The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.

Leeches and oligochaetes comprise a monophyletic group of annelids, the Clitellata, whose reproduction is characterized by simultaneous hermaphroditism. While most clitellate species reproduce by cross-fertilization, self-fertilization has been described within the speciose genus Helobdella. Here we document the reproductive life histories and reproductive capacities for three other Helobdella species. Under laboratory conditions, both H. robusta and H. octatestisaca exhibit uniparental reproduction, apparently reflecting self-fertility, and suggesting that this trait is ancestral for the genus. However, the third species, H. austinensis, seems incapable of reproduction by self-fertilization, so we inferred its reproductive life history by analyzing reproduction in breeding cohorts. Comparing the reproductive parameters for H. robusta reproducing in isolation and in cohorts revealed that reproduction in cohorts is dramatically delayed with respect to that of isolated individuals, and that cohorts of leeches coordinate their cocoon deposition in a manner that is not predicted from the reproductive parameters of individuals reproducing in isolation. Finally, our comparisons of reproductive capacity for individuals versus cohorts for H. robusta, and between different sizes of cohorts for H. austinensis, reveal differences in resource allocation between male and female reproductive roles that are consistent with evolutionary theory.

Behaviour-change interventions have been consistently considered an essential part of comprehensive HIV, STI and unintended pregnancy prevention. In 2015, the World Health Organization reviewed and assessed existing evidence on brief behavioural interventions, leading to the publication of Brief sexuality-related communication: recommendations for a public health approach. This guideline recommends the use of brief behaviour intervention and communication programmes to promote sexual health and to prevent HIV, STIs, and unintended pregnancies in primary health services, particularly sexual and reproductive health services.

While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the 'bag of worms' phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant females causes fetal death and increased piglet mortality, but there is substantial variation in the extent of reproductive pathology between individual dams. This study used RNA-sequencing to characterize the whole blood transcriptional response to type 2 PRRSV in pregnant gilts during the first week of infection (at 0, 2, and 6 days post-inoculation), and attempted to identify gene expression signatures associated with a low or high level of fetal mortality rates (LFM and HFM; n = 8/group) at necropsy, 21 days post-inoculation. The initial response to infection measured at 2 days post-inoculation saw an upregulation of genes involved in innate immunity, such as interferon-stimulated antiviral genes and inflammatory markers, and apoptosis. A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed. By day 6 the pattern had reversed, with a drop in innate immune signaling and an increase in the expression of genes involved in cell division and T cell signaling. Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points. Among the 15 DEGs upregulated in LFM gilts on all three days were several genes involved in platelet function, including integrins ITGA2B and ITGB3, and the chemokine PF4 (CXCL4). LFM gilts exhibited a higher baseline expression of interferon-stimulated and pro-inflammatory genes prior to infection, and of T cell markers two days post-infection, indicative of a more rapid progression of the immune response to PRRSV. This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.

Endocrine profiling is an increasingly utilized tool for detecting pregnancies in wild populations of mammals. Given the difficulty in calculating reproductive rates of Pacific walruses (Odobenus rosmarus divergens) the use of endocrine techniques for determining pregnancy rates could be particularly useful for management of the population. The goals of this study were to 1) determine if progesterone and total estrogen concentrations in ovarian tissues of female walruses could be used to determine reproductive state and 2) determine if walruses undergo a functional postpartum estrus, as is seen in other pinnipeds. Ovaries were collected from female walruses (n = 13) hunted in subsistence hunts by Alaska Native communities. Females were categorized as postpartum, full-term pregnant, pregnant diapause or unbred. Total estrogen concentrations were greatest in unbred (n = 2) and pregnant (n = 2) females. Progesterone concentrations were also nominally larger in unbred (n = 2) than pregnant (n = 2) and postpartum (n = 9) animals. Small samples sizes precluded the use of statistical comparisons among groups. Corpora lutea tissue samples in this study did not reflect the presence of a postpartum estrus in the month of May as postpartum females yielded lower total estrogen concentrations than unbred or pregnant animals. Both unbred animals were in a state of pseudopregnancy, which has not been physiologically described for this species before. The progesterone profiles in late (59 ng/g) and early (140 ng/g) pregnancy were lower than expected and fell within the range of the postpartum females (36-210 ng/g), suggesting low production of the hormone by the corpus luteum during these phases of pregnancy. Profiling reproductive hormones in free-ranging walruses demonstrates that an endocrine approach may be a valuable tool for determining reproductive status of females, however increased sample sizes and time of year must be considered to accurately separate pregnant versus pseudopregnant individuals.

Recently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection.

Porcine reproductive and respiratory syndrome (PRRS) is endemic in most pork producing countries. In Chile, eradication of PRRS virus (PRRSV) was successfully achieved in 2009 as a result of the combined efforts of producers and the animal health authorities. In October 2013, after several years without detecting PRRSV under surveillance activities, suspected cases were confirmed on a commercial swine farm. Here, we describe the PRRS epidemic in Chile between October 2013 and April 2015, and we studied the origins and spread of PRRSV throughout the country using official surveillance data and Bayesian phylogenetic analysis. Our results indicate that the outbreaks were caused by a PRRSV closely related to viruses present in swine farms in North America, and different from the strain that circulated in the country before 2009. Using divergence time estimation analysis, we found that the 2013-2015 PRRSV may have been circulating in Chile for at least one month before the first detection. A single strain of PRRSV spread into a limited number of commercial and backyard swine farms. New infections in commercial systems have not been reported since October 2014, and eradication is underway by clearing the disease from the few commercial and backyard farms that remain positive. This is one of the few documented experiences of PRRSV introduction into a disease-free country.

Porcine reproductive and respiratory syndrome (PRRS), caused by PRRS virus (PRRSV), is a serious contagious disease in the swine industry. At present, there are no effective control strategies against PRRSV. Thus, there is an urgent need for new treatment regimens that have efficacious antiviral activity to compensate for vaccines. The anti-infective effect of Cryptoporus volvatus has previously been demonstrated in Tradational Chinese Medicine. In this report, we expected to identify a new anti-PRRSV agent in the aqueous extract of C. volvatus, by employing a combination of modern chromatographic purification techniques and indirect immunofluorescence assay (IFA). Our results showed that C. volvatus extracts from every separation step differed in their inhibitory potency on PRRSV. One anti-PRRSV component designated as CM-H-L-5 was isolated from water-soluble fraction of C. volvatus. The inhibition induced by CM-H-L-5 occurred in a dose-dependent manner. CM-H-L-5 appeared to be a low-molecular-weight polyol fragment with amide groups and carboxylic acid groups. Collectively, our findings imply that CM-H-L-5 from the aqueous extract of C. volvatus has the potential to be used for anti-PRRSV therapy.

In this study, we tested the hypothesis that rectal immunization with a VCG-based chlamydial vaccine would cross-protect mice against heterologous genital Chlamydia trachomatis infection and Chlamydia-induced upper genital tract pathologies in mice. Female mice were immunized with a C. trachomatis serovar D-derived subunit vaccine or control or live serovar D elementary bodies (EBs) and the antigen-specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined by evaluating the intensity and duration of genital chlamydial shedding following intravaginal challenge with live serovar E chlamydiae. Protection against upper genital tract pathology was determined by assessing infertility and tubal inflammation. Rectal immunization elicited high levels of chlamydial-specific IFN-gamma-producing CD4 T cells and humoral immune responses in mucosal and systemic tissues. The elicited immune effectors cross-reacted with the serovar E chlamydial antigen and reduced the length and intensity of genital chlamydial shedding. Furthermore, immunization with the VCG-vaccine but not the rVCG-gD2 control reduced the incidence of tubal inflammation and protected mice against Chlamydia-induced infertility. These results highlight the potential of rectal immunization as a viable mucosal route for inducing protective immunity in the female genital tract.

SUMOylation is a reversible post-translational modification that regulates the function of target protein. In this study, we first predicted by software that the multiple proteins of porcine reproductive and respiratory syndrome virus (PRRSV) could be sumoylated. Next, we confirmed that Nsp1β, Nsp4, Nsp9, Nsp10 and nucleocapsid (N) protein of PRRSV could interact with the sole SUMO E2 conjugating enzyme Ubc9, and Ubc9 could be co-localized with Nsp1β, Nsp4, Nsp9 and Nsp10 in the cytoplasm, while with N protein in both the cytoplasm and nucleus. Finally, we demonstrated that N protein could be sumoylated by either SUMO1 or SUMO2/3. In addition, the overexpression of Ubc9 could inhibit viral genomic replication at early period of PRRSV infection and the knockdown of Ubc9 by siRNA could promote the virus replication. These findings reveal the SUMOylation property of PRRSV N protein and the involvement of Ubc9 in PRRSV replication through interaction with multiple proteins of PRRSV. To our knowledge, this is the first study indicating the interplay between SUMO modification system and PRRSV.

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a devastating pig disease present all over the world. The remarkable genetic variation of PRRSV, makes epidemiological and molecular analysis of circulating viruses highly important to review current diagnostic tools and vaccine efficacy. Monitoring PRRS viruses supports modern herd management by explaining the source of found viruses, either internally or externally from the herd. No epidemiological or molecular study has been published on circulating PRRS-viruses in the Netherlands, since the early nineties. Therefore, the objective of this study is to investigate circulating PRRS-viruses in the Netherlands in 2014, 2015 and 2016 on a molecular level by sequencing ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7. The results demonstrate that the 74 PRRSV strains belong to PRRSV-1, but the diversity among strains is high, based on nucleotide identity, individual ORF length and phylogenetic trees of individual ORFs. Furthermore, the data presented here show that the phylogenetic topology of some viruses is ORF dependent and suggests recombination. The identity of the strain of interest might be misinterpreted and wrong conclusions may be drawn in a diagnostic and epidemiological perspective, when only ORF5 is analyzed, as performed in many routine sequencing procedures.

Long-term studies of individual animals in nature contribute disproportionately to our understanding of the principles of ecology and evolution. Such field studies can benefit greatly from integrating the methods of molecular genetics with traditional approaches. Even though molecular genetic tools are particularly valuable for species that are difficult to observe directly, they have not been widely adopted. Here, we used molecular genetic techniques in a 10-year radio-telemetric investigation of the western diamond-backed rattlesnake (Crotalus atrox) for an analysis of its mating system and to measure sexual selection. Specifically, we used microsatellite markers to genotype 299 individuals, including neonates from litters of focal females to ascertain parentage using full-pedigree likelihood methods. We detected high levels of multiple paternity within litters, yet found little concordance between paternity and observations of courtship and mating behavior. Larger males did not father significantly more offspring, but we found evidence for size-specific male-mating strategies, with larger males guarding females for longer periods in the mating seasons. Moreover, the spatial proximity of males to mothers was significantly associated with reproductive success. Overall, our field observations alone would have been insufficient to quantitatively measure the mating system of this population of C. atrox, and we thus urge more widespread adoption of molecular tools by field researchers studying the mating systems and sexual selection of snakes and other secretive taxa.

Reproductive aging may impact the vaginal microbiome and genital tract mucosal immune environment and contribute to genital tract health in women living with and at-risk for HIV infection.

Pseudokirchneriella subcapitata is a sickle-shaped freshwater green microalga that is normally found in unicellular form. Currently, it is the best known and most frequently used species of ecotoxicological bioindicator because of its high growth rate and sensitivity to toxicants. However, despite this organism's, our knowledge of its cell biology-for example, the patterns of nuclear and cytoplasmic division in the mitotic stage-is limited. Although it has been reported that P. subcapitata proliferates by popularity forming four daughter cells (autospores) through multiple fission after two nuclear divisions, here, we report two additional reproductive patterns by which two autospores are formed by binary fission ("two-autospore type") and eight autospores are formed by multiple fission ("eight-autospore type"). Moreover, we found that cell reproductive patterns differed markedly with the culture conditions or with exposure to either of two typical toxicants, potassium dichromate (K2Cr2O7) and 3,5-dichlorophenol (3,5-DCP). The eight-autospore type occurred at the highest frequency in the early phase of culture, but it disappeared under 3,5-DCP at 2.0 mg/L. Under 0.3 mg/L K2CrO7 (Cr(VI)) the eight-autospore type took substantially longer to appear than in control culture. The two-autospore type occurred only in the late phase of culture. To our knowledge, this is the first detailed evaluation of the reproductive patterns of P. subcapitata, which changed dramatically in the presence of toxicants. These findings suggest that observation of the reproductive patterns of P. subcapitata will help to elucidate different cell reactions to toxicants.

Fomites might be responsible for virus introduction in swine farms, highlighting the importance of implementing practices to minimize the probability of virus introduction. The study's objective was to assess the efficacy of different combinations of temperatures and holding-times on detecting live PRRSV and PEDV on surfaces commonly found in supply entry rooms in swine farms. Two PRRSV isolates (MN 184 and 1-4-4 L1C variant) and one PEDV isolate (NC 49469/2013) were inoculated on cardboard and aluminum. An experimental study tested combinations of four temperatures (20°C, 30°C, 40°C, and 50°C) and six holding-times (15 minutes, 60 minutes, 6 hours, 12 hours, 24 hours, and 36 hours) for the presence of the viruses on each surface type. After virus titration, virus presence was assessed by assessing the cytopathic effects and immunofluorescence staining. The titers were expressed as log10 TCID50/ml, and regression models; half-lives equations were calculated to assess differences between treatments and time to not detect the live virus. The results suggest that the minimum time that surfaces should be held to not detect the virus at 30°C was 24 hours, 40°C required 12 hours, and 50°C required 6 hours; aluminum surfaces took longer to reach the desired temperature compared to cardboard. The results suggest that PRRSV 1-4-4 L1C variant had higher half-lives at higher temperatures than PRRSV MN 184. In conclusion, time and temperature combinations effectively decrease the concentration of PRRSV and PEDV on different surfaces found in supply entry rooms in swine farms.

Maternal and neonatal mortality in Bihar, India was far higher than the aspirational levels set out by the Sustainable Development Goals. Provider training programs have been implemented in many low-resource settings to improve obstetric and neonatal outcomes. This longitudinal investigation assessed diagnoses and management of postpartum hemorrhage (PPH), hypertensive disorders of pregnancy, birth asphyxia (BA), and low birth weight (LBW), as part of the CARE's AMANAT program in 22 District Hospitals in Bihar, between 2015 and 2017. Physicians and nurse mentors conducted clinical instruction, simulations and teamwork and communication activities, infrastructure and management support, and data collection for 6 consecutive months. Analysis of diagnosis included 11,259 non-referred and management included 11,800 total (non-referred and referred) admissions that were observed. Data were analyzed using the chi-square test for trend. PPH was diagnosed in 3.7% with no significant trend but diagnosis of hypertensive disorders increased from 1.0% to 1.7%, (ptrend = 0.04), over the 6 months. BA was diagnosed in 5.8% with no significant trend but LBW diagnoses increased from 11% to 16% (ptrend<0.01). Among PPH patients, 96% received fluids, 85% received uterotonics and 11% received Tranexamic Acid (TXA). There was a significant positive trend in the number of patients receiving TXA for PPH (6% to 13.8%, ptrend = 0.03). Of all neonates with BA, there were statistically significant increases in the proportion who were initially warmed, dried, and stimulated (78% to 94%, ptrend = 0.02), received airway suction (80% to 93%, ptrend = 0.03), and supplemental oxygen without positive pressure ventilation (73% to 86%, ptrend = 0.05). Diagnoses of hypertensive disorders and LBW as well as initial management of BA increased during the AMANAT program. However, underdiagnoses of PPH and hypertensive disorders relative to population levels remain critical barriers to improving maternal morbidity and mortality.

Treating insects with a lower oxygen atmosphere before and during exposure to radiation can mitigate some of the negative physiological effects due to the irradiation. The irradiation of pupae under oxygen-reduced environment such as hypoxia or anoxia is routinely used in the sterile insect technique (SIT) of some tephritid species as it provides radiological protection. This treatment allows to have the sterile pupae already in sealed containers facilitating the shipment. SIT is an environment friendly control tactic that could be used to manage populations of Drosophila suzukii in confined areas such as greenhouses. The objectives of this study were to assess the effect of irradiation on the reproductive sterility in D. suzukii males and females under low-oxygen atmosphere (hypoxia) and atmosphere conditions (normoxia). Additionally, we assessed the differences in radiological sensitivity of pupae treated under hypoxia and normoxia conditions. Finally, the effect on emergence rate and flight ability of the irradiated D. suzukii adults exposed to doses that induced >99% of sterility were assessed. Pupae needed a 220 Gy irradiation dose to achieve >99% of egg hatch sterility in males irrespective of the atmosphere condition. For females the same level of sterility was achieved already at 75 Gy and 90 Gy for the normoxia and hypoxia treatments, respectively. Radiation exposure at 170 and 220 Gy under the two atmosphere treatments did not have any effect on the emergence rate and flight ability of D. suzukii males and females. Therefore, hypoxia conditions can be used as part of an area-wide insect pest management program applying SIT to facilitate the protocols of packing, irradiation and shipment of sterile D. suzukii pupae.

Although suggested that "fetal" cell-free DNA (cfDNA) is derived from trophoblast cells, the exact origin is unclear. The studies in this report sought to demonstrate that placental tissue releases cfDNA in parallel with cell death, that the size range of cfDNA is similar to that found in maternal plasma, and that the cfDNA fragments are able to stimulate a proinflammatory cytokine response. Placentas were harvested from near term pregnant CD-1 mice and cultured in DMEM/Ham's F12/FBS media in 8% or 21% O2. After centrifugation to remove cells and cellular debris, the cfDNA was extracted from the media and quantified by DNA spectrophotometry. The cfDNA fragments were sized using a 1.5% TAE gel. Cell death was quantified by lactate dehydrogenase assay; and tissue homogenates were used to quantify caspase activity and BAX expression. Cultured RAW-264.7 macrophage cells were used to determine IL6 stimulation by cfDNA. The cfDNA levels released in 8% O2 (placental normoxia) were not significantly different from explants cultured in 21% O2 (placental hyperoxia). The cfDNA fragments ranged in size from < 100 -< 400 bp. The cfDNA release increased when cultured with LPS, whereas it decreased with trolox (vitamin E analog). Explant release of cfDNA increased in parallel with cell death. The cfDNA release and cell death of trophoblast appears to involve components of the apoptosis signaling pathway as suggested by LPS enhancement of placental caspase activity, suppression of cfDNA release by a pan-caspase inhibitor and the trend toward increased Bax protein expression. Studies with cultured macrophage cells confirmed the ability of cfDNA to stimulate an IL6 response. In summary, these studies have confirmed the ability of placental tissue to release significant amounts of cfDNA, a phenomenon that appears to be mediated, at least in part, by apoptosis; and that the cfDNA released by the placental explants is able to stimulate a significant proinflammatory response. Thus, these studies provide support for the hypothesis that cell-free fetal DNA released by placental tissue potentially plays a mechanistically important role during the events leading to the onset of parturition.