Apis mellifera differs from all other social bees in its gonad phenotype and mating strategy. Honey bee queens and drones have tremendously enlarged gonads, and virgin queens mate with several males. In contrast, in all the other bees, the male and female gonads are small, and the females mate with only one or very few males, thus, suggesting an evolutionary and developmental link between gonad phenotype and mating strategy. RNA-seq comparisons of A. mellifera larval gonads revealed 870 genes as differentially expressed in queens versus workers and drones. Based on Gene Ontology enrichment we selected 45 genes for comparing the expression levels of their orthologs in the larval gonads of the bumble bee Bombus terrestris and the stingless bee, Melipona quadrifasciata, which revealed 24 genes as differentially represented. An evolutionary analysis of their orthologs in 13 solitary and social bee genomes revealed four genes with evidence of positive selection. Two of these encode cytochrome P450 proteins, and their gene trees indicated a lineage-specific evolution in the genus Apis, indicating that cytochrome P450 genes may be involved in the evolutionary association of polyandry and the exaggerated gonad phenotype in social bees.The honey bee,  This highly eusocial lifestyle, characterized by large colonies composed of at least one highly reproductive queen and hundreds of subfertile or sterile workers, represents an irreversible major evolutionarily transition5, as the morphologically distinct queen and worker castes have become mutually completely dependent on one another. The development of such physical castes not only strengthened the queen’s reproductive monopoly, but also promoted division of labor and task specialization in the worker caste, which enabled the highly eusocial Hymenoptera to establish very large colony sizes.The establishment of a highly eusocial form of life occurred independently in two closely related lineages of bees, the honey bees and the stingless beesVespa and Vespula) and in the honey bees (genus Apis). While the females of the facultatively social or primitively eusocial bees and wasps generally mate with only a single male (monandry), the queens of several highly eusocial species are known to mate with more than two males (polyandry), which, in cases of extreme polyandry, can be 20 or more6. Polyandry has fitness consequences for the workers, as it diminishes the within-colony genetic relatedness, and thus, affects the structure of the genetic conflict over male production and sex ratio allocation8. As monandry is the ancestral condition9, the multiple mating systems and their associated male traits have drawn attention and generated controversial arguments about the adaptive significance of multiple mating in the highly eusocial Hymenoptera10. Among the eight hypotheses proposed for the evolution of polyandry in social insects11, the currently most accepted one is that it strengthens the colonies’ resistance against parasite or pathogen infections12. Polyandry, however, is not a simple categorical trait that stands in contrast with monandry, but there is a very strong skew in the number of recorded mating events13. Specifically, in only 0.2% of the known eusocial hymenopteran species the queen mates with more than three males, and mating frequencies with over 20 males are extremely rare6.This transition in the level of social organization was accompanied by a change in the queens’ mating strategy that occurred independently in several lineages of the ants, in two lineages of wasps 15. Hence, while on the one hand, A. mellifera is the best-established model organism for functional and structural aspects of the complex social organization in insects, the entire genus Apis strongly differs from all the other social bees in terms of its gonad morphology and mating behavior.The Western honey bee, 17, while the ovaries of an adult worker consist of only 2–12 ovarioles each19. In contrast, in all the other bees, the females have only 3–4 ovarioles per ovary21, including the primitive eusocial bumble bees (Bombini) and the highly eusocial stingless bees (Meliponini). Together with the solitary to facultatively eusocial orchid bees (Euglossini) and the honey bees (Apini), these four tribes form a monophyletic clade, the corbiculate bees22, and thus, the question is: why and how did the ovarian architecture of the honey bees become so different from all the other bees? Or put in other terms, what may have been the evolutionary conditions or drivers that favored the origin of the exaggerated ovary phenotype in the genus Apis?An adult honey bee queen has very large ovaries, each composed of 100–150 serial units (ovarioles), and referred to as a transgressive (exaggerated) ovary phenotypeBombus terrestris, and the stingless bee, Melipona quadrifasciata, for a comparativ transcriptomics approach against our prior RNA-seq data generated for the honey bee queen, worker, and drone larval gonads23.To address the this question we decided to take an EvoDevo approach where we contrasted the larval gonad transcriptomes for three bee species that differ in gonad architecture and reproductive strategies within a well defined phylogenetic background. For this, we generated RNA-seq libraries of larval gonads of the bumbe bee, B. terrestris (n = 3) and the stingless bee M. quadrifasciata (n = 3) were similar to those simultaneously obtained for the honey bee larval gonads 23, consisting of between 29,304 and 41,890 fragments per million, where more than 83% of these good quality reads could be mapped to the respective reference genomes of the three species. The alignment scores were high for A. mellifera, varying between 83 and 87.4%, and also for B. terrestris (79% to 86%), but were lower for M. quadrifasciata (60.8% to 63%). Hence, while the 15 RNA-seq libraries generated for this study had similar numbers in terms of good quality reads, they differed in their mapping rates. This difference is likely due to the better annotation quality of the honey bee24 and bumble bee genomes25, compared to that of the M. quadrifasciata genome26.In terms of read numbers, the RNA-seq results for the larval gonads of the bumble bee A. mellifera reads , and here it is important to note that we used a less stringent cutoff level for differentially expressed genes (DEGs) than in our prior study on these honey bee larval gonad transcriptomes, where we had set the cutoff at Padj < 0.0123. The reason for adopting the less stringent DEG level in the current study was that for the subsequent hierarchical clustering against the bumble bee and stingless bee transcriptomes and the evolutionary analysis we needed a sufficiently large gene set of clear orthologs. This less stringent analysis revealed 5,546 genes as differentially expressed among the three types of honey bee larval gonads, including a central set of 870 genes that was common to all three contrasts  associated with the conservation of a high ovariole number in the queen caste, in contrast with the reduced ovariole number in the workers due to the massive programmed cell death in their larval gonads23, and (b) differentially expressed also between the two sexes, independent of caste in the female sex. A Gene Ontology (GO) analysis for these 870 DEGs returned 566 genes with GO information 27. In both cases we performed reciprocal BLAST searches against the honey bee genes. The RPKM counts (reads per kilobase per million mapped) for these orthologs were then retrieved from the respective RNA-seq data. We opted to use the RPKM normalization method because it eliminates the influence of gene length and variation in sequencing counts for comparisons between RNA-seq libraries28.For the bumble bee we obtained the orthologs of this gene set from the B. terrestris and M. quadrifasciata genomes, this was only possible for the comparison with the bumble bee, because many of the 870 honey bee DEGs showed unclear orthology relationships in the stingless bee genome. For B. terrestris, we obtained a total of 806 orthologs for the 870 A. mellifera DEGs, and the hierarchical clustering analysis performed in JMP Genomics software showed that the two species are clearly separated . Hence, we decided to perform the joint analysis for the three bee species with a reduced set of 45 DEGs that represent enriched GO categories  performed on the RPKM counts of these 45 genes in the 15 RNA-seq libraries produced a similar result Fig. . The two29. This test assumes that evolutionary patterns can change quickly and lead to independent evolutionary rates between branches. The analysis was done with the four fully sequenced Apis species serving as test branch against the other nine bee species from the families Apidae, Megachilidae, and Halictidae as background.We next retrieved the nucleotide and amino acid sequences of the respective orthologs for the 24 divergently represented genes shown in Fig. p value < 0.05. These were as follows: CYP9Q3, CYP6AQ1, PHGDH, and ACADM, encoding, respectively, Cytochrome P450 9e2, Cytochrome P450 6AQ1 × 1, D-3 phosphoglycerate dehydrogenase, and the mitochondrial medium-chain specific acyl-CoA dehydrogenase. Among these genes, CYP9Q3 (GB43728) and CYP6AQ1 (GB52023) showed lower RPKM counts in the larval gonads of A. mellifera and M. quadrifasciata compared to B. terrestris  showed low RPKM counts for A. mellifera queens and workers and higher counts in honey bee drones, as well as in the bumble bee and the stingless bee, but the opposite was the case for ACADM (GB42141), which showed higher counts in all honey bee samples and also in the stingless bee, contrasting with lower count numbers for the bumble bee.In this set of 24 divergently represented genes, four showed evidence of positive selection Table , denoted30, applying the models and rate substitution parameters listed in Table CYP9Q3, which had the highest index for positive selection, showed only a minor divergence with respect to the Apoidea phylogenetic tree31, and this divergence is related to the positioning of A. florea  to the A. mellifera CYP6AQ1 amino acid sequence. For A. mellifera CYP9Q3, the most similar D. melanogaster CYPs were CYP9F2 and CYP6A13 . These Drosophila CYPs, especially CYP6G2 and CYP6G1, are involved in interesting biological functions, such as juvenile hormone biosynthesis in the case of CYP6G2, and for CYP6G1, in DDT and imidacloprid resistance35.This divergent result for the ent Fig. , potentiApis mellifera, but in all species of the genus Apis36, and the high number of ovarioles and testioles in the gonads of both queens and drones may be evolutionarily and developmentally coupled. As this stands in contrast to all other social bees, including the highly eusocial stingless bees, which essentially have a monandrous mating system37, we proposed to test this by a comparative transcriptome analysis of the larval gonads of A. mellifera queens, workers, and drones23, against those of B. terrestris and M. quadrifasciata larvae as the ancestral state.Our study was driven by the hypothesis that the extremely polyandrous mating system in honey bees, not only in the Western honey bee, A. mellifera genes that represented DEGs for all the three gonad contrasts  biosynthesis35. These functional annotations in Drosophila are of interest with respect to honey bee biology. On the one hand, JH is a major driver in honey bee caste development and also for the behavioral maturation of adult workers49. On the other hand, imidacloprid is a neonicotinoid that is strongly associated with colony losses50, including sublethal effects on the learning and memory of honey bee workers, which affect the homing of foragers as they return from nectar and pollen resources. Furthermore, JH has recently been shown to play a critical role in the brain-reproduction energetics tradeoff that distinguishes the honey bees from the bumble bees51.Our BLASTP searches against the 52. Together with our results that indicate positive selection acting on the CYP6AQ1 gene in the Apis lineage, selection on JH signaling-related genes in the evolution of sociality has recently also been demonstrated in a comprehensive comparative genomics study in halictid bees53. These authors looked for evidence of positive selection in genes associated with lineages that have independently made the transition from a solitary to a eusocial lifestyle, versus relaxed selection in those lineages where the females reverted from eusociality to secondarily become solitary nesting. Under this premise, four genes showed such contrasting evidence. Two of these, apolipoprotein and hexamerin 110, are abundant insect hemolymph proteins related to JH binding and transport.JH is a fundamental hormone in the insects’ life cycles, orchestrating molting, metamorphosis, and reproduction, and it is a key driver for the adaptive expression of characters, including the castes of social insects53 could identify evidence of positive (diversifying) or relaxed selection in genes or gene families that are related to important transitions in the evolution of insect sociality shows the strength of comparative genomics or transcriptomics when performed under a well-defined phylogenetic background. Though not all these genes may be directly associated with important functions, such as JH signaling, they can heuristically guide future research efforts on how natural selection may have shaped developmental and behavioral plasticity54, which is key to the queen-worker polyphenism in social insects.In conclusion, the fact that we and othersM. quadrifasciata) were obtained during the Southern summer season (January/February) from brood combs of three hives kept in the Experimental Apiary of the University of São Paulo in Ribeirão Preto, Brazil. The larval stage equivalent to the fifth-instar early feeding stage (L5F1) of honey bees used in our prior study23 is the early third instar (L3.1) of Melipona bees55. Bumble bee (B. terrestris) larvae were obtained during the Northern summer season (June/July) from three commercial Koppert pollination hives . These hives were kept in the Baden-Württemberg State Bee Research Institute at the University of Hohenheim, Stuttgart, Germany. The developmental schedule listed in56 was used for the identification and dissection of third instar B. terrestris larvae, which is the stage corresponding to the L5F1 stage of A. mellifera, for which RNA was extracted simultaneously and at the location23.Stingless bee larvae , and the manufacturer’s protocol for the use with small tissue quantities was used for RNA extraction: TRIzol (500 μL), chloroform (100 μL), glycogen (5 μg), and overnight precipitation (− 80 °C) with isopropanol (250 μL). After solvent evaporation, the RNA was resuspended in 20 μL RNase-free water. The number of gonads composing each A. mellifera sample is reported in23. For B. terrestris, each of the three biological samples consisted of 40 larval gonad pairs, and for M. quadrifasciata, the number of gonad pairs was 50 for each of the three samples. Sample quality control, library preparation, and high-throughput sequencing protocols were all performed simultaneously by CeGaT GmbH , as previously described23. Paired-end sequencing was carried out on a NovaSeq6000 (Ilumina) platform, with read lengths of 2 × 100 bp and an output of 6 Gb per sample.The larval gonads of all three species, https://github.com/nf-core/rnaseq). It included the MultiQC v1.6 program57 and FASTQC v0.11.8 to determine FASTQ file quality, and after removal of low-quality reads, adapter sequences were trimmed with Trim Galore 0.5.0. The raw trimmed sequences of each of the three B. terrestris and M. quadrifasciata larval gonad RNA-seq libraries were submitted to NCBI .The initial RNA sequencing data analysis was done using the Nextflow-based RNA-Seq pipeline release 1.2 , and the Melipona quadrifasciata genome . An evaluation of the mapped RNA-seq reads was done with RSeQC v2.6.459, and for read counting of the features , FeatureCounts v1.6.260 was used. The read counts were then normalized to RPKM to remove potential technical bias in the RNA-seq data, such as depth of sequencing and gene length. All programs were used with default settings for mismatch allowance.Next, using the HISAT2 v2.1.0 aligner23. The focal genes for the comparison across the three bee species were defined by a Gene Ontology (GO) enrichment analysis on the 870 A. mellifera larval gonad DEGs performed using Blast2GO implemented in OmicsBox . Enrichment significance was assessed using Fisher’s Exact Test. This resulted in a set of 45 genes, for which the B. terrestris orthologs were retrieved from GenBank using the NCBI BLASTP tool. For M. quadrifasciata, the respective BLASTP searches were done in the Hymenoptera Genome Database (hymenopteragenome.org)27. Ortholog tables with the respective gene_id 61 were used to find and manually confirm the corresponding gene_ids. Their orthology status was confirmed by reciprocal BLASTP searches against the honey bee genome.The starting point of the analysis were the 870 DEGs Fig.  of the hA. mellifera and 3 each for B. terrestris and M. quadrifasciata). A principal component analysis and a hierarchical cluster analysis on the RPKM counts were performed in JMP Genomics 12.2.0 software (SAS).Using the respective gene_id, the RPKM counts for each gene were retrieved from the 15 transcriptomes , two bumble bee species (Bombus terrestris and B. impatiens), the stingless bee Melipona quadrifasciata, the orchid bee Eufriesea mexicana, the carpenter bee Ceratina calcarata, and the anthophorid Habropoda laboriosa. These 10 species all belong to the family Apidae. In addition, we included Megachile rotundata (Megachilidae) and two Halictidae, Lasioglossum albipes and Dufourea novaeangliae. The respective nucleotide sequences were aligned using ClustalW and visualized in Aliview62. The software geneious (https://www.geneious.com) was used to convert the nucleotide sequences into consensus amino acid sequences, and a codon alignment analysis was performed using Pal2Nal .For the 24 genes that showed clear contrasts for the honey bee gonad transcriptomes against those of the bumble bee and the stingless bee in the hierarchical clustering analysis, BLAST tools (BLASTP and then BLASTN) were used to extract the corresponding nucleotide sequences (CDS) of their orthologs from the genomes of 13 bee species deposited in GenBank and the Hymenoptera Genome Database27 Table . These i29, implemented in the DataMonkey server (https://datamonkey.org/busted). BUSTED checks for synonymous and non-synonymous substitutions and calculates the dN/dS value for each gene. Additionally, it computes the dN/dS value for the respective gene within each species and calculates a p value for the gene in the comparison between the test branch versus the background branches. With this, a dN/dS-value is attributed to each gene, and a result for evidence of selection (p < 0.05) is given. This avoids relying on dN/dS-values alone, as these have been shown to have limited power in detecting positive selection63. In our analysis, the four species of the genus Apis served as test branch against nine other bee species.To reveal whether genes or specific sites may have experienced episodes of positive selection, the codon alignment was used as input to an evolutionary test performed in BUSTED30. Modeltest implemented in MEGA was used to identify the evolutionary model that best described the amino acid substitution pattern for each data set. The model with the lowest BIC scores (Bayesian Information Criterion) was the used for the construction of unrooted trees based on Maximum Likelihood. Models, BIC scores, as well as the G+ and I+ values used for gene tree construction are listed in Table Evolutionary trees were generated for the four genes with evidence of positive selection using MEGA7 softwareNot applicable. As no chordate species were used, the Committee for the Use of Animals in Experiments (CEUA) of FMRP-USP declared the study protocol as exempt from regulation.Supplementary Information.


Lissotriton vulgaris meridionalis and the role of drinking troughs in supporting viable breeding populations within a rural landscape interested by traditional husbandry and agriculture. Our genetic analysis highlighted the conservation value and the potential stepping-stone function of artificial aquatic sites in the dispersal of the species and for the gene flow maintenance. Indeed, populations of drinking troughs show allelic richness and heterozygosity levels comparable to those from natural ponds and there is no great evidence of genetic bottlenecks. A complex system of artificial aquatic sites and few natural wetlands was identified sustaining a well-structured network of demes highly interconnected with themselves and natural aquatic sites. The conservation of the identified genetic clusters may be useful to prevent further population declines and future loss of genetic diversity within the study area characterized by scarce natural wetlands that frequently dried because of agricultural practices and strong seasonality. Site-specific protection measures are needed to contrast the progressive disappearance of drinking troughs observed in the last years in Italy because of the abandonment of traditional farming practices in favour of modern agriculture and intensive farming.Amphibians are particularly sensitive to habitat loss and fragmentation caused by the intensification and modernization of farming occurring in the second half of the twentieth century in the Mediterranean basin. However, artificial water bodies, associated with traditional husbandry, proved to be important surrogate for amphibian feeding and reproduction. Here, multilocus genotypes were used to investigate the spatial population structure of  This is particularly true for the Mediterranean basin, a biodiversity hotspot, in which the current mosaic of agricultural lands, traditional pastures, evergreen woodlands and maquis habitats, is the result of anthropogenic activities over millennia3. Particularly, traditional agricultural systems, derived from well-integrated and sustainable management practices, being spatially heterogeneous, have supported a high level of biodiversity4. However, in the second half of the twentieth century, the intensification of modern agricultural practices has increased habitat loss and fragmentation, spatial homogenization, and a reduction in biodiversity5. In general, amphibians are particularly sensitive to habitat destruction and fragmentation that have a negative impact at a demographic and genetic level6 with habitat connectivity playing a key role in their viability at the regional scale7. Indeed, agriculture and urban development activities may create barriers to amphibian dispersal9 resulting in isolated subpopulations more subject to increased risk of inbreeding and local extinction11, and/or less likely to be recolonized by dispersers from neighbouring subpopulations12. In an isolated population, a decrease in the immigration rate of individuals from other populations (i.e. gene flow) will inevitably produce a genetic diversity loss6. This happens when population size is reduced13 and the isolation among populations is so strong that inbreeding and genetic drift are no longer counterbalanced by gene flow14. These negative effects are further exacerbated by the fact that amphibians, due to their habitat specificity, strict ecophysiological requirements15, low dispersal abilities, and philopatry16, do not behave as single, infinitely large panmictic population17 but as patchy demes representing unique genetic entities despite that geographic proximity. In addition, due to their biphasic life cycle in aquatic and terrestrial habitats, amphibians are vulnerable to the alteration of both environments and to any factors reducing population connectivity18. Despite human activities negatively impact amphibian populations, many studies in the Mediterranean highlighted the importance of artificial water bodies, associated with traditional agriculture and cattle watering, for amphibian feeding and reproduction22. This suggests that such artificial facilities, often connected to natural water bodies, may represent important surrogate breeding habitats for amphibians23. Indeed, the ability of amphibians to colonise artificial aquatic sites  has been well documented worldwide25. Moreover, they may act as stopovers during dispersal events, thereby facilitating amphibians’ movements by reducing effective inter-aquatic systems distances27. However, there are no studies on their role and importance as for the population genetic conservation and for stepping-stone function in the dispersal process and gene flow maintenance.The European landscape is the result of the dynamic interaction between people and their natural environments28. In this context, microsatellite markers have proved to be sensitive indicators of change in dispersal rates and suitable tools for addressing questions related to population subdivision and interconnection even at fine scale29. Studies on the individual movements and the spatial scale of dispersal among subpopulations are needed in addition to basic demographic research for improving our knowledge of amphibian population dynamics and sustainability, and to assist landscape management and conservation planning30. However, species genetic management is often overlooked in conservation programmes due to lack of resources or expertise31. However, the determination of genetic relationships among populations may be particularly useful to detect areas of high conservation priority, dispersal corridors, population declines and individuals and/or populations fundamental for augmentation/translocation or reintroduction programs32. Here, we described the spatial population structure of the Italian smooth newt Lissotriton vulgaris meridionalis (Linnaeus 1758) in a rural landscape of Central Italy (Latium) within a patchy system interested by infrastructural and agricultural development  were used to (i) evaluate genetic diversity within and among artificial and natural breeding sites; (ii) infer population structure in the study area; (iii) detect migration and gene flow among demes; (iv) establish whether drinking troughs may provide alternative and/or complementary aquatic habitats for amphibian conservation in rural landscapes.With increasing anthropogenic habitat modification, it is particularly important to understand the genetic consequences of changes in landscape composition and configurationGenotypic data were obtained from 464 individuals, excluding six samples that had more than 20% missing genotypes. One locus (Lm_013) was also excluded from data analyses because of a large amount of missing data, due to an amplification failure.Estimated null allele frequencies were not significant, indicating the absence of null alleles, large allele dropout and false homozygotes at any locus in all populations.He) ranged from 0.048 to 0.955, and observed heterozygosity (Ho) from 0.048 to 1.00  conformed to Hardy–Weinberg equilibrium after Bonferroni correction, with ten populations showing a slight deviation at one locus  to 10.6 (FER) in artificial sites and from 5.4 (RP) to 9.7 (RER) in natural water bodies. Allelic richness varied from 4.4 (FP) to 8.4 (FER) in drinking troughs and from 5.0 (RP) to 8.1 (RIC) in natural sites (Table He) heterozygosity varied from 0.507 (FT) to 0.659 (FER) in artificial sites, and from 0.547 (RP) to 0.681 (RR) in natural sites. Observed heterozygosity (Ho) ranged from 0.515 (FT) to 0.668 (FER) in artificial sites and from 0.550 (RP) to 0.670 (LE) in natural sites . Expected , except for three populations  showing FIS estimates significantly greater than zero after Bonferroni correction . No significant differences were observed in He values within each site pair represented by a natural and an artificial site .Heterozygosity did not vary between artificial and natural sites  to 0.239 (FT-FB) with an average FST = 0.066. FST values were significantly higher than zero in 202 of 210 cases after sequential Bonferroni correction for multiple comparisons. FST was not significant in three out five natural/artificial site pairs . Overall, low to moderate levels of population differentiation were observed except for FT showing the highest FST values (Table Pairwise Nm) between populations, calculated directly from the FST values  among pairs of all populations were significantly correlated with the geographical distance between localities . However, geographical distance explained less than 14% of the genetic variation (r2 = 0.137) in the study area  explaining about 43% of the genetic variation (r2 = 0.428) . No close relatives were present within the five site pairs  identified 5 clusters. The resulting scatterplot showed evidence of fair separation among clusters Fig. a, with sDAPC revealed a pronounced genetic differentiation among the five artificial-natural site pairs Fig. b, highliThe model-based clustering method suggested that the model with K = 5 (where K is the number of population genetic clusters) was substantially better than alternative models.Five clusters were identified with FT was split from the other populations. FB-RR and FP-RP formed two clusters on their own. All the other populations clustered in two distinct groups corresponding to north-western and south-eastern geographical areas Fig. .Figure 4Mean membership coefficients for all individuals into the north-western and south-eastern demes was generally high, with a mean of 0.796 (SD = 0.160) and 0.751 (SD = 0.235), respectively. A few populations , located on the borders of these two clusters, showed membership coefficients less than 70% and thus may be considered as admixed demes Figs. , 5.FigurThe hierarchical analysis of molecular variance (AMOVA) confirmed the genetic structure detected by clustering methods, with 86.73% of genetic variation resided within populations, 7.89% distributed among populations within clusters, and 5.38% of the variance possibly explained by differentiation among the five clusters Table . AlthougThe hypothesis of a recent bottleneck was tested with infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model (TPM). We observed the occurrence of a bottleneck event in 7 out of 21 populations under IAM model  detected recent migrations. In 20 instances, the migration rate was greater than 0.04 indicating the occurrence of substantial contemporary gene flow. In 12 cases, migration rates were > 0.1 suggesting that more than 10% of the population is composed by recent immigrants. The highest level of gene flow was mostly detected in populations that were geographically closed and belonging to the same genetic cluster  within a rural area of Central Italy (Latium).To our knowledge, this is the first study to examine the distribution of genetic variation and the spatial genetic structure of Italian smooth newt  comparable to those from the closest brook. As the level of genetic diversity reflects population’s adaptive and evolutionary potential36, this finding demonstrates the importance of artificial habitats for the persistence of amphibians especially where original habitats have been degraded or lost20.Populations showed a similar genetic diversity with no differences between natural and artificial wetlands, comparable to other urodeles and temperate amphibians37, resulting in large genetic differentiation among populations that may facilitate local adaptation or cause genetic drift38. To date, for L. vulgaris meridionalis, genetic data are only available from17, who found moderate to high differentiation levels among close populations. In our study, genetic differentiation among populations was moderate (average FST = 0.066), thus indicating the existence of a network of populations connected by dispersing individuals39. This suggests that drinking troughs contributed to sustain gene flow, decreasing local genetic divergence, and mitigating genetic drift and population inbreeding40. Our findings are in agreement with previous fine-scale studies of urodeles employing microsatellite markers43.Smooth newts are highly philopatric, with short dispersal abilityFST-values not consistent with the geographic distribution . This may be due to the relatively longer timespan since isolation and the higher habitat fragmentation characterising FT and FP-RP demes. It is likely that, in the past, the study area consisted of a unique unfragmented woody landscape with many natural aquatic systems used by newts for reproduction. In the 1950s, wide-scale human impact caused habitat loss and fragmentation isolating some demes (i.e. FT and FP-RP) in less favourable areas. The transition forests-arable lands or grazed-cultivated lands, and the construction of roads may have removed suitable terrestrial shelters and artificial breeding sites used for reproduction44, affecting the connectivity of local populations, dispersal, and genetic diversity45. Our suppositions are in agreement with previous studies documenting the negative effects of habitat fragmentation on local genetic variability and connectedness in amphibians48.Some populations showed higher pairwise 50. Indeed, we found a significant IBD pattern between populations separated by a maximum distance of 16.7 km (FFA-FER) and 17.8 km (FB-FE). We thus confirm the limited dispersal ability of L. vulgaris meridionalis even at small spatial scale43, whose average dispersal distance has been estimated to be 400–500 m51. However, this moderate correlation coefficient detected suggests that further processes other than distance may have structured populations’ genetic composition48. Indeed, dispersal capability is known to be not only determined by species’ intrinsic biology but also by landscape features and habitat quality53. In this context, it is likely that natural and artificial water bodies have increased survival probability and facilitated juvenile dispersal and adult breeding migration43. Drinking troughs, in particular, seem to act as stepping-stone system connecting distant natural breeding sites and building a network able to face potential isolation.We also found a correlation between genetic and geographical distance measures. Isolation by distance is expected to shape genetic variation, even on small spatial scales, leading to populations more differentiated from each other with increasing distance54.Five statistically significant population clusters with a moderate genetic differentiation have been identified. Gene flow seems to be extensive among populations within each cluster determining quite genetically homogeneous assemblages, with a genetic variation attributable to within-populations differences. Moreover, the five artificial-natural site pairs were genetically interconnected representing each a unique nearly panmictic deme. Such a positive association confirms the potential of drinking troughs to contrast drift and local loss of genetic diversity providing a network of permanent aquatic sources used for breeding and migration55. This probably reflects the counterbalance of different effects currently ongoing or acted in the past. On one hand, natural limits to dispersal and population-level processes may have affected site fidelity and low vagility increasing genetic distance56; on the other, gene flow among pristine habitats, once widespread in the study area, may have reduced genetic differentiation. Two clusters drifted apart probably because of landscape features alone or in combination with topographical differences. Indeed, both natural barriers and human-induced environmental changes affect occasional migration events among distant populations in L. vulgaris meridionalis hindering gene flow and resulting in a strong genetic structure43.Three clusters had a very similar cohesiveness concordant with the geographic distribution evidencing a significant positive relationship between genetic differentiation and geographical distance41. Tolfa Mountains conform to this landscape scenario being characterized by large patches of natural habitats interspersed with areas subject to traditional agricultural and husbandry activities without absolute geographic barriers limiting individual exchange among populations57.Five populations showed evidence of mixed genetic ancestry. Such admixed populations are expected in systems where geographical structure exists but there are no barriers to dispersal58.Signals of current migration were found within clusters but not among populations from different sites. Interestingly, migration was unidirectional and influenced by the hydrology of the site (permanent vs. temporary) rather than its typology . Surprisingly, not only natural but also artificial permanent aquatic sites acted as sources of individuals. The observed configuration of water network and the connectivity among drinking troughs, and between drinking troughs and natural aquatic systems, suggests the existence of a metapopulation with a source-sink configuration, in which recolonization following local extinctions, and natural restocking after demographic contractions are likely to occur42 resulting in a genetic bottleneck effect at local scale. This also suggests that populations did not recently experience severe effects of natural and anthropogenic pressures, or such pressures were mitigated by the positive impact of the observed configuration of water network. RT, a brook population subject to periodic drought, seems to have experienced a recent bottleneck probably counterbalanced by individuals migrating from neighbouring natural and artificial sites. FFA and FBG drinking troughs populations probably suffered inadequate management practices, associated with mechanical and chemical cleaning activities and water extraction19. Such operations may cause early desiccation and metamorphosis failure of larvae, with massive mortalities every year and consequent repercussions on the occurrence and abundance of species at local scale20 and a decline in genetic variation13.We found evidence of a slight occurrence of bottleneck events . Amphibian populations are known to fluctuate from year to year with a demographic and connectivity instability57 and the current landscape. Drinking trough populations probably derived from individuals in dispersal from neighbouring natural sites subject to a relatively fast drift known to affect small and isolated populations59. Since 15–20 generations are needed for signals of genetic effects to be observable60, the genetic structure inferred is more likely to represent a snapshot of past landscape configuration and composition rather than contemporary ones61.Our study revealed that a complex system of artificial permanent aquatic sites and few natural wetlands can sustain a well-structured network of amphibian demes that seem to be quite connected. The current landscape matrix suggests that the observed genetic structure may not reflect present-day gene flow but an intermediate situation between the historic scenario after the 1950sL. vulgaris meridionalis conservation. The Maremmana cow seems to act as an ecosystem engineer (i) directly by structuring landscape features (i.e. increasing habitat heterogeneity) and (ii) indirectly by providing suitable artificial sites crucial for newt genetic diversity maintenance. We emphasize how drinking troughs may represent an interesting tool for promoting individual dispersal and gene flow, reinforcing metapopulation patterns observed in the study area. Intervention measures are timely to prevent the progressive abandonment of drinking troughs observed in the last 50 years in Italy due to expansion of modern and intensive farming63. At the local scale, conserving and managing the existing natural sites as well as incentivization of traditional farming accompanied by drinking trough restoration, preservation and proper management will increase the availability of water supplies to both the Maremmana cow and newts, preventing further population declines within the study area. Particularly, the correction of trap effects, invasive species eradication, cleaning practices respectful of newt phenology, intervention to avoid disrepair and permeability loss23 will help to preserve habitat suitability maximizing the effectiveness of drinking troughs as stepping-stones. At the regional scale, habitat suitability of both natural and artificial water bodies could be improved by increasing site connectivity64. Indeed, the co-occurrence of aquatic sites differing in sizes and other features  in the landscape is known to favour species’ dispersal reinforcing their metapopulations64.Our study highlights the significant role played by the Maremmana cow in 19.Our conclusions suggest that amphibian protection and social rural and cultural values preservation can be simultaneously achieved combining conservation biologists’ efforts and local livelihood needs65.The study area falls within the Maremma district, extended throughout Latium and Tuscany regions. Fieldwork was carried out in the Comprensorio Tolfetano-Cerite-Manziate  a Special Protection Area , spanning 70,000 ha, included in the European Ecological Natura 2000 network. It is characterized by a high biodiversity value representing one of the highest wilderness areas in Italy despite the proximity to several main cities and its rural profile interested by traditional agriculture and husbandry. A mosaic of pastures, cultivated lands, and woodlands characterizes the vegetation landscape, mainly consisting of riverine woods, wide areas covered by bushy pastures and mesophilous forests66. Such artificial aquatic sites are particularly used by the Maremmana cow (Bos primigenius taurus), a robust and frugal native breed well adapted to difficult environments living freely in the marshes and maquis of the south coastal area of Italy for more than 1500 years68.In this area, natural wetlands are scarce and are frequently dried due to agricultural practices and their strong seasonality. Indeed, they are characterized by a short hydroperiod, especially in summer, because of high temperatures and irregular rainfalls. However, artificial drinking troughs built by farmers after World War II to facilitate livestock watering , are particularly widespread. Drinking troughs are small, shallow (20–60 cm depth) habitats, with rectangular shape and vertical walls, often in connection with natural permanent or semi-permanent aquatic systemsL. vulgaris meridionalis and a distribution across the study area aimed at minimizing spatial autocorrelation  were identified through orthophotos in Google Earth Pro software (version 7.3.1.4507). Overall, 7 natural aquatic sites  and 51 functioning drinking troughs were detected within an area covering approximately 18,000 ha. We focused on all 7 natural sites and on 14 drinking troughs  of a natural brook/pond  in 2015–2016 years. Since cohorts can vary in genetic composition, we avoided sampling the same site at different times during the yearTissue samples were obtained with no anaesthetic and by careful handling of animals. After this procedure, individuals were temporarily housed in tanks filled with water for approximately 30 min to verify their return to normal activity and released at the same point of capture. No individuals died or were otherwise harmed during samplings. This study was conducted according to the ethical scientific guidelines described by the American Society of Ichthyologists and Herpetologists (ASIH), The Herpetological League (HL) and the Society for the Study of Amphibians and Reptiles (SSAR).Experimental protocols were approved by Italian Ministry of the Environment that gave LV the permit [0001255/PNM] to manipulate amphibians and collect tissue samples.All methods are reported in accordance with ARRIVE guidelines.71. Three multiplex PCRs  were performed to save time and reagents. PCRs were carried out using Qiagen Multiplex PCR Kit in an 8 μL mix composed of 3.50 μL HotStarTaq Master Mix, 0.70 μL Qsol, 1.50 μL DNA, 0.20 μL each primer (10 μM), brought to volume with H2O. Amplification conditions for all loci included an initial denaturing at 95 °C for 15 min; 40 cycles of denaturing at 94 °C for 30 s, annealing temperature of 55 °C for 1.30 min, and 72 °C extension for 1 min; and a final 10 min extension at 72 °C. Two replicates for each sample at each locus were executed to confirm the genotypes.Total genomic DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN) and QIAcube robotic workstation, then amplified at 12 microsatellite loci (STRs) optimized for this speciesForward primers for each locus were 5’-labelled with a fluorescent dye for detection (Applied Biosystems). PCR products were electrophoresed with internal size standard  in an ABI Prism 3130XL sequencer and allele sizes were scored using the software GeneMapper v.5.0 (Applied Biosystems).72.The presence of null alleles was checked by Micro‐Checker v.2.2.374. Allelic richness was computed in Fstat 2.9.375. Arlequin v.3.5.276 was used to estimate observed (HO) and expected (HE) heterozygosity and to test genotypic frequencies at each population and locus for statistically significant deviations from Hardy–Weinberg equilibrium (HWE). We used a Monte Carlo approximation of the Fisher’s exact test77 and a standard Bonferroni correction for multiple comparisons . The Markov chain algorithm was run for 100,000 steps following 10,000 dememorization steps. The pairwise probability of linkage disequilibrium was estimated using a Fisher’s exact test implemented in Genepop v.4.278 with 10,000 steps following 1000 dememorization steps.Genetic diversity indices  were calculated for each population by GenAlex v.6.5Analysis of variance (ANOVA) was used to reveal differences in expected heterozygosity as a function of site type . Moreover, the Wilcoxon matched-pairs test was performed to compare heterozygosity values within each site pair represented by a natural and an artificial site. Statistical tests were performed using STATISTICA software (version 8.0 for Windows) with alpha set at 5%.75. Pairwise significance tests for FST79 were performed by permutation and resampling of multilocus genotypes among pairs of samples with 10,100 randomizations allowed for a table-wide significance at the 5% nominal level after standard Bonferroni corrections .Fstat 2.9.3 was used to perform a global test of overall population differentiation (not assuming HWE within populations)Nm estimates, derived from the fixation coefficient FST80.The degree of gene flow among demes, in terms of success of immigrants, was determined using FST values81 were correlated with geographical distances to test for patterns of spatial subdivision and isolation by distance (IBD) using a Mantel test with 1000 randomizations (implemented in GenAlex v.6.5). We performed two tests for IBD separately, on (i) all populations, and on (ii) populations with the exclusion of potential outliers (i.e. populations showing higher genetic distance despite the similar geographic distance in respect to the others).Pairwise population linearized 82 LR estimator by 2 to give a maximum value of 1 and minimum of − 1.Pairwise relatedness analysis was carried out to assess the presence of close relatives in our populations. We performed the analysis (implemented in GenAlex v.6.5) for each population and for the five artificial-natural site pairs using Lynch & Ritland’s84. We used “find.clusters” function to identify the optimal number of clusters. k-means algorithm was run comparing different clustering solutions using Bayesian Information Criterion (BIC) values with increasing values of k. The optimal number of clusters was selected corresponding to the lowest BIC. Then, we determined the number of PCs to retain considering the cumulated variance explained by the eigenvalues of the PCA. After the cross-validation step, DAPC was performed with the retention of 60 PCs (> 80% of variance explained) and four discriminant functions. A further DAPC was conducted to verify genetic differentiation within the five artificial-natural site pairs previously identified, with the retention of 60 PCs and four discriminant functions.A discriminant analysis of principal components (DAPC) was performed to assess the genetic divergence among populations and provide a description of the genetic clustering based on the largest between‐group and smallest within‐group variances in allele coefficients (R package “adegenet”)85. We ran 15 independent simulations of 1,000,000 iterations  for each value of K85 between 1 and 21. The admixture model was applied assuming gene flow among populations and allowing for correlated allele frequencies across populations. We performed two different models: (i) the default mode for STRUCTURE that uses only genetic information to infer population structure, and (ii) the LOCPRIOR model that uses sampling locations as prior information to assist the clustering.We performed Bayesian clustering analysis to characterize the population genetic structure of aquatic systems across the study area and assess the geographical scale of population differentiation using STRUCTURE v2.3.4K was selected based on the natural log of Pr(X|K) values and the ∆K criterion86 provided by STRUCTURE HARVESTER v.0.6.9487. Replicate runs were averaged using CLUMPP v.1.188 and plotted in DISTRUCT v.1.189 to generate graphical displays representing the mean membership of each individual over the replicate runs.Appropriate 90, implemented in Arlequin v.3.5.276, was performed on FST values to examine the distribution of genetic variation at three hierarchical levels: within populations, among populations within demes, and among demes. We assigned each population to a cluster based on its largest proportion of membership and then examined hierarchical genetic distribution considering only populations with a mean membership ≥ 0.7 and excluding admixed populations. The significance of the variance components was tested using 10,000 permutations.Considering the genetic clusters inferred from the Bayesian analyses as panmictic demes, an Analysis of Molecular Variance (AMOVA)91 was used to test for evidence of recent bottlenecks in each population. The main assumption of this test is that allele frequency distribution results from an equilibrium between mutation and genetic drift92. In non-bottlenecked populations, the expected heterozygosity at mutation-drift equilibrium (Heq) equals the measured Hardy–Weinberg equilibrium heterozygosity (He). Instead, in case of recent bottlenecks, a correlative reduction of allelic diversity and heterozygosity at polymorphic loci occurs and the mutation-drift equilibrium is temporarily disrupted: He will be significantly greater than Heq, calculated from the number of alleles sampled93. The calculation of Heq depends on the mutation model considered analysing the loci92. In the present study, Heq values were calculated under three mutation models: the infinite allele model (IAM)94, the Stepwise Mutation Model (SMM)95 and a combination of those two extreme hypotheses, the two-phase model 96. Significant deviations in observed heterozygosity across loci for each population were detected using a two-tailed Wilcoxon sign-rank test97 that allows the detection of the heterozygosity excess due to the faster loss of low frequency alleles and thus determines whether a population has been recently bottlenecked. Moreover, a qualitative descriptor of allele frequency distribution was used to infer bottlenecks. This method compares allele frequency distribution to the distribution expected at mutation-drift equilibrium (l-shaped distribution), considering that population bottlenecks cause a characteristic mode-shift distortion in the distribution of allele frequencies at selectively neutral loci97.The software BOTTLENECK v1.2.0291. Since the IAM is mainly recommended for allozyme data and microsatellites are often described as being closer to the single step mutation model91 or to the Two-Phase Mutation model96, we may consider the results obtained under SMM and TPM more reliable.For fewer than 20 loci (i.e. 11 loci in our study), the Wilcoxon’s test is the most appropriate and powerful98, which employs a Bayesian MCMC approach to identify migrants or recent descendants of migrants based on transient linkage disequilibrium among multilocus genotypes from different source populations. This program was run following the recommendation of99 concerning convergence issues. We performed 10 separate runs, with different seed numbers, using 107 iterations, with a burn-in of 106 and a sampling frequency of 1000. This run length was sufficient for the posterior probability to achieve convergence. The MCMC mixing parameters were set to 0.50, 0.80 and 0.25, for allele frequency, level of inbreeding and migration rate, respectively. These delta values resulted in acceptance ratios between 40 and 60% to maximize log-likelihood values98. Migration rates were averaged over the 10 independent runs and compared to average migration rates of randomly permuted data set, generated to assess significance. Estimated migration rates were considered significant when the 95% confidence interval (CI) did not overlap with the 95% CI of the randomly permuted data.We estimated recent migration rates between localities using the program BAYESASS 3.0Supplementary Information.


Erythrocytes have been thoroughly investigated as drug delivery systems for a wide range of therapeutic molecules and using different kinds of loading methods, outstanding the osmosis-based methods as the most used ones. Most of them involve too much handling of blood components and the immediate obtention of fresh blood. Based on our group’s considerable experience in dialysis-based carrier erythrocyte preparation, this study details a simple method based on hypotonic dilution and subsequent resealing that has been developed for stavudine using packed erythrocytes from a local blood bank. Properties of the obtained carrier erythrocytes were studied in comparison to those prepared by dialysis. Erythrocytes’ morphology, osmotic fragility, hematological parameters, and in vitro release profiles were evaluated. Loaded erythrocytes obtained with the proposed method did not show impaired properties in comparison with those obtained with our reference method, provided that the buffer composition remained the same. In the present work, we have optimized a simplified method for erythrocytes’ drug loading, which can use blood transfusion products and could be easily automatized and scalable. Since the 70s to nowadays, erythrocytes have been explored for their application as vehicles of different foreign molecules ,2,3,4. TThe shape, membrane fragility, flexibility, and hematological indices are essential erythrocyte parameters. However, during the cargo process, they may undergo irreversible changes, causing erythrocytes to be eliminated more rapidly from the bloodstream. Those properties, together with the intrinsic characteristics of the charged molecule, lead to important differences in its encapsulation efficiency and release profile ,8.Throughout this time, different erythrocyte loading methods have been proposed, from electroporation to cell-penetrating peptide-mediated ones ,10,11. OThe loading methods applied in clinics are mainly based on hypotonic dialysis and need specific devices or facilities . HypotonStavudine is a nucleoside reverse transcriptase inhibitor used as an antiretroviral drug that has a very short half-life and high water solubility . This woBesides the simplification of the loading procedure, in this work, we propose to use erythrocytes from the blood bank. Although the most common origin of the native erythrocytes in this kind of procedure is fresh autologous erythrocytes ,17,18, tv:v) against a hypotonic buffer with osmolality of ~90 mOsm/kg for 30 min at 4 °C. Resealing was achieved by subsequent incubation against 30 mL of hypertonic (~500 mOsm/Kg) buffer for 15 min at 37 °C. Resealed erythrocytes were washed twice with isotonic Hanks-PBS buffer in order to remove the non-entrapped drug.Different osmosis-based methods have been proved . Loaded v:v) of packed erythrocytes is prepared in hypotonic buffer containing the drug and incubated at 4 °C. Next, hypertonic buffer is added to this mixture, and a resealing step is performed at 37 °C in order to restore the tonicity and incubation. Final washings with isotonic medium are also included to remove the non-entrapped drug excess.Regarding the hypotonic dilution method, a suspension of 70% (Previous studies developed by our group let us establish 30 min as the optimum time for hypotonic incubation in order to maximize the stavudine loading efficacy in human blood bank erythrocytes. v:v), and the time for resealing, as shown in Once the conditions for this first step were established, and considering resealing as one of the most critical stages to avoid drug loss, studies focused on optimizing the resealing stage. Two factors were studied: the ratio volume of erythrocyte dispersion:hypertonic buffer  was replaced by 5-mmol/L KH2PO4, 5-mmol/L K2HPO4  (conditions Nc), and the loaded erythrocytes obtained were characterized and compared with the results obtained with the usual hypotonic buffer.In the next step, going further with the aim of simplifying the encapsulation procedure, the hypotonic medium composition was modified to avoid complex macromolecules inclusion. Based on previous studies, our usual hypotonic buffer  at 10,900 rpm for 5 min, and stavudine was quantified in the loaded erythrocytes by UFLC. Results of drug encapsulation are expressed as drug concentration estimated in loaded erythrocytes and also standardized by the number of cells counted for each individual experience.Encapsulation efficiency (EE) is calculated as the percentage of the total available drug that is entrapped inside, as follows:® column . The mobile phase was an aqueous solution of 0.1% formic acid and acetonitrile (97:3) with a flow rate of 0.50 mL/min at 45 °C. Detection wavelength was 265 nm. Sample treatment was carried out by precipitation with 30% trichloroacetic acid (TCA) in a 10:1 ratio (sample:TCA) and filtration through a pore size of 0.22 µm prior to analysis.Stavudine was determined using a UFLC chromatograph  equipped with a PDA SPD-M20 detector and a C18 KinetexThe osmotic fragility of native erythrocytes and stavudine-loaded carrier erythrocytes was determined according to Dacie’s method as described elsewhere . CarrierStandard hematological parameters in the control and loaded erythrocytes, such as hematocrit (HCT), hemoglobin (HGB), total red blood cell count (RBCS), red cell distribution width (RDW), mean corpuscular volume (MCV) mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), were estimated by spectrophotometry and impedance analysis using a Horiba Medical’s Yumizen hematology autoanalyzer . Packed erythrocytes were fixed with glutaraldehyde and then postfixed in osmium tetroxide. After ethanol dehydration with a graded ethanol series, samples were critical point-dried and sputter-coated with gold. Images obtained with a Jeol IT500 Scanning Electron Microscope  were analyzed for a qualitative evaluation of changes in morphology, and quantitative analysis of erythrocyte sizes was performed with ImageJ software version 1.53 .In vitro release was performed by including the loaded erythrocytes in a regenerated cellulose semipermeable membrane . In order to guarantee the sink conditions, 300 µL of stavudine carrier erythrocytes were incubated for 24 h at 37 °C in 3 mL of isotonic medium . Aliquots were removed at predefined times (5 min to 24 h), and the drug released was analytically determined by UFLC. p < 0.05.For the statistical analysis of the encapsulation results and hematological parameters, analysis of variance was performed using SPSS statistical software version 28.0.1.1 . The differences were considered statistically significant when In the present work, we have compared dialysis and hypotonic dilution methods for stavudine encapsulation in ghost erythrocytes in order to confirm the potential of the simpler hypotonic dilution procedure. The influence of the resealing conditions and the hypotonic buffer composition in the key parameters of the carrier erythrocytes were studied.The results for drug encapsulation are compiled in v:v), and the interaction between the two is also significant (p < 0.001). Regarding the hypotonic dilution methods, ANOVA results reflect statistically significant differences in the encapsulated concentration in the diverse conditions studied for both the time factor and the ratio factor  in most of the parameters studied, except for RDW, MCV, and HCM, which are significantly affected by the resealing ratio. Analysis of variance confirmed that there are no statistically significant differences , as it can be seen in the corresponding figure.After quantitative analysis, measurements of the sizes of loaded and control erythrocytes confirmed  that theStavudine release studies from hypotonic dilution-loaded erythrocytes showed similar profiles to the previous reference method based on hypotonic dialysis . RegardiThe A conditions, which combine the least resealing time and hypertonic buffer volume, result in a higher release rate and a high maximum of almost 80% in the period studied, which is reached at 6 h. These results are consistent with other results already observed for these conditions, while the curve profiles observed for B, C, and D are similar, maintaining about 50% of the encapsulated drug inside throughout the entire interval studied.According to these profiles, it could be suggested that a better degree of erythrocyte resealing could affect the drug release rate and that the proposed hypotonic dilution method even seems to prevent drug leakage better than the previous reference method.v:v) ratio (conditions Nc).Considering that previous results pointed out C conditions as the preferential resealing procedure, the simplified hypoosmotic buffer was tested by using a resealing of 30 min with a 1:0.5 erythrocyte dispersion:hypertonic buffer (10 cells) than those obtained for C conditions.Regarding stavudine encapsulation, the simplified buffer led to a lower drug concentration (0.20 ± 0.08 mg/mL) and efficiency (9.22 ± 3.94%), but when standardized by cell count, we can observe higher values  for all the studied parameters, except for RBCS count (number/µL), RDW and CHCM which could be attributed to the excessively low osmolality of this hypotonic buffer.Comparative statistical analysis of hematological parameters observed for Nc conditions-loaded erythrocytes also showed similar profiles to C-loaded ones, providing sustained drug levels, maintaining around half of the drug encapsulated inside them for 24 h , whereas other features, such as release profiles, are maintained.In summary, these preliminary results obtained for erythrocytes loaded with NIt seems that the use of a simpler loading buffer with relevant lower values of osmolality could not conveniently preserve the loaded erythrocytes’ properties, although the new conditions should be more thoroughly explored to confirm this assumption.In this work, taking our standard dialysis-based loaded erythrocytes procedure as a starting point and reference, we have compared and optimized a hypotonic dilution method for stavudine encapsulation. v:v) of 1:0.5 being the most adequate among those studied. Results showed that erythrocytes obtained present suitable properties for their use as drug delivery systems with the resealing conditions of 30 min and the ratio volume of erythrocyte dispersion: hypertonic buffer (Moreover, we have used packed erythrocytes provided by a local blood bank in all the procedures studied, thus confirming the feasibility of using this kind of product as the source for preparing loaded erythrocytes as drug delivery systems.The stavudine ghost erythrocytes developed constitute a controlled release drug delivery system that may prolong the plasma residence time of stavudine and increase its access to macrophage reservoirs, improving the success of the fight against AIDS infection.Preliminary assays for the use of a simpler hypotonic medium to avoid the use of macromolecules have been carried out. In view of the obtained results, this alternative needs to be studied more in-depth.In summary, we have demonstrated that loaded erythrocytes can be obtained from blood transfusion products with a really easy-to-perform method that implies minor handling. The use of transfusion products as raw material and the simplicity of the newly proposed method would increase the successful automation of these carrier systems’ obtention and allow compliance with quality requirements, which broadens the horizons of applicability of these systems and their clinical translation.


As microorganisms are very sensitive to changes in the lake environment, a comprehensive and systematic understanding of the structure and diversity of lake sediment microbial communities can provide feedback on sediment status and lake ecosystem protection. Xiao Xingkai Lake (XXL) and Xingkai Lake (XL) are two neighboring lakes hydrologically connected by a gate and dam, with extensive agricultural practices and other human activities existing in the surrounding area. In view of this, we selected XXL and XL as the study area and divided the area into three regions  according to different hydrological conditions. We investigated the physicochemical properties of surface sediments in different regions and the structure and diversity of bacterial communities using high-throughput sequencing. The results showed that various nutrients  and carbon  were significantly enriched in the XXLD region. Proteobacteria, Firmicutes, and Bacteroidetes were the dominant bacterial phyla in the sediments, accounting for more than 60% of the entire community in all regions. Non-metric multidimensional scaling analysis and analysis of similarities confirmed that β-diversity varied among different regions. In addition, the assembly of bacterial communities was dominated by a heterogeneous selection in different regions, indicating the important influence of sediment environmental factors on the community. Among these sediment properties, the partial least squares path analysis revealed that pH was the best predictor variable driving differences in bacterial communities in different regions, with higher pH reducing beta diversity among communities. Overall, our study focused on the structure and diversity of bacterial communities in lake sediments of the Xingkai Lake basin and revealed that high pH causes the β-diversity of bacterial communities in the sediment to decrease. This provides a reference for further studies on sediment microorganisms in the Xingkai Lake basin in the future. As one of the important components of global aquatic ecosystems, lakes play key roles in global hydrological processes, biodiversity maintenance, and biogeochemical cycles owing to their ability to process nutrients over long hydraulic retention times ,2,3. AltLake sediments, which have been demonstrated to be major reservoirs of carbon and other materials, involve high biomass and microbial taxa richness . SedimenXingkai Lake is an important boundary lake between China and Russia, containing various ecological functions. There are many wetlands, swamps, and farmlands in the Xingkai Lake basin. Rapid shrinkage and fragmentation have occurred in the Xingkai Lake Basin in the past fifty years . FurtherIn our study, we divided the study area into three regions according to the special topography of Xiao Xingkai Lake and Xingkai Lake. Our objectives were: (1) to investigate the physical and chemical characteristics of sediments in different regions; (2) to explore the community assembly processes and the community structure and diversity; (3) to determine the factors driving bacterial community structure and diversity. Our research can provide a scientific basis for the restoration and development of the lake ecosystem of Xingkai Lake in the future.Xingkai Lake (XL), extending from 131°58′ E to 133°07′ E and 45°01′ N to 45°34′ N, is located at the border between China and Russia . Xiao XiThe hydrological conditions in the Xingkai Lake Basin are complex. The upper reaches of XXL are fed by agricultural irrigation rivers, and the lower reaches are connected to XXL through dams. Hydrological connectivity plays a crucial role in the biodynamic and physicochemical variables of connected lakes. In view of this, we divided this area into three regions according to their special hydrological conditions C: the riw/v) and finally determined with a TOC analyzer. For specific operation steps, please refer to [All sediment samples were freeze-dried to constant weights, and roots and stones were removed. Subsequently, these samples were softly grinded in a mortar and then were passed through a 2 mm sieve. Sediment pH was measured in 2.5:1 mixtures of deionized water and soil using a pH meter . Total crefer to . Labile refer to ,26. Tota® High Fidelity PCR Master Mix , 0.2 μM of forward and reverse primers, and approximately 10 ng of template DNA. Thermal cycling comprised 30 cycles of initial denaturation at 98 °C for 1 min, followed by denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s, and finally 72 °C for 5 min. Sequencing was employed on an Illumina NovaSeq platform  and 250 bp paired-end reads were generated.Sediment DNA was extracted from 0.5 g wet sediment using a Power Soil DNA Isolation Kit . The extracted DNA was then preserved in −80 °C until the next step. Specific primer pairs  combined with the adapter sequence and barcode sequence were used to amplify the distinct regions (V3–V4) of the bacterial 16S rRNA genes. All PCR reactions were performed with 15 μL of Phusionhttps://qiime2.org (accessed on 7 July 2022)). Paired-end reads were merged using FLASH and were denoised using DADA2 in QIIME 2 [The 16S rRNA sequencing data were analyzed using QIIME 2  needs to be further calculated to disentangle explicit processes. The RCbray is calculated as the difference between the observed Bray-Curtis dissimilarity and its null distribution. RCbray > +0.95 reveals the dispersal limitation; RCbray < −0.95 represents the homogenizing dispersal and |RCbray| < 0.95 refers to “undominated” processes including drift, dispersal, diversification, and weak selection [Stegen proposed a framework to assess the effects of ecological drift, selection, and dispersal in regulating community assembly using null models ,30. Hereelection ,35. All p < 0.01  were remained to construct the network edges [To evaluate the species coexistence of microbial communities in sediments, the metacommunity cooccurrence networks of all three regions  were constructed. The ASVs selected were the same as we used in community assembly. Robust correlations with Spearman’s correlation coefficient (ρ) > 0.8 and rk edges ,37. AfteOne-way ANOVA was employed to compare the differences in sediment physical and chemical properties using SPSS . Non-metric multi-dimensional scaling (NMDS) analysis based on Bray–Curtis dissimilarity and analysis of similarities (ANOSIM) were conducted to evaluate the complexity of the community composition and compare the differences between sampling regions. Both NMDS and ANOSIM were performed in R v.4.0.5 using the “vegan” package. The α-diversity was calculated using the “picante” package. In this study, β-diversity was quantified as the average distance of group members to the group centroid in the vegan package based on the Bray–Curtis distances . Then, wThe sediment physicochemical properties varied among the different sampling regions . SpecifiThe sediment bacterial community composition exhibited a distinct variation among different sampling regions . As showp = 0.001) analysis, these sampling regions contained different bacterial community structures (p < 0.05) indicated significant differences among different sampling regions . This suggested that TS affected sediment bacterial community structure.The assembly process of the microbial community was assessed by the phylogenetic turnover of the bacterial communities, which was primarily evaluated through the β nearest taxon indices (βNTI); the main results are presented in 95 and 1 b. Specif95 and 1 c. Moreov95 and 1 d. We als95 and 1 . The resCo-occurrence networks were constructed to investigate the biotic interactions across microbial communities in different sediment environments . OverallConsidering the roles of biotic and abiotic factors, we conducted partial least squares (PLS) path analysis to explore how these factors determined microbe community diversity in the sediment. First, we examined the correlation between environmental factors and network topological features and community diversity . The resIn our study, we noticed the enrichment of nutrients and carbon at the downstream dam of Xiao Xingkai Lake (region XXLD). This may be due to the reason that despite the input of nutrient-rich water sources for farmland irrigation and drainage, the upstream impact of water flow is not conducive to the deposition of these elements. Alterations in natural flow regimes cause changes in upstream and downstream hydrological and ecological conditions ,42. In tOverall, building dams alters biogeochemical cycles, such as interrupting the flow of organic carbon, altering nutrient balance, and altering oxygen and thermal conditions. The consequences of this may not be immediately apparent. Due to the complexity and uniqueness of aquatic ecosystems, it is difficult to make precise predictions about the impact of a particular dam. Due to the inflow of massive agricultural water in the Xingkai Lake Basin, nutrients have gradually accumulated and enriched in the XXL. Cyanobacteria can thrive in conditions of high nutrient concentration and low water flow . Therefobray) to quantify different community assembly processes, finding that deterministic processes dominated within all the regions. For bacterial communities, stochastic and deterministic processes play important roles in community assembly, with determinism sometimes dominating and stochasticity sometimes more important [Assessing the relative significance of deterministic and stochastic processes on community assembly is important for identifying factors affecting community composition . We emplmportant ,48. Furtmportant ,49. For mportant ; the incmportant . In addimportant ; stochasmportant . One potmportant ,55. To gmportant , it is emportant , organicmportant , salinitmportant , availabmportant , and TN mportant  are impomportant . Sulfidemportant ,63,64. Hmportant ,65,66, wmportant ,67,68. TMicrobial co-occurrence networks provide important insights into microbial community properties. Our results showed that in different regions, the bacterial community exhibited different degrees of network properties, among which region XLD exhibited relatively stronger network interactions. Different environmental factors had a significant impact on these network properties . RelevanIn microbial ecology, the mechanisms of community diversity have been extensively explored . Here, wMicrobial community diversity and phylogeny differences likely result from many different factors, the most critical of which are those characteristics that define the physical and chemical environments of communities . In sediIn our study, we investigated the bacterial community structure and diversity characteristics in the sediments of Xiao Xingkai Lake and Xingkai Lake and how these characteristics responded to changes in the sediment environment. We found that the sediments in the Xingkai Lake Basin exhibited different physicochemical properties under different hydrological conditions, and the impact of the dam significantly lead to the enrichment of carbon, nitrogen, and phosphorus. Proteobacteria, Firmicutes, and Bacteroidetes were the prevalent bacterial phyla in sediments. Variations in nutrient loads , carbon  and pH lead to changes in sediment microbes, with pH being the most important factor determining differences in sediment bacterial community diversity across regions with different hydrological conditions. In conclusion, our findings have important implications for a comprehensive description of the biogeochemical processes of bacteria in sediments and ecological restoration and construction in the Xingkai Lake area. In the future, we should also focus on the impact of lake eutrophication on sediments and microbial communities in Xingkai Lake.


Neural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition.Here, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction.In addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules.We infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs. Neuronal development from iPSC offers the potential to discover molecular pathways governing neurological pathologies and provide screens for target and therapeutic discovery. Furthermore, recent studies have demonstrated that iPSC-derived neurons reflect key aspects of clinical phenotype of the individual from whom the iPSCs were derived in a patient-specific manner , openingvia blockade of BMP/NODAL signaling through release of inhibitors and other inductive and permissive signals originating from the organizer and flanking epidermis  coated plates. Differentiation of iPSCs was carried out as previously described (Three human induced pluripotent stem cell (hiPSC) lines , derived from neurotypic males from ages ranging between 35 to 55 years, were used to generate two time series namely 0–48 h (h) and 0–8 days (d). These iPSC lines were maintained in Essential 8™ medium (Thermo Fisher) without antibiotics at 37°C, 5% COescribed . Brieflyescribed . Thus, T2.2.RNA was extracted at time points: day 0 (before the addition of differentiation media) and days 1, 2, 3, 4, 6 and 8 after induction. RNA was harvested and lysed with Trizol reagent  and isolated by centrifugation with 100% Chloroform, following by 100% isopropanol and lastly by 75% ethanol. The RNA was purified with Qiaquick PCR purification kit  and quantified with the NanoDrop 1,000 Spectrophotometer (Thermo scientific). The quality control was assessed by analyzing the RNA with the Agilent RNA 6000 nano Kit  in combination with the Agilent 2,100 Bioanalyzer system.RNAseq libraries were prepared with the Truseq RNA Kit. Briefly, mRNA was purified from total RNA followed by cDNA synthesis. Subsequently, the cDNA was end-paired, A-tailed and custom indexing adapters were ligated. Samples were size selected and pooled for sequencing. Libraries were multiplexed at 8 samples per lane and sequenced in a Hiseq 2,500 Sequencing System (Ilumina) to a depth of 20–30 million reads per sample.2.3.For CRISPRi experiments, iPSCs bearing a dCas9-KRAB construct under control of the inducible tetO promoter and integrated into the AAVS1 locus  were pas2.4.The primers were designed with Primer3Plus (v. 0.4.0) software . Gene se2.5.Approximately 100,000 cells were plated on each well of Nunclon delta surface 96 well plates (Thermo Fisher148761) and fixed at different time points with 4% paraformaldehyde (PFA)  in PBS 1X for 15 min at room temperature. The wells were washed 3 times with 1X PBS and permeabilized and blocked by incubation with 4% normal donkey serum  in 1X PBST. The nuclei were stained by incubation with Hoechst 33342 . Cells were incubated with specific dilutions of mouse monoclonal and rabbit polyclonal primary antibodies together according to the provider specifications . ValidatImages were acquired with a 20X objective with the Cell insight CX5 High Content Screen Platform . Expression was quantified using the bioapplication Cell Health Profiling from the iDev software package (Thermo Fisher). Hoechst staining was used to assess cell viability. Specific staining intensity, shape and size parameters were established to identify positive and negative labeled cells. A total of 3 wells were analyzed per primary antibody pair with 61 acquired fields per well. The means of the percentages of positive cells at each time point were statistically compared by a two-way ANOVA test with 95% confidence interval with Bonferroni correction with Prism package of GraphPad software.2.6.RNA-seq fastq files were aligned to the Human Reference Genome (version hg38) and FPKM counts (Refgene) were generated using the Omicsoft Aligner OSA; . Only ge2.7.Protein–protein interaction (PPI) analysis was performed using STRING database in Cytoscape.2.8.TRANSFAC® data was used initially as the source for Transcription factor (TF)–target gene information. However, curation of TRANSFAC was over-represented by the large number of immune system and disease studies, thus the analysis using TRANSFAC data produced spurious results indicating a major role of JUN, FOS and STATs in neural induction. To overcome this problem, ChIP-seq data on human embryonic stem cells (ESCs) from 3.3.1.In this study, transcriptomic data were harvested throughout 0–48 h and 0–8 days of neural induction of human iPSCs. These datasets were then used to infer the transcriptional regulatory mechanisms operative throughout the early stages of neural induction. Monotonically increasing number of DE genes  in both Stem cell differentiation is accompanied by the increase in reactive oxygen species (ROS) and oxidative stress  and thisTo understand the temporal transcriptional regulation of neural induction, knowledge-based lists of pluripotency genes, neural induction transcription factors and neuroectoderm markers were curated  and anal3.2.We further constructed PPI networks using all DE TFs to provide a global view of the dynamic changes in transcriptional regulation throughout neural induction. In 0–48 h dataset , upregulMany of the functional modules identified in the 0–48 h time series were also evident in the 0–8 days time series, but strikingly the TF membership of the modules was only partially conserved . Thus, p3.3.Our studies identified OTX2 as one of the most precociously expressed transcription factors during neural induction . OTX2 isIn order to validate the causality implied by our analysis of the dynamic transcriptional landscape during neural induction, we used CRISPRi to knock down (KD) expression of OTX2 , indicat4.During the course of cortical neural induction, iPSCs go through a series of cell states, starting from the pluripotent stem cells and culminating in multipotent neuroectodermal stem cells, which then undergo further maturation before terminal differentiation into cortical neurons. Our results show the transcriptional and regulatory mechanisms that are associated with this transition across time. BMP signaling is required for the maintenance of pluripotency in human stem cells  and blocvia control of central carbon metabolism through the pentose phosphate pathway and glutamine metabolism  induced by specific mutations in PSEN1, PSEN2 or APP proteins. It has been established that transcription factors NRF2 and REST play a crucial role in defining cell fate outcomes in FAD , 2022. NMuch of our understanding of the gene regulatory pathways underlying the initial steps of neuronal development is largely based on animal models, particularly in Xenopus and mouse. We summarize here, some of the most salient observations. In Xenopus, Foxd4 is expressed in the neuroectodermal precursors and promotes the expression of Gmnn, Sox11 and Zic2. Foxd4, Gmnn, Zic2 and Sox11 block non-neural induction by inhibiting BMP/WNT pathways and their targets . The defStudies in human iPSCs have enabled identification of important markers and drivers of neural differentiation including PAX6, ZEB2, ZNF521, NESTIN and NR2F2 . In humahttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223624 accession ID: GSE223624.The original contributions presented in the study are publicly available. Data is submitted in GEO repository. This data can be found here: SG: data analysis and drafting of manuscript. LP: data acquisition. MF: data acquisition and analysis. HR: data acquisition and critiquing of manuscript. DM: data analysis. NB: experimental design and interpretation, drafting, and critiquing of manuscript. SS: data analysis and interpretation, drafting, and critiquing of manuscript. All authors contributed to the article and approved the submitted version.The authors wish to acknowledge funding support from NIH grants, R01 LM012595 (SS), OT2 OD030544 (SS), R01 HL106579-07 (SS) and the Joan and Irwin Jacobs endowed professorship (SS), SENESCYT PhD scholarship (LP), and Mano Maurya in the Subramaniam laboratory for discussions on analytical methods used.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.