination site and the resident promoter PC oriented towards the variable cassette array  and double-stranded DNA (attI site) depending on their structure and sequence respectively   is formed .,15.attC ttI site ,17. In c) Figure   Figure . This sppromoter . The recd Figure , which cd Figure . SeveralttC site . Host cettC site . The mosresponse ,28. Suchresponse .Escherichia coli strains. Paradoxically, our knowledge of the SCI of the V. cholerae pathogenic strain remains predominantly descriptive more than 20 years after its discovery and despite its paradigmatic role in the field. Here, for the first time, we designed experimental assays to study cassette recruitment dynamics directly in the V. cholerae SCI. We delivered cassette substrates using conjugation, but also through natural transformation since V. cholerae is known to be naturally competent and to exchange DNA in this way. We measured significant cassette insertion rates mediated by the sole endogenous integrase. We confirmed that this integrase expression is due to the SOS activation probably triggered by the single-stranded cassette delivery during conjugation and transformation processes  is critical for cassette recruitment at the attIA site of the V. cholerae SCI. The impact of RecAVch on cassette recombination is SOS-independent and seems specific to the attIA × attC reaction mediated by the integrase of V. cholerae, IntIA. Indeed, the RecAVch protein did not influence neither attC × attC recombination mediated by IntIA nor cassette recombination mediated by the MI Class 1 integrase, IntI1. Moreover, unlike that of IntI1, the V. cholerae SCI IntIA is not active in other bacterial hosts . Altogether, these results suggest that, in contrast to MIs, some specific host factors can regulate cassette recombination in SCIs. Therefore, MIs might have been selected to be independent of such host factors. In addition to their association with transposons and conjugative plasmids, this evolutionary trait may explain the large MI dissemination among bacteria and the antibioresistance expansion.By performing Escherichia coli and Vibrio cholerae strains were cultivated at 37°C in Luria Bertani (LB) media. V. cholerae and E. coli strains containing a plasmid with a thermo-sensible origin of replication were grown at 30°C. Thymidine (Thy) and diaminopimelic acid (DAP) were supplemented, when necessary, to a final concentration of 0.3 mM. Glucose (Glu), l-arabinose (Ara) and isopropyl-β-d-thiogalactopyranoside (IPTG) were added respectively at final concentrations of 10, 2 mg/ml and 0.8 mM. To induce the Ptet promoter, anhydrotetracycline (aTc) was supplemented into the media to a final concentration of 1 μg/ml. In the case of E. coli strains, antibiotics were added at the following concentrations: carbenicillin (Carb), 100 μg/ml, chloramphenicol (Cm), 25 μg/ml, kanamycin (Km), 25 μg/ml and spectinomycin (Sp), 50 μg/ml. V. cholerae strains were cultivated with the same antibiotic concentrations except in the case of Cm and Sp, that were supplemented at a final concentration of 5 μg/ml and 100 μg/ml respectively. When V. cholerae strains were cultivated in presence of glucose, the later concentration of Sp was increased 2-fold (200 μg/ml).The different strains and plasmids that were used in this study are described in ΔrecA, N16961 ΔrecA ΔattIA, N16961 ΔrecA ΔattIA::attI1, N16961 hapR+ lexA(ind-) and N16961 hapR+ lexA(ind-) ΔrecA. To this purpose, we used different variants of the pMP7 vector, respectively pB203, pK590, pK584 and p6780  expressing vector . The exribed in . The helr (CarbR ), which aadA7attC recombination site. An advantage of using this vector is its relatively small length (1817 bps) which enables to better mimicking the integron cassettes  and grown in the presence of Sp and Ara (0.2%) respectively to maintain the pBAD43 vector and to allow the expression of the integrase. In the case of the VchrecA and EcrecA complemented strain, IPTG was also added in the media. The donor strain was grown in parallel in presence of DAP. When both donor and recipient cultures reach an OD600 nm of 0.7–0.8, recipient V. cholerae strains were washed by centrifugation at 3000 rcf for 6 min and resuspension of the pellet in 1 ml of LB. 1 ml of each donor and recipient cultures were then mixed and centrifuged at 3000 rcf for 6 min. The obtained pellet was re-suspended in a droplet of LB and spread on a 0.45 μm filter placed on MH DAP, Ara plates and incubated at 37°C for 3 h. After incubation, the filter was re-suspended in 5ml of LB and this suspension was used to spread appropriate dilutions on MH, Cm, Sp, Glu and MH, Sp, Glu plates. After 2 days of incubation at 37°C, the recombination frequency was calculated as the ratio of CmR clones over the total number of recipient colonies that grew on MH, Sp, Glu plates. Note that in the case where we did not detect recombination events for one replicate, we calculate the recombination rates as the ratio of the mean of recombinant clones over the mean of total recipient clones obtained for the different replicates. In this case no error bars are represented on our graphs.This assay has been previously described  and implV. cholerae natural transformation was previously described . The culture was grown at 30°C until they reached an OD600nm of 0.5. One milliliter of cells was then centrifuged  and resuspended in 1 ml of M9 minimal medium supplemented with MgSO4 (32 mM) and CaCl2 (5 mM) and Sp (100 μg/ml). Tubes containing 50–80 mg of chitin  were inoculated with 0.5 ml of washed cells and 0.5 ml of fresh M9 medium supplemented with MgSO4, CaCl2 and Sp, vortexed, and grown 48 h at 30°C with shaking. Cultures were then washed  and resuspended in an equal volume of fresh M9 medium supplemented with MgSO4, CaCl2, Sp and anhydrotetracycline (aTc) to induce integrase expression. The cultures were incubated again for 30 min at 30°C with shaking. Then, 2 μg of plasmid DNA (the pD060 vector) were added to the cultures and incubated for 36 h at 30°C with shaking. After incubation, bacteria were detached from chitin by vortexing vigorously for 30 s. The obtained bacteria suspension was used to spread appropriate dilutions on MH, Cm, Sp and MH, Sp plates. After 2 days of incubation at 37°C, the recombination frequency was calculated as the ratio of CmR clones over the total number of recipient colonies that grew on MH, Sp plates in the same manner as for suicide conjugation assay.In this study, we used the same pSW23T vector (pD060) that was used in the suicide conjugation assay. escribed . Here, wattIA site of the SCI we used 5778 and SWend primers. These primers hybridize respectively in a sequence upstream of attIA in V. cholerae chromosome 2 or downstream of aadA7attC in the pSW23T vector. For E. coli and V. cholerae strains transformed with the pSU38Δ or on pBAD43 vectors that harbor recombination sites, SWbeg and MFD or 1704 and SWend primers respectively were used to amplify one junction of the co-integrate. Finally, to detect insertion of pSW23T in the genome of V. cholerae, either at secondary sites or into the VCR sites of the SCI, we performed random PCR amplification. For these, we performed a first randomized PCR reaction using degenerate 1863 and 2405 primers. The 2405 primer hybridizes upstream of the attC sites on pSW23T plasmids. Due to the presence of degenerate nucleotides in the 1863 primer, low hybridization temperatures were used, first, 30°C during 5 cycles and after, 40°C during 30 cycles. The obtained amplified DNA fragments were subjected to a second PCR reaction in order to enrich for PCR products corresponding to cassette insertion. For this purpose, we used primers 1865 and 1388. These primers hybridize respectively to the fixed part of the degenerated 1863 primer and upstream (but closer than 2405) of the attC sites on pSW23T plasmids. Recombination points were precisely determined by sequencing PCR products using primer 1366. The 1366 primer hybridizes upstream (but closer than 1388) of the attC sites on pSW23T plasmids. For each condition assay, at least three PCR reactions were purified and sequenced to confirm the insertion point.For each condition assay, at least 16 recombinant clones were isolated on appropriate plates and analyzed by PCR. For this, we performed different PCR reactions. In order to determine precisely if the pSW23T vector has been inserted into the attC sites are carried by a replicative plasmid. The vectors that we used  replicate unidirectionally and the aadA7attC recombination sites they carry have been cloned so that the recombinogenic bottom strand is located either on leading or lagging strand template (‘lead’ or ‘lag’ orientation). V. cholerae strains were transformed with these unidirectional-replicative vectors and the IntIA expressing vector  or with the empty version of the plasmid . As p7523 and p7546 have both a thermo-sensitive origin of replication, the cells were cultivated overnight at 30°C. The next day, cultures were diluted (1:100) and incubated for 5h at 30°C in presence of Cm and Carb to maintain both pTSC29 and pBAD18 plasmid. In order for recombination to take place, L-arabinose was also added to the media at a concentration of 0.2% to allow the expression of the integrase. After 5h of incubation, appropriate dilutions of the cultures were plated in parallel on MH Cm, Carb, Glu and MH Carb, Glu plates and were incubated for two days at 42°C. Since pTSC29 vector cannot replicate at 42°C, the selection on Cm plates at this temperature allows the recovery of only the clones in which aadA7attC site have been recombined by IntIA. Recombination rates were calculated as for suicide conjugation assays, by considering the ratio of CmR clones over the total number of recipient clones that grew on MH Carb, Glu plates. In order to determine if cassette insertion occurs into the attIA site of the SCI of V. cholerae, we performed PCR reactions on at least eight random isolated clones using 5778 and 573 or 5778 and MFD primers (respectively used for ‘lead’ and ‘lag’ orientation of the bs of aadA7attC site on pTSC vector).This assay was previously described  and alloV. cholerae SCI constitute large reservoirs of functions for their host bacteria. In such massive integrons, several recombination sites can constitute potential targets for cassette insertion (attIA primary recombination site and/or the numerous VCR sites). In a previous study, recombination assays that were performed in V. cholerae aimed at evaluating cassette insertion in recombination sites carried on plasmids  are delivered on a single-stranded form in a recipient V. cholerae strain containing a vector expressing the IntIA integrase. Once delivered, attC-containing plasmids cannot replicate and can therefore be assimilated to a non-replicative integron cassette. The unique way for these synthetic cassettes to be maintained is to be inserted in the V. cholerae host genome by a recombination between the cassette attC sites and the SCI attI or attC sites.Large SCIs such as the plasmids . Here, won assay  and devee Figure . The pla–3) of the pSW23TaadA7::attC vector using the N16961 wt V. cholerae strain  for both assays  strain in which the SOS response is not inducible. Indeed, in this strain, the SOS regulon genes are constitutively repressed because of the presence of the uncleavable LexAA91D version of the LexA repressor (ΔintIA and lexA(ind-) mutant strains and as expected we obtained a very high recombination frequency . The RecA protein is functionally conserved among bacterial species  when using the wt parental strain as recipient strain. We observed a decrease of more than two orders of magnitude in the recombination rates (7.7 × 10–5) in the corresponding N16961 ΔrecA mutant strain . These results mean that the RecAVch protein favors insertion of cassettes in the attIA site of V. cholerae SCI. As expected, no recombination event was detected in the N16961 ΔrecA control strain that carries the empty pBAD43 vector  to complement the VchrecA deletion. The E. coli and V. cholerae RecA proteins are highly similar, but concentrate their variations in their C-terminal part  mutant, in which the SOS response is non-inducible, we did not observe any effect on recombination efficiency compared to the wt strain  ΔrecA mutant, we still observe a large decrease of more than two orders of magnitude in the recombination rates  and lexA(ind-) ΔrecA strains). These results allow us to demonstrate that the RecAVch effect on IntIA mediated recombination is independent on the SOS regulon proteins. We also determined the role of the RecAVch protein during cassette recombination in attIA when this site is located on a plasmid  and transformed with an attIA-containing plasmid. Once again, we obtained a decrease of two orders of magnitude . Here, we did not observe any differences in the recombination rates obtained with ΔattIA and VchΔattIA ΔrecA mutant strains. To confirm the cassette insertion in VCRs, we performed random PCR reactions . As the recombination rates into VCR sites is similar between both strains . We obtained a significant rate of recombination for both ΔattIA and VchΔattIA ΔrecA strains and we did not observe any impact of the RecAVch protein on aadA1attC × aadA7attC reaction catalyzed by IntIA in V. cholerae  or on a plasmid (MI attC sites).To confirm this absence of RecAe Figure . TogetheVch protein on cassette recombination was striking since it has been previously shown that the RecAEc protein does not influence the recombination reactions catalyzed by IntI1 in E. coli . Secondly, we performed this assay in V. cholerae strains deleted for the attIA site (V. cholerae N16961 ΔattIA) and transformed with an attI1-carrying plasmid. We observed, using these two assays, that the frequencies of attI1 × attC reaction catalyzed by IntI1 were similar for both ΔattIA and ΔattIA ΔrecA recipient strains . These vectors replicate unidirectionally and the aadA7attC site that they carry was cloned in two orientations, so that the bottom strand of the attC site is either present on the lagging or leading strand template , bs attC site folding from ssDNA is favored .In all previously presented assays, we used horizontal gene transfer mechanisms as a means for delivering the single-stranded recombinogenic bottom strand of the e Figure . When thd Figure . When thxtrusion   in conditions where IntIA is overexpressed. However, we were also able to detect a significant level of cassette insertion (>10–6) catalyzed by expression of the sole endogenous integrase. We also detected cassette insertion events catalyzed by the endogenous integrase by delivering cassettes during natural transformation. The entry of single-stranded cassettes by natural gene transfer processes activates the SOS response sufficiently to express the endogenous integrase at levels allowing the proper insertion of these incoming cassettes. We therefore reproduce the whole natural integron pathway in which single-stranded integron cassettes entry, mediated by HGT processes, activates their proper insertion in the integron platform , these events can be selected and evolutionarily fixed if cassette expression leads to an advantageous new function. Thus, the knowledge of the precise cassette functions in SCI would allow us to trace back the environmental conditions of cell growth and the evolutionary history of SCI-containing cells.Interestingly, by analyzing the insertion events, we demonstrated that despite the presence of about 180 VCR sites in the large array of cassettes, the ollowing ,48. Noneollowing ,50. Firsollowing . Third, gression . NeverthattC substrates depends on several cellular processes including conjugation, replication or DNA topology  during the SOS response. Such regulation is ensured by the binding of the LexA repressor in the promoter region of Pint , which are recombined. Altogether, our results indicate that the RecA protein is a host factor specifically involved in the cassette recruitment in the attIA site of the V. cholerae SCI during environmental stress conditions. Finally, the RecA protein acts at several steps during the integron recombination process . ThiI Figure .Vch on integron recombination could thus be the consequence of an indirect effect. Using a V. cholerae mutant strain lexA(ind-) in which the SOS response is constitutively repressed, we showed that SOS response abrogation did not affect recombination rates and therefore that the observed RecAVch effect on attIA × attC recombination does not involve any other protein belonging to the SOS regulon. Besides, we know that the E. coli RecA protein differs from the V. cholerae RecA protein in the extreme C-terminal part and that this part is known to modulate interactions with regulatory proteins  or double-stranded (replicative assay), we observed an important effect of the RecAVch protein in attIA × attC recombination. Altogether, these results show that the observed RecAVch effect is not linked to the regulation of attC site folding. We therefore suppose that the attIA × attC synaptic complex formed during the recombination could impede the aHJ resolution by blocking replication fork progression. The effect of RecA would be to bind the ss attC sites and help to maintain the attC site in a non-folded single-stranded form destabilizing the synaptic complex and favoring the replicative resolution step. Note that we did not observe this RecAVch effect on attC × attC recombination suggesting a differential configuration between both attC × attC and attIA × attC complexes. Indeed, synapses formed during both reactions are known to be different since involving respectively two flexible bs attC sites versus a flexible bs attC and a stiffer ds attI  in E. coli impeding this reaction to efficiently occur. This is not surprising since, even though they show some slight variations in the C terminal part, RecAEc and RecAVch present a very high level of identity  and all these reactions are efficient in V. cholerae. Therefore, while SCI cassette recombination activity seems restricted to a given host, MI recombination seems efficient in different species suggesting a host factor recruitment more stringent for SCIs compared to MIs. This is consistent with the observed widespread MIs dissemination among bacterial species. The evolutionary success of class 1 MIs clearly reflects the fact that they are functional in a wide range of bacterial hosts. This could result from a co-evolution between the integrase IntI1 and its attI1 site to ensure that cassette recombination takes place in absence of specific accessory proteins. Both IntI and attI level of divergence, 45% between IntIA and IntI1 , to replace trans-acting host factors necessary for the attI1 × attC reactions. For instance, DRs of attI1 site acting as topological filters (In contrast, RecA is not involved in any reactions mediated by IntI1 (neither in nd IntI1  and the ts’ DRs, , that cots’ DRs, . These Deactions . HowevertI sites  and, for any DRs . Upon mo filters  could thVibrio cholerae, responsible for seven major pandemics since 1817, the latter of which being still ongoing (attIA site of the SCI contained in V. cholerae where they are expressed and selected if providing an advantage to the V. cholerae strain. The natural bacterial competence has already been demonstrated for at least 80 species of bacteria but remains little explored and is probably underestimated (Vibrionaceae, where SCIs are mainly found and constitute very plastic regions (Vibrionaceae species.HGT contributes to the emergence of pathogens and the spread of virulence factors, and also enables many disease-causing bacteria to rapidly evolve in response to environmental pressures such as antibiotic use . Among H ongoing . Cassettstimated ,70. Nota regions ,72. Heregkab412_Supplemental_FileClick here for additional data file.


In dairy cattle populations in which crossbreeding has been used, animals show some level of diversity in their origins. In rotational crossbreeding, for instance, crossbred dams are mated with purebred sires from different pure breeds, and the genetic composition of crossbred animals is an admixture of the breeds included in the rotation. How to use the data of such individuals in genomic evaluations is still an open question. In this study, we aimed at providing methodologies for the use of data from crossbred individuals with an admixed genetic background together with data from multiple pure breeds, for the purpose of genomic evaluations for both purebred and crossbred animals. A three-breed rotational crossbreeding system was mimicked using simulations based on animals genotyped with the 50 K single nucleotide polymorphism (SNP) chip.For purebred populations, within-breed genomic predictions generally led to higher accuracies than those from multi-breed predictions using combined data of pure breeds. Adding admixed population’s (MIX) data to the combined pure breed data considering MIX as a different breed led to higher accuracies. When prediction models were able to account for breed origin of alleles, accuracies were generally higher than those from combining all available data, depending on the correlation of quantitative trait loci (QTL) effects between the breeds. Accuracies varied when using SNP effects from any of the pure breeds to predict the breeding values of MIX. Using those breed-specific SNP effects that were estimated separately in each pure breed, while accounting for breed origin of alleles for the selection candidates of MIX, generally improved the accuracies. Models that are able to accommodate MIX data with the breed origin of alleles approach generally led to higher accuracies than models without breed origin of alleles, depending on the correlation of QTL effects between the breeds.Combining all available data, pure breeds’ and admixed population’s data, in a multi-breed reference population is beneficial for the estimation of breeding values for pure breeds with a small reference population. For MIX, such an approach can lead to higher accuracies than considering breed origin of alleles for the selection candidates, and using breed-specific SNP effects estimated separately in each pure breed. Including MIX data in the reference population of multiple breeds by considering the breed origin of alleles, accuracies can be further improved. Our findings are relevant for breeding programs in which crossbreeding is systematically applied, and also for populations that involve different subpopulations and between which exchange of genetic material is routine practice.The online version contains supplementary material available at 10.1186/s12711-021-00637-y. Genomic evaluation facilitates the accurate selection of genetically superior individuals as early as their DNA samples are obtained . GeneticFor an accurate genomic prediction, a large population of individuals with both phenotypes and genotypes is needed, which may not be possible for all traits and/or all breeds –6. In suWhen multiple breeds are combined to form a reference population, predictions rely on the SNP-QTL LD across breeds. However, LD may be different , 12, or Crossbreeding emerges as an efficient strategy for dairy cattle breeding to achieve better productivity and robustness at the animal as well as the system level. The improved performance is due to the use of specific combining abilities and heterosis . In dairAn appealing approach to make use of data of admixed individuals in genomic prediction is to incorporate breed proportions in genomic prediction models. Makgahlela et al.  extendedIn this article, we propose a methodology that is suitable for genomic prediction using a reference population of multiple purebred and admixed individuals. Through simulations, we investigated the impact of the correlation of QTL effects between the breeds, and the heritability of the trait on the accuracy of genomic prediction using different approaches: (i) treating the combined data as a single homogeneous population, (ii) considering breed-specific SNP effects with or without accounting for correlations between the breeds, and (iii) considering priors that lead to the use of region-specific correlations among the breeds.Genotype data at 51,477 loci were available for animals from each of the three dairy cattle breeds: Danish Holstein (HOL), Swedish Red (RED) and Danish Jersey (JER), from which a subset of 1050 (HOL and RED) or 220 (JER) individuals formed the base populations for this study. The SNPs that were fixed for the same allele in all three breeds were removed. For computational reasons, only the SNPs  on first five chromosomes were considered. A plot summarising the principle component analysis of genomic relationships among all animals was depicted to assess the genetic relationships between the pure breeds  or 220 (JER) individuals (generation 0–G0), of which 50 (HOL and RED) or 20 (JER) were assigned as males and the rest as females. The purebred populations were generated by mating sires and dams from the same breed  is the lambda parameter . RecombiThe total number of QTL was set at 250, which were selected randomly among the SNPs that satisfied Even if additive and dominance effects of QTL are the same in different breeds, the difference in QTL allele frequencies may cause substitution effects of QTL  to diffei , respectively. The k, in breed j. The QTL effects were scaled such that the mean of the breed-specific genetic variances  is 100 at G0. A random residual Each individual had two alleles  at each locus, inherited from its dam and sire. The breed origin of each allele for all loci was traced back to pure breeds at G0, and was known without error. The breeding value of each individual j = H,R,J and k QTL. The genetic correlations between HOL–RED, HOL–JER and RED–JER were 0.88, 0.75 and 0.78, respectively, for a correlation between QTL of 1.00, over 10 replicates and at G0. These genetic correlations were 0.45, 0.38 and 0.38 for a correlation between QTL of 0.50, and 0.22, 0.19 and 0.19 for a correlation between QTL of 0.25, respectively. The differences between QTL effect correlations and genetic correlations were due to the difference in QTL allele frequencies between the breeds. The correlations between QTL allele frequencies of HOL–RED, HOL–JER and RED–JER were 0.33, 0.22 and 0.41, respectively. The correlations between SNP allele frequencies were 0.47, 0.32 and 0.46. The QTL effect correlations of 0.50 and 0.25 are consistent with the reported genomic correlations (genetic correlations estimated based on available SNP sets) between some cattle breeds for milk [True (simulated) genetic correlations between the breeds were computed from the genetic variances, nt}j≠j′)  at k QTLfor milk , 25 and for milk , respectGenerations 6,7 and 8 (G6–G8) were used to form reference populations, while generation 9 (G9) was used to form validation populations. Hence, 660 JER individuals, and 3150 individuals from each of the HOL, RED and MIX were available for forming reference populations to estimate SNP effects.The SNP effects were estimated using different reference populations: (i) a single pure breed , (ii) combined data of multiple pure breeds (HOL + RED + JER), and (iii) combined data of multiple pure breeds and admixed (MIX) individuals. The MIX dataset was either used as a different “breed”, assuming homogeneous SNP effects across all breeds (HOL + RED + JER + MIX), or truly treated as an admixed population considering the breed origin of alleles (BOA) approach and uncorrelated (uncor) or correlated (cor) SNP effects between the breeds (HOL + RED + JER + MIX uncor/cor) , 56, 57.The prediction of breeding values for each pure breed was performed using: 1) the estimated SNP effects from their own breed (within-breed prediction), (2) the estimated SNP effects from each of the other breeds (across-breed prediction), (3) the estimated SNP effects from a combined reference population (multi-breed prediction) and (4) the estimated SNP effects from a combined reference population considering the BOA approach. The breeding values were predicted by multiplying SNP effects with allele dosages, with (4) or without (1–3) considering breed origin of alleles. These same strategies (1–4) were used to predict the breeding values of admixed individuals, with the addition of fixed breed contributions in multi-breed prediction [see model , multiple breeds data without considering breed origin of alleles as “combined”, and multiple breed’s and MIX data considering breed origin of alleles as “BOA”.n depends on the reference population size, and l is the number of SNPs. Model  to accoS subsets each with df) and a scale parameter (S): A Bayesian approach was considered in the parameter estimation, which requires assigning prior distributions to the unknowns of the model. Analyses were carried out separately for each trait. To investigate the impact of assuming a heterogeneous (co)variance of SNP effects between different genome regions, three region sizes were considered based on a fixed number of SNPs; 1 SNP, 100 SNPs and the whole genome (WG). Region sizes of 1 SNP and WG can be regarded as BayesA and SNP-best linear unbiased prediction (SNP–BLUP) ) when us , 30. TheS subsets each with j : In the analyses using model , all genentclass1pt{minimaIn both models  and 2),,2), resij, and lth SNP in breed j. Only one j = H,R,J and The hyper-parameters of the prior distributions for the variance components were derived from the simulated genetic (co)variances and residual variances at G0 as follows. For the analysis using model  assuming,l1-pj,l . Here, \entclass1pt{minimaThe Markov-chain Monte Carlo (McMC) algorithm was used to obtain samples of each parameter from its full-conditional posterior distribution. The chain length for the analyses consisted of 50,000 cycles, of which the first 10,000 were discarded as burn-in. Every 10th sample of the post burn-in cycles was kept for posterior analysis, yielding 4000 posterior samples. The mean value of the posterior samples was used as the estimate of each parameter. All the analyses were performed using self-written scripts in Julia .Prediction accuracy was assessed as the correlation between true and predicted breeding values of validation individuals  at G9. The accuracies of prediction using different data sets and models to estimate SNP effects were compared for each trait, QTL correlation and region size, separately. The accuracies of prediction for different region sizes were compared for each data set and model, trait and QTL correlation, separately. All comparisons were performed using a two-sided paired t-test, for which accuracies were paired across each replicate for the same validation population. A Bonferroni correction was used to control the type 1 error rate of 0.05.Accuracies for all scenarios and all region sizes are given in Additional file Accuracies were lowest when using SNP effects from any of the pure breeds to predict the breeding values of admixed individuals. For the high heritability trait, predictions using SNP effects of HOL, RED and JER yielded accuracies of 0.411, 0.275 and 0.114, respectively, when the region size was 1 SNP Fig. .Fig. 3AcThe efficiency of the methods considering breed origin of alleles in model training became more apparent as the correlation of the simulated QTL effects between the breeds decreased , yielded much lower accuracies than within-breed predictions. This was true even when the simulated QTL effects had a correlation of 1.00. It should be noted that, although the simulated QTL effects were identical in the scenario with a correlation of 1.00, some QTL were not segregating in each breed Table . The resFor within-breed predictions, both family relationships and LD between SNPs and QTL contribute to accuracy –39. For Although the simulated traits in this study were relatively polygenic, the variance structure at the SNP level may be different from that at the QTL level along the genome , 43, favFor a high heritability trait and purebred analysis, accuracies obtained for different region sizes were generally ranked as 100 SNPs > 1 SNP > WG. It was shown earlier by simulations , 35 and If the studied population is small, it might be challenging to establish a large reference population, and in turn the accuracy of genomic prediction might also be limited . For breecreased .In a study based on real genotypes of imputed sequence variants , an appealing strategy is to apply multi-trait methods where the same trait in different breeds is considered as different but two correlated traits, e.g. , 25. In If a large amount of commercial farm data for admixed populations becomes available, it can help to improve selection accuracy by expanding the data size for each pure breed population . Such daIncluding the data of an admixed population (MIX) along with the data of pure breeds in the reference population led to higher accuracies than the combined reference population of pure breeds. The JER benefited relatively more from adding MIX data. Because we mimicked a rotational cross breeding system, at each generation, admixed population dams were sired by a purebred individual. Consequently, when an admixed female was mated with a purebred male, the offspring had an entire paternal chromosome from a pure breed, and a maternal chromosome including large chunks of the (i) admixture of all breeds and (ii) the pure breed of the maternal grand-sire. This means that, at each generation following G1, pure breeds were not equally represented in the genome of admixed individuals. Consider a single admixed individual at generation 6. That individual has an expected breed composition for a maternal chromosome of roughly 28% JER, 16% HOL, and 56% RED, and for a paternal chromosome of %100 HOL. Those proportions change to 14% JER, 58% HOL, and 28% RED for a maternal chromosome, and 100% JER for a paternal chromosome at generation 7, and to 57% JER, 29% HOL, and 14% RED for a maternal chromosome, and 100% RED for a paternal chromosome at generation 8. Because a full rotation cycle of three generations (G6–G8) was considered when forming the reference populations, each pure breed was represented in the MIX data almost equally. Thereby, the reference population size increased almost equally for all breeds by adding MIX data to the combined data of three breeds, HOL + RED + JER + MIX. As one would expect, JER benefited more from this increase in data size, since it is the breed with the smallest pure breed reference population. It should be noted that the validation individuals of RED had the grand-sires which were also the sires of MIX at G8, and G8 was included in the reference population. Hence, although the data size increased almost equally for each breed, the added value of this may not be equal for all the breeds.More elaborative ways to include individuals with admixed genetic background in the genomic evaluations, were proposed. Makgahlela et al.  fitted aModels accounting for breed origin of each SNP allele, rather than genome-wide breed proportions estimated from pedigree or markers, have been proposed, and were shown to improve genomic predictions for simple 2 or 3-way crosses. Those studies applied either univariate whole genome regression models at the SNP level ignoring that the SNP effects might be correlated between the pure breeds , 55, or Our results did not show any clear evidence of the benefit of accounting for correlations among the breeds when MIX data were used with the BOA approach, even for the breed (JER) with a small reference population, for which one would expect more gain in accuracy compared with breeds with a large reference population (HOL and RED). A possible explanation of the unobserved benefit for JER could be due to this breed being genetically distinct from HOL and RED , and theThe differences in LD pattern and phase persistency across different breeds  may resuvan den Berg et al.  showed tentclass1pt{minimaEstimation of the breed composition of individuals with admixed genomic background is of relevance for genomic prediction, because if it is not accounted for, it may lead to spurious estimates of SNP effects . In realGenetic background, as well as environment, alters the effects of QTL , 19. Thuh2), and the effective number of chromosomal segments segregating in the population , and also for populations involving different subpopulations between which exchange of genetic material has become routine practice .The aim of this simulation study was to provide a model allowing the inclusion of data from individuals with an admixed genetic background in genomic evaluations, while accounting for the differences in marker effects for each purebred population in the gene pool. Combining pure breeds’ and admixed population’s data in a multi-breed reference population was beneficial for the estimation of breeding values for pure breeds with a small reference population. For the admixed population, combining all available data  and realizing a combined genomic evaluation led to higher accuracies than considering BOA for selection candidates only and using breed-specific SNP effects estimated separately in each pure breed. Including data from admixed individuals in the reference population of multiple breeds by considering BOA, accuracies were further improved. Our findings are relevant for breeding programs in which crossbreeding is systematically applied  for a trait with high heritability h2 = 0.40. Table S4. Accuracies for admixed individuals (MIX) for a trait with low heritability h2 = 0.05. Table S5. Accuracy for the low heritability h2 = 0.05 trait, using only 250 QTL and region size of 1 SNP.


Paragonimus species are highly prevalent in various regions of China. The study's objective is to isolate and identify Paragonimus from natural habitats and compare the phylogenetic diversity of Paragonimus in southern Yunnan province, China. Metacercariae of Paragonimus was isolated from crabs, and morphologic identification was performed by microscopy. Metacercariae were injected into experimental Paragonimus free Sprague Dawley rats. After 114 days, adult worms and eggs were isolated from multiple organs. Morphologic identification confirmed the initial identification. DNA was extracted from 5 adult worms, and molecular characterization was performed by amplification and sequencing of CO1 and ITS2 regions, followed by phylogenetic analysis. Out of 447 crabs captured, 186 crabs were found to be infected. A total of 4 species of Paragonimus was observed from naturally infected crabs. Paragonimus microrchis (2), Paragonimus heterotremus (1), Paragonimus proliferus (1), and Paragonimus skrjabini (1) were isolated and identified. A total of 32 sequences were downloaded from the National Center for Biotechnology Information, and 5 sequences generated in the study were used for phylogenetic analysis. In the phylogenetic tree of the CO1 gene, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini were clustered with the same species, and the confidence values of their branches were >95%. A congruent phylogenetic relationship was observed with the ITS2 phylogenetic tree. In the phylogenetic tree constructed with the combined dataset of CO1 and ITS2 datasets, Paragonimus proliferus, Paragonimus heterotremus, and Paragonimus skrjabini clustered with the same species, and their branch confidence values were >94%. Paragonimus microrchis clustered with Paragonimus bangkokensis in both datasets. Phylogenetic analysis revealed robustness of the double loci method as against the single-locus method with either CO1 or ITS2 alone. Paragonimus species isolated from the southern Yunnan province, China, was phylogenetically diverse, and the analysis revealed the clustering of multiple species of Paragonimus isolated from different geographic locations. Paragonimus are important parasites causing zoonotic infections in vertebrates, including human beings. They are of socioeconomic importance since the route of infection is mainly food-borne [Paragonimus is relatively complex that requires a minimum of three hosts, including a definitive host and two intermediate hosts [Paragonimus have their own predilection for infecting specific genera of snails and crabs and hence the epidemiological prevalence of different species of Paragonimus is determined by the existence of suitable hosts. Some species of Paragonimus have been identified only from intermediate hosts in certain geographies which suggests infection and maintenance in nonhuman mammals [Trematodes belonging to the genus od-borne . The lifte hosts . The firte hosts . Differe mammals , 4.Paragonimus is rich in species diversity that has been reported from varied geographies, including Asia, Africa, and Central and South America spanning both tropical and temperate climates [Paragonimus and mainly comprises 3 species complexes. Paragonimus westermani is the most commonly reported species complex found in South and South East Asia [Paragonimus heterotremus species has been predominantly isolated and reported from South East Asia while the Paragonimus skrjabini complex has been reported from China and East Asia [Paragonimus ohirai and Paragonimus mexicanus possesses many delicate branches, while that of Paragonimus westermani has 6 simple lobes [The genus climates . The speast Asia . Paragonast Asia , 8. Usuale lobes , 10. Thele lobes . There wle lobes . One of le lobes .DNA sequence data can be used to construct phylogenetic trees that can be used to infer evolutionary trends, including insights into speciation and geographic spread , 14. FurParagonimus have been reported worldwide, of which China has the largest distribution and number of species [Paragonimus species. Moreover, the diagnosis of paragonimiasis is difficult due to nonspecific clinical symptoms [Paragonimus species from nonmammalian hosts will help understand the genetic variation and also suggest suitable lifestyle modifications for populations at risk of acquiring paragonimiasis. Yunnan province has the most endemic species of Paragonimus due to its mountainous plateau topography and unique geographical environment [Paragonimus species isolated from crabs in southern Yunnan province, China.Over 50 species of the  species . Due to  species , 18, Chiironment . Hence, Paragonimus metacercariae were isolated from naturally infected primary freshwater crabs belonging to the genus Indochinamon, the second intermediate hosts, from Tongchang Town, Jinping County, Yunnan Province, China. The habitat included fast-moving streams with no adjacent vegetation. The identification of the secondary hosts was done according to the classification method of “Chinese Medical Crustaceans” [Paragonimus were counted under the microscope and a part of the sediment with the metacercariae were fixed with 70% absolute ethanol and stored in a refrigerator at 4°C with 96% ethanol for molecular biological experiments.The taceans” . The craParagonimus. After 114 days of injection, the SD rats were dissected to isolate the cysts, eggs, and adult worms from the muscles, abdomen, liver, thoracic cavity, and lungs of SD rats. The isolated adult worms were used for genomic DNA extraction and for preparing permanent slides for microscopic confirmation by fixing them onto glass slides with alcohol, formalin, and acetic acid.Freshly isolated, live metacercariae were then injected intraperitoneally (15 metacercariae per rat) into paragonimiasis-negative Sprague Dawley (SD) rats . All the animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . All experimental protocols were approved by the Animal Care and Use Committee of Kunming Medical University (reference: KMMU2015002). Experimental animal inoculation was performed based on the sampling point. After subcutaneous injection, five SD rats were sacrificed on the second, fourth, and sixth week to confirm infection of rats with cercariae of The different characteristic features such as the shape, body size, measurement, and position of the suckers were used for microscopic and morphologic characterization.µL of distilled water. The extracted total genomic DNA was quantified and stored in the refrigerator at−20°C until further use.Genomic DNA was extracted from both the adult worms in SD rats and the metacercariae extracted from crab using QIAamp DNA Mini kit . The whole process was carried out in strict accordance with the manufacturer's instructions. The final elution of DNA was done with 100 µL volume with 2 µL template DNA corresponding to 0.1 ng and 1 µL of primers (10 µmol/µL), 2.5 µL of 10X PCR buffer, 1 µL of 10 mM deoxynucleotide triphosphates (dNTP), 0.1 µL (0.5 units) of Taq enzyme (5 U/ul), and 17.4 µL of PCR grade water. The setup of PCR was done in an ice bath. The PCR amplification was conducted in TaKaRa PCR instrument , and the amplification conditions were as follows: initial denaturation of 95°C for 3 minutes followed by 35 cycles of denaturation at 93°C for 1 minute, annealing at 48°C (for CO1)/60°C (for ITS2) for 1 minute, and extension at 72°C for 1 minute followed by a final extension at 72°C for 5 minutes. The expected length of the PCR fragments was 500–750 base pairs. Detection of PCR amplified products was done by agarose gel electrophoresis with 1.5% agarose gel immersed in 1.0% Tris-Acetate-EDTA buffer stained with ethidium bromide. The purity and quantity were estimated by imaging the gel in a gel documentation system (Bio-Rad company).Polymerase chain reaction (PCR) was performed with primers targeting a fragment of the CO1 gene and the ITS2 region synthesized by Shanghai Bioengineering Co., Ltd. The primers used for amplifying CO1 gene fragments were CO1F-5′GAGGTGTATGTCCTGATTTTGCC-3′ and CO1R-5′GACCTCACCCAATGACCCTGCAACA3′, and the primers for amplifying ITS2 gene fragments were ITS2F-5′GGGTACCGGATCACTCGCTCGGTG3′ and ITS2R-5′GGGGATCCTGGTTGCCTTAGTCTCCGC3′ . PCR wasThe PCR products were then subjected to bidirectional sequencing using the same PCR primers by Shanghai Biotechnology Co., Ltd. (Hitachi fluorescent DNA sequencer SQ-3000). The forward and reverse sequences were then manually curated and aligned with the Dnastar v7.1 software, and the consensus sequence was used for bioinformatic analysis.The initial quality check of the sequences was done by checking the coverage and alignment with previously submitted sequences in National Center for Biotechnology Information (NCBI) using the BLAST tool. Previously submitted sequences of CO1 and ITS2 were retrieved from NCBI and compared with the sequences obtained in this study with ClustalX software with default parameters. Phylogenetic analysis was done as per the Kimura 2-parameter model. The neighbor-joining method (NJ) and maximum parsimony (MP) method were used to construct the phylogenetic tree. The genetic distances of the Kimura-2-parameter model were calculated, considering all substitutions and missing/gaps as unambiguous changes. Test of phylogenetic accuracy was done with bootstrap replicates of 1000. The analysis was performed with MEGA5.0 software. The cut-off value for the consensus tree was set to 75%.A total of 447 crabs captured from the stream in Tongchang Town, Jinping County, Yunnan Province, China, were included for this study. Out of 447 crabs, 186 crabs were found to be infected. A total of 551 metacercariae were isolated and identified from the 186 crabs (2.96 per crab). The size of the metacercariae was found to be 0.419 mm ∗ 0.398 mm. The average thicknesses of the outer wall and inner wall were found to be 0.004 mm and 0.012 mm, respectively, using the microscopic technique. Further, the larvae were surrounded by two larger excretory cysts and intestinal branches.After experimental infection, from each SD rat, 2 to 4 adult worms were retrieved . Adult wParagonimus: Paragonimus proliferus, Paragonimus microrchis, Paragonimus heterotremus, and Paragonimus skrjabini.Morphological identification of the metacercariae isolated from the infected crabs and adult worms from the SD rats led to the identification of 4 species of Paragonimus from southern Yunnan province were used for molecular phylogenetic analyses. The sequences were aligned with those of several related species within genus Paragonimus obtained from GenBank. PCR amplification of the CO1 and ITS2 regions, followed by agarose gel electrophoresis, revealed amplicons of about 500 and 750 bp in length, respectively. BLAST searches using our sequences as queries found matches with 100% coverage in GenBank. Further, only the CO1 sequence of Paragonimus microrchis differed slightly from previously published sequences. All the other sequences obtained in this study revealed 100% identity to previously published sequences.A total of 5 samples  of T was very high, with an average of 43.2%, far exceeding the base composition of ITS2, which is consistent with the abundant base A and base T content of the mitochondrial genome   . Paragonkokensis . A congrgkokensi .Paragonimus species, wherein 45 genera and 311 species have been identified till 2018 [Paragonimus species in southern Yunnan province, China. In the current study, out of the sequenced samples, 2 were Paragonimus microrchis and 1 each of Paragonimus heterotremus, Paragonimus skrjabini, and Paragonimus proliferus.China is known for the endemic diversity of ill 2018 . A previill 2018  and the ill 2018 . This nuill 2018 . Hence, Since the differential identification features of cercariae are not well marked, most often, the species identification is done at the metacercariae stage from the secondary intermediate hosts . In thisParagonimus consisting of Paragonimus siemensis and Paragonimus westermani in cluster 1; Paragonimus skrjabini, Paragonimus miyazakii, Paragonimus proliferus, Paragonimus macrorchis, and Paragonimus heterotremus in cluster 2; and Paragonimus microrchis, Paragonimus bangkokensis, Paragonimus harinasutai, Paragonimus iloktsuensis, and Paragonimus sadoensis in cluster 3. Extensive branching was observed in cluster 2 wherein Paragonimus skrjabini isolated from different regions in China showed distinct branches. Further, Paragonimus heterotremus clustered into a stable branch with Paragonimus heterotremus from South East Asia clustering into a single monophyletic branch group distinct from the Paragonimus heterotremus from India. Similarly, Paragonimus proliferus also clustered as a monophyletic group in most cases (except in NJ trees constructed by ITS2). The relationship between Paragonimus macrorchis and the first two branches is not clear. However, in the MP phylogenetic tree of ITS2 datasets, CO1 and ITS2 combined datasets, Paragonimus heterotremus tends to be clustered into one branch.Phylogenetic analysis with previously published sequences and the sequences generated in this study revealed concordance between the single locus (CO1 or ITS2) and double loci (CO1 and ITS2) phylogenetic analysis. The phylogenetic analysis revealed the presence of 3 distinct clusters of Paragonimus macrorchis and Paragonimus heterotremus were clustered together, which is supported by 91%.By comparing the results of constructing adjacent NJ and MP phylogenetic trees from CO1, ITS2, CO1, and ITS2 datasets, it is obvious that different datasets and tree-building methods have a great influence on the robustness of phylogenetic trees. In phylogenetic trees constructed from single-gene datasets, the number of branches with more than 75% support is less than that constructed from joint datasets, especially self-exhibition, which deserves significant improvement. The phylogenetic tree constructed by the MP method using CO1 and ITS2 datasets has high robustness, and Paragonimus spp. reported in this study, all the species were previously reported from China. A study by Lou et al. utilized similar approaches in identifying the phylogenetic diversity of Paragonimus in China and reported clustering of isolates based on geographic location, which was in accordance with the current study [Paragonimus skrjabini was previously reported from Gansu, Shanxi, Yunnan, Guangxi, Guizhou, Sichuan, and Jiangxi provinces of China. In contrast, P. heterotremus has been reported only from Yunnan and Guangxi provinces [Among the nt study . Paragonrovinces .Paragonimus, where there is no consensus on identification with both morphological and molecular characteristics, phylogenetic analysis helps in determining the phylogeographies [Paragonimus species among individuals consuming freshwater crabs is scarce. A recent study reported that 8 adult humans were infected with P. heterotremus, which were recovered from their lung masses on examination in Xishuangbanna, Yunnan [Phylogenetic analysis has varied values in different organisms. In graphies , 27. The, Yunnan . In suchThe study has certain limitations. The study sites were localized to southern Yunnan province in China; hence, the results may not be extended to other parts of China. Furthermore, we used the maximum parsimony method, which is infrequently used despite the robustness mainly because of the nature of assumptions deployed in the method. Nevertheless, the maximum likelihood method used in other studies also has drawbacks.Paragonimus from southern Yunnan province, China. Phylogenetic analysis using the polymorphisms in two different loci revealed considerable variations in the species of Paragonimus identified in different geographic locations with clustering of Paragonimus based on geographic location.In conclusion, we obtained 4 morphologically different species of


Ailuropoda melanoleuca) are endangered animals and are uniquely inhabitant in China. These rare animals have gradually developed bamboo-eating adaptability through persistent evolution. Intestinal microbes play an important role in the digestion, absorption, metabolism, and development of giant pandas especially by facilizing the degradation of bamboo polysaccharides such as cellulose. Currently, genes directly related to cellulose degradation have not been identified in the genome of giant panda, and cellulose digestion is therefore likely dependent on intestinal microbes. This study analyzed the changes in intestinal microbial structure of giant pandas  in different developmental stages. The impact was also assessed with the changes in food composition probed into the succession regularity of intestinal microbes and the activities of intestinal flora on the digestion and utilization of cellulose in bamboo.Giant pandas (Ailuropoda melanoleuca) is indispensable for the healthy development of giant pandas. In this study, we analysed the diversity of bacteria and fungi in the intestines of six giant pandas (two pandas in each development stage) with a high-throughput sequencing technique to expand the relative variation in abundance of dominant microbes and potential cellulose-degradation genera in the intestines of the giant pandas and to explore the correlation between dominant microbial genera in the intestines and cellulose digestion activities of giant pandas. The results showed that the intestinal bacterial diversity of young giant pandas was higher than that of sub-adult and adult giant pandas, and Shannon’s diversity index was about 2.0. The intestinal bacterial diversity of giant pandas from sub-adult to adult (mature stage) stage showed an increasing trend, but the intestinal fungal diversity showed no considerable regular relations with their ages. The microbial composition and abundance of giant pandas changed in different developmental stages. Pearson correlation analysis and path analysis showed that there was a close relationship between the dominant microbes in the intestines of giant pandas, and the interaction between microbial genera might affect the cellulose digestion ability of giant pandas. Generally, the digestibility of cellulose degraders in pandas was still insufficient, with low enzymic activity and immature microbial structure. Therefore, the utilization and digestion of bamboo cellulose still might not be a main source of energy for pandas.The interaction between intestinal microbial flora and giant pandas ( Intestinal microbes are critical in nutrient absorption ,2,3, metThe adaption to bamboo diet and cellulose digestibility by giant pandas is closely related to the floral structure of intestinal bacteria and fungi and is partially influenced by external environments such as altitude and season ,14. HoweRecently, several studies have been conducted to identify key roles of the dynamic changes of intestinal flora in the healthy development of giant pandas, especially in the different stages of cub development. Guo et al.  found thHowever, there are few reports on the dynamic changes in intestinal flora structure in different stages  of giant pandas and the succession regularity of intestinal microbes in the whole growth and development process of giant pandas. In addition, in the long-term bamboo-eating process, it is still questionable whether the giant panda domesticates intestinal predominant microbes through the high content of cellulose in bamboo and its correlation with cellulose digestion.In this study, a total of six captive giant pandas at different growth stages  with similar living environments were fed with a bamboo diet. The structure and abundance of intestinal bacteria and fungi of giant pandas at different stages of growth were analysed by high-throughput sequencing. Their cellulose digestibility, the change regularity of intestinal floral structure, and its relationship with cellulose digestibility during the growth and development of giant pandas were further studied in order to provide scientific insights into the feeding and protection of giant pandas.In this study, six captive giant pandas  in different stages of development were kept in Shanghai Zoo and Shanghai Wild Animal Park. During the sampling period (from August 2017 to February 2018), the sampled giant pandas were all in good condition during the total of two sampling processes, and there were no abnormalities such as illness or mucus excrement, and no medication was taken during this study. Some of the key characteristics of giant pandas are listed in Fresh stool samples of 6 captive giant pandas were collected and were numbered from S1–S6. We collected the stool in an enclosure to avoid environmental interference every morning for five consecutive days in each sampling month (August 2017 and February 2018). The stool was stored in frozen condition and unfrozen with ice bags before use. For each panda sample, a sterile spoon was used to collect 10 g/d of the pollution-free core of each stool sample, which had been collected and stored in the freezing condition during the aforementioned 5 consecutive days. The cores were fully mixed as one new stool sample of the giant panda and were used to extract the total DNA of microbes for microbial diversity analysis. We also stored every stool sample at 4 °C, and the cellulase activity was assessed.R2 = 0.9935. The production of glucose released by hydrolysing a total of 1 μg faecal sample in 1 h was defined as 1 enzyme activity unit (U), and the determination conditions were modified appropriately by referring to the relevant literature of soil cellulase activity analysis [A total of 5 g of giant panda stool stored in cold condition was taken into a 50 mL Erlenmeyer flask, and 5 mL of 1% carboxymethyl cellulose solution was added. Afterward, 5 mL of acetate buffer solution (pH 5.5), was added to the culture and the mixture was kept at 37 °C for 72 h before measuring the enzymatic activity at 540 nm by DNS colorimetry method. We set up parallel experiments (n = 3) and made no-substrate control group tests (using distilled water to replace the carboxymethyl cellulose solution). The glucose standard curve used was y = 0.6099 × −0.0059, analysis ,22® DNA Isolation Kit  kit and the concentration was determined using NanodropTM 2000 Spectrophotometer  before storage at −20 °C. Bacteria were amplified using 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers in a master mix of 20 μL: 5 × Fastpfu Buffer 4 μL, 2.5 mM dNTPs 2 μL, FastPfu Polymerase 0.4 μL, primers each 0.8 μL, BSA 0.2 μL, template 10 ng, and supplemented with ddH2O. Specifically, fungi were amplified using ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (2043R) (5′-GCTGCGTTCTTCATCGATGC-3′) in a reaction mix of 10 × Buffer 2 μL, 2 μL of 2.5 mM dNTPs, 0.2 μL of rTaq Polymerase, 0.8 μL of each primer, 0.2 μL of BSA, and 10 ng of template, filled with ddH2O. Reaction thermoprofile include 95 °C for 3 min, 95 °C for 30 s, 55 °C for 30 s, 72 °C for 45 s (27 cycles for bacteria and 35 cycles for fungi), 72 °C for 10 min, 10 °C to end the reaction. The amplified products were further purified and separated using 2% agarose gel.Frozen samples were used to extract DNA using PowerSoilhttp://drive5.com/uparse/, accessed on 15 October 2017), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analysed by RDP Classifier  against the silva 16S rRNA database and UNITE ITS database using confidence threshold of 70% [Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform was performed according to the standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database. Raw fastq files were demultiplexed and quality-filtered using QIIME (version 1.17) with the following criteria: (i) The 300 bp reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window, discarding the truncated reads that were shorter than 50 bp. (ii) Exact barcode matching, 2 nucleotides mismatch in primer matching, reads containing ambiguous characters were removed. (iii) Only sequences that overlapped longer than 10 bp were assembled according to their overlap sequence. Reads that could not be assembled were discarded. Operational Units (OTUs) were clustered with 97% similarity cut-off using UPARSE  analysed by Pearson correlation and direct path coefficient (Piy) were obtained by regression analysis processed by SPASS 22.0. The indirect path coefficient was calculated with the product of riy and Piy.The microbial α-diversity in the representative data was calculated with OTU abundance, the dominant microbial taxa were calculated at the genus level, and the cellulase data were analysed by OriginPro 8.0. All data were expressed by the mean value and standard error (n = 3), with significant difference expressed as The library coverage was more than 99.8%, and the coverage was substantial for the microbial diversity . AccordiAccording to Shannon’s diversity and Simpson’s diversity indices, the intestinal bacterial diversity of giant panda cubs was higher than that of sub-adults and adults, and the average value of cub samples was approximately 2.0. After the shift from milk feeding to bamboo feeding, Shannon’s diversity index of intestinal bacteria in giant pandas of about 1 year old was as high as 2.25 (on average). On the other hand, Shannon’s diversity index of intestinal bacteria in giant pandas, which developed at 1 and a half years old was similar to that of sub-adults and adults. Interestingly, the diversity and heterogeneity of intestinal bacteria between sub-adult and adult giant pandas were similar.The analysis of fungal diversity in stools of giant pandas at different developmental stages are outlined in Therefore, changes in the diversity of gut microbes were linked to the growth of giant panda, especially the bacteria. During the stage of cub and the prime of life, in which the metabolizing conditions were stronger, the diversity of gut bacteria was higher than that of other developmental stages. The fluctuation in diversity of fungi was the opposite to that of bacteria in pandas, which suggests that there might be a strict relationship between bacteria and fungi in the gut within a limited space. The dominant bacterial genera gradually occupied a large proportion, whereas the fungal genera were evenly distributed in pandas as their age increased. This indicates that specific microbial structure might be shaped with the growth of individuals and intestinal bacteria, and these more likely interact with their hosts.Streptococcus (accounting for 35.2–38.1%), Escherichia–Shigella (22.3–26.1%), Clostridium_sensu_stricto_1 (11.5–12.3%), Lactobacillus (11.3–12.2%), and Turicibacter (4–5%). The most dominant bacterial genera in the intestine during the sub-adult period were Escherichia–Shigella (37.2–41.4%), Clostridium_sensu_stricto_1 (25.7–30.8%), Streptococcus (21.5–22.7%), Terrisporobacter (4–5%), and Serratia (0.5~1%). On the other hand, the dominant bacterial genera in the intestine during adulthood were Streptococcus (33.3–62.3%), Escherichia–Shigella (31~36.4%), Clostridium_sensu_stricto_1 (3.5~22.6%), Turicibacter (0–1%), and Klebsiella (0–0.7%) (The predominant genera of bacteria in the intestine of giant panda cubs during the panda period were (0–0.7%) .Others contain some microbes whose relative abundance is less than 0.05 in samples. Both a and b represent sampling time.Trichosporon (1.2–16.1%), Trimmatostroma (6.2–12.6%), Candida (1–8.5%), and Unclassified_f_norank_o_Pleosporales (6.8–9%). The sub-adult giant panda intestinal dominant fungal genus was Unclassified_f_Montagnulaceae , Unclassified_p_Ascomycota (15.1–20.4%), Trimmatostroma (15.1–19.1%), Unclassified_f_norank_o_Pleosporales (6.5–6.6%), and Unclassified_f_Apios (3.5–5.2%). The dominant genera of intestinal fungi in adult giant pandas were Unclassified_o_Pleosporales (3.8–26.4%), Leptoxyphium (2.5–24.6%), Shiraia (2.8–19%), Unclassified_f_norank_o_Pleosporales (3.2–13.5%), and Unclassified_f_Montagnulaceae (4.8–10.5%) , 8–10.5%) .It could be seen that the dominant genus of bacteria in the intestine of giant pandas was predominant, but the proportion of the dominant genus of fungi generally did not exceed 25%, which was consistent with the results of diversity. The dominant genera of intestinal bacteria of giant pandas in different developmental stages were mainly Streptococcus, Escherichia–Shigella and Clostridium_sensu_stricto_1, and there were differences in the specific percentages of different developmental stages .A specific trend was obviously in the order of Streptococcus > Escherichia-Shigella > Clostridium_sensu_stricto_1 in the intestinal tract of young cubs. The order in the sub-adult intestinal tract was Escherichia-Shigella > Clostridium_sensu_stricto_1 > Streptococcus whereas in the adult intestine, it was Streptococcus > Escherichia-Shigella > Clostridium_sensu_stricto_1. In addition, the intestinal dominant bacterial structure was similar between different individuals at the same developmental stage. It was evident that the dominant bacterial structure in the panda’s intestine may be the result of long-term evolution of the giant panda.Lactobacillus was also dominant in the intestines, accounting for 35.6%. This was primarily attributed to the giant panda cubs, which were fed with breast milk or other dairy products as their main food source. In addition, some small amounts of genera were also distributed by staged characteristics; for example, Actinobacillus, Romboutsia and Sphingobacterium prefer to colonize in cubs such as Enterobacter, and Klebsiella are dominant in both cubs and sub-adults. The Serratia, Pantoea and Raoultella were only found in sub-adults such as Weissella and Vagococcus were only found in adults.For the giant panda cubs, Streptococcus showed a trend of decreasing at first and then increasing during the development of giant pandas. The amount of Streptococcus was rich in the sub-adult-to-adult development stage; however, it was strange that the abundance of Streptococcus in the cub to sub-adult stage decreased at first. It was accompanied by the increase in abundance of other bacteria such as Clostridium_sensu_stricto_1, Escherichia-Shigella, etc. Whether the interaction between bacterial genera or other external factors had the potential to affect the status of these predominant genera requires further study. The abundance of Clostridium_sensu_stricto_1 changed with the age of giant pandas with the order of sub-adults > adults > cubs. The population quantity of Escherichia-Shigella was stable during the development of giant pandas, especially after the sub-adult stage.The structure of intestinal dominant fungi showed no considerable changes with age. The proportion of dominant fungi in different development stages and different individuals varies greatly, and the fungal populations were rich and evenly distributed with low predominance, consistent with the results of α diversity of fungal community in pandas.PCA analysis indicated that the flora structure of bacteria and fungi in the intestines of giant pandas in each development stages were similar . InteresAs shown previously, the structure of bacterial community in pandas was gradually stabilized with growth. The status of the dominant genera was also determined, and changes in their abundance had staged characteristics, whereas the fungal community in pandas was very likely influenced by environmental factors, because the twin cubs or twin sub-adults that held the same living and raising conditions, had more similar fungal components.During the growth of giant pandas, the intestinal cellulase activity changes obviously in different periods ; the celCombining the above-mentioned results of intestinal dominant bacterial flora structure, it was found that the abundance of the dominant bacteria genera also changed greatly during the growth from cub to sub-adult, and the abundance remained stable or increased during the development from sub-adult to adult (such as S5). In order to further understand the key role of dominant microbes in digesting cellulose and the corresponding interaction effect of intestinal microbes in giant pandas, the cellulase activity of giant pandas was correlated with the relative abundance of several intestinal dominant microbes, and then the following results were obtained by correlation analysis and path analysis .Escherichia-Shigella, Streptococcus and Clostridium_sensu_stricto_1 and Unclassified_f_Apiosporaceae. In addition, there was a close relationship between the panda intestinal microbes, including bacteria and bacteria, fungi and fungi, and bacteria and fungi. A negative correlation between Streptococcus and Escherichia-Shigella with a correlation coefficient of −0.859 (p < 0.05) was observed. The Unclassified_o_Pleosporales was negatively correlated with Unclassified_f_Montagnulaceae; however, it was positively correlated with Leptoxyphium (p < 0.05). Streptococcus was significantly negatively correlated with Unclassified_f_Apiosporaceae (p < 0.05).According to the results of Pearson correlation analysis, it was noticed that the dominant intestinal bacteria and fungi that were closely related to the panda cellulase activity include Escherichia-Shigella (x1), Streptococcus (x2), Unclassified_f_Apiosporaceae (x3), and Clostridium_sensu_stricto_1 (x4) was conducted  and independent variables onducted . The intbacillus  and Sphiacterium  were in Aspergillus, Penicillium and Trichoderma, were also found in pandas [Therefore, during the long-term domestication of bamboo-eating pandas, the structure of functional microbial communtiy became complex and remained immature. Although the dominant bacterial genera and their potentially active interaction were positively correlated to cellulose metabolism, some cellulose-metabolising degraders were represented poorly in pandas. Furthermore, some fungal genera that are the main degraders of complex cellulose in nature, such as n pandas ,27 partiIn order to reasonably explore the succession regularity of intestinal microbes during their growth and development, different development stages in the long-life cycle of pandas  were investigated under similar growth environments. Twins were selected with similar physiological conditions and in the same development stage to maintain homogeneity. We found diversity and structurally characterized abundance of intestinal microbes in giant pandas at different developmental stages. Compared with other development stages, the intestinal bacterial flora structure of giant panda cubs was more complex, which may be related to the complex dietary shift process and active metabolism of giant panda cubs . TherefoHowever, the complexity of intestinal bacterial structure of older adult giant pandas decreased . TherefoShiraia mainly exists on bamboo, gradually and stably colonizing the gut of pandas with bamboo diets. Species Aspergillus, Penicillium and Trichoderma species can degrade lignocellulose in plants and have been found in the gut microbiome of our samples by sequencing [Erythrobasidium and Trimmatostroma are abundant in the faeces and expand with the development of pandas, and most of these fungi in the intestines are pathogenic of plant origin [During the development of giant pandas, the change in bacterial flora abundance was contrary to that of fungi. Therefore, it was speculated that there is a certain competitive relationship between bacteria and fungi in the limited space of the micro-ecosystem in the intestines of giant pandas to keep the system steady ,32. The quencing . In addit origin ,36, but t origin ,38. Thert origin . AccordiStreptococcus, Escherichia-Shigella and Clostridium_sensu_stricto_1, which is consistent with the research of Zhang et al. [Streptococcus, as was observed by Wang et al. [Streptococcus increasing from 14.6% to 47.6%, especially after the cubs change feeding habits into a high-cellulose diet. Clostridium increased gradually during the giant pandas’ specialization of eating bamboo and especially occupied a dominant position in the intestine of sub adults’, who had just started to consume low-energy food [Escherichia–Shigella always maintained a high proportion of the intestinal bacterial community, and it was indeed correlated with many essential energy metabolism pathways in panda [The dominant bacteria genera in the intestines of giant pandas at different developmental stages were mainly g et al. . From thg et al. ,40. Howeg et al. , can degrgy food . The Escin panda . There win panda ,43. Whilin panda .On the whole, during the growth and development of giant pandas, the giant pandas in the development stage from sub-adult to adult carried the most stable intestinal flora structure, with no obvious difference between genders. In the actual process of feeding and managing giant pandas, human activities such as changing captivity, which may slightly affect the physiological health of giant pandas, should be scheduled at this stage as far as possible. However, the complexity of intestinal microbial structure of giant panda cubs was very high, even higher than that of adult pandas, which may be due to many influencing factors of intestinal flora of cubs, such as physiological environment in their maternal body, the external environments after birth and dietary shift process, which can change the structure of intestinal flora of cubs and even extend to the whole life . The vulFor a long time, giant panda cubs have adapted to bamboo in the stage of dietary shift . In contStreptococcus processed a similar fluctuation to it. However, both the abundance of Streptococcus and the cellulase activity decreased in cubs when they began to consume a bamboo diet, which suggests that Streptococcus was likely an important genus to metabolize cellulose in supplementary food but not in bamboo in cubs. Although Clostridium also kept a certain amount and were reported to have the ability to assist cellulose degradation [Pseudomonas, Paenibacillus and Sphingobacterium among bacteria and Aspergillus, Penicillium and Trichoderma among fungi were represented with low abundance in pandas. In general, according to the cellulase activity in pandas, the digestibility of gut microbes to bamboo cellulose was still poor, though the adults increased their enzymic activity with bamboo diet.Considering changes in endocellulase activity in pandas, the dominant radation ,43, unforadation . In factStreptococcus increased, and it was shown that a positive correlation with the cellulase activity in adult pandas with bamboo diet depended on the interaction of other microflora, although it also preferred to use oligosaccharides such as Escheriachia-Shigella [Studies have found that in the process of adapting to bamboo eating, giant pandas not only accumulated dominant flora which had potential for cellulose degradation but also maximized the use of nutrients such as protein, amino acid and fatty acid in food through other flora or evolved metabolism ,48. It wShigella  and dietStreptococcus, Escherichia-Shigella and Clostridium_sensu_stricto_1. The structure of intestinal dominant fungi carried no obvious regularity with age. During the growth of giant pandas, the activity of intestinal cellulase was obviously different in different periods with the order of cubs > adults > sub-adults. The sub-adult stage was the turning point, and its intestinal microbial structure may determine its ability to digest and absorb bamboo cellulose, protein and other components in the later growth and development stage when they have vigorous metabolism. However. bamboo cellulose is still not a useful substrate for the dominant intestinal microbes of giant panda to obtain energy and nutrition after eating a bamboo diet.Taken together, the intestinal microbial structure of giant pandas varied at different developmental stages. The diversity of intestinal bacteria was high in the cub stage, and the structure of bacterial flora tends to be stable after the consumption bamboo diet during adaptation to eating bamboo. In the adult development stage, the complexity of the intestinal bacterial structure of giant pandas may be at its highest point, which was also the most vigorous period of giant pandas. The flora abundance of fungi showed an opposite trend to that of bacteria. In the limited space of the micro-ecosystem in the intestines of giant pandas, there was a certain strict relationship between bacteria and fungi, which maintained the system steady. The dominant bacterial genera in the intestines of giant pandas showed obvious dominance and at different developmental stages were mainly


Better protection can be provided during neurosurgery due to the establishment of somatosensory-evoked potential (SEP) and motor-evoked potential (MEP) monitoring technologies. However, some studies have showed that inhaled halogenated anesthetics have a significant impact on neurophysiological monitoring.A total of 40 consecutive patients undergoing neurosurgery were randomly assigned to two groups receiving inhaled anesthetics, either desflurane or sevoflurane. Multiples levels  of anesthetics were administered at minimum alveolar concentration (MAC), and then the latencies and amplitudes of SEPs and MEPs were recorded.P < 0.05). The SEP latencies of both the upper and lower limbs in the desflurane group were significantly longer, and the SEP amplitudes were significantly lower than those in the sevoflurane group (P < 0.05). The MEP amplitudes in the desflurane group were significantly lower than those in the sevoflurane group (P < 0.05), only the amplitudes of the upper limbs at 0.3 MAC did not vary significantly.SEP and MEP signals were well preserved in patients who underwent neurosurgery under general anesthesia supplemented with desflurane or sevoflurane at concentrations of 0.3, 0.6 and 0.9 MAC. In each desflurane or sevoflurane group, the amplitudes of SEPs and MEPs decreased and the latencies of SEPs were prolonged significantly as the MAC increased , Clinical Trials identifier ChiCTR2100045504 (18/04/2021).The study registered on the Chinese Clinical Trial Registry ( Neurophysiological monitoring has a history of over 30 years and has been widely used in surgery for spinal cord, skull base, functional area tumors and epilepsy. Clinical methods for monitoring the nervous system include the wake-up test, somatosensory-evoked potentials (SEPs) and motor-evoked potentials (MEPs). The wake-up test is recommended as the golden standard, and can directly reflect the function of the nervous system during surgery. However, this test usually takes considerable time during surgery and frequently leads to psychological disorders in patients. Additionally, it cannot be used for continuous intraoperative monitoring and cannot detect suspicious nerve damage in time. Wake-up tests cannot be performed in patients with neuromuscular disease, weak muscle strength, and uncooperative behavior, nor in children and patients with mental disorders. SEP monitoring is currently a very popular method in neurosurgery, that can reflect the full function of the somatosensory conduction pathway. Decreased amplitudes or prolonged latencies of SEPs suggest some damage to brain action . MEP monA previous study revealed that intraoperative anesthetics have some significant effects on SEPs and MEPs. For instance, muscle relaxants have the greatest effect, followed by inhaled anesthetics and intravenous anesthetics , 4. TotaThis study protocol was approved by the Institutional Ethics Committee of the Second Affiliated Hospital of Kunming Medical University . All methods were carried out in accordance with relevant guidelines and regulations and with CONSORT recommendations. The study was registered in the Chinese Clinical Trial Registry . Forty consecutive patients were enrolled in the study, and written informed consent was obtained from each patient. The inclusion criteria were adult patients  scheduled to undergo general anesthesia for elective neurosurgery expected to last at least 3 h. All patients were neurologically normal at clinical examination, as determined preoperatively by the neurosurgeon. Exclusion criteria were patients less than 18 years old or more than 60 years old, an ASA grade greater than II, surgical time less than 3 h, preoperative neurological deficits or neuromuscular transmission dysfunction, cortical stimulation contraindications , a preoperative left ventricular ejection fraction less than 30%, significant aortic regurgitation or cardiac arrhythmias, severe obstructive or restrictive ventilatory dysfunction, preoperative infection, emergency surgery, current pregnancy or lactation period, known allergy to halogenated anesthetics, and participation in another trial. Additionally, any patient who required unexpected emergency resuscitation during the operation, such as a patient who experienced massive bleeding (> 3000 ml), severe anaphylaxis, and cardiac arrest, was also excluded. Finally, any patient for whom monitoring  could not be successfully performed due to the nature of the surgical site was also excluded.The randomization of the study (1:1) was performed by the narcotic drug administrator in the Department of Anesthesia, using internet-based randomization software. The morning of the surgery, blinded halogenated anesthetics  were delivered to the anesthesiologist in charge of the patient. Study anesthetics were only identified by the assigned patient number. Intraoperative electrophysiological data were analyzed and recorded by one neuroelectrophysiological specialist. Importantly, the neuroelectrophysiological specialist remained blinded to the anesthetic allocation and the inspiratory concentration when collecting data.All included patients were allowed solid foods up to 6 h before surgery and fluids up to 2 h before surgery. Patients received 1 to 2 mg midazolam  intravenously after entering the operating room. Standard monitoring for this surgery included a one-lead electrocardiogram, pulse oximetry, noninvasive blood pressure, urine output, oropharyngeal temperature, invasive radial arterial pressure, central venous pressure, inspiratory and expiratory gas concentrations, and continuous electroencephalography  to monitor the depth of anesthesia. For the induction of anesthesia, intravenous 0.3 μg/kg sufentanil , 2 mg/kg propofol , 0.7 mg/kg rocuronium  were administered. After tracheal intubation, a bite block was used to protect the teeth and tongue. Mechanical ventilation with oxygen/air was performed, and Fio2 and respiratory parameters were adjusted to maintain SpO2 in 95-100% and PETC02 in 30-35 mmHg. Body temperature was kept between 36 and 37 degrees Celsius by thermal means. General anesthesia was maintained by intravenous pumping of propofol (75 to 125 μg/kg/min), and remifentanil (0.1 to 0.2 μg/kg/min) and volatile anesthetic either sevoflurane  or desflurane  depending on anesthetic allocation. However, no further doses of neuromuscular blocking agents were subsequently used. Propofol and remifentanil concentrations were adjusted intraoperatively to target bispectral index (BIS) values between 40 and 60. All anesthetics were administered according to the patient’s actual weight. During the operation, blood pressure was maintained between 80 and 120% of the preoperative baseline level, and the heart rate was more than 50 times per min. Any incidence of low heart rate and low blood pressure was treated as appropriate with cardiovascular active drugs such as noradrenaline, ephedrine and atropine.All neurophysiological monitoring measurements were performed and recorded by the same trained neurophysiologist using the same equipment each time and without knowing which anesthetic method was chosen. Multipulse transcranial electric stimulation was achieved using the Cadwell Cascade TCS-1 evoked potential machine . The machine delivered a constant voltage to evoke a compound muscle action potential, which was performed after craniotomy and incision of the meninges. The electrodes were placed in accordance with international EEG standards  .SEPs were elicited by bilateral stimulation of the median nerves at the wrist in the upper limbs and the posterior tibial nerves at the ankle in the lower limbs using subdermal needle electrodes. Continuous single pulse electrical stimulation with a stimulation intensity of 10-35 mA was presented at a rate of 2-10 Hz. SEPs were recorded through needle electrodes placed over the cortex at the C3’ and C4’ areas, defining two channels: the Cz active electrode, in the midline of the scalp 2 cm behind the earlobe, and the midfrontal (Fpz) reference, approximately 10 cm before the sagittal line of two external auditory meatuses . The eleNeurogenic MEPs were obtained by stimulating the motor cortex through sterile platinum needle electrodes inserted by the neurosurgeons. Stimulating electrodes were placed 2 cm in front of the SEP cortical electrodes at the Cz area along the motor cortex . The groAfter the anesthesia depth was stable, a train-of-four twitch assessment was performed using a stimulator at the ulnar nerve. The variation in myokymia induced by the abductor pollicis muscle was measured by a TOF-watch SX accelerometer with a pulse width of 200 s, frequency of 2 Hz, and current of 60 mA. Muscle relaxation recovered completely only when the TOF value (T4/T1) was greater than 90%. The first set of SEP and MEP recordings was obtained approximately 60 min after anesthesia induction. Then, the concentration of inhaled anesthetics was adjusted so that the end-expiratory concentration would be 0.3, 0.6 and 0.9 MAC successively. Higher concentrations of volatile anesthetic agents were not tested to avoid excessive decreases in blood pressure. Each concentration was maintained for at least 10 min, which was regarded as a steady state. After stopping the surgical operation, the latencies and amplitudes of all SEPs and MEPs at each concentration were recorded. SEP and MEP data were collected only during noncritical stages of the surgery and when no extrinsic disturbances, such as diathermy, were present. The measurements were analyzed by the neurophysiological technologist without knowledge of the inhalational agent or its respective MAC. In the end, we took the average of at least two values in each record.To obtain better statistics, we included 20 patients in each group, and the amplitudes and latencies of all SEPs and MEPs were recorded and analyzed. The normality of the data distribution was tested using the Kolmogorov-Smirnov test. SPSS statistics 21.0 software was used for statistical analysis. The data are presented as the mean and SD for amplitude and latencies of SEP and MEP signals. The paired sample T-test method was used for intragroup data comparison, while the independent sample T-test method was used for intergroup comparison. A probability value less than 0.05 was considered significant.P > 0.05), at the introduction of each agent in either group. The mean infusion rate of propofol and remifentanil before the administration of each inhaled anesthetic being studied was kept standardized. Intraoperative combined monitoring of evoked potentials was successfully completed in all patients, without related complications  caused by monitoring. Additionally, no neurological deficits occurred after the operation and all patients had normal postoperative neurological examinations results. The physiological data are listed in Table The 40 neurosurgical patients were monitored with intraoperative somatosensory evoked potentials and motor evoked potentials. There were no statistically significant differences in the preoperative baseline biological parameters, such as sex, age, weight, blood pressure, heart rate, PaO2, PETCO2, temperature and disease type ; only the SEPs of the upper limbs at 0.3 MAC in the sevoflurane group did not vary significantly (Table P < 0.05)  (Table P < 0.05); the amplitudes in both upper and lower limbs in the desflurane group were significantly lower than those in the sevoflurane group at 0.6 MAC (P < 0.05); and the latencies of both upper and lower limbs in the desflurane group were significantly longer and their amplitudes were significantly lower than those in the sevoflurane group at 0.9 MAC (P < 0.05)  Table . When mo5) Table .P < 0.05). The inhibitory effect on MEP amplitudes in the upper limbs did not vary significantly, but only at 0.3 MAC, between the sevoflurane and desflurane groups , desflurane and sevoflurane seem to have no significant suppressive effect on MEP latencies in either the upper or lower limbs. Similar results were demonstrated by Chin Ted Chong et al. , who shoHowever, there are some limitations in our research due to the constraints of experimental conditions. For example, Ushirozako H et al.  demonstrThis study is the first to comprehensively compare the effects of two inhaled anesthetics, sevoflurane and desflurane, on somatosensory evoked potentials and motor evoked potentials. The results demonstrated that SEP and MEP monitoring could be carried out successfully under the usual doses of sevoflurane and desflurane for general anesthesia. However, to obtain more accurate monitoring results and to avoid interfering with the judgment of the operator, the concentration of inhaled anesthetics was recommended to be below 0.6 MAC. Furthermore, the inhibitory effect of desflurane was significantly stronger than that of sevoflurane at the same concentration. When monitoring SEPs or MEPs during operation, it is recommended that sevoflurane be given priority. Finally, monitoring both the upper and lower limbs simultaneously is recommended to avoid confounding factors.