Tamias) species, has more than any other state or country, occupying habitats ranging from chaparral to the high peaks of the Sierra Nevada. Chipmunks host zoonotic pathogens including Yersinia pestis, Anaplasma phagocytophilum, relapsing fever (RF) Borrelia spp., Borrelia burgdorferi, and spotted fever group (SFG) Rickettsia species. Chipmunk species are often not differentiated by public health workers, yet different species utilize different ecological niches and may have intrinsically different capacities for maintaining vector-borne pathogens and infecting vectors. We surveyed over 700 individuals from nine species of chipmunks throughout California for exposure to and infection by Y. pestis, A. phagocytophilum, RF Borrelia spp., Borrelia burgdorferi, and SFG Rickettsia species. DNA of all five pathogens was found and all chipmunks except Merriam’s chipmunk (T. merriami) were PCR-positive for at least one of the pathogens. Anaplasma phagocytophilum was most common  in Sonoma chipmunks (T. sonomae) from Marin county and B. burgdorferi most common  in redwood chipmunks (T. ochrogenys) from Mendocino county. RF Borrelia spp. was detected in 2% (6/297) of redwood chipmunks in Mendocino county and 10% (1/10) of both least (T. minimus) and lodgepole (T. speciosus) chipmunks in the western Sierra. Exposure to SFG Rickettsia spp. was found in the Northern Coastal region  and in the northern and western Sierra in several species of chipmunks. Y. pestis infection was found only in the western Sierra—in a yellow-pine (T. amoenus) and a long-eared (T. quadrimaculatus) chipmunk. Though more data are needed to thoroughly understand the roles that different chipmunk species play in disease transmission, our findings suggest that some chipmunk species may be more important to the maintenance of vector-borne diseases than others within each geographic area.California, with 13 chipmunk ( Tamias spp.) are small, terrestrial, diurnal mammals characterized as a genus by rapid reproductive rates and high population densities [T. palmeri) is federally endangered [T. bulleri) is classified as vulnerable by the IUCN [Chipmunks  Borrelia spp., Borrelia burgdorferi, the agent of Lyme disease, and spotted fever group (SFG) Rickettsia species [Y. pestis is potentially fatal in chipmunks as well as humans [A. phagocytophilum, RF Borrelia spp., B. burgdorferi and SFG Rickettsia spp. are zoonoses but not known to cause clinical disease in chipmunks [Chipmunks host important zoonotic pathogens in the western United States including  species –8. Y. pes humans , while Ahipmunks –13.T. merriami), to the high peaks of the Sierra Nevada where the alpine chipmunk  can be found. In the eastern-central Sierra Nevada, ranges of the lodgepole (T. speciosus), least (T. minimus), yellow-pine (T. amoenus), Allen’s (T. senex), and Uinta chipmunks (T. umbrinus) overlap at elevations between approximately 1800 and 2700m. On the western slope of the central Sierra Nevada, the long-eared (T. quadrimaculatus), lodgepole, Allen’s and Merriam’s chipmunk are found at elevations close to 1800m [Neotoma spp.), deer mice (Peromyscus spp.), and other sciurids such as the western grey squirrel (Sciurus griseus), California ground squirrel (Otospermophilus beecheyi), and Douglas squirrel (Tamiasciurus douglasii) [In California, which is home to 13 species of chipmunks, chipmunks occupy diverse habitats ranging from coastal chaparral which is home to Merriam’s chipmunk  Chipmuuglasii) , 16–23.Chipmunk species can be difficult for the lay-person to differentiate and are often referred to generically in the public health arena, without differentiating by species , 9, 24. A. phagocytophilum, B. burgdorferi, RF Borrelia spp. SFG Rickettsia spp. and Y. pestis, sampling over 700 individuals from nine species of chipmunks throughout California. We evaluated for differences in infection and exposure prevalence by geographic region and by chipmunk species, and we investigated relationships between prevalence and chipmunk species diversity and richness at each site. Test agreement between evidence of exposure and active infection was also explored.In order to address these gaps, we undertook a survey in chipmunks for the vector-borne pathogens Tamias spp.) were covered under California Department of Fish and Wildlife scientific collecting permit #SC-854. The study spanned multiple applicable protocols which were approved by the University of California, Davis Institutional Animal Care and Use Committee. The most recent protocol is #18179.Trapping and sampling of chipmunks (2) was taken using sterile scissors, stored in 70% ethanol, and kept cool until it could be refrigerated.  Each animal was given an individually numbered metal ear tag prior to release. Additional museum samples were collected by US Forest Service in 2010, 2012 and 2013 in the western Sierra Nevada  and donated to the UC Davis Museum of Wildlife and Fish Biology. Blood and ear snips were collected from these chipmunks post-mortem. All work with animals was performed in compliance with the UC Davis or Humboldt State University IACUC, and under valid scientific collecting permits from California Department of Fish and Wildlife, the U.S. Forest Service, and landowners where applicable.Chipmunks were live-trapped using Sherman  or Tomahawk  traps baited with oats and peanut butter or molasses and birdseed. Trapping took place opportunistically at 16 study sites distributed across 12 California counties  between October 2005 and August 2015. Animals were trapped both during daytime and nighttime hours and traps were checked early morning, twice during the day and in the late evening. Animals were anesthetized with up to 40mg/kg ketamine and 4mg/kg xylazine delivered subcutaneously. Each animal was identified to species in the field when possible, and the age (juvenile or adult) and sex were determined. Blood was collected from the retroorbital sinus or the lateral saphenous vein into EDTA blood collection tubes  and kept cool until plasma was separated by centrifugation at 3000rpm for 10 minutes. Whole blood and plasma were then stored at -20°C to -80°C until use. A small piece of marginal ear tissue  and then applied to A. phagocytophilum, B. burgdorferi or R. rickettsii antigen slides . Slides were incubated at 37°C with moisture for 45 min, washed three times in PBS and then incubated at 37°C for 45 minutes with fluorescein isothiocyanate-labeled rabbit anti-rat immunoglobulin G heavy and light chain antibodies  diluted in PBS at 1:25. Slides were washed three additional times in PBS and, during the third wash, incubated with two drops of eriochrome black for 2 minutes. Positive (previously tested rodents) and negative (PBS and negative rodent serum) controls were included in each run. Samples were considered positive if fluorescence was detected in the appropriate distribution for each pathogen.Indirect immunofluorescent assays (IFA) were performed to evaluate for exposure to A. phagocytophilum, Y. pestis, RF Borrelia spp. and SFG Rickettsia spp.) or ear tissue (for B. burgdorferi) using a kit  following manufacturer’s instructions. Real-time PCR for A. phagocytophilum [Y. pestis [Borrelia spp. [B. burgdorferi [Rickettsia spp. [A. phagocytophilum, RF Borrelia spp., and SFG Rickettsia spp. was not performed on all chipmunks due to insufficient sample volumes. PCR for B. burgdorferi was performed on all chipmunks from which ear tissue samples were collected. A subset of chipmunks from the north coastal region of California  and from the Sierra Nevada was evaluated for active infection by Y. pestis. These two regions were selected for Y. pestis surveillance as multiple species of animals, including small mammals, from these areas have been found to be seropositive for this pathogen [Chipmunks were assessed for active infection by real-time TaqMan PCR. DNA was extracted from 100μl of whole blood . For calculating prevalence, geologically, climatically, and botanically related sites were grouped to include counties north of Lake Tahoe (Nevada and Sierra counties) classified as “northern Sierra”, counties south of Lake Tahoe and west of the Sierra crest classified as “western Sierra” and counties south of Lake Tahoe and east of the Sierra crest classified as “eastern Sierra”. Additionally, to compare pathogen prevalence between the Sierra Nevada mountains and the northern coastal region of California, two super-regions were created for statistical analyses: all Sierra regions were combined into “Sierra Nevada” and all counties north of San Francisco were combined into “north-coastal”. Pearson’s chi-square tests were used to assess prevalence differences between regions and between chipmunk species. Because one species (Allen’s chipmunk) has a range that spans two regions, Fisher’s exact tests were used to assess differences in sero- and PCR-prevalence between Allen’s chipmunks from north-coastal California (Humboldt county) and the Sierra Nevada. Fisher’s exact tests were also used to assess the significance of disease prevalence levels between sympatric chipmunk species with different uses of space  in the Sierra Nevada. Cohen’s Kappa coefficients were calculated to assess agreement between PCR and serology [Data were maintained in Excel  and analyzed in R  of chipmunks being PCR-positive for B. burgdorferi, 1.4% (8/591) being PCR-positive for RF Borrelia, and 66/308 (21.4%) being seropositive, although the serology cross-reacts among the Borrelia species. For other pathogens, 8.4% (50/595) of chipmunks were A. phagocytophilum PCR-positive and 118/526 (22.4%) were seropositive; 0.7% (4/581) were SFG Rickettsia spp. PCR-positive and 110/415 (26.5%) were seropositive; and 0.9% (2/230) were Y. pestis PCR-positive (serology was not performed for Y. pestis).A total of 709 chipmunks from nine species was sampled between September 2005 and August 2015 . The patRickettsia spp., 0.088 (0–0.175) for A. phagocytophilum, 0.098 (0.001–0.195) for RF Borrelia spp., and 0.337 (0.148–0.526) for B. burgdorferi.Agreement between serological and PCR results was low, with Kappa equal to -0.015 (95% CI: -0.032–0.002) for SFG A. phagocytophilum PCR-detection (p = 0.015), with the highest PCR-prevalence in Marin  and no A. phagocytophilum PCR-positive results in Santa Cruz or the eastern or western Sierra. On serology, regions again differed significantly (p = 0.004), and the highest seroprevalence was in Marin , although serological evidence of A. phagocytophilum was detected in Santa Cruz and the eastern Sierra  and in the western Sierra, with a significantly higher PCR-prevalence  detected in Mendocino . There was evidence of exposure to Borrelia spp. in all northwestern regions and the northern Sierra; Mendocino had the highest seroprevalence , although the difference between regions was not significant (p = 0.086).Infection with Rickettsia spp. were found only in a few individuals sampled in Del Norte, Mendocino and the western Sierra, with Del Norte having the highest PCR-prevalence , and no significant differences among regions (p = 0.393). Mendocino had the highest seroprevalence , with additional seropositive animals in Humboldt, Marin, Mendocino, and the northern Sierra; differences in seroprevalence were significant (p = 0.0002). Two individuals from the western Sierra were PCR-positive for Y. pestis.Spotted fever group A. phagocytophilum) had significantly higher levels of infection with both A. phagocytophilum and B. burgdorferi when compared to chipmunks sampled from the Sierra Nevada (p = 0.026 for A. phagocytophilum and p < 0.0001 for B. burgdorferi) but no significant differences for PCR-prevalence of RF Borrelia, SFG Rickettsia spp. or Y. pestis. Seropositivity for A. phagocytophilum, Borrelia spp. and SFG Rickettsia spp. was also higher in the north-coastal super-region than the Sierra Nevada (p < 0.0001).When analyzed as a whole, chipmunks from the north-coastal super-region  ranged from one to five with the highest number of chipmunk species being found in the northern and western Sierra, while Menhinick’s index (D) ranged from a low of 0.06 in Mendocino to a high of 0.49 in the Northern Sierra. No significant correlations were found between disease prevalence and either chipmunk species richness, S or D, or chipmunk species diversity.A. phagocytophilum was highest in the Sonoma chipmunk  and differences in PCR-prevalence among species were significant  in the Sonoma chipmunk; the differences found between species were significant  and lodgepole chipmunks; differences among species were not significant (p = 0.065). However, highly significantly (p < 0.0001) more B. burgdorferi DNA was found in the redwood, long-eared, Siskiyou (T. siskiyou) and Allen’s chipmunks than in other species, with the redwood chipmunk having the highest PCR-prevalence . Although only the yellow-pine, lodgepole, redwood, Allen’s and Merriam’s chipmunks were Borrelia seropositive, differences in seroprevalence were not significant (p = 0.162). Three species were positive by PCR for SFG Rickettsia spp.: the yellow-pine, redwood and Siskiyou chipmunks; although differences in PCR among species were not significant (p = 0.589), differences in serology were (p = 0.015), with antibodies found in the yellow-pine, redwood, Sonoma and Allen’s chipmunks. Only two individuals were PCR positive for Y. pestis: one yellow-pine and one long-eared chipmunk.All chipmunk species except for Merriam’s chipmunk were PCR-positive for at least one of the pathogens. Mirroring the regional analyses, active infection by Rickettsia spp. (p < 0.001) compared to individuals from the Sierra Nevada, while there were no significant differences between the seroprevalence of A. phagocytophilum or Borrelia spp., nor for any of the pathogens evaluated by PCR.We examined one species, Allen’s chipmunk, which is distributed across the north-coastal super-region east into the Sierra Nevada. North-coastal individuals were significantly more likely to be seropositive for SFG Borrelia spp. (p = 0.013) in arboreal species, higher seroprevalence of Borrelia spp. (p = 0.037) in arboreal and mixed chipmunk species, and higher seroprevalence of SFG Rickettsia spp. (p = 0.003) in mixed chipmunks.Five species of chipmunks were sampled from both the northern and western Sierra Nevada—yellow-pine, Allen’s, long-eared, least and lodgepole . These fBorrelia spp. and A. phagocytophilum. Exposure to SFG Rickettsia spp. was also common, despite DNA in blood being extremely rare, presumably because the DNA is sequestered in the target cells for SFG rickettsiae, which are endothelium. Chipmunks from the northern coastal region were more likely to have evidence of vector-borne pathogen infection than were chipmunks from the Sierra Nevada. Our results are in agreement with previous, more geographically restricted, studies of vector-borne zoonotic diseases in California chipmunks [At least five vector-borne, zoonotic pathogens are present from the northern coast of California to the eastern Sierra in at least eight chipmunk species. Testing revealed substantial PCR- and seropositivity for both hipmunks , 40, 41 Borrelia spp. PCR because ear tissue was not routinely collected in earliest sampling and Y. pestis serology because the antigen was unavailable. The poor correspondence between PCR and serology could be an outcome of reservoir hosts mounting meager antibody responses during infection with pathogens to which they are essentially tolerant, as described previously for tick-borne pathogens in woodrats [Borrelia burgdorferi has clearly been shown to induce differential antibody responses across different reservoir species [Here we used PCR and serology for most pathogens in order to evaluate both active infection of individuals as well as exposure and subsequent antibody production, which is a tool that gives useful insight into population responses to pathogens and herd immunity. The exceptions were woodrats . Borreli species –45. ThesA. phagocytophilum, with high PCR- and seroprevalence, relatively persistent infection for at least 4 months, and competence to infect the Pacific black-legged tick [B. burgdorferi in California, including B. burgdorferi sensu stricto and B. bissettiae in Siskiyou chipmunks, [A. phagocytophilum and B. burgdorferi, chipmunks play a lesser role in the ecologies of these pathogens east of the Rocky Mountains, where white-footed mice (P. leucopus) account for the majority of infected ticks [T. sibericus), which is becoming established in Europe, has been shown with higher tick infestation loads and B. burgdorferi sensu lato PCR-prevalence (primarily B. afzelii) than the recognized reservoirs [As expected, there was abundant evidence of exposure to agents of Lyme disease and anaplasmosis in chipmunks from areas where humans are also found infected . Redwoodged tick . Numerouipmunks,  as well ipmunks, . In conted ticks , 49. Theservoirs .B. hermsii, transmitted by the soft tick Ornithodoros hermsi, which occurs at higher altitudes (450 to 2400 m) and feeds preferentially on squirrels and chipmunks [Tamiasciurus hudsonicus), yellow-pine chipmunks, and meadow voles (Microtus pennsylvanicus) and clinical disease in the pine squirrels [Borrelia in chipmunks, active infection was detected on the coast in redwood chipmunks, in addition to infection in least and lodgepole chipmunks in the Sierra Nevada. More work to differentiate among chipmunk species in their reservoir capacity is needed. This includes additional testing within fine spatial scale enzootic foci, reporting chipmunks to species, and additional experimental trials of multiple species of chipmunks.The geographic distribution of cases of relapsing fever in California is much more restricted than for Lyme disease cases, reported most often in the Lake Tahoe and Mammoth Lakes regions of the Sierra Nevada . Cases ihipmunks . These thipmunks . Experimquirrels . Statewiquirrels , and PCRRickettsia spp. in chipmunks although most reactive chipmunks had antibodies but not DNA, making it impossible to determine which bacterial species they were exposed to. Rickettsiae are poorly sampled using PCR of blood because the target tissue is less accessible than blood, suggesting greater yield could be expected if sampling was done of whole carcasses. There are numerous co-circulating spotted-fever group rickettsial species, including R. rhipicephali, R. montanensis, and R. rickettsii [R. rhipicephali [R. rickettsii and an isolate was acquired from a yellow-pine chipmunk in Montana, although infection was not prolonged beyond a week [R. rickettsii [Rickettsia spp. in a tick can inhibit transovarial transmission of a second species [We also found considerable evidence for ckettsii –57. Rodeicephali . Chipmund a week . In theockettsii , while p species .M. californicus), deer mice (P. maniculatus), and chipmunks experience less lethal disease but not prolonged infection and thus do not represent classical “reservoirs” [Martes americana) fed primarily on chipmunks, golden-mantled ground squirrels , and California ground squirrels. As we did not have access to antigen for serological surveillance, the very low PCR-prevalence is not unexpected given the limited duration of infection and relatively high rates of fatality in chipmunks. This suggests that seroprevalence would be low as well [Chipmunks are also known hosts of plague in California. Ground squirrels function as highly susceptible “amplifying hosts” and are responsible for most observed outbreaks, while voles  on redwood chipmunks, one of the chipmunk species that occurs along the coast in areas typically not inhabited by any other chipmunk species [Rickettsia spp. than coastal Allen’s chipmunks where there is minimal range overlap among chipmunk species, also arguing against increased host diversity supporting more disease and suggesting that a species’ ecological importance may change depending on location.In contrast, Sierra Nevada chipmunks overlap, although fine-scale space use varies: Allen’s and long-eared chipmunks are almost exclusively terrestrial, yellow-pine chipmunks are primarily terrestrial but also commonly climb into brush and small trees up to 2m tall, while the least and lodgepole chipmunks exhibit both terrestrial and arboreal tendencies –39. In t species . We alsoA. phagocytophilum and B. burgdorferi [I. pacificus ticks have been documented on tree trunks up to about a meter above ground [Chipmunks are moderate sized rodents with longer lifespans than some reservoirs, such as white-footed mice, and are heavily infested with ticks and fleas in diverse parts of the world. They may achieve a higher population density than western gray squirrels, the western reservoir of gdorferi , 71 but gdorferi , they mae ground  but haveThis study comprises the most far-reaching survey of vector-borne zoonotic diseases in California chipmunk species to date. The key findings provide several jumping-off points for future studies that will continue to increase our understanding of the complex ecological role that these small mammals play in disease maintenance and transmission in California.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


H. japonica.In contrast to the Western Palearctic and Nearctic biogeographic regions, the phylogeography of Eastern-Palearctic terrestrial vertebrates has received relatively little attention. In East Asia, tectonic events, along with Pleistocene climatic conditions, likely affected species distribution and diversity, especially through their impact on sea levels and the consequent opening and closing of land-bridges between Eurasia and the Japanese Archipelago. To better understand these effects, we sequenced mitochondrial and nuclear markers to determine phylogeographic patterns in East-Asian tree frogs, with a particular focus on the widespread H. japonica populations, including two main clades of Late Miocene divergence (~5 Mya). One occurs on the northeastern Japanese Archipelago (Honshu and Hokkaido) and the Russian Far-East islands , and the second one inhabits the remaining range, comprising southwestern Japan, the Korean Peninsula, Transiberian China, Russia and Mongolia. Each clade further features strong allopatric Plio-Pleistocene subdivisions (~2-3 Mya), especially among continental and southwestern Japanese tree frog populations. Combined with paleo-climate-based distribution models, the molecular data allowed the identification of Pleistocene glacial refugia and continental routes of postglacial recolonization. Phylogenetic reconstructions further supported genetic homogeneity between the Korean H. suweonensis and Chinese H. immaculata, suggesting the former to be a relic population of the latter that arose when the Yellow Sea formed, at the end of the last glaciation.We document several cryptic lineages within the currently recognized H. japonica around the Sea of Japan. Our findings urge for important taxonomic revisions in East-Asian tree frogs. First, they support the synonymy of H. suweonensis  and H. immaculata . Second, the nominal H. japonica  represents at least two species: an eastern (new taxon A) on the northern Japanese and Russian Far East islands, and a southwestern species (n. t. B) on southern Japanese islands and possibly also forming continental populations. Third, these continental tree frogs may also represent an additional entity, previously described as H. stepheni Boulenger, 1888 . A complete revision of this group requires further taxonomic and nomenclatural analyses, especially since it remains unclear to which taxon the species-epitheton japonica corresponds to.Patterns of divergence and diversity were likely triggered by Miocene tectonic activities and Quaternary climatic fluctuations (including glaciations), causing the formation and disappearance of land-bridges between the Japanese islands and the continent. Overall, this resulted in a ring-like diversification of The online version of this article (doi:10.1186/s12862-016-0814-x) contains supplementary material, which is available to authorized users. To do so, we sequenced fast-evolving mitochondrial and nuclear markers from samples across most of the range of H. japonica and estimate divergence times between all major lineages. To complement the phylogeographic analyses and get insights into the location of glacial refugia, we estimated the current and past distribution for the widespread H. japonica using Species Distribution Modelling approaches. Given that tree frogs are generally declining and threatened in many regions, our results also have potentially useful implications for the future management of populations.In this paper, we aim to understand the diversification patterns of East Asian tree frogs and whether divergences correspond to major biogeographic events in the region. Specifically, we undertake a phylogeographic survey of H. japonica from 34 populations were considered . Two samples from H. immaculata (newly collected) and seven from H. suweonensis  were included. Details on the origin of specimens are available from Additional file Tissue or oral  swab samples were obtained from museum samples  and field captured frogs. For the latter, DNA was obtained from muscle or liver, or from non-lethal sampling , in which case the animals were immediately released afterwards. A total of 96 specimens of cytochrome-b (cyt-b), in all individuals as described [α-Fibrinogen intron 1 . At least eight clones were sequenced per sample in order to capture the two alleles of heterozygotes.We sequenced ~900 bp of the mitochondrial escribed . For nucprimers: ) in a recyt-b haplotypes with PhyML [cyt-b sequences of H. suweonensis . We performed a similar analysis for the nuclear α-Fibrinogen haplotypes, using a HKY + I model (MrAIC) and included our published sequences for H. suweonensis . For thspecies, ; GenBankquenced, ; GenBankX182365; ). For boHyla by Stöck et al. [cyt-b sequences, available for seven European taxa (3–11 per taxon) with a resolved phylogeny and known divergence times . Importantly, the cyt-b tree is concordant with the most robust phylogenetic analyses for these species published to date, based on thousands of SNPs [H. meridionalis (9.7 Mya), of the H. arborea group (6.1 Mya) and between H. molleri and H. orientalis (3.7 Mya), using normally distributed priors with a standard deviation of 1 My. This enables to effectively span a large range of prior distributions to encompass the posterior distributions obtained by Stöck et al. [Dxy) between and within the main mtDNA clades with DNAsp [α-Fibrinogen haplogroups.We estimated divergence times between the main East-Asian tree frog lineages with BEAST , using tk et al. . For thi of SNPs . We applk et al.  for thesk et al.  to verifth DNAsp . Given iH. japonica populations, we built networks of mitochondrial and nuclear haplotypes using TCS [α-Fibrinogen, we considered insertion-deletion (indel) polymorphism and the two alleles of each sample separately.Finally, to explore the genetic structure of sing TCS , withoutH. japonica , Bio2 , Bio3 , Bio5 , Bio12 , Bio15 , and Bio19 . We used a jackknife analysis for estimation of relative contributions of the variables to the Maxent model. The mask applied for the distribution modelling extends from 90 to 155° N and 20 to 65° E.For the contemporary niche predictions, we used 1008 localities of cyt-b was very informative: we sampled 67 haplotypes . For the nuclear α-Fibrinogen, we distinguished 28 haplotypes with 42 variable sites, including 22 parsimony-informative.Mitochondrial and nuclear trees recovered similar haplogroups, although topologies differed in respect to the lower informativeness of the nuclear marker, which accordingly also featured low branch supports. The mitochondrial H. immaculata sequences unambiguously clustered with those of H. suweonensis, forming a genetically homogeneous monophyletic clade .Dxy from 0.036 to 0.070, Additional file The first clade (A) is restricted to the central and northern parts of the Japanese Archipelago, Kunashir and Sakhalin Islands (pop. 1–6), and is further subdivided into four main lineages ((ii).Dxy from 0.017 to 0.042, Additional file the second clade (B) has a wide distribution across the southern parts of the Japanese Archipelago and the mainland pop. 7–34), with marked subdivisions , Tsushima, Sado, and Jeju islands, most of the Korea Peninsula, the shallow Yellow Sea, and the southernmost Manchuria.The ecological niche modeling had high AUC test values 0.935 ± 0.005) and showed significance for the binomial omission test, indicating a good performance of the model. The predicted potential niche model under the current climate conditions is shown in Fig.  ± 0.005 H. japonica. The latter represents a species group, composed of two major genetic clades (corresponding to two new taxa A and B) with pronounced subdivisions. Figure Mitochondrial and nuclear phylogenetic analyses of tree frogs from the East-Asian mainland and the Japanese Archipelago revealed three main lineages, including cryptic genetic diversity within cyt-b suggested a Late Miocene divergence (~6.5 Mya) of the common ancestor of H. immaculata + H. suweonensis from the lineage leading to all current nominal H. japonica. This branching corresponds to the geological period when the Philippine Sea Plate (PHS) resumed its subduction, forming large volcano-tectonic depressions across the Korean Strait (separating the Japanese Archipelago from the Korean Peninsula) and the Ryuku Arc , eventud China; . These gd China; , fishes d China; , and espd China; . In accoH. japonica harbors high cryptic diversity throughout its range with multiple Miocene and Plio-Pleistocene diversifications. This genetically diverse taxon can first be divided into two main mitochondrial clades . Further population genetic investigations will probably inform on the accurate distribution of these taxa and on their genetic interactions across contact zones.Our findings show that the widespread des Fig. , with thulations . The oldulations . This saa rugosa , Buergerbuergeri  and Bufofo toads . From ouH. japonica mtDNA-clade further presents allopatric subdivisions, of Plio-Pleistocene divergence (2–3 Mya). In clade A (n. t. A), the complex topography of central of northern Japan, combined with Pleistocene climatic fluctuations, may have promoted this strong geographical structure. Especially, subclade A1, occurring on Hokkaido, Kunashir and Sakhalin Islands may have become isolated during unsuitable Quaternary glacial periods, as suggested by the CCSM past distribution model  shows that continental populations diverged from southern Japanese ones about 3 Mya. The mitochondrial differentiation was also accompanied by the formation of distinct nuclear haplogroups. This pattern suggests that tree frogs did not use land-bridges across the Korea Strait, which connected southern Japan with the Korean mainland during the last glacial cycles Fig. . For exaHyla [H. immaculata/H. suweonensis . Additional nuclear loci will help solving the nuclear phylogeny of H. japonica clades.Contrasting with our phylogenetic data, previous allozyme genetic distances  suggesteHyla . HoweverH. japonica can also be considered as a case of ring diversification. This type of diversification involves parapatric or allopatric differentiation of populations along a stepping-stone-like geographic continuum around a major dispersal barrier, preventing gene flow between non-neighbouring demes. In our case, populations diversified around the Sea of Japan, stretching from Sakhalin to southern Honshu on the one hand (n. t. A), and from Eastern Russia, China, Korea and onto the Southern Japanese Archipelageo on the other hand (n. t. B). As recently emphasized, ring-like phylogeographic patterns might be more common than previously assumed [H. japonica’s diversity around the Sea of Japan and test whether the northern, terminal mtDNA lineages A1 (n. t. A) and B5 (n. t. B) interact and “close” this ring (i.e. between our loc. 1 and 28). Moreover, this pattern should be tested in other organisms, where cryptic diversity may have arisen by similar biogeographic processes.Interestingly, the phylogeographic pattern of  assumed , 56, esp assumed . Ancestr assumed , 59, a sH. japonica lineages (subclades B4 and B5) supports multiple glacial refugia across the Korean Peninsula. In this region, glacial conditions were supposedly milder  were not shared by any Korean H. suweonensis, suggesting limited dispersal and/or recent divergences.The lack of sequence differentiation between the Chinese ons Fig. . Thus, pica Fig. . Future H. japonica , which can be considered a superspecies, including at least two main taxa . Our findings are consistent with Li et al. [H. japonica A” in [H. japonica B” in [Hyla lineages of similar or lower degree of divergence are recognized as different species [Our results stress the need for systematic revisions of East-Asian tree frogs. First, we provide evidence for cryptic speciation within  species , 66, and species .H. japonica populations should be considered a separate species, H. stepheni (or its junior synonym H. ussuriensis), distinct from Japanese ones [. [H. japonica and H. ussuriensis can be considered synonyms. In contrast, our present data rather suggest that these continental and southern Japanese tree frogs form monophyletic mitochondrial lineages, as old as about 3 Mya , with distinct, although paraphyletic, nuclear haplogroups. Interestingly, experimental crosses between “H. ussuriensis” (from mainland Korea and Tsushima island) with southern Japanese “H. japonica” (from Hiroshima) suffered from lower metamorphosis rates and inferior offspring fitness compared to controls, which might indicate post-zygotic isolation [Second, there has been an ongoing debate on whether continental ese ones . Based o ones [.  argued tsolation . AdditioH. japonica into two taxonomic entities (n. t. A and n. t. B) raises the question to which one the species-epitheton japonica, coined by Günther ([Hyla arborea”) with database record “RMNH.RENA.1701, Hyla arborea, 17 ex., collected by: Siebold & Bürger, locality: Japan” . While these frogs are extensively described by Temminck and Schlegel (p. 112 in [Hyla arborea”), their work contains no further geographic details. Siebold was a resident physician at the Dutch trading post on Dejima Island in Nagasaki. Yet, in 1826, he traveled at least to Edo (now Tokyo). Therefore, types of H. japonica may have originated from the vicinities of Nagasaki (inhabited by n. t. A), but it cannot be excluded that the types were collected in the vicinities of Tokyo (inhabited by n. t. B), or elsewhere in Japan, as zoological and botanical material was also obtained from local people from several parts of Japan [japonica belongs, cannot be conclusively answered.The split of the nominal Günther Günther  as wellGünther  and dep. 112 in ; as “Hylof Japan , 70. In H. immaculata and H. suweonensis belong to one and the same tree frog species, as recently suspected based on few mitochondrial sequences [H. suweonensis Kuramoto, 1980 [H. immaculata Boettger, 1888 [H. japonica superspecies. The use of faster-evolving markers, such as microsatellites, will allow to better understand the recent history of H. immaculata populations, and define management units for their conservation.Finally, both our mitochondrial and nuclear datasets unambiguously supported that equences . From outo, 1980  a juniorer, 1888 . This liThe phylogeography of Eastern tree frogs revealed cryptic diversity in a ring-like manner, and potential speciation of several deeply-diverged lineages. These divergences likely result from past tectonic events and paleoclimatic changes, and their effects on the formation and disappearance of land-bridges between the topographically complex Japanese Archipelago and the Korean Peninsula, paralleling the situation in other terrestrial vertebrates. Furthermore, by the identification of multiple glacial refugia and continental routes of post-glacial recolonization, we demonstrated that Quaternary glaciations also had a strong genetic impact on the evolution of East-Asian anuran amphibians. For this part of the Palearctic realm, it is usually assumed that such effects are limited compared to other biogeographic regions of the northern hemisphere.


Trypanosoma cruzi parasites are the causative agents of Chagas disease. These parasites infect cardiac and gastrointestinal tissues, leading to local inflammation and tissue damage. Digestive Chagas disease is associated with perturbations in food absorption, intestinal traffic and defecation. However, the impact of T. cruzi infection on the gut microbiota and metabolome have yet to be characterized. In this study, we applied mass spectrometry-based metabolomics and 16S rRNA sequencing to profile infection-associated alterations in fecal bacterial composition and fecal metabolome through the acute-stage and into the chronic stage of infection, in a murine model of Chagas disease. We observed joint microbial and chemical perturbations associated with T. cruzi infection. These included alterations in conjugated linoleic acid (CLA) derivatives and in specific members of families Ruminococcaceae and Lachnospiraceae, as well as alterations in secondary bile acids and members of order Clostridiales. These results highlight the importance of multi-‘omics’ and poly-microbial studies in understanding parasitic diseases in general, and Chagas disease in particular. T. cruzi infection perturbs the fecal microbiome and metabolome, indicating functional changes affecting the gastrointestinal lumen. Our results support further investigation into the role of the microbiota-parasite interaction in gastrointestinal Chagas disease pathogenesis.Host-parasite interactions are usually studied as a binary system, without considering the role of the host microbiota. This work integrates microbiome research into the study of gastrointestinal Chagas disease. We show that  Trypanosoma cruzi are protozoan parasites endemic to Central and South America. They cause a range of cardiac and gastrointestinal manifestations collectively known as Chagas disease. With increasing travel and immigration, infected individuals are also now found worldwide. Six to seven million people are T. cruzi-positive, thirty to forty percent of which will develop symptomatic disease decades after their initial exposure to the parasite. Cardiac symptoms are the most common; these include conduction abnormalities, arrhythmias, aneurysms, and heart failure leading to death. Clinically apparent gastrointestinal Chagas disease is less prevalent; gastrointestinal Chagas disease is associated with enlargement of the esophagus and/or colon , leading to pain, dysphagia, altered intestinal transit, altered nutrient intake, and constipation  library reference (bottom). (B) Cholic acid molecular network. (C) Comparable cholic acid levels in infected and uninfected mice. (D) m/z 357.281 RT 337s. (E) m/z 357.281 RT 371s. (F) m/z 358.285 RT 371s. (G) m/z 358.285 RT 387s. (H) m/z 375.291 RT 393s. *, p<0.05 .((DOCX)Click here for additional data file.S9 FigA) Phylum level. (B) Class level. (C) Order level. (D) Family level. Each bar represents a given mouse.((DOCX)Click here for additional data file.S10 FigA) and with only samples and pooled QC (B). Blank samples are distinctly different from all other samples, while pooled QC are in the middle of the PCoA. Distinct clustering of high parasite burden samples is highlighted with dotted oval, where permitted by the viewing angle.Various views and principal coordinates of PCoA plots with samples, blanks and pooled QC samples ((DOCX)Click here for additional data file.


Solenopsis invicta) on the native tick, small mammal and pathogen community in southeast Texas. Using a replicated large-scale field manipulation study, we show that small mammals were more abundant on treatment plots where S. invicta populations were experimentally reduced. Our analysis of ticks on small mammal hosts demonstrated a threefold increase in the ticks caught per unit effort on treatment relative to control plots, and elevated tick loads (a 27-fold increase) on one common rodent species. We detected only one known human pathogen (Rickettsia parkeri), present in 1.4% of larvae and 6.7% of nymph on-host Amblyomma maculatum samples but with no significant difference between treatment and control plots. Given that host and vector population dynamics are key drivers of pathogen transmission, the reduced small mammal and tick abundance associated with S. invicta may alter pathogen transmission dynamics over broader spatial scales.Invasive species may impact pathogen transmission by altering the distributions and interactions among native vertebrate reservoir hosts and arthropod vectors. Here, we examined the direct and indirect effects of the red imported fire ant ( Previous studies have most commonly implicated the invader in altering species relationships in ways that support vector-borne pathogen transmission and, therefore, increase disease risk. For example, a widespread, invasive shrub increases human risk of ehrlichiosis because it provides habitat for deer that host infected ticks , and dened voles .Solenopsis invicta) on tick, small mammal and pathogen communities in southeast Texas. Ticks and small mammals transmit and maintain numerous zoonotic pathogens that are significant public health concerns. Solenopsis invicta are known to predate small mammals [Solenopsis invicta are also associated with reductions in tick populations [S. invicta populations across an area of historic invasion, we expected that S. invicta predation and avoidance behaviour by mammals and ticks would lead to decreased mammal, tick and pathogen abundance in plots where S. invicta were in high density relative to treatment plots where S. invicta were experimentally suppressed.Here, we investigate the potential impact of the invasive red imported fire ant  [The manipulative experiment occurred at two field sites separated by over 160 km in southeast Texas: Attwater Prairie Chicken National Wildlife Refuge (APCNWR) and a private ranch in Goliad County (GRR). Each field site was partitioned into two treatment plots and two control plots. Treatment plots were chemically treated with Extinguish Plus™  for twateri) ; controltwateri) ; see theRickettsia and Borrelia .Small mammals and their attached ticks were collected using seed-baited Sherman live traps . Three line transects (approx. 20 m apart), each with 20 traps spaced 10 m apart, were spread across each of the four plots at both field sites, resulting in a total of 60 traps per plot and 240 traps per site. Small mammal trapping was conducted for two consecutive nights each month . All captured mammals were marked with an ear tag, identified to species and inspected for ticks, which were removed, identified and stored in 70% ethanol. Off-host tick presence was assessed via drag sampling . On-host ticks were tested for infection with microbes in the genera χ2-distribution. Association between pathogen infection of ticks  and S. invicta treatment was tested with a Fisher's exact test.We used general linear mixed models assuming a negative binomial error distribution to analyse counts of mammals and on-host ticks across treatment and control plots. We used a zero-inflated (ZI) model if it fit the data better (i.e. lower Akaike information criteria) than the same model that did not account for ZI. All models were implemented in program R (v. 3.2.2) in the package glmmADMB (v. 0.8.3.2). Site  and season  were added to models as random intercepts . Sampling effort (effective trap nights) per transect was included in the model using the offset function. Significance of all treatment coefficients was assessed through a log-likelihood ratio test of nested models assuming a 3.S. invicta-suppressed treatment plots (p < 0.001). The effect was consistent among the three most commonly sampled mammal species . Our model predicted a 2.0-fold increase in S. hispidus captured on treatment relative to control plots (p < 0.001). Effect sizes were slightly lower for B. taylori  and R. fulvescens .The majority (64.1%) of small mammals captured were from nt plots . Our modAmbylomma maculatum (99.6%) with the exception of one nymphal Ixodes scapularis (0.4%). The rodent species most heavily parasitized by ticks were S. hispidus , Chaetodipus hispidus (7.7%), R. fulvescens (7.7%) and B. taylori (1.4%). Our model predicted a threefold increase in the number of on-host ticks caught per unit effort on treatment relative to control plots (p = 0.01). When the number of rodents captured during a sampling night was included in the model with the offset function, the model still predicted an increase in the number of ticks on treatment plots, but this effect was no longer significant (p = 0.45). This suggests that the effect of a greater number of on-host ticks on treatment plots was primarily driven by an increased capture rate of small mammals along treatment transects. To directly investigate tick loads across treatment and control plots, we modelled the number of ticks per host individual in S. hispidus and R. fulvescens, two well-sampled  and highly parasitized species in our data. Tick loads did not vary significantly across plots in S. hispidus .Ninety-eight mammals (8.7% of captures) were parasitized by a total of 237 ticks, including 142 larvae and 95 nymphs . Nearly all ticks were Rickettsia species, of which 34 (27.0%) tested positive . Most rickettsial sequences had high homology to species regarded as endosymbionts . A total of seven A. maculatum samples were infected with the human pathogen R. parkeri . The proportion of ticks infected with R. parkeri was not different between treatment and control plots (p > 0.05). A total of 83 tick samples were tested for infection with Borrelia species of which B. lonestari was found in a single A. maculatum nymph on an APCNWR treatment plot .A total of 126 individual tick nymphs and larval pools removed from mammals were tested for infection with 4.S. invicta . Notably, our study did not investigate other potentially important hosts that support ticks at the larval and nymph stage , or adult-stage ticks , which may also affect tick abundance. It is possible that lower small mammal abundance could increase the frequency of ticks feeding on alternative hosts, including humans, thus increasing disease risk . We obs invicta , possiblst ticks , consistsk (e.g. ).S. invicta on native small mammal and tick populations have important potential implications for the transmission of tick-borne pathogens, which represent significant public health concerns. Small mammals such as S. hispidus, which was heavily parasitized in this study, are reservoirs for numerous tick-borne pathogens including those in the genera Borrelia, Rickettsia, Anaplasma and Babesia, as well as multiple viruses [S. invicta-suppressed areas are expected to intensify contact rates between ticks and hosts, facilitating pathogen transmission. Indeed, increasing host abundance is one of the main drivers of tick-borne disease emergence [R. fulvescens on treatment plots directly increase vector-host ratios, potentially resulting in increased tick-borne pathogen transmission [The cascading effects of  viruses . Increasmergence . Higher smission .R. parkeri, which was present in 1.4% of larvae and 6.7% of nymphs. Rickettsia parkeri is a spotted fever group Rickettsia long associated with A. maculatum and recently associated with human disease in the United States [R. parkeri infection in our study is low compared with recent research in Virginia , these S. invicta have pervasive impacts on the ecosystems they invade [S. invicta suppression may have on tick-borne disease dynamics in some systems. Our work implies that during its invasion S. invicta may have produced ecosystem cascading effects that could lead to decreased vector, host and pathogen abundance.While y invade , includiy invade , land ma


Poecilia formosa) is the result of a hybridization of the Atlantic molly (P. mexicana) and the sailfin molly (P. latipinna) approximately 120,000 years ago. As a gynogenetic species, P. formosa needs to copulate with heterospecific males including males from one of its bisexual ancestral species. However, the sperm only triggers embryogenesis of the diploid eggs. The genetic information of the sperm donor typically will not contribute to the next generation of P. formosa. Hence, P. formosa possesses generally one allele from each of its ancestral species at any genetic locus. This raises the question whether both ancestral alleles are equally expressed in P. formosa. Allele-specific expression (ASE) has been previously assessed in various organisms, e.g., human and fish, and ASE was found to be important in the context of phenotypic variability and disease. In this study, we utilized Real-Time PCR techniques to estimate ASE of the androgen receptor alpha (arα) gene in several distinct tissues of Amazon mollies. We found an allelic bias favoring the maternal ancestor (P. mexicana) allele in ovarian tissue. This allelic bias was not observed in the gill or the brain tissue. Sequencing of the promoter regions of both alleles revealed an association between an Indel in a known CpG island and differential expression. Future studies may reveal whether our observed cis-regulatory divergence is caused by an ovary-specific trans-regulatory element, preferentially activating the allele of the maternal ancestor.The all-female Amazon molly ( Poecilia formosa) is an all-female species with unusual unisexual reproduction, called gynogenesis. P. formosa originated from two bisexual ancestral species, the Atlantic molly, P. mexicana, and sailfin molly, P. latipinna, probably via a single hybridization event at least 120,000 years ago . The transformed fluorescence ratio K’ was highly consistent among triplicates both for the cDNA and gDNA. The coefficient of variation (CV) of K’ was between 0.01% and 3.17% for cDNA and between 0.04% and 1.28% in gDNA . The avearα in P. formosa, one heterozygous marker (site 1974) was evaluated using Real-Time PCR. The mixes of plasmids  were used as standard curve and ASE calculation was conducted using the transformed fluorescence ratio K’. The ASE ratios  in ovary, brain, and gill in P. formosa are presented in P. mexicana-derived allele in P. formosa ovary was up to 0.770 (sd = 0.02), while these percentages in brain and gill were 0.462 (sd = 0.04) and 0.407 (sd = 0.09), respectively . In our study, this allelic bias in ovary was reproducible among all four biological replicates.To investigate ASE of ectively . Hence, P. mexicana-derived allele to P. latipinna-derived allele). They never significantly deviated from a 1:1 ratio.The frequency of the two heterozygous alleles in the gDNA assessment ranged from 0.48 to 0.54 . We applied the same method to gDNA of P. formosa, where–given the heterozygous status of this species [The transformed fluorescence ratio K’ method proved to accurately quantify the allele frequency in pooled DNA samples . However species ,12–14–thP. formosa [arα. ASE in P. formosa was estimated in several tissues, i.e., ovary, brain, and gill, using Real-Time PCR. Brain and gill did not show significant differential allelic expression. However, in ovarian tissue, the ASE of the two alleles showed a ratio of 7 to 3 (P. mexicana-derived to P. latipinna-derived), i.e., the allele derived from the maternal ancestor P. mexicana was expressed at level about twice as high as the other allele. A previous study on triploid Medaka (Oryzias latipes) reported that the maternally inherited allele was never silenced or suppressed, while one paternal allele was completely suppressed in the liver [P. mexicana-derived) exhibited higher expression than the paternal allele (P. latipinna-derived) only in ovarian tissue. Mechanisms and functions of tissue-specific ASE are still not well understood [arα we suggest tissue-specific epigenetic studies and investigations of methylation sites in the putative promoter region of arα among tissues.All unisexual vertebrates seem to be of hybrid origin  and have formosa ,13,46–48 formosa . Here, whe liver . In catthe liver . In our derstood . HoweverCYP1A2 gene in human liver [arα gene are unrelated: While total expression was similar among ovary and brain, but less in gill [P. formosa [arα in the ovary of P. formosa did not show a statistically significant difference relative to its ancestral species. However, those data revealed for ovary tissue some tendency towards higher expression in P. latipinna and lower expression in P. mexicana, with intermediate expression of P. formosa. Interestingly, in our study on the all-female P. formosa, the P. mexicana-derived allele  is more expressed than the P. latipinna-derived allele  specifically in the female gonad. As we identified potentially functionally relevant differences in the upstream putative promoter regions of the two alleles (see above), this allelic bias could constitute a cis-regulatory divergence [P. mexicana-derived allele and hence causes the observed allelic bias.Previous studies indicate that one cannot generally assume a correlation between total mRNA expression and ASE, as, e.g., shown for the an liver . Also in in gill , allelic in gill ,50–52. A formosa , the totvergence . PerhapsXiphophorus [arα in P. formosa could, however, not be evaluated in the context of this study, as the specimens analyzed so far from both parental species were fixed for respective species-specific alleles with identical coding and promoter region.Allelic bias in interspecies hybrids has been thoroughly investigated in the teleost fish genus hophorus –56. In tP. formosa, both alleles are apparently still suited to serve the biological function of this gene. To our knowledge, this is the first study of ASE in a clonal, hybrid vertebrate. Our data provides an example of tissue-specific allelic bias in a species characterized by a combination of two genomes of fundamentally different origin. It would be interesting to discover how widespread allelic bias is in the transcriptomes of this and other clonal species.As ASE does not have a significant impact on the total mRNA expression in S1 FigPolymorphic nucleotide positions (A) and the prediction of CpG islands and CpG sites (B) in the promoter region of androgen receptor alpha.(PDF)Click here for additional data file.


In this review, we overview the most important contributions of vibrational spectroscopy based techniques in the study of probiotics and lactic acid bacteria. First, we briefly introduce the fundamentals of these techniques, together with the main multivariate analytical tools used for spectral interpretation. Then, four main groups of applications are reported: (a) bacterial taxonomy (Subsection 4.1); (b) bacterial preservation (Subsection 4.2); (c) monitoring processes involving lactic acid bacteria and probiotics (Subsection 4.3); (d) imaging-based applications (Subsection 4.4). A final conclusion, underlying the potentialities of these techniques, is presented. Lactobacillus and Bifidobacterium. Among the beneficial effects of probiotic strains, one can mention immunological stimulation, improvement of digestion and absorption, synthesis of vitamins, inhibition of potential pathogens, decrease of cholesterol levels, lowering of gas distension, and restoration of the normal flora after antibiotic therapy . Th. (2000)  designed. (2012)  obtainedet al. (2000) [on-line analyze both substrates  and products (ethanol and lactic acid) of alcoholic and lactic fermentations. In turn, Sivakesava et al. (2001) [at-line monitoring the production of biomass, glucose and lactic acid by L. casei. Suitable spectral wavenumber regions were selected to calibrate PLS and PCR models.The concentrations of lactose, galactose, lactic acid and biomass in fermentation processes were determined using combined approaches of FTIR and PLS, and HPLC as reference method ,98. In a. (2000)  develope. (2001)  used midet al. (2002) [L. rhamnosus RW9595M cultures by using second derivative NIR spectra and Grassi et al. (2013 and 2014) [S. thermophilus and L. delbrueckii subsp. bulgaricus to obtain information about quality parameters, including bacterial counts, titratable acidity, pH, lactose, galactose and lactic acid concentrations. Oberreuter et al. (2000) [Saccharomyces cerevisiae/Hanseniasporauvarum and L. acidophilus/S. thermophilus.Macedo . (2002)  defined nd 2014) ,103 moni. (2000)  also detet al. (2011) [Staphylococcus aureus in milk, as well as the growth interaction between S. aureus and Lactococcus lactis ssp. cremoris. Both species were investigated as pure cultures and in co-cultures after inoculation into UHT milk. Definition of PLS based models using plate counting as reference method, enabled an accurate and sensitive detection of the mentioned microorganisms directly from the FTIR and Raman spectra [Nicolau . (2011)  detected spectra .et al. (2002) [i.e., lactose, galactose, and lactate), and a secondary network to predict a qualitative process state variable describing critical process phases . This approach improves the quality control of yoghurt fermentation by simultaneously predicting both quantitative and qualitative process’ variables. Navraätil et al. (2004) [on-line monitoring fermentations leading to the production of yogurt and fimjölk, a Swedish yogurt-like sour milk, under industrial conditions. First, PCA was carried out and then, the first PCs were used for on-line generation of a plot indicating the evolution of the fermentation process. With that background, an acceptable PLS model was defined to predict pH and titratable acidity [Cimander . (2002)  followed. (2004)  used NIR acidity .et al. (2009) [et al. (2013) [Da Cruz . (2009)  develope. (2013)  used Ramet al. (2014) [L. delbrueckii subsp. bulgaricus after dehydrating microorganisms in the presence of galactooligosaccharides and lactulose. The relative composition of microorganisms  throughout the kinetics of growth was assessed by multiparametric flow cytometry, using carboxyfluorescein diacetate and propidium iodide probes. PCA performed on the second derivative NIR spectra showed three groups along PC1, corresponding to the lag, exponential and stationary phases of growth, which explained 99% of the total variance. Along PC2, two groups were observed, corresponding to damaged and viable cells [Santos . (2014)  monitorele cells .L. plantarum, L. brevis, L. paracasei, L. fermentum, Pediococcus pentosaceus and S. thermophilus. As a result of fermentation, sorghum prolamins are hydrolyzed and in vitro digestibility is facilitated. The fermentation process can be followed by FTIR. In a first paper, Correia et al. (2005) [L. fermentum, L. bulgaricus, L. lactis, Pediococcus pentosaceus and P. cerevisiae and also with a mixture of L. bulgaricus and S. thermophilus from a commercial yogurt. The increase of titratable acidity, total protein content and free aminoacids, and the decrease of reducing sugars, soluble proteins and starch during fermentation were analyzed using traditional chemical methods and confirmed by 1H NMR and FTIR, thus supporting their use to follow these fermentation processes [Sorghum is generally fermented with commercial strains of . (2005)  inoculatrocesses . In a larocesses .et al. (2004) [Johnson . (2004)  investiget al. (2015) [Aliakbarian . (2015)  used NIRi.e., sugars, ethanol) and also other quality parameters such as phenolic composition and volatile compounds. All these parameters can be followed during wine production by using FTIR and chemometric analysis [Infrared spectroscopy has been widely applied in winemaking to quick perform a quality control of the product at all stages of the process. This analysis includes assessing both substrate and product concentrations  and leads to a decrease of moisture, starch and lipid content and an increase of protein conntrations. Therefore, FTIR analysis can be used to investigate the changes experienced by rice during fermentation [Other interesting applications of FTIR have been used in Asia. Rice fermentation is an important step in the elaboration of rice paper because it changes physicochemical and morphological properties of rice flour and also influences the final product. This fermentation step results from the activity of different genera of lactic acid bacteria  [Certain species of lactobacilli are associated with meat spoilage. Different authors used NIR to detect them. Argyri . (2010)  correlat. (2010) . In a la. (2010) . Similar. (2010) ,122,123.et al. (2011) [i.e., L. plantarum, Leuconostoc mesenteroides and L. sakei) in the 1100–2500 nm region. PCA enabled a clear discrimination among the three species and PLS enabled a successful quantification in the range 3–9 log CFU/mL, and also the possibility of distinguishing between spoilage (7–9 log cfu/mL) and non-spoilage concentration levels (3–6 log cfu/mL) [Camara-Martos . (2011)  used NIR cfu/mL) .et al. (2012) [L. crispatus to inhibit the pathogenic foodborne bacteria Camylobacter jejuni. The harmful effect of L. crispatus is given by lactic acid. Spectral features of C. jejuni and L. crispatus enabled the accurate prediction of bacterial viability and also, the discrimination of C. jejuni according to the treatment received. When both microorganisms are grown in co-culture, the metabolism was dominated by Lactobacillus prior to the killing of C. jejuni [McKinney . (2012)  used FTI. jejuni .Other authors demonstrated the reliability of vibrational spectroscopies to determine the capacity of lactic acid bacteria to remove harmful concentrations of heavy metals .et al. (2010) [L. rhamnosus IMC 501 for 10 days. He et al. (2014) [et al. (2013) [et al. (2014) [Hyperspectral chemical imaging is a broad term referring spatially resolved spectral information obtained through different techniques, namely Raman, FTIR, NIR and fluorescence. Using these techniques for investigation of lactic acid bacteria and probiotics is very scarce and most of publications are focused on the effects of microorganisms on eukaryotic cells or tissues rather than on bacteria themselves. For example, Giorgini . (2010)  reported. (2014)  reported. (2013)  quantifi. (2014)  observed. (2014) .Vibrational spectroscopic techniques were born as almost exclusive physical and chemical tools generally employed to resolve the chemical structure of compounds having a reduced number of atoms. The development of strong equipment and computational resources certainly contributed to the growth of novel research areas at the interface between Biosciences and Physical-Chemistry. The enormous advantage of these techniques is that they do not require external reagents  and sample preparation is minimal, which is particularly relevant for industrial applications. However, the inherent complexity of biological samples requires a strong background on mathematical and statistical tools (Chemometrics) for spectral interpretation. Unfortunately, sometimes this is not a common skill among researchers with biological background.In this review, we put into relevance the use and potentialities of this approach in studying lactic acid bacteria and probiotics both at an industrial and at a research level. Considering the great importance of probiotics market, the systematic industrial implementation of these techniques could be much more explored than it is. In regard to research, it is our opinion that using vibrational spectroscopic techniques to investigate lactic acid bacteria and probiotics represents a relatively novel approach far from being fully explored and where a lot of things still remain to be done.


Macrophage migration inhibitory factor (MIF), a pleiotropic proinflammatory cytokine, has been showed to be associated with the immunopathogenesis of many diseases. Recent study demonstrated that MIF promoted tumorigenesis and tumor progression and played a critical role in various kinds of human cancer including head and neck squamous cell carcinoma(HNSCC). Hence, in this paper we retrospected the relationship between MIF and angiogenesis, epithelial-mesenchymal transition (EMT), inflammation, immune response, hypoxia microenvironment, and discussed whether it is a promising biomarker for diagnosis and supervisor of HNSCC. Head and neck squamous cell carcinoma (HNSCC), which accounts for 90 % of head and neck cancers, has been reported as the sixth common cancer worldwide and the third most common cancer in developing nations. –3 DespitMacrophage migration inhibitory factor (MIF), a T-cell-derived factor, was firstly thought to inhibit the migration of macrophages in experiments designed to characterize delayed-type hypersensitivity and hence derived its name in 1966.  Later, tMIF lies on the human genome (22q11.2), regulated by the two polymorphic sites (CATT repeat at -794 and a single nucleotide polymorphism at -173 (G/C)) in the promoter region. [MIF is highly conserved across phylogeny, there is a remarkable feature of the human MIF gene that is the presence of a microsatellite repeat (CATT)5-8 within the 5′ promoter region. [The  region. –17 Thoug region.  It is as region.  A detail region. S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-MIF is a molecule comprised of 115 amino acids with a molecular weight of 12.5 kDa.  In the aisoxazole acetic acid methyl ester (ISO-1), an inhibitor of DDT, could decrease wild-type or mutant MIF activity in human and murine mononuclear cells. So there was a hypothesis that MIF also displayed some enzymatic activity. via cyclin D1 transcription and subsequent phosphorylation of the Rb gene and prostaglandin E2 production. [Although MIF was found in the 1960s, the cell surface receptor for MIF was identified until more than 35 years later.  CD74 wasduction. , 26 Therduction.  Besides,duction.  The longduction.  Intriguiduction.  In this duction.  whereas duction. via activation of HMGB1/TLR4/NF-κB axis. [The nuclear factor - kappa B (NF-κB) which plays a vital role in carcinogenesis is involved in the MIF associated signaling pathways. In nasopharyngeal carcinoma (NPC) cell lines, C666-1, MIF/IL-8/ CXCR2 signaling could enhance the growth of the tumor spheres. And NF-κB inhibitor parthenolide could inhibit the gene expression of IL-8.  Lv et alκB axis.  In the nκB axis. in vitro and in vivo. It was shown that MIF secretion was inhibited by high anti-inflammatory concentration of steroids. [The notion of MIF as a proinflammatory protein was firstly confirmed by the studies about delayed hypersensitivity and further evaluated in the mouse model of septic shock.  It was iteroids.  Comparedteroids.  Bacher eteroids.  As mentiteroids.  Moreoverteroids.  Additionteroids. + T cells in the peritumoral tissue. [via CXCR2-dependent chemotaxis and trigger the inflammatory activity by eliciting neutrophils’ release of C-C Motif Ligand 4 (CCL4) and matrix metalloprotease 9 (MMP9) in human hypopharyngeal carcinoma cell line FaDu. [in vitro, Liao et al. inferred that MIF contributed to lymph node metastasis by upregulating IL-8 expression. [The role of MIF in inflammation underlined its position in the development of HNSCC. In laryngeal carcinoma samples, Kindt et al. emphasized that the elevated MIF level was associated with a slightly decreased abundance of CD3 tissue.  In anothne FaDu.  Both CCLne FaDu. –42 Li etne FaDu.  With obsression. +Gr-1+ myeloid cells by elevating production of MIF via HIF-1α/HIF-2α-dependent ways in HNSCC. [via ERK activity. [MIF. [The crosstalk between tumor cells and microenvironment of the host is an important driving force in the selection of clone that is prone to invasion and metastasis. Hypoxia, resulting from rapid tumor growth in the absence of accompanying blood supply, is a critical symbol and determinant of tumor microenvironment. To maintain growth advantage, tumor cells under hypoxia microenvironment enhanced expression of hypoxia-inducible factor-1 (HIF-1), which favors anaerobic tumor growth, resistance to therapy, and metastatic adaptation.  HIF-1, an HNSCC.  Fu et alctivity.  Baugh ety. [MIF.  Mladenovy. [MIF.  Increasiy. [MIF. via p53-dependent manner in vitro and in vivo. [Conversely, MIF also contributed to stabilize HIF-1. No et al. suggested that a ternary complex formed by HIF-1α, MIF, and CSN9 signalosome subunit 5 (the bridge between HIF-1α and MIF) was necessary to prevent degradation of HIF-1α under aerobic condition.  Oda et ain vivo. However, Larsen et al. found that inhibition of HIF-1α, HIF-2α using siRNA had no effect on hypoxia-induced MIF secretion in MCF-7 breast cancer cells, which hinted that there existed other hypoxia-induced regulatory mechanisms for the up-regulation of MIF. The authors considered that NF-κB and C/EBPβ signaling pathways were association with this phenomenon. Analogous to normal tissues, tumors require vessels to sustainably supply nutrients and oxygen as well as evacuate metabolic wastes and carbon dioxide simultaneously. Indeed, no matter synthetic or catabolic metabolism is excessively exuberant in tumors. Hence, it is necessary to induce new blood vessels to meet tumor cells metabolism. Angiogenesis, as a vital mode to achieve this goal, is the formation of new blood vessels from the existing vasculature by sprouting. ,58 OpposAccumulated evidence has proved that MIF was associated with tumor angiogenesis. When the B lymphoma mouse model was administrated with monoclonal antibody (mAb) specific for MIF, Chesney et al. observed a marked reduction in B lymphoma growth. However, profound analysis showed that anti-MIF antibody unaffected B-cell lymphoma proliferation. Reciprocally, microvascular endothelial cells were sensitivity to the mAb, whose proliferation was dependent on MIF. This result suggested that the antitumor effect caused by anti-MIF resulted from MIF suppression on angiogenesis.  Subsequevia using anti-CD74 shRNA, the SCCVII cells showed a significant decrease in producing VEGF compared to the control, which damaged the capacity of promotion angiogenesis. [Vascular endothelial growth factor (VEGF) is the well-known angiogenesis inducer.  One possgenesis.  IL-8, angenesis. EMT is a process that polarized epithelial cells convert into motile mesenchymal phenotype which is characterized by suppression of the adherent protein E-cadherin and overexpression mesenchymal markers, such as N-cadherin and vimentin. –66 In thMIF has been deemed one of the factors triggering EMT. In A549 lung adenocarcinoma cells, Keshamouni et al. identified that MIF was up-regulated during TGF-β induced EMT with quantitative differential proteomic analysis.  FunamizuKip1via the PI3K/Akt pathway. [Kip contributed to regulate cell cycle progression from G1 to S phase. [via the AMPK/mTOR signaling pathway. [Research evidence supported that the activation of MIF was involved in cell proliferation and apoptosis.  During cpathway.  Both theS phase.  Thus, thS phase.  In renalS phase.  SiRNA agS phase.  MIF-KD sS phase.  And partS phase.  MIF inhiS phase.  CPSI-130S phase.  Xia et apathway.  In maligpathway.  Park et pathway.  Liu et apathway. p53, one crucial tumor suppressors, is the most commonly silenced or mutated gene in cancer. [via p53-dependent manner. [ cancer.  Generall cancer. –90 Hudso cancer.  Subseque cancer. –94 Howev cancer.  And Jab1 cancer. , 97 Besi manner.  Reciproc manner. beclin1 induced hepatocellular carcinoma, lung adenocarcinoma, mammary hyperplasia, and lymphoma in the mice. The constitutive activation of PI3k/Akt pathway by mutations might stimulate the process. Tamoxifen, used to treat certain types of breast cancer, was considered to activate autophagy by up-regulation beclin1. Thus, autophagy is considered as a double-edged sword for its role may be altered during tumor progression. [Autophagy is another manner that manipulates the survival and death of cell. Since it's essential in the degradation of accumulated damaged or long-lived and superfluous organelles which are toxic for cells with the lysosomal machinery, autophagy has been characterized as an intracellular catabolic pathway that plays vital role in maintenance cellular homeostasis.  In generression. , 104in vitro cell culture and in vivo mice experiments. [MIF-induced cytokines, such as IL-1β, TNF-α, have been confirmed to be involved in autophagy. Chuang et al. found that MIF could induce autophagy in hepatocytes through ROS generation.  Chen et riments.  In breasriments.  In Liu eriments. Taken together, the relationship beteewn MIF and HNSCC has been established by a great deal of studies, however, the mechanism is still ambiguous and further research is needed. We summarize the relationship between them mentioned above in the Figure in vitro. Collectively, they proposed that MIF could be a potential tissue biomarker for OSCC. [in vitro and xenograft tumor growth in vivo by targeting MIF. [With the analysis of quantitative immunohistochemistry, Kindt et al. found that MIF staining intensity significantly increased in hypopharyngeal squamous cell carcinoma samples compared to tumor-free epithelia, low-grade dysplasia or high-grade dysplasia.  They hador OSCC.  Liu et aing MIF.  In laryning MIF. Firstly, MIF was ascribed as a proinflammatory factor. Further studies have demonstrated that MIF is involved in the progression of tumors including HNSCC. The expression of MIF in HNSCC samples has been proved to be related to the clinical outcomes of patients. The higher MIF level in serum was also found in patients with HNSCC. MIF can regulate HNSCC cells proliferation, apoptosis, invasion, and metastasis though its pleiotropic roles in mediating hypoxia response, angiogenesis, and EMT. The inhibition of MIF can restrict the progression of tumors. This evidence hints that MIF may be a potential biomarker of HNSCC.As mentioned above, for the roles of MIF in supporting cancer, there exists possibility that it can be a therapeutic target for cancer. Indeed, the restriction of MIF showed the efficacy of cancer treatment. The inhibition of MIF in gallbladder cancer cell line by ISO-1 and 4-IPP or its specific siRNA led to a decrease in the colony forming ability.  ISO-1, t


Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis.Sexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes.We focused on P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin.Our microsatellite data revealed that P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings.Our results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with The online version of this article (10.1186/s13293-018-0172-z) contains supplementary material, which is available to authorized users. Pelophylax [Squalius [Misgurnus [Hypseleotris [Squalius alburnoides complex suggests that perpetuation of hybrid males in populations depends on highly complex reproductive mechanisms [Pelophylax and Hypseleotris. It is not yet clear what their origin is and what enables the maintenance of male unisexuality in these systems.Unisexual animals are model systems for understanding the evolution and maintenance of sex and recombination despite the costs that may arise , 2. Intelophylax –10 and fSqualius , Misgurnisgurnus , 13, andeleotris . An examchanisms . Here, hHybridogenesis, combining elements of both clonal and sexual reproduction, seems to be the most promising reproductive mode for the maintenance of male unisexuality as separate, sex-specific lineages . HybridsPelophylax esculentus complex consists of two sexual species, the pool frog Pelophylax lessonae (genomic composition LL) and the the marsh frog P. ridibundus (RR), and their hybrid taxon, the edible frog P. esculentus (RL). In most parts of Western and Central Europe, P. esculentus lives in sympatry with P. lessonae, in what is known as the “L-E system” [lessonae (L) genome from their germline and transmit the ridibundus (R) genome to their gametes. Hybridity is restored in each generation by heterospecific crosses between P. esculentus and P. lessonae, the donor of the L genome. When a hybrid male mates with a LL or RL female, only female offspring originates. Thus, males originate from matings between hybridogenetic females and heterospecific sexual P. lessonae males producing both hybrid sexes [The  system” . In thisid sexes .P. esculentus exclude the ridibundus genome, transmit their lessonae genome, and mate with P. ridibundus to perpetuate the hybrid lines [P. ridibundus and only P. esculentus males. Such populations have been reported from the Czech Republic [P. esculentus males from this population type transmit only the lessonae genome, others only the ridibundus genome, and some males even produce both ridibundus and lessonae gametes [P. ridibundus females and P. esculentus males usually two genotypes originate: if the ridibundus (R) eggs are fertilized by sperm with an R genome, RR females originate, while the combination of R eggs an L sperm gives rise to only hybrid (LR) males [P. esculentus. Consequently, there has been, and still might be, the possibility for de novo formation of P. esculentus males via ongoing primary hybridizations between the two sexual species, thus leaving open the question of their stable persistence in natural populations.Conversely, in the R-E system, most id lines –23. SpecRepublic , GermanyRepublic , 9, 25, Republic , and PolRepublic –29. In c gametes , 30. FroR) males , 30, 31.This study was aimed to (1) explore the origin of all male lineages in populations of the R-E system, i.e., whether male unisexuality evolved from one or several hybridization events, and (2) to understand how all-male hybrids are able to persist without hybrid females in natural populations. In pursuing these goals, we sampled 249 water frogs from 16 populations along the upper Oder River. Based on 17 microsatellite markers, we investigated (a) the mode of asexual reproduction, (b) the genetic variability of clonally transmitted genomes, and (c) their genealogical relationships to homotypic genomes from adjacent populations.Pelophylax taxa were caught with a hand net at 16 locations during years 2002 and 2008. Pelophylax kurtmuelleri from Greece, a sister species of European P. ridibundus, was used as an outgroup in a phylogenetic tree construction. Males were distinguished from females by the presence of vocal sacs and nuptial pads. Taxon affiliation was determined on the basis of external morphological characters [All European water frogs were collected in the upper Oder River drainage , glucose-6-phosphate isomerase , glycerol-3-phosphate dehydrogenase , l-lactate dehydrogenase , phosphoglucomutase , and phosphogluconate dehydrogenase , were analyzed by horizontal potato starch gel electrophoresis [a” fastest to “e” slowest according to their mobility. Samples which revealed unclear patterns were reprocessed.For genotype determination, six allozyme loci previously identified to be diagnostic for ere used , 33. Appere used ) using aphoresis . Subsequphoresis –37. StaiGenomic DNA was extracted using a NucleoSpin commercial kit (Macherey-Nagel GmbH and Co.) with the epMotion 5075 automated pipetting system (Eppendorf). We amplified 17 microsatellite loci: Ga1a19, Re2caga3, Re1Caga10, RICA1b6 , RlCA1b5P. lessonae and P. ridibundus were checked for potential genotyping errors with Micro-Checker version 2.2.3 [HW) genotypic equilibrium. We found one locus (Res20) in P. lessonae and four loci  in P. ridibundus with a potential presence of null alleles, and therefore applied a correction for null alleles at these loci following Wagner et al. [AN), observed heterozygosities (HOBS), and expected heterozygosities (HEXP) within populations.Due to the presence of null alleles, raw microsatellite genotypes of on 2.2.3 . This meon 2.2.3 . In eachr et al. . The aftr et al.  was usedlessonae and ridibundus genomes according to the allele species specificity known from the literature [Based on the definition of hybridogenetic reproduction, diploid hybrid water frogs from Central Europe transmit one haploid chromosome set clonally to gametes (therefore termed a “hemiclone” ), whereaterature . Then, tridibundus and lessonae genomes were included in our analyses, we used only lessonae genomes of hybrids from the L-E system populations because in this system we were not interested in the origin of ridibundus genomes. We determined a hemiclone by a multilocus genotype (MLG), defined by the identical combination of alleles found in our microsatellite analyses. A minimum of three samples exhibiting the same allele composition was a clear indication that the genome was inherited clonally  and did not originate from a sexual donor [lessonae and ridibundus genomes of P. esculentus (MLG’s) separately. To diploidize these haploid genotypes for GeneClone, we simply doubled lessonae and ridibundus genomes. Detailed information about the programs used are given below.While for hybrids from R-E system populations both al donor . As mostIn order to determine ploidy levels, all individuals were analyzed by flow cytometry using blood samples following the standardized methodology . A drop P. esculentus lineages, we analyzed genetic relationships between hybrids and their parental species on the basis of allele frequencies of up to 17 microsatellite loci using Structure v. 2.3.1 [In order to determine the origin of the all-male v. 2.3.1 , GenAlExv. 2.3.1 , Populatv. 2.3.1 , and Genv. 2.3.1 .P. esculentus was analyzed with a model-based clustering method as implemented in Structure to infer population structure based on genotypic data consisting of unlinked markers. Structure analyses included datasets of 177 individuals  repeats. The probability of the used admixture model was tested for clusters K = 1 − 10. The most probable number of K populations was estimated using log-likelihood ln P(D) according to Evanno et al. [The origin of o et al. , with Sto et al. .PSEX values using the program GeneClone. When the same genotype is detected more than once, PSEX expresses the probability that this MLG has been derived from distinct reproductive events [PSEX probability taking into account the FIS estimated of the dataset [FIS was also estimated on the basis of the round-robin method, and further used to estimate a corrected PGEN, calculated as the unique MLG probability [PSEX [PSEX in the program, we considered 10 loci from which both genomes were amplified , i.e., MLGs with missing alleles were eliminated , we used multilocus matches analysis as implemented in the program GenAlEx. In order to estimate the genetic relatedness between individual genomes, we compared allele frequencies, heterozygosity, and polymorphism estimates with the program GenAlEx. A centred principal component analysis (PCA) was applied to examine clustering of individuals based on total variation of microsatellite allele frequencies without scaling of alleles. For the PCA, we converted a list of 275 MLG’s from 17 loci  were codistance  calculatP. lessonae, 42 individuals of P. ridibundus, and 98 individuals of P. esculentus using six diagnostic allozyme loci  and two polymorphic allozyme loci ; in P. ridibundus, two monomorphic  and four polymorphic loci  were found. In P. esculentus, all six loci were polymorphic. The allelic products of three loci  displayed an atypical expression in 12 individuals. Here, four P. ridibundus possessed alleles characteristic of the lessonae genome in G3pdh and Pgm, one P. lessonae possessed alleles characteristic of the ridibundus genome in Pgm, and seven P. esculentus possessed either two lessonae or two ridibundus specific alleles in both Gpi and Pgm  that are assumed to be specific for P. lessonae [P. ridibundus [P. lessonae and P. ridibundus samples.Overall, all 17 microsatellite loci were polymorphic among 249 lessonae , 40, andP. lessonae  and P. ridibundus . The presence of one or two alleles per locus  representing hybrid P. esculentus, which is in accordance with results from the determination based on diagnostic markers  included lessonae-specific MLGs obtained from P. esculentus in R-E populations while cluster 3 (red and blue symbols) grouped P. ridibundus and ridibundus-specific MLGs of P. esculentus from R-E populations.The PCA identified three groups of microsatellite MLGs, the first two principle components accounted for 62.15% (axis 1) and 11.47% (axis 3) of genetic variation, respectively  designated as ODERL1.In the dataset which contained missing data for some loci, GenAlEx estimated a total of 185 MLGs among all lessonae genomes, all originating from P. esculentus of R-E male populations, were represented by only a single MLG. The PSEX value of this MLG ranged from 0.15 to 1.50E−41, indicating clonal inheritance of ODERL1.Considering only MLGs with complete allelic data, GenClone generated 122 MLGs among 143 genotypes. Twenty-one P. ridibundus of both sexes and only males of P. esculentus  live outside the valley. Since 2001, no hybrid female was detected among 377 hybrid individuals in localities of the R-E system  supports its clonal inheritance.First, our extensive sampling did not reveal the presence of id males . The seches Fig. a, b. Morridibundus and lessonae allelic richness in all-male P. esculentus revealed that 13 loci within the ridibundus genome were polymorphic with the observed allelic frequencies (0.036–0.842). Together with the abovementioned observation that single ridibundus MLGs were not shared among individuals at all, it is reasonable to assume that these ridibundus genomes came from recombinant eggs of sexually reproducing P. ridibundus. In the lessonae genome, however, we observed only monomorphic loci with allele frequencies of 1.00. Therefore, it is proven that the lessonae genome is clonally inherited and represents a single clone  is new finding. Most animal systems with female unisexuality comprise multiple clonal MLGs, as spined loach fishes of the genera Cobitis [Poeciliopsis [Phoxinus eos-neogaeus complex [Poecilia formosa, populations show a fairly high level of clonal diversity [Pelophylax populations of the L-E system, where both hybrid males and females coexist, the diversity of transmitted ridibundus clones is high [The characteristic of the l et al. . However Cobitis  and Poeciliopsis  and the  complex . Even whiversity . In Pelo is high , 69.Pelophylax water frogs in particular, hinge on the formation of a specific genome from a particular sexual species. In European water frogs, the genome of P. lessonae represented by the MLG ODERL1 seems to play a key role in the formation of all-male P. esculentus lineages. Supporting evidence comes from rare Central- and West-European mixed populations, where all-male P. esculentus are triploid and originate from clonal sperm with a diploid lessonae genome and haploid ridibundus eggs [Apparently, the existence and maintenance of all-male unisexuality among animals in general, and in dus eggs .P. lessonae genepool, this clone does not cluster with other lessonae-specific MLGs Additional file 2:Table S2. DNA microsatellite data file for the 17 loci used in the study. (PDF 996 kb)Additional file 3:Table S3. Allozyme data file for the six loci used in the study. (PDF 514 kb)Additional file 4:Table S4. Species-specificity of microsatellite alleles used in this study. (PDF 259 kb)Additional file 5:Table S5. Summary statistics of microsatellite alleles found in P. lessonae, P. ridibundus, and P. esculentus from the R-E system. (PDF 317 kb)Additional file 6:Figure S1: Phylogenetic tree of DA distance of 10 microsatellite loci reconstructed in Populations . One terminal branch represents one individual: Green color – P. lessonae, yellow color - L genome from P. esculentus, red colour – P. ridibundus, blue color – R genome from P. esculentus, violet color – P. kurtmuelleri. (JPEG 3388 kb)Additional file 7:Table S6. Multilocus Genotypes (MLGs) derived from P. lessonae, P. ridibundus and P. esculentus from the R-E system based on 17 microsatellite loci. (PDF 316 kb)


Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.  •Robust and enriched motor neurogenesis and astrogliogenesis from human iPSCs•VCP-mutant motor neurons show TDP-43 mislocalization and ER stress as early pathogenic events•VCP-mutant astrocytes exhibit a cell-autonomous survival phenotype•VCP-mutations perturb the ability of astrocytes to support motor neuron survival Hall et al. use iPSCs to examine the sequence of events by which motor neurons degenerate in a genetic form of ALS. They find that astrocytes, a type of supportive cell, also degenerate under these conditions. The ALS-causing mutation disrupts the ability of astrocytes to promote survival of motor neurons. VCP mutations account for 2% of familial ALS cases  , which is ubiquitously expressed and contributes to myriad cellular functions in a cofactor-dependent manner. VCP functions include maintenance of protein homeostasis, mitochondrial quality control, and apoptosis  is a rapidly progressive and fatal neurological condition characterized by degeneration of MNs (motor neurons). Over 20 distinct gene mutations have been identified, although their collective functions have not yet converged on a singular molecular pathway. Autosomal-dominant LS cases , compara(TDP-43) . The VCPpoptosis . Wild-typoptosis , includipoptosis .While undeniably valuable both animal-based models and many cell-based ones have thus far relied on VCP overexpression or knockdown in non-human or non-neuronal cells, which may fail to precisely capture the clinical pathophysiological state. Consequently, there is a need for accurate characterization of how mutant VCP affects human MNs and ACs (astrocytes). To examine early pathogenic events in VCP-related ALS, we employed patient-specific iPSCs and robust ontogeny-recapitulating methods of directed differentiation to enriched populations of both spinal cord MNs and ACs. By integrating this human experimental platform with cellular phenotyping assays, we uncovered early sequential pathogenic events in VCP-mutant MNs. Additionally, we identified VCP-mutant cell-autonomous AC pathology and a non-cell-autonomous effect of mutant ACs on both control and mutant MNs.Using established reprogramming methods , four cl−8, log-rank test , revealing a significant increase in the VCP-mutant d17 MNs compared to control  and dendrites  and ChAT, respectively Di. A sigassembly A. Analysand GRM7 B.Having identified cell death, synapse loss, and transcriptional perturbations of genes encoding ion channels and synapse structure and/or assembly, we next addressed VCP mutation-related functional consequences by examining electrophysiological properties of our MNs on multi-electrode arrays (MEAs). Electrical activity began to appear on individual electrodes during the first few days following differentiation as random spiking. Over subsequent maturation of control MNs, uncoordinated local bursting behavior was observed on active electrodes that eventually became synchronized across the whole active network . These pMislocalization of TDP-43 from the nucleus to cytoplasm correlates with cellular toxicity . To explm) was examined using the fluorescent cationic dye TMRM (tetramethylrhodamine methyl ester)  A. VCP-mus of Δψm B, while  systems . We next systems Ci. We fo systems Cii. Furt systems D. TogethWe next evaluated the contribution of VCP-mutant ACs in our model. Using cross-sectional end point analysis, we found no significant difference in survival between control and VCP-mutant ACs . However, recognizing the proliferative capacity of ACs in their differentiated state, we reasoned that this might allow an underlying survival phenotype to escape detection when using a cross-sectional approach. Therefore, we again employed the more sensitive real-time longitudinal-imaging platform and indeed detected a significant increase in risk of cell death of the VCP-mutant ACs compared to control ACs Ai. It ism was observed in d14 VCP-mutant ACs compared to controls  mutation has been shown to cause a cell-autonomous survival phenotype in hiPSC-derived MNs . The preutations . It is nutations Di would In summary, our study demonstrates the potential of integrating directed differentiation of iPSCs with time-resolved phenotyping assays as a pre-clinical model to confidently identify primary molecular pathogenic events in ALS patient-derived MNs, together with cell autonomous and non-cell-autonomous contributions of mutant ACs in this context. Our findings could thus help drive the development of therapies addressing specific disease-initiating mechanisms and raise the prospect of targeting ACs as a strategy to ameliorate disease progression in ALS.Informed consent was obtained from all patients and healthy controls in this study. Experimental protocols were all carried out according to approved regulations and guidelines by UCLH’s National Hospital for Neurology and Neurosurgery and UCL’s Institute of Neurology joint research ethics committee (09/0272).∗C) and ThermoFisher Scientific (cat. number A18945) respectively.Informed consent was obtained from all patients prior to skin biopsy. Dermal fibroblasts were cultured in OptiMEM +10% FCS medium. The following episomal plasmids were transfected for iPSC generation: pCXLE hOct4 shp53, pCXLE hSK, and pCXLE hUL (Addgene), as previously reported . DetailsInduced PSCs were maintained on Geltrex (Life Technologies) with Essential 8 Medium media (Life Technologies), and passaged using EDTA . All cell cultures were maintained at 37°C and 5% carbon dioxide.For any experiment, the average experimental unit is calculated, then the average across all the clones is taken, so the variation shown is the biological variation across biological replicates . The data are checked for normality, and we utilized an unpaired t test with post hoc correction for multiple testing.C.E.H., Z.Y., M.C., G.E.T., S.G., and R.P. conceived and designed the experiments. C.E.H., Z.Y., M.C., G.E.T., S.G., and R.P. wrote the manuscript with contributions from all coauthors; C.E.H., R.L., N.L., and C.S. developed the computational pipelines and analyzed the RNA-seq data. S.J.C., D.M.K., and R.B. conducted and/or supervised electrophysiological profiling of MNs. A.L. performed and supervised the synaptic analyses. A.S. performed and supervised analysis of the longitudinal imaging data. S.H.Y.L. and L.M.M. conducted and analyzed electron microscopy. Project design and concepts were developed by S.G. and R.P., who supervised the work.