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Understanding of the present-day genetic diversity, population structure, and evolutionary history of tree species can inform resource management and conservation activities, including response to pressures presented by a changing climate. Cercis canadensis (Eastern Redbud) is an economically valuable understory tree species native to the United States (U.S.) that is also important for forest ecosystem and wildlife health. Here, we document and explain the population genetics and evolutionary history of this deciduous tree species across its distributed range. In this study, we used twelve microsatellite markers to investigate 691 wild-type trees sampled at 74 collection sites from 23 Eastern U.S. states. High genetic diversity and limited gene flow were revealed in wild, natural stands of C. canadensis with populations that are explained by two major genetic clusters. These findings indicate that an ancient population bottleneck occurred coinciding with the last glacial maximum (LGM) in North America. The structure in current populations likely originated from an ancient population in the eastern U.S. that survived LGM and then later diverged into two contemporary clusters. Data suggests that populations have expanded since the last glaciation event from one into several post-glacial refugia that now occupy this species’ current geographic range. Our enhanced understanding benchmarks the genetic variation preserved within this species and can direct future efforts in conservation, and resource utilization of adaptively resilient populations that present the greatest genetic and structural diversity.

Chinese alligator (Alligator sinensis) is an endangered freshwater crocodilian endemic to China, which experienced a severe bottleneck about 30 years ago. In this study, we developed locus-specific primers to investigate the polymorphism of 3 major histocompatibility complex (MHC) loci in 3 Chinese alligator populations, in combination with 6 neutral microsatellite markers as a contrast. We found the genetic trace for the bottleneck effect on the endangered Chinese alligator: the low allelic diversity (2 alleles at each locus), the low nucleotide substitution rate (no more than 0.009) at all sites, the deviation from Hardy-Weinberg Equilibrium/heterozygote deficiency, and the significant Tajima’s D values, indicating the MHC class I and class II loci being at different stages of bottleneck. We also obtained 3 pieces of evidence for balancing selection on this severely bottlenecked reptile: an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions, the mean synonymous substitution rate of MHC exons significantly higher than mean nucleotide substitution rate of introns, and the differentiation coefficient FST of MHC loci significantly lower than that of microsatellite loci. Consequently, we emphasize that the Chinese alligator holds a pretty low adaptive ability and requires scientific conservation strategies to ensure the long-term population development.

The Tibetan antelope (chiru, Pantholops hodgsoni) is one of the most endangered mammals native to the Qinghai-Tibetan Plateau. The population size has rapidly declined over the last century due to illegal hunting and habitat damage. In the past 10 years, the population has reportedly been expanding due to conservation efforts. Several lines of evidence suggest that the Tibetan antelope has undergone a demographic bottleneck. However, the consequences of the bottleneck on genetic diversity and the post-bottleneck genetic recovery remain unknown. In this study, we investigate the genetic variation of 15 microsatellite loci from two Tibetan antelope populations sampled in 2003 (Pop2003) and 2013 (Pop2013). A higher level of genetic diversity (NA, 13.286; He, 0.840; PIC, 0.813; I, 2.114) was detected in Pop2013, compared to Pop2003 (NA, 12.929; He, 0.818; PIC, 0.789; I, 2.033). We observe that despite passing through the bottleneck, the Tibetan antelope retains high levels of genetic diversity. Furthermore, our results show significant or near significant increases in genetic diversity (He, PIC and I) in Pop2013 compared with Pop2003, which suggests that protection efforts did not arrive too late for the Tibetan antelope.

MicroRNAs (miRNAs) are small non-coding RNA molecules whose primary function is to regulate the expression of gene products via hybridization to mRNA transcripts, resulting in suppression of translation or mRNA degradation. Although miRNAs have been implicated in complex diseases, including cancer, their impact on distinct biological pathways and phenotypes is largely unknown. Current integration approaches require sample-matched miRNA/mRNA datasets, resulting in limited applicability in practice. Since these approaches cannot integrate heterogeneous information available across independent experiments, they neither account for bias inherent in individual studies, nor do they benefit from increased sample size. Here we present a novel framework able to integrate miRNA and mRNA data (vertical data integration) available in independent studies (horizontal meta-analysis) allowing for a comprehensive analysis of the given phenotypes. To demonstrate the utility of our method, we conducted a meta-analysis of pancreatic and colorectal cancer, using 1,471 samples from 15 mRNA and 14 miRNA expression datasets. Our two-dimensional data integration approach greatly increases the power of statistical analysis and correctly identifies pathways known to be implicated in the phenotypes. The proposed framework is sufficiently general to integrate other types of data obtained from high-throughput assays.

Bison (Bison bison) are one of the few terrestrial megafauna to survive the transition into the Holocene and provide a unique opportunity to study a species on a broad spatiotemporal scale. Today, bison are primarily managed in small and isolated herds with little known about their ancestral ecology. We studied the carbon and nitrogen isotopes of Northern Great Plains bison from the terminal Pleistocene and throughout the Holocene to gain insight into their paleoecology. This time span is contemporary with the first population bottleneck experienced by bison at the end of the Pleistocene and includes the second bottleneck which occurred in the late 19th century. Results were compared with modern bison herd isotopic values from Theodore Roosevelt National Park (TRNP). Patterns of isotopic variation found in bison over time indicate significant (δ13C p = 0.0008, δ15N p = 0.002) differences in diet composition and correlate with climate throughout the Holocene. Isotopic relationships described here reveal the plasticity of ancient bison in unrestricted rangelands during periods of climatic fluctuations. Managers at TRNP and elsewhere should pursue opportunities to expand bison range to maximize forage opportunities for the species in the face of future environmental change.

Genetic diversity is the raw foundation for evolutionary potential. When genetic diversity is significantly reduced, the risk of extinction is heightened considerably. The long-snouted seahorse (Hippocampus guttulatus) is one of two seahorse species occurring in the North-East Atlantic. The population living in the Ria Formosa (South Portugal) declined dramatically between 2001 and 2008, prompting fears of greatly reduced genetic diversity and reduced effective population size, hallmarks of a genetic bottleneck. This study tests these hypotheses using samples from eight microsatellite loci taken from 2001 and 2013, on either side of the 2008 decline. The data suggest that the population has not lost its genetic diversity, and a genetic bottleneck was not detectable. However, overall relatedness increased between 2001 to 2013, leading to questions of future inbreeding. The effective population size has seemingly increased close to the threshold necessary for the population to retain its evolutionary potential, but whether these results have been affected by sample size is not clear. Several explanations are discussed for these unexpected results, such as gene flow, local decline due to dispersal to other areas of the Ria Formosa, and the potential that the duration of the demographic decline too short to record changes in the genetic diversity. Given the results presented here and recent evidence of a second population decline, the precise estimation of both gene flow and effective population size via more extensive genetic screening will be critical to effective population management. 16-02-01-FMP-54, MAR-01.04.02-FEAMP-0029, UID/Multi/04326/2020 info:eu-repo/semantics/publishedVersion

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Maintaining standing genetic variation is a challenge in human-dominated landscapes. We used genetic (i.e., 16 short tandem repeats) and morphological (i.e., length and weight) measurements of 593 contemporary and historical brown trout (Salmo trutta) samples to study fine-scale and short-term impacts of different management practices. These had changed from traditional breeding practices, using the same broodstock for several years, to modern breeding practices, including annual broodstock replacement, in the transnational subarctic Pasvik River. Using population genetic structure analyses (i.e., Bayesian assignment tests, DAPCs, and PCAs), four historical genetic clusters (E2001A-D), likely representing family lineages resulting from different crosses, were found in zone E. These groups were characterized by consistently lower genetic diversity, higher within-group relatedness, lower effective population size, and significantly smaller body size than contemporary stocked (E2001E) and wild fish (E2001F). However, even current breeding practices are insufficient to prevent genetic diversity loss and morphological changes as demonstrated by on average smaller body sizes and recent genetic bottleneck signatures in the modern breeding stock compared to wild fish. Conservation management must evaluate breeding protocols for stocking programs and assess if these can preserve remaining natural genetic diversity and morphology in brown trout for long-term preservation of freshwater fauna.

The bald eagle (Haliaeetus leucocephalus) once experienced near-extinction but has since rebounded. For decades, bald eagles near the Wisconsin River, USA, have experienced a lethal syndrome with characteristic clinical and pathological features but unknown etiology. Here, we describe a novel hepacivirus-like virus (Flaviviridae: Hepacivirus) identified during an investigation of Wisconsin River eagle syndrome (WRES). Bald eagle hepacivirus (BeHV) belongs to a divergent clade of avian viruses that share features with members of the genera Hepacivirus and Pegivirus. BeHV infected 31.9% of eagles spanning 4,254 km of the coterminous USA, with negative strand viral RNA demonstrating active replication in liver tissues. Eagles from Wisconsin were approximately 10-fold more likely to be infected than eagles from elsewhere. Eagle mitochondrial DNA sequences were homogeneous and geographically unstructured, likely reflecting a recent population bottleneck, whereas BeHV envelope gene sequences showed strong population genetic substructure and isolation by distance, suggesting localized transmission. Cophylogenetic analyses showed no congruity between eagles and their viruses, supporting horizontal rather than vertical transmission. These results expand our knowledge of the Flaviviridae, reveal a striking pattern of decoupled host/virus coevolution on a continental scale, and highlight knowledge gaps about health and conservation in even the most iconic of wildlife species.

Socioeconomic and climatic factors are modifying fire regimes with an increase of fire frequency and extension. Unfortunately, the effects of recurrent fires on biological processes that ultimately affect the genetic diversity of animal populations are mostly unknown. We examined genetic patterns of diversity in the wall lizard Podarcis guadarramae in northern Portugal, one of the European regions with the highest percentage of burnt land. This species is a small saxicolous lizard as it inhabits natural outcrops and artificial stone walls, likely in recurrent-fire landscapes. We genotyped nine microsatellites from ten populations selected according to a gradient in fire recurrence, and compared genetic diversity indexes and demographic patterns among them. At the population level, we hypothesize that a high level of mortality and population bottlenecks are expected to reduce genetic heterozygosity in sampled localities affected by recurrent fires. Alternatively, genetic signatures are expected to be absent whether fire did not cause high mortality. Regardless of levels of mortality, we expect a gain in genetic diversity whether recurrent fires facilitate lizard dispersal and migration due to the increased quality of the habitat for wall lizards. At the regional level, we examine whether a recurrent fire regime may disrupt the spatial structure of populations. Our results showed an increase in genetic diversity in recurrently burnt populations, and a decline in longer-unburnt populations. We did not detect bottleneck effects in repeatedly-burnt populations. High genetic diversity in recurrent fire populations suggests a high dispersion rate between adjacent metapopulations and perhaps immigration from outside the fire boundary. At the regional level, lizard populations show low differentiation and weak genetic structure, suggesting no effects of fire. This study confirms field-based censuses showing that recurrent-fire regimes give ecological opportunities to wall lizards that benefit from habitat openness.

The gharial (Gavialis gangeticus) is a critically endangered crocodylian, endemic to the Indian subcontinent. The species has experienced severe population decline during the twentieth century owing to habitat loss, poaching, and mortalities in passive fishing. Its extant populations have largely recovered through translocation programmes initiated in 1975. Understanding the genetic status of these populations is crucial for evaluating the effectiveness of the ongoing conservation efforts. This study assessed the genetic diversity, population structure, and evidence of genetic bottlenecks of the two managed populations inhabiting the Chambal and Girwa Rivers, which hold nearly 80% of the global gharial populations. We used seven polymorphic nuclear microsatellite loci and a 520 bp partial fragment of the mitochondrial control region (CR). The overall mean allelic richness (Ar) was 2.80 ± 0.40, and the observed (Ho) and expected (He) heterozygosities were 0.40 ± 0.05 and 0.39 ± 0.05, respectively. We observed low levels of genetic differentiation between populations (FST = 0.039, P G’ST = 0.058, P D = 0.016, P M ratio (Chambal = 0.31 ± 0.06; Girwa = 0.41 ± 0.12) suggested the presence of a genetic bottleneck in both populations. The mitochondrial CR also showed a low level of variation, with two haplotypes observed in the Girwa population. This study highlights the low level of genetic diversity in the two largest managed gharial populations in the wild. Hence, it is recommended to assess the genetic status of extant wild and captive gharial populations for planning future translocation programmes to ensure long-term survival in the wild.

Aphis gossypii, one of the most important agricultural pests in the world, can cause serious economic losses in the main crop-producing areas. To clarify issues such as the genetic differentiation, genetic structure, and demographic history of A. gossypii populations, we used 10 nuclear microsatellite loci (SSR) and two mitochondrial gene sequences (COI and Cytb) to investigate genetic diversity and population structure of A. gossypii populations that were collected from 33 sampling sites in China from different climatic zones. SSR and mtDNA data suggested low to moderate levels of genetic diversity. A star-shaped network of mtDNA haplotypes indicated that the maternal ancestor of China cotton aphids likely originated in Xinjiang. The POPTREE, STRUCTURE and principal coordinate analysis (PCoA) revealed two genetic clusters: an eastern and a western region group. Isolation by distance (IBD) results showed a positive correlation between geographic distance and genetic distance in the vast eastern region but not in the western region. Neutrality testing and mismatch distribution analysis provided strong evidence for a recent rapid expansion in most populations. Genetic bottleneck was not detected in A. gossypii populations of China. The present work can help us to develop strategies for managing this pest.

Characterising genetic diversity and structure of populations is essential for effective conservation of threatened species. The Greater Spotted Eagle (Clanga clanga), a large and globally vulnerable raptor, is extinct or in severe decline in most of its previous range in Europe. We assessed whether the remnants of European population are genetically impoverished, and isolated from each other. We evaluated levels of genetic diversity and population structuring by sequencing mitochondrial pseudo-control region and 10 introns from various nuclear genes, and estimated length diversity in 23 microsatellite markers. The European population has expanded since the late Pleistocene, and does not exhibit signs of a recent population bottleneck. The global genetic diversity in Europe was rather similar to that detected in other similar species. Microsatellites suggested shallow but significant differentiation between the four extant populations in Estonia, Poland, Belarus and Russia (Upper Volga region) populations, but introns and mtDNA showed that only the Estonian population differed from the others. Mitochondrial diversity was highest in the northernmost Estonian population, introns suggested lower diversity in Upper Volga, microsatellites indicated equal diversity among populations. A recent bottleneck was detected in Poland, which is consistent with the observed repopulation of the region. We conclude that significant gene flow and high genetic diversity are retained in the fragmented Greater Spotted Eagle populations; there is currently no need for genetic augmentation in Europe.

Although largemouth bass Micropterus salmoides has shown its extremely economic, ecological, and aquacultural significances throughout the North American and Asian continents, systematic evaluation of genetic variation and structure of wild and cultured populations of the species is yet to be documented. In this study, we investigated the genetic structure of M. salmoides from 20 wild populations and five cultured stocks across the United States and China using eight microsatellite loci, which are standard genetic markers for population genetic analysis. Our major findings are as follows: (1) the result of Fst showed largemouth bass had high genetic differentiation, and the gene flow indicated the genetic exchange among wild populations is difficult; (2) AMOVA showed that 14.05% of the variation was among populations, and 85.95% of the variation was within populations; (3) The majority of largemouth bass populations had a significant heterozygosity excess, which is likely to indicate a previous population bottleneck; (4) Allelic richness was lower among cultured populations than among wild populations; (5) Effective population size in hatcheries could promote high levels of genetic variation among individuals and minimize loss of genetic diversity; China’s largemouth bass originated from northern largemouth bass of USA. The information provides valuable basis for development of appropriate conservation policies for fisheries and aquaculture genetic breeding programs in largemouth bass.

The fire blight pathogen, Erwinia amylovora (EA), causes significant economic losses in rosaceae fruit crops. Recent genome sequencing efforts have explored genetic variation, population structure, and virulence levels in EA strains. However, the genomic aspects of population bottlenecks and selection pressure from geographical isolation, host range, and management practices are yet unexplored. We conducted a comprehensive analysis of whole genome sequences of 41 strains to study genetic diversity, population structure, and the nature of selection affecting sub-population differentiation in EA. We detected 72,741 SNPs and 2,500 Indels, representing about six-fold more diversity than previous reports. Moreover, nonsynonymous substitutions were identified across the effector regions, suggesting a role in defining virulence of specific strains. EA plasmids had more diversity than the chromosome sequence. Population structure analysis identified three distinct sub-groups in EA strains, with North American strains displaying highest genetic diversity. A five kilobase genomic window scan showed differences in genomic diversity and selection pressure between these three sub-groups. This analysis also highlighted the role of purifying and balancing selection in shaping EA genome structure. Our analysis provides novel insights into the genomic diversity and selection forces accompanying EA population differentiation.

Detecting how historical and contemporary factors contribute to genetic divergence and genetic structure is a central question in ecology and evolution. We examine this question by intergrating population genetics with ecological niche modelling of Litsea auriculata (Lauraceae), which is endangered and native to east China. Geographical and environmental factors including climatic fluctuations since the last glacial maximum (LGM) have also contribute to population demography and patterns of genetic structure. L. auriculata populations underwent expansion after divergence and dramatically decreased to the current small size with relative population bottlenecks due to climate changes. Populations separated by physical geographical barrier including geographic distance and Yangtze River, as a result contemporary gene flow among L. auriculata populations showed drastic declines in comparison with historical gene flow, resulting in a high level of population divergence. Thus, patterns of genetic structure of L. auriculata can result from both geographic and environmental factors including climate changes. This information is helpful in forming conservation strategies for L. auriculata in China.

Since domestication, population bottlenecks, breed formation, and selective breeding have radically shaped the genealogy and genetics of Bos taurus. In turn, characterization of population structure among diverse bull (males of Bos taurus) genomes enables detailed assessment of genetic resources and origins. By analyzing 432 unrelated bull genomes from 13 breeds and 16 countries, we demonstrate genetic diversity and structural complexity among the European/Western cattle population. Importantly, we relaxed a strong assumption of discrete or admixed population, by adapting latent variable models for individual-specific allele frequencies that directly capture a wide range of complex structure from genome-wide genotypes. As measured by magnitude of differentiation, selection pressure on SNPs within genes is substantially greater than that on intergenic regions. Additionally, broad regions of chromosome 6 harboring largest genetic differentiation suggest positive selection underlying population structure. We carried out gene set analysis using SNP annotations to identify enriched functional categories such as energy-related processes and multiple development stages. Our population structure analysis of bull genomes can support genetic management strategies that capture structural complexity and promote sustainable genetic breadth.

Elementary flux modes (EFMs) are non-decomposable steady-state pathways in metabolic networks. They characterize phenotypes, quantify robustness or identify engineering targets. An EFM analysis (EFMA) is currently restricted to medium-scale models, as the number of EFMs explodes with the network's size. However, many topologically feasible EFMs are biologically irrelevant. We present thermodynamic EFMA (tEFMA), which calculates only the small(er) subset of thermodynamically feasible EFMs. We integrate network embedded thermodynamics into EFMA and show that we can use the metabolome to identify and remove thermodynamically infeasible EFMs during an EFMA without losing biologically relevant EFMs. Calculating only the thermodynamically feasible EFMs strongly reduces memory consumption and program runtime, allowing the analysis of larger networks. We apply tEFMA to study the central carbon metabolism of E. coli and find that up to 80% of its EFMs are thermodynamically infeasible. Moreover, we identify glutamate dehydrogenase as a bottleneck, when E. coli is grown on glucose and explain its inactivity as a consequence of network embedded thermodynamics. We implemented tEFMA as a Java package which is available for download at https://github.com/mpgerstl/tEFMA.

The White-winged Flufftail (Sarothrura ayresi) population is listed as globally Critically Endangered. White-winged Flufftails are only known to occur, with any regularity, in the high-altitude wetlands of South Africa and Ethiopia. Threats to the species include the limited number of suitable breeding sites in Ethiopia and severe habitat degradation and loss both in Ethiopia and South Africa. Toll-like receptors (TLRs) are increasingly being studied in a variety of taxa as a broader approach to determine functional genetic diversity. In this study, we confirm low genetic diversity in the innate immune regions of the White-winged Flufftail similar to that observed in other bird species that have undergone population bottlenecks. Low TLR diversity in White-winged Flufftail indicates that this species is more likely to be threatened by changes to the environment that would potentially expose the species to new diseases. Thus, conservation efforts should be directed towards maintaining pristine habitat for White-winged Flufftail in its current distribution range. To date, no studies on immunogenetic variation in White-winged Flufftail have been conducted and to our knowledge, this is the first study of TLR genetic diversity in a critically endangered species.

Gayal (Bos frontalis) is a semi-wild and endangered bovine species that differs from domestic cattle (Bos taurus and Bos indicus) and its genetic background remains unclear. Here, we performed whole-genome sequencing of one Gayal for the first time, with one Red Angus cattle and one Japanese Black cattle as controls. In total, 97.8 Gb of sequencing reads were generated with an average 11.78-fold depth and >98.44% coverage of the reference sequence (UMD3.1). Numerous different variations were identified, 62.24% of the total single nucleotide polymorphisms (SNPs) detected in Gayal were novel and 16,901 breed-specific nonsynonymous SNPs (BS-nsSNPs) that might be associated with traits of interest in Gayal were further investigated. Moreover, the demographic history of bovine species was first analyzed and two population expansions and two population bottlenecks were identified. The obvious differences among their population sizes supported that Gayal was not B. taurus. The phylogenic analysis suggested that Gayal was a hybrid descendant from crossing of male wild gaur and female domestic cattle. These discoveries will provide valuable genomic information regarding potential genomic markers that could predict traits of interest for breeding programs of these cattle breeds and may assist relevant departments with future conservation and utilization of Gayal.

Near Eastern wild boars possess a characteristic DNA signature. Unexpectedly, wild boars from Israel have the DNA sequences of European wild boars and domestic pigs. To understand how this anomaly evolved, we sequenced DNA from ancient and modern pigs from Israel. Pigs from Late Bronze Age (until ca. 1150 BCE) in Israel shared haplotypes of modern and ancient Near Eastern pigs. European haplotypes became dominant only during the Iron Age (ca. 900 BCE). This raises the possibility that European pigs were brought to the region by the Sea Peoples who migrated to the Levant at that time. Then, a complete genetic turnover took place, most likely because of repeated admixture between local and introduced European domestic pigs that went feral. Severe population bottlenecks likely accelerated this process. Introductions by humans have strongly affected the phylogeography of wild animals, and interpretations of phylogeography based on modern DNA alone should be taken with caution.

Naturally occurring stable isotope ratios in animal tissues allow estimation of species trophic position and ecological niche. Measuring multiple isotopes of migratory species along flyway bottlenecks offers the opportunity to sample multiple populations and species whose tissues carry information at continental scales.We measuredδ2H,δ18O,δ13C,δ15N in juvenile feathers of 21 bird species captured at a migratory bottleneck in the Italian Alps.We examined if trends in individual isotopes reflected known migratory strategies and whether dietary (δ13C-δ15N) and spatially-explicit breeding origin (δ2H-δ18O) niche breadth (NB) differed among long-distance trans-Saharan (TS), short-distance (IP) and irruptive (IR) intra-Palearctic migrants, and whether they correlated with reported populations long-term trends.In both TS and IP groups, speciesδ2H declined with capture date, indicating that northern populations reached the stopover site later in the season, following a Type-I migration strategy. Values ofδ2H indicated that breeding range of TS migrants extended farther north than IP and IR migrants. The breeding season was longer for IP migrants whoseδ13C andδ15N values declined and increased, respectively, with time of capture.Average species dietary NB did not differ among migratory groups, but TS migrants displayed wider breeding origin niches, suggesting that long-distant migration is linked to broader ecological niches. Isotope origin NB well reflected species geographic range extent, while dietary NB did not correlate with literature accounts of species’ diet. We found no relationship between species breeding NB and population trends in Europe, implying that conditions in the wintering grounds could be more important for the long-term persistence of species.We demonstrate that isotopic measurements of passerines migrating through a bottleneck represents a unique opportunity to investigate large-scale life-history phenomena relevant to conservation.

The Griffon vulture was once a widespread species across the region of Southeast Europe, but it is now endangered and in some parts is completely extinct. In the Balkan Peninsula the largest Griffon vulture inland population inhabits the territory of Serbia. We present, for the first time, the genetic data of this valuable population that could be a source for future reintroduction programs planned in South-eastern Europe. To characterize the genetic structure of this population we used microsatellite markers from ten loci. Blood samples were collected from 57 chicks directly in the nests during the ongoing monitoring program. We performed a comparative analysis of the obtained data with the existing data from three native populations from French Pyrenees, Croatia, and Israel. We have assessed the genetic differentiation between different native populations and determined the existence of two genetic clusters that differentiate the populations from the Balkan and Iberian Peninsulas. Furthermore, we analysed whether the recent bottleneck events influenced the genetic structure of the populations studied, and we found that all native populations experienced a recent bottleneck event, and that the population of Israel was the least affected. Nevertheless, the parameters of genetic diversity suggest that all analysed populations have retained a similar level of genetic diversity and that the Griffon vulture population from Serbia exhibits the highest value for private alleles. The results of this study suggest that the Griffon vulture populations of the Balkan Peninsula are genetically differentiated from the populations of the Iberian Peninsula, which is an important information for future reintroduction strategies.

Despite full immunoprophylaxis, mother-to-child transmission (MTCT) of Hepatitis B Virus still occurs in approximately 2–5% of HBsAg positive mothers. Little is known about the bottleneck of HBV transmission and the evolution of viral quasispecies in the context of MTCT. Here we adopted a newly developed tag linkage deep sequencing method and analyzed the quasispecies of four MTCT pairs that broke through immunoprophylaxis. By assigning unique tags to individual viral sequences, we accurately reconstructed HBV haplotypes in a region of 836 bp, which contains the major immune epitopes and drug resistance mutations. The detection limit of minor viral haplotypes reached 0.1% for individual patient sample. Dominance of “a determinant” polymorphisms were observed in two children, which pre-existed as minor quasispecies in maternal samples. In all four pairs of MTCT samples, we consistently observed a significant overlap of viral haplotypes shared between mother and child. We also demonstrate that the data can be potentially useful to estimate the bottleneck effect during HBV MTCT, which provides information to optimize treatment for reducing the frequency of MTCT.

According to the nearly-neutral theory of evolution, the relative strengths of selection and drift shift in favour of drift at small population sizes. Numerous studies have analysed the effect of bottlenecks and small population sizes on genetic diversity in the MHC, which plays a central role in pathogen recognition and immune defense and is thus considered a model example for the study of adaptive evolution. However, to understand changes in genetic diversity at loci under selection, it is necessary to compare the genetic diversity of a population before and after the bottleneck. In this study, we analyse three fragments of the MHC DQA gene in woolly mammoth samples radiocarbon dated to before and after a well-documented bottleneck that took place about ten thousand years ago. Our results indicate a decrease in observed heterozygosity and number of alleles, suggesting that genetic drift had an impact on the variation on MHC. Based on coalescent simulations, we found no evidence of balancing selection maintaining MHC diversity during the Holocene. However, strong trans-species polymorphism among mammoths and elephants points to historical effects of balancing selection on the woolly mammoth lineage.

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

Broomcorn millet (Panicum miliaceum L.) was domesticated in northern China at least 7,000 years ago and was subsequentially adopted as a cereal in many areas throughout Eurasia. One such locale is Areni-1 an archaeological cave site in Southern Armenia, a region that has an important history in crop domestication. The rich botanical material found at Areni-1 includes grains identified by morphology as broomcorn millet that were 14C dated to the medieval era (873 ± 36 CE and 1118 ± 35 CE). To retrace the demographic history of these broomcorn millet samples, we used ancient DNA extraction and hybridization capture enrichment to sequence and assemble three chloroplast genomes from the Areni-1 grains and then compared these sequences to 50 modern chloroplast genomes. Overall, the chloroplast genomes contained a low amount of diversity and little inference on broomcorn demography could be made. However, in a phylogeny the chloroplast genomes separated into two clades with strong bootstrap support, similar to what has been reported for nuclear DNA from broomcorn millet. In a haplotype network, the chloroplast genomes of two accessions of wild (undomesticated) broomcorn millet contained a relatively large number of variants, 11 SNPs. These SNPs were not present in the domesticated varieties, suggesting these wild accessions may not be directly related to the lineages that underwent domestication or that broomcorn millet may have undergone a domestication bottleneck resulting in lost diversity in the chloroplast genome. These results demonstrate that broomcorn millet from archaeological sites can preserve DNA for at least 1000 years and serve as a genetic resource to study the domestication of this cereal crop.

Rhynchaenus maculosus is an emerging insect pest with an increasingly serious tendency. Lack of biology information results in the bottleneck of integrated management of this pest. To facilitate an available design of integrated pest management strategy, biology of R. maculosus, including voltinism, life cycle, distribution, and damage has been investigated. Results reveal that R. maculosus is oligophagous and distributes in Heilongjiang, Jilin, and Liaoning provinces, China. This pest produces one generation per year (univoltinism) and overwinters as adults in leaf litter. From mid-April to late-April, active overwintering adults emerge from overwintering sites. The next generation of adult R. maculosus appears from mid-May to early June until mid-August to early September when the beetles move into the overwintering places. The entire time span of adult occurrence ranges from 315.6 ± 3.6 to 336.4 ± 3.2 days (Mean ± SD). Larvae undergo 3 instars with a total duration of 20 to 23 days. R. maculosus larvae feed on Q. wutaishanica and Q. mongolica without host-specific preference between the two host species, but do not harm Q. acutissim. Three species of larval parasites were collected and identified as Braconidae sp., Eulophidae sp., and Ceraphronidae sp. Biological information of R. maculosus provides essential insights for design and implementation of integrated management of this pest.

Lignin is a heterogeneous aromatic polymer responsible for cell wall stiffness and protection from pathogen attack. However, lignin represents a bottleneck to biomass degradation due to its recalcitrance related to the natural cell wall resistance to release sugars for fermentation or further processing. A biological approach involving genetics and molecular biology was used to disrupt lignin pathway synthesis and decrease lignin deposition. Here, we imaged three-dimensional fragments of the petioles of wild type and C4H lignin mutant Arabidopsis thaliana plants by synchrotron cryo-ptychography. The three-dimensional images revealed the heterogeneity of vessels, parenchyma, and fibre cell wall morphologies, highlighting the relation between disturbed lignin deposition and vessel implosion (cell collapsing and obstruction of water flow). We introduce a new parameter to accurately define cell implosion conditions in plants, and we demonstrate how cryo-ptychographic X-ray computed tomography (cryo-PXCT) provides new insights for plant imaging in three dimensions to understand physiological processes.

The Japanese macaque (Macaca fuscata) is native to the main islands of Japan, except Hokkaido, and is the most northerly living non-human primate. In the Chubu Sangaku National Park of the Japanese Alps, macaques live in one of the coldest areas of the world, with snow cover limiting the availability of preferred food sources. Winter is typically a bottleneck for food availability potentially resulting in marked energy deficits, and mortality may result from famine. However, streams with groundwater upwelling flow during the winter with a constant water temperature of about 5 °C are easily accessible for Japanese macaques to search for riverine biota. We used metabarcoding (Cytochrome c oxidase I) of fecal samples from Japanese macaques to determine their wintertime diet. Here we provide the first robust evidence that Japanese macaques feed on freshwater biota, including brown trout, riverine insects and molluscs, in Chubu Sangaku National Park. These additional food sources likely aid their winter survival.

Genome sequencing has reinvigorated the infectious disease research field, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from different lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversification in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identified and confirmed by two independent methods with solid support. Still, recombination reveals a weaker effect on M. bovis diversity compared with mutation (overall r/m = 0.037). The differential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further effort in future comparative genomics studies for which WGS of large datasets from different epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n = 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identification of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.

Typically, large ungulates show a single seasonal peak of heart rate, a proxy of energy expenditure, in early summer. Different to other large ungulates, wild boar females had peak heart rates early in the year (at ~ April, 1), which likely indicates high costs of reproduction. This peak was followed by a trough over summer and a secondary summit in autumn/early winter, which coincided with the mast seeding of oak trees and the mating season. Wild boars counteracted the effects of cold temperatures by decreasing subcutaneous body temperature by peripheral vasoconstriction. They also passively gained solar radiation energy by basking in the sun. However, the shape of the seasonal rhythm in HR indicates that it was apparently not primarily caused by thermoregulatory costs but by the costs of reproduction. Wild boar farrow early in the year, visible in high HRs and sudden changes in intraperitoneal body temperature of females. Arguably, a prerequisite for this early reproduction as well as for high energy metabolism over winter is the broad variety of food consumed by this species, i.e., the omnivorous lifestyle. Extremely warm and dry summers, as experienced during the study years (2017, 2018), may increasingly become a bottleneck for food intake of wild boar.

The history of the domestication of rice is controversial, as it remains unknown whether domestication processes occurred once or multiple times. To date, genetic architecture and phylogenetic studies based on the rice nuclear genome have been extensively studied, but the results are quite different. Here, we found interesting results for different selections in Oryza sativa based on comprehensive studies of the rice mitochondrial (mt) genome. In detail, 412 rice germplasms were collected from around the world for variant architecture studies. A total of 10632 variants were detected in the mt genome, including 7277 SNPs and 3355 InDels. Selection signal (πw/πc) indicated that the selection sites in Oryza sativa L. ssp. japonica were different from those of Oryza sativa L. indica rice. The fixation index (FST) was higher between indica and japonica than between indica and wild rice. Moreover, haplotype and phylogenetic analyses also revealed indica clusters and japonica clusters that were well separated from wild rice. As mentioned above, our studies indicate that the selection sites of the indica type were different from those of the japonica type. This means that indica and japonica have experienced different domestication processes. We also found that japonica may have experienced a bottleneck event during domestication.

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

Color is a widely used communication channel in the living world for a variety of functions ranging from sexual communication to warning colors. A particularly rich spectrum of colors appears on the wings of many butterflies. The males of lycaenid butterflies often exhibit a conspicuous blue coloration generated by photonic nanoarchitectures on their dorsal wing surfaces. Using UV-VIS spectroscopy, we investigated the spatio-temporal variations of this coloration for Polyommatus icarus butterflies, considering an interval of more than 100 years and a geographical range spanning Europe (west) and Asia (east). The blue coloration in Hungary is very stable both within a year (three broods typical in Hungary) and within the period of 100 years (more than 300 generations). East-west geographic variation was investigated among 314 male P. icarus butterflies. In agreement with earlier genetic and morphometric studies, it was found that the western males are not divided in distinct lineages. Clear differences in coloration were found between the eastern and western groups, with a transition in the region of Turkey. These differences are tentatively attributed to bottleneck effects during past glaciations.

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

Mesenchymal stromal cells (MSC) are used in cell therapy, but results depend on the unknown quality of cell populations. Extended culture time of MSC increases their senescent levels, leading to a critical loss of cell fitness. Here, we tested the suitability of MSC-sorting based on their FACS autofluorescence profile, for a rapid and non-invasive method of senescent cell elimination. Cells were classified in low- (LA) and high- (HA) autofluorescence groups, and results compared to the original MSC population (control). Three days after sorting, cells were screened by replicative senescence markers (cell volume, SA-β-Gal assay and gene/protein expression) and MSC differentiation assays. The transcriptional profiles of sorted MSC were also analyzed by RNA‐Seq. Compared to control, LA cells had 10% lower cell volume and autofluorescence, and 50% less SA-β-Gal + cells. Instead, HA cells had 20% higher cell volume and autofluorescence, and 120% more SA-β-Gal + cells. No changes in replicative senescence and differentiation potentials were observed between all groups. However, 68 genes (16 related to senescence) were significantly differentially expressed (DEG) between LA and other groups. Biological network of DEG identified CXCL12 as topological bottleneck. In summary, MSC sorting may have practical clinical implications to enhance the results of MSC-based therapies.

Alzheimer’s disease (AD), the most prevalent form of dementia, is a progressive and devastating neurodegenerative condition for which there are no effective treatments. Understanding the molecular pathology of AD during disease progression may identify new ways to reduce neuronal damage. Here, we present a longitudinal study tracking dynamic proteomic alterations in the brains of an inducible Drosophila melanogaster model of AD expressing the Arctic mutant Aβ42 gene. We identified 3093 proteins from flies that were induced to express Aβ42 and age-matched healthy controls using label-free quantitative ion-mobility data independent analysis mass spectrometry. Of these, 228 proteins were significantly altered by Aβ42 accumulation and were enriched for AD-associated processes. Network analyses further revealed that these proteins have distinct hub and bottleneck properties in the brain protein interaction network, suggesting that several may have significant effects on brain function. Our unbiased analysis provides useful insights into the key processes governing the progression of amyloid toxicity and forms a basis for further functional analyses in model organisms and translation to mammalian systems.

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species.

Microalgae are unicellular photosynthetic organisms considered as potential alternative sources for biomass, biofuels or high value products. However, their limited biomass productivity represents a bottleneck that needs to be overcome to meet the applicative potential of these organisms. One of the domestication targets for improving their productivity is the proper balance between photoprotection and light conversion for carbon fixation. In the model organism for green algae, Chlamydomonas reinhardtii, a photoprotective mechanism inducing thermal dissipation of absorbed light energy, called Non-photochemical quenching (NPQ), is activated even at relatively low irradiances, resulting in reduced photosynthetic efficiency. Two pigment binding proteins, LHCSR1 and LHCSR3, were previously reported as the main actors during NPQ induction in C. reinhardtii. While previous work characterized in detail the functional properties of LHCSR3, few information is available for the LHCSR1 subunit. Here, we investigated in vitro the functional properties of LHCSR1 and LHCSR3 subunits: despite high sequence identity, the latter resulted as a stronger quencher compared to the former, explaining its predominant role observed in vivo. Pigment analysis, deconvolution of absorption spectra and structural models of LHCSR1 and LHCR3 suggest that different quenching efficiency is related to a different occupancy of L2 carotenoid binding site.

Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics. The possibility to transfer the readout signal from the protein to the DNA level with an oligonucleotide-conjugated antibody increased the sensitivity of protein assays by orders of magnitude and enabled new multiplexing strategies. A bottleneck in the generation of larger oligonucleotide-conjugated antibody panels is the low conjugation yield between antibodies and oligonucleotides, as well as the lack of product purification methods. In this study, we combined a non-site-directed antibody conjugation technique using copper-free click chemistry with ion-exchange chromatography to obtain purified single and double oligonucleotide-conjugated antibodies. We optimized the click conjugation reaction of antibodies with oligonucleotides by evaluating crosslinker, reaction temperature, duration, oligonucleotide length, and secondary structure. As a result, we were able to achieve conjugation yields of 30% at a starting quantity as low as tens of nanograms of antibody, which makes the approach applicable for a wide variety of protein analytical assays. In contrast to previous non-site-directed conjugation methods, we also optimized the conjugation reaction for antibody specificity, confirmed by testing with knockout cell lines. The advantages of using single or double oligonucleotide-conjugated antibodies in regards to signal noise reduction are shown within immunofluorescence, proximity ligation assays, and single cell CITE-seq experiments.

Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, our approach involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N-acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated and subsequently used to develop a glycan microarray for high-throughput, fluorescence-based screening of glycan-binding proteins. Taken together, these results confirm our combined synthesis strategy as a new, user-friendly route for supplying chemically defined human glycans simply by combining biosynthetically-derived precursors with enzymatic remodeling.

Atmospheric carbon dioxide concentrations have been rising during the past century, leading to ocean acidification (OA). Coastal and estuarine habitats experience annual pH variability that vastly exceeds the magnitude of long-term projections in open ocean regions. Eastern oyster (Crassostrea virginica) reproduction season coincides with periods of low pH occurrence in estuaries, thus we investigated effects of moderate (pH 7.5, pCO2 2260 µatm) and severe OA (pH 7.1, pCO2 5584 µatm; and 6.7, pCO2 18480 µatm) on oyster gametogenesis, fertilization, and early larval development successes. Exposure at severe OA during gametogenesis caused disruption in oyster reproduction. Oogenesis appeared to be more sensitive compared to spermatogenesis. However, Eastern oyster reproduction was resilient to moderate OA projected for the near-future. In the context of projected climate change exacerbating seasonal acidification, OA of coastal habitats could represent a significant bottleneck for oyster reproduction which may have profound negative implications for coastal ecosystems reliant on this keystone species.

Single-cell RNA sequencing (scRNA-seq) resolves heterogenous cell populations in tissues and helps to reveal single-cell level function and dynamics. In neuroscience, the rarity of brain tissue is the bottleneck for such study. Evidence shows that, mouse and human share similar cell type gene markers. We hypothesized that the scRNA-seq data of mouse brain tissue can be used to complete human data to infer cell type composition in human samples. Here, we supplement cell type information of human scRNA-seq data, with mouse. The resulted data were used to infer the spatial cellular composition of 3702 human brain samples from Allen Human Brain Atlas. We then mapped the cell types back to corresponding brain regions. Most cell types were localized to the correct regions. We also compare the mapping results to those derived from neuronal nuclei locations. They were consistent after accounting for changes in neural connectivity between regions. Furthermore, we applied this approach on Alzheimer’s brain data and successfully captured cell pattern changes in AD brains. We believe this integrative approach can solve the sample rarity issue in the neuroscience.

Liriomyza is a large genus that includes polyphagous and invasive species (L. trifolii, L. sativae, and L. huidobrensis), and oligophagous species such as L. Chinensis in China. Effective control of these invasive and oligophagous species is not easy due to the fast invasion rate, interspecific competition, and pesticide resistance. In this study, we investigated population genetics of five Liriomyza species L. trifolii, L. sativae, L. huidobrensis, L. bryoniae, and L. chinensis based on COI and EF-1a genes, and microsatellite DNA. These five Liriomyza species revealed highly conservative characteristics in the COI gene among populations collected from different geographical regions and host plants. By contrast, the mutation rate of the EF-1a gene was higher than COI, and phylogenetic tree based on EF-1a showed that haplotypes of L. trifolii and L. sativae were not distinguished well. Genetic differentiation in microsatellite loci was obvious among the five species. Our results also indicated that geographic isolation had a greater impact on genetic differentiation in L. trifolii than the host plant. Populations of L. trifolii in China showed a high to moderate level of genetic differentiation and they had divided into two groups representing the coastal areas of southern China and northern regions. The genetic diversity of the southern group was higher than the northern group. We speculated that the invasion of L. trifolii likely occurred in southern regions of China and then spread northward. Bottleneck analyses revealed that the L. trifolii population in China was in a steady growth period.

Human limbus-derived stromal/mesenchymal stem cells (hLMSC) can be one of the alternatives for the treatment of corneal scars. However, reliable methods of storing and transporting hLMSC remains a serious translational bottleneck. This study aimed to address these limitations by encapsulating hLMSC in alginate beads. Encapsulated hLMSC were kept in transit in a temperature-conditioned container at room temperature (RT) or stored at 4 °C for 3–5 days, which is the likely duration for transporting cells from bench-to-bedside. Non-encapsulated cells were used as controls. Post-storage, hLMSC were released from encapsulation, and viability-assessed cells were plated. After 48 and 96-hours in culture the survival, gene-expression and phenotypic characteristics of hLMSC were assessed. During transit, the container maintained an average temperature of 18.6 ± 1.8 °C, while the average ambient temperature was 31.4 ± 1.2 °C (p = 0.001). Encapsulated hLMSC under transit at RT were recovered with a higher viability (82.5 ± 0.9% and 76.9 ± 1.9%) after 3 (p = 0.0008) and 5-day storage (p = 0.0104) respectively as compared to 4 °C (65.2 ± 1.2% and 64.5 ± 0.8% respectively). Cells at RT also showed a trend towards greater survival-rates when cultured (74.3 ± 2.9% and 67.7 ± 9.8%) than cells stored at 4 °C (54.8 ± 9.04% and 52.4 ± 8.1%) after 3 and 5-days storage (p > 0.2). Non-encapsulated cells had negligible viability at RT and 4 °C. Encapsulated hLMSC (RT and 4 °C) maintained their characteristic phenotype (ABCG2, Pax6, CD90, p63-α, CD45, CD73, CD105, Vimentin and Collagen III). The findings of this study suggest that alginate encapsulation is an effective method of hLMSC preservation offering high cell viability over prolonged durations in transit at RT, therefore, potentially expanding the scope of cell-based therapy for corneal blindness.

An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.

Previous studies have shown that forced expression of core cardiogenic transcription factors can directly reprogram fibroblasts to induced cardiomyocyte-like cells (iCMs). This cardiac reprogramming approach suggests a potential strategy for cardiomyocyte regeneration. However, a major challenge of this approach remains the low conversion rate. Here, we showed that ensuring expression of four cardiogenic transcription factors (i.e. Gata4 (G), Hand2 (H), Mef2c (M), and Tbx5 (T)) in individual fibroblasts is an initial bottleneck for cardiac reprogramming. Following co-transduction of three or four retroviral vectors encoding individual cardiogenic transcription factors, only a minor subpopulation of cells indeed expressed all three (GMT) or four (GHMT) factors. By selectively analyzing subpopulations of cells expressing various combinations of reprogramming factors, we found that co-expression of GMT in individual fibroblasts is sufficient to induce sarcomeric proteins. However, only a small fraction of those cells expressing GMT were able to develop organized sarcomeric structures and contractility. In contrast, ensuring expression of GHMT markedly enhanced the development of contractile cardiac structures and functions in fibroblasts, although its incremental effect on sarcomeric protein induction was relatively small. Our findings provide new insights into the mechanistic basis of inefficient cardiac reprogramming and can help to devise efficient reprogramming strategies.