Due to persistent infections of human central nervous system (CNS), polyomaviruses have been identified as one of the risk factors for brain tumor development. Human BK virus is of signiﬁcant interest due to its experimental neuro-oncogenic potential and the possible association with CNS neoplasms. However, the results of different studies are discrepant. In the present study, we aimed to investigate the prevalence of BK virus genome and quantify BK viral load in Iranian patients with primary and metastatic brain malignancies.To assess the prevalence of BK virus sequences, a total of 58 fresh brain tumors were examined by quantitative real-time PCR. The BK viral load was determined as viral copy number per cell. -3±0.27×10-3 (range 0.01×10-3- 0.8×10-3) copies per cell.Of the 58 brain tumor samples BK tumor antigen (TAg) sequences were detected in 26 (44.8%) of cases. In primary brain tumors, BK virus sequences were recognized more frequently in schwannomas (15.5%) and meningiomas (12.1%). The mean BK virus TAg copy number in positive cases was 0.20×10Taken together, in the present study low copy numbers of BK virus TAg gene was detected in brain tumor cells, which can indicate that BK virus may contribute to tumor induction by indirect mechanisms or neuro-persistence of this virus without any pathological consequences. Central nervous system (CNS) tumors are diverse groups of neoplasms originating from the brain, spinal cord or meninges and accounting for 2% of all adulthood malignancies . Brain tIn addition, intracerebral, subcutaneous and intravenous inoculation of these viruses into new world monkeys’ results in astrocytoma, glioblastoma and neuroblastoma , 6. PolyLow levels of polyomaviruses are maintained in individuals with competent immune system, but following immunodeficiency or immunosuppression, these viruses replicate at very high levels and lead to disease. Due to having tumor antigen (TAg) oncogenes, human polyomaviruses can interact with tumor suppressor proteins including retinoblastoma protein (pRb) and p53 and offer a possible mechanism of oncogenicity. However, current evidence only supports the role of MCPyV as a human carcinogen, lead to Merkel cell carcinoma (MCC) . In MCCs, parts of the virus genome, in particular TAg oncogene sequences; have been integrated into the host cell DNA . The firSince that reports, there have been numerous studies on the role of BK virus in the development of human CNS tumors. However, the results of various studies are inconsistent and the role of BK virus in CNS tumor development is still highly controversial , 13. ThePatients and Samples: The present study was performed on 58 patients with brain tumor referred to 2 neurosurgical reference Hospitals of Tehran, Shariati Hospital and Khatam Al Anbia Hospital, from November 2012 to January 2014. After obtaining written informed consent, fresh biopsy specimen of brain tumor were surgically removed and immersed in RNALater solution . Fresh biopsy specimens were immediately transferred to the study site on the same day of surgery and stored in a -70° C freezer. Patients with primary and metastatic brain tumor were included in the study. Patients with positive test for human immunodeficiency virus type 1 (HIV-1) antigen or antibody and subjects who received immunosuppressive therapy were excluded from investigation. This study was approved by the Ethics Committee of Tehran University of Medical Sciences (Project code: 92-01-30-20990) and informed consent was obtained from all participants.DNA Extraction: The QIAamp DNA mini kit  was used to extract DNA from the tissue samples based on the manufacturer’s instructions. NanoDrop spectrophotometer was used to estimate the concentration and quality of the extracted DNA . Concentrations of DNA extracted from tissue were in the range of 450 - 500 ng/μl and their A260/A280 ratio was in the range of 1.7 to 1.9 indicating optimum concentration and purity.BK virus Quantitative Real-Time PCR: A TaqMan real-time PCR method was used to assess the presence of BK virus TAg oncogene and to determine the amount of viral load using the Rotor-Gene® Q Real-time PCR System  according to a previously described procedure . The recombinant plasmids were extracted from the grown colonies and then confirmed by sequencing. To determine the copy number of the human RNase P gene, a conserved region of this gene was amplified using specific primers . The χ2-test was used to evaluate the associations between categorical variables. Normal distribution of the variables was analyzed by Kolmogorov–Smirnov test. Analysis of continuous variables was performed using the independent-samples t-test /Mann–Whitney U test and ANOVA / Kruskal-Wallis test. A p-value of ≤0.05 was considered to be statistically significant.This study included 58 brain tumor samples. Regarding brain tumor origin, 54 (93.1%) were primary and four (6.9%) metastatic brain tumors. Demographic, clinical and histopathologic parameters of BK positive and negative patients are presented in -3 ±0.27×10-3 (0.01×10-3 – 0.8. 10-3-copies per cell). In the current investigation, due to lower frequency of other tumors compared with schwannoma and meningioma we placed them in a new group called others for statistical analysis. The mean BK virus TAg gene copy number was higher in schwannoma tumors compared with others and meningioma groups , meningioma (20.7%) and glioblastoma multiform (12.1%) were the most common types of tumors. The prevalence pattern observed in this study is consistent with the reports of brain tumor prevalence from the developing countries, and with the results of the most recent systematic review of 10802 brain tumor cases in Iran . In the n tumors . AnalysiIn a study conducted by Martin et al., BK virus genome was identified in 57% of meningioma tumors; these findings might be relevant with our results in terms of BK virus prevalence rate in meningiomas . In thisIn a positive agreement with a previous study, current investigation revealed a low copy number of BK virus TAg gene per cell in primary and metastatic brain tumors . In addiTaken together, in the present study low copy numbers of BK virus TAg gene was detected in tumor cells, which can indicate that BK virus may contribute to tumor induction by indirect mechanisms or neuro-persistence of this virus without any pathological consequences. These results should urge further worldwide epidemiological and virological studies to distinguish the possible role of BK virus in tumor formation from simple persistent viral replication.


We aimed to demonstrate the benefits of using chickpea as energy-protein feed in diets for bulls belonging to a native Italian breed (Maremmana) organically reared. Compared to the control diets containing barley, the dietary inclusion of chickpea improved the growth rate and carcass conformation of bulls, reduced the feeding costs, and did not impair the plasma metabolic parameters and meat quality. These results suggest that chickpea may allow a sustainable performance improvement of native breeds within their traditional farming systems.Longissimus thoracis et lumborum 7 days after slaughter. The chickpea-fed animals showed a significantly greater average daily gain (1064 vs. 1168 kg/day), a shorter growing phase (364 vs. 335 days), and a better carcass conformation. Plasma metabolites and meat quality were not influenced by the treatments. The better growth performance and carcass quality of the chickpea fed bulls resulted in a higher economic profit for the chickpea-based diets. Results suggest that chickpea may allow sustainable performance improvement of native breeds within their traditional farming systems.We assessed the effects of inclusion of chickpea from 24 to 21%, as feed basis, in diets for organically reared bulls. Sixteen young bulls  belonging to a native Italian breed (Maremmana) were randomly assigned to two dietary treatments. The control diets were based on mixed grass hay, maize meal, and barley meal. In the experimental diets, barley was equally substituted by locally produced chickpea. Animals were weighed every 2 weeks until the prefixed slaughtering weight (630 kg). Plasma metabolites were measured at the 1st, 7th, and 14th month of the experiment. Chemical composition, colour, shear force, and water holding capacity of meat were assessed on  As a result of agriculture industrialization, several European native cattle breeds have suffered a dramatic decrease in numbers in favour of a few, highly selected, more productive breeds . All theCicer arietinum L, kabuki types) is attractive in Mediterranean context because of its low input requirement and resistance to drought joined to a long sowing window [Chickpea cropping  on a dry matter (DM) basis. In the chickpea diets, barley was equally replaced for chickpea at 223 g/kg DM of CP . All conBarley (cultivar Explora) and chickpea (cultivar Principe) were locally produced (5.9 and 2.1 kg/ha respectively for barley and chickpea). The concentrates  were ground and fed twice daily (h 0800–1500) at the established amount, while the hay was supplied ad libitum . The animals were individually housed into adjacent pens with concrete solid floor and straw bedding equipped with feed manger and water bowls. Longissimus thoracis et lumborum (LT) muscle was removed from the right side of each carcass. In the meat laboratory, the LT muscles were cut into 3–4 cm slices, vacuum packaged, matured at 4 °C until 7 days post-mortem, and then analysed. Each animal was weighed at group formation and thereafter at 2-week intervals along with measurement of the hay intake calculated as the difference between hay offered and refused. At the 1st, 7th, and 14th months, coinciding with the farm official sanitary routine inspections, blood samples of each animal were collected into Li-heparinized vacuum tubes  by coccygeal venipuncture performed by a skilled and authorized veterinarian. The samples were immediately cooled in a thermo-isolate icebox and centrifuged (3000 rpm × 15 min at 4 °C) on the field within 30 min. Recovered plasma was transported to the laboratory on dry ice, and then stored at −20 °C until analysed. At the target weight (630 kg), the animals were deprived of feed but not of water for 12 h, transported to the abattoir, and then weighed, stunned, and slaughtered according to the EU Regulation. After dressing and 24 h chilling at 4 °C, the carcasses were weighed, visually scored according to the EUROP classification for conformation  and fatness , and The feed samples were analysed according to the methods of Association of Official Analytical Chemists  to detePlasma samples were assayed for glucose, total cholesterol, triglycerides, total protein, urea, creatinine, ß-hydroxybutyric acid (BHBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP). Analyses were performed by using standard commercial kits and a UV spectrophotometer  adopting the kits manufacturer recommended procedures . The meat samples were analysed (three replicates/sample) for chemical composition  and total and insoluble collagen according to AOAC  and ModzAn input–output budgeting procedure was used to calculate the costs and incomes associated to the two dietary treatments. The actual farm gate-price was used for both inputs and outputs. The feed costs (€/kg as fed) were mixed grass hay 0.12, maize meal 0.26, barley 0.22, chickpea 0.26, vitamin and mineral premix 1.50, and refer to feed-grade and organic certified feedstuffs. The amount of feedstuff consumed, the labour cost, and the other variable costs were calculated for each animal based on the length in days of each growing phases. Labour cost per head was computed based on an hourly wage of 7.30 € and 3 min of labour per day. Daily costs for machinery and health and hygiene products and services were estimated at 0.12 and 0.06 €/head, respectively. The interests on working capital were based on a 5% money legal interest rate. The cost per kg of weight gain was calculated by dividing total costs by total weight gain. Income achieved per animal was computed based on the selling value of carcass at slaughter, which varied according to carcass weight and conformation. The EU subsidies (60.26 €/head ) were alThe net energy for maintenance and growth (ENmg) and the intestinal digestible protein fractions  values of the diets were estimated based on the chemical composition of the feedstuffs . Data weThe barley and chickpea diets had a roughly similar energy content while the CP levels were constantly higher for the chickpea diets . The PDIp < 0.05) and 3 , and, as an overall effect, for the total ADG (p < 0.001). Consistently, the length of phase 1 and of the finishing phase did not differ among the groups, while phase 2 (p < 0.05), phase 3 (p < 0.05), and the total length of the growing period  were shorter for the chickpea-fed animals that also showed younger age at slaughtering (p < 0.001). The effects of the dietary inclusion of chickpea on the animal growth performances are in p < 0.05) according to the age-related trend observed for growing bulls [The concentrations of plasma metabolites are summarized in ng bulls ,38,39.p < 0.05) in chickpea-fed bulls, whereas total protein was unaffected by the diet. The total cholesterol was significantly lower in the chickpea group (p < 0.05), while no differences were observed for glucose and triglycerides as well as for the blood activity of circulating liver enzymes, and electrolytes concentration. The plasma concentration of urea and creatinine and AP activity were higher (p < 0.01) but had greater (p < 0.01) fatness score. Meat quality was unaffected by the diet, except drip loss was higher in the barley group (p < 0.01), and cooking loss was higher in the chickpea group (p < 0.01). The slaughtering traits and meat quality as influenced by the diet are shown in The economic performances associated to each dietary treatment are given in 2O emissions and mitigating the carbon footprint of cropping systems [Organic and locally produced chickpea presents two main benefits compared to similarly produced barley: It is richer in protein, so it can help meet the animals’ protein requirement according to the organic standards, and fixes atmospheric nitrogen, so contributing to reduced N systems . In this systems . By cont systems ,43.As reviewed by Bampidis and Christodoulou , the scaThe ultimate measurement of the efficiency of a diet for beef cattle is not only given by weight gain, but also by the effects on carcass quality. Theoretically, a higher dietary protein level might result in greater muscle development  and indeThe values observed for meat quality parameters were in line with previous reports for Maremmana bulls of comparable age ,62,63,64The dietary inclusion of chickpea increased the income per head due both to the reduction of feeding costs and to the higher price paid for the better-conformed carcass, namely the main drivers of beef farm profitability . The EurSubstitution of barley for chickpea in diets for young native bulls improved the growth rate and carcass conformation, reduced the feeding costs, and did not impair the plasma metabolic parameters and meat quality. Overall, the introduction of chickpea in amount ranging from 24 to 21%, as feed basis, appears to guarantee better long-term economic sustainability of meat organic production by using native cattle. We conclude that chickpea represents an attractive energy-protein feed, allowing sustainable performance improvement of native breeds within their traditional farming systems and so contributing to the proper management and conservation of semi-natural habitats as well as to agroecological production of animal foods.


Schizophrenia is ranked among the top 25 leading causes of disability worldwide in 2013 which resulting in social and economic burden. By observing patients with schizophrenia one year before and after switching from oral antipsychotics (OAPs) to once-monthly paliperidone palmitate (PP1M), we can better understand the change of total costs in schizophrenic patients, including direct costs and indirect costs, after switching treatment patterns.A total of 100 schizophrenic (ICD-10) patients from Shandong Mental Health Center were collected from December 2016 to June 2019. Treatment modalities, health care resource utilization and costs were compared before and after switching directly from oral antipsychotics to PP1M.Of the 82 patients included in the main analyses, treatment with PP1M resulted in an increase in direct costs of 31.92% (P < 0.01), an increase in medicine costs of approximately 142% (P < 0.01), and a reduction in hospital costs of 68.15% (P > 0.05). There was no significant increase in total costs (P = 0.25), while 31.92% increase in direct costs (P < 0.01), and 35.62% decrease in indirect costs (P < 0.01) after conversion to PP1M. Compared with before administration of PP1M, patients with ≥ 1 inpatient stay in 1 year Pre-PP1M treatment with OAPs (n = 32) had a 20.16% decrease in direct costs (P < 0.01), a 144% increase in medicine costs (P < 0.01), and a significant 72.02% decrease in hospital costs (P < 0.01). The observed reduction in the number of hospitalizations  and inpatient stays  and after transition to PP1M resulted in a reduction in hospitalization costs (P < 0.01).Switching from OAPs to PP1M decreased the household workforce burden without increasing clinical healthcare costs. Direct costs were significantly reduced in patients with ≥ 1 inpatient stay in 1 year pre-PP1M treatment with OAPs after the switch, which decreased by improving adherence to therapy and reducing the number and length of hospital stays, suggesting that those patients may benefit after switching to PP1M. Schizophrenia is a remitting and relapsing psychiatric disorder, characterized by profound disruptions in thinking, language, perception, and self-perception . SchizopIn addition, long-acting injectable antipsychotics (LAIs) have been shown to increase medication adherence , 16, redA total of 100 patients with schizophrenia (ICD-10) who could be followed for at least one year in Shandong Mental Health Center were collected from December 2016 to June 2019. Patients with ≥1 hospitalization experience in one year before mirror point were included in the statistical analysis. The inclusion criteria were as follows: (a) between 18 and 65 years old; (b) subjects with the course of the disease ≥1 year; (c) subjects were in stable condition  total score < 70 or Clinical Global Impression-Severity (CGI-S) score of ≤3 (mild ill); (d) subjects with at least one illness exacerbation or hospitalization (PANSS score ≥70) in the year prior to enrollment; (e) the date of the first observation of a diagnosis of schizophrenia during this period was identified as the initial date; (f) subjects received paliperidone palmitate at their own will; (g) taking antipsychotic drugs ≥6 months within in one year before mirror point. The exclusion criteria were as follows: (a) pregnant or lactating women; (b) alcoholism or substance abuse; (c) patients with severe physical diseases and organic brain disease;(d) patients who could not be followed for least 1 year; (e) patients with PP1M no less than 4 times/year. We obtained data from hospital information management system. The study protocol was reviewed and approved by Ethical Committee (2017R22).This was a mirror-image study. The day on which PP1M commenced was set as the “mirror point”, as show in Table Until now, direct costs were obtained through the databases. At present, objective indirect cost assessment tools are lacking. Through literature review,indirect cost is mainly evaluated by purely descriptive method or by the author’s own design of burden questionnaire,which was quantified in monetary form or in days of loss , 27. In Statistical analyses were performed using 23.0 SPSS software. Demographic characteristic including age, gender and mean duration of disease were described at baseline. Means, medians, and standard deviations (SDs) were used for continuous variables with T-test, while counts and percentages were used for categorical variables. Pair Wilcoxon signed-rank tests were performed to compare differences in direct (indirect)and the total costs between the pre/post-PP1M periods in this 1-year mirror-image study. Pair T tests were performed to compare differences in number of hospitalization and inpatient stays during the period before and after PP1M. A total of 100 patients with schizophrenia who transitioned from oral risperidone or oral paliperidone to PP1M were included in the study. 18 patients fell off, including that 7 patients lost contact, 2 patients became agitated and could not tolerate side effects, and 9 patients were unwilling to continue use PP1M for personal reasons. Patients with ≥1 hospitalization experience in one year before mirror point were 32.Of the 82 patients included in the main analyses, the average age was 32.2 (SD=1.2) years old and the mean duration of disease was 57.5 months (SD=40.6)53.66% of the patients were female and the mean CGI-S score was 2.83 (SD=0.39).Compared with pre-PP1M, medicine costs increased significantly post-PP1M (P < 0.01), while the hospitalization costs decreased 68.15% from ¥9,155 to ¥2,915 ( P > 0.05). To sum up, direct costs increased significantly from ¥17,457 to ¥23,030 (P < 0.01), as shown in Table Compared with pre-PP1M, medicine costs in inpatient stay costs increased post-PP1M (P < 0.01), and the hospital costs decreased significantly from ¥25,656 to ¥7,179 (P < 0.01). An obvious decrease in direct costs of ≥1 hospitalization in 1 year pre-PP1M treatment with OAPs were observed (P < 0.01), as shown in Table After transition to PP1M, The number of hospitalizations and length of inpatient stay did not decrease significantly. Inpatient length of stay and number of hospitalizations  and caregiver care costs (P ≤0.01) reduced post-PP1M. Consequently, indirect costs also significantly reduced (P < 0.01) Table .The direct costs of the patients increased (P < 0.01), while the indirect costs decreased significantly (P < 0.05) after the switch to PP1M, by the comparison of the pre-PP1M period. Overall, the total costs to patients was no significant increased (P > 0.05), as shown in Table versus ¥23,030, P < 0.01). However, patients who have had ≥1 hospitalization experience in the year before the mirror point will have different consequences. Surprisingly, the decline in hospital costs could completely offset the increase in medicine costs, resulting a decrease in direct costs . We also found the same results with other research [Nowadays, oral antipsychotics remain the first-line pharmacologic treatment option for patients with schizophrenia. However, a high discontinuation rate is a known problem with oral antipsychotic treatment . LAIs arresearch . In a miresearch , 1272 Koresearch , the useAt the same time, schizophrenia patients affected by the mental symptom caused high productivity loss, and resulted in high productivity losses to patients and their families. In addition, the burden of schizophrenia, in addition to direct medicine costs also had a profound impact on caregivers and patient families . CurrentIn contrast to prior studies that included a comparison of OAPs to PP1M using a cohort design, this study used a mirror-image design to compare patients before and after the initiation of PP1M. The mirror-image design is a study that compare the results before and after the medication changes (from OAPs to PP1M). The focus here is to compare the economic results of dressing changes for patients with schizophrenia before and after. There are not many studies in this direction, and previous literature review studies have focused on summarizing direct costs associated with schizophrenia. There has been a lack of emphasis on indirect costs estimation. Furthermore, China has no similar results at present. Therefore, this study focuses on the economic burden of direct and indirect costs when Chinese patients with schizophrenia switch from OAP to PP1M.However, the finding of this study should be interpreted in the context of some limitations. First, part of our database (indirect costs) was based on patient-provided data, and its accuracy cannot be guaranteed. Second, the sample size was relatively small, and the data may not be representative of the entire Chinese population. Therefore, in the follow-up study, we will further cooperate with other regions in China to carry out follow-up research and evaluation.Comparing patients one year before and after switching from OAPs to PP1M, the direct costs increased and the indirect costs decreased, leading to a smaller difference in total costs after switching to PP1M. This suggests that patients switching from OAPs to PP1M can improve patient productivity, reduce caregiver burden and can not increase the costs of health care in the clinic. For patients with ≥1 inpatient stay one year prior to PP1M, the hospital costs decreased which were associated with the reduction of number of hospitalization and inpatient stays, and the medicine costs increased which was fully offset by lower hospital costs after switching from OAPs to PP1M. The reduction of direct costs suggests that patients with hospitalization experience obtain greater economic benefits by changing from OAPs to PP1M.


Data from 840 Holstein-Friesian cows (1321 lactations) were used to evaluate trends in fat-to-protein ratios in milk (FPR), and the use of FPR as an indicator of energy balance (EB). The fat-to-protein ratio was negatively related to EB, and this relationship became more negative with increased parity. Regression slopes describing linear relationships between FPR and EB differed over time, although trends were inconsistent. Similarly, ‘High’ FPR scores in milk (≥1.5) were consistently associated with a greater negative energy balance, milk yields, body weight loss, and plasma non-esterified fatty acid concentrations; however, their relationships with dry matter intake did not follow a clear trend. Although FPR can provide an indication of EB at a herd level, this analysis suggests that FPR cannot accurately predict the EB of individual cows. A statistical re-assessment of aggregated individual cow data was conducted to examine trends in fat-to-protein ratio in milk (FPR), and relationships between FPR and energy balance  in Holstein-Friesian dairy cows of different parities, and at different stages of lactation. The data were collected from 27 long-term production trials conducted between 1996 and 2016 at the Agri-Food and Biosciences Institute (AFBI) in Hillsborough, Northern Ireland. In total, 1321 lactations , derived from 840 individual cows fed mainly grass silage-based diets, were included in the analysis. The energy balance was calculated daily and then averaged weekly for statistical analyses. Data were further split in 4 wk. intervals, namely, 1–4, 5–8, 9–12, 13–16, and 17–20 WIM, and both partial correlations and linear regressions (mixed models) established between the mean FPR and EB during these periods. Three FPR score categories  were adopted and the performance and EB indicators within each category were compared. As expected, multiparous cows experienced a greater negative EB compared to primiparous cows, due to their higher milk production relative to DMI. Relatively minor differences in milk fat and protein content resulted in large differences in FPR curves. Second lactation cows displayed the lowest weekly FPR, and this trend was aligned with smaller BW losses and lower concentrations of non-esterified fatty acids (NEFA) until at least 8 WIM. Partial correlations between FPR and EB were negative, and ‘greatest’ in early lactation , and gradually decreased as lactation progressed across all parities . With increasing parity, daily EB values tended to become more negative per unit of FPR. In primiparous cows, regression slopes between FPR and EB differed between 1–4 and 5–8 WIM (−54.6 vs. −47.5 MJ of ME/day), while differences in second lactation cows tended towards significance (−57.2 vs. −64.4 MJ of ME/day). Irrespective of the lactation number, after 9–12 WIM, there was a consistent trend for the slope of the linear relationships between FPR and EB to decrease as lactation progressed, with this likely reflecting the decreasing milk nutrient demands of the growing calf. The incidence of ‘High’ FPR scores was greatest during 1–4 WIM, and decreased as lactation progressed. ‘High’ FPR scores were associated with increased energy-corrected milk (ECM) yields across all parities and stages of lactation, and with smaller BW gains and increasing concentrations (log transformed) of blood metabolites  until 8 WIM. Results from the present study highlight the strong relationships between FPR in milk, physiological changes, and EB profiles during early lactation. However, while FPR can provide an indication of EB at a herd level, the large cow-to-cow variation indicates that FPR cannot be used as a robust indicator of EB at an individual cow level.  The energy balance (EB) in dairy cows can be defined as the difference between energy consumed from feed (input) and energy expended for maintenance, production, activity, and pregnancy (output). In early lactation, high-yielding dairy cows are often unable to consume sufficient nutrients to meet their energy requirements for milk production and, consequently, enter into a period of negative EB (NEB) , which iA number of indicators have been used to provide an indirect assessment of EB, and to assist with the early detection of metabolic or management problems at a herd level. Changes in BW have been used as an indicator for EB, with the increasing availability of automated weighing systems on commercial farms making this approach more accessible ,10. HoweBlood metabolites, such as beta-hydroxy butyrate (BHB) and non-esterified fatty acids (NEFA) are also frequently advocated as indicators of the energy status in dairy cows, with their concentrations being related to lipid mobilization. Negative EB in early lactation is characterized by high concentrations of both NEFA and BHB , with thIn contrast to blood, milk samples can be obtained daily during a normal milking routine, with milk composition known to change with the energy status ,19. ManyFrequent limitations of many studies, which have examined FPR as an indicator of EB, are the relatively small number of cows involved, and the use of EB proxies that may not provide a robust indicator of EB . In addition, there is little information available on how effective FPR is as an indicator of EB for cows of different parities and at different stages of lactation. Therefore, the aim of the present study is to use a large dataset to explore the usefulness of FPR to act as an indicator for EB in Holstein-Friesian dairy cows offered predominantly grass silage-based diets, and to examine how changes in the FPR vs. EB relationship changes with the lactation number and within the stage of lactation during the first 20 weeks in milk (WIM). This study involved a statistical re-assessment of aggregated individual cow data obtained from 27 studies, which were conducted between 1996 and 2016 at the Agri-Food and Biosciences Institute (AFBI) in Hillsborough, Northern Ireland. The results of the majority of these studies have been published in peer-reviewed scientific papers, conference proceedings, technical reports, and a Ph.D. Thesis . A minimn = 6) involved a comparison of two cow genotypes, only data from Holstein-Friesian cows were included within this analysis. In addition, individual cows within experiments were excluded from the analysis if they were on an experiment for less than the first 90 days’ post calving . Data recorded during the first three days of lactation were excluded from the analysis, while data were included in the analysis up until a maximum of 140 DIM . In addition, cows with a lactation number > 6 were excluded from the analysis. A total of 79 treatments were examined across the 27 studies, with the majority of treatments examining the impact of diet and/or management strategies on cow performance. While, a number of studies (n = 631), with these cows having a mean Predicted Transmitting Ability (PTA2018) for milk fat plus protein yield of 10 (S.D. 14.0) kg, and a mean Profitable Lifetime Index (PLI2018) of GBP 84 (S.D. 154.2). The conversion rate (British pounds to US dollars) at the time when genetic data were sourced was GBP 1 = USD 1.2652. The final dataset represented 840 individual cows, and comprised data from 1321 individual lactations, 474 of which were primiparous, with the remainder multiparous (second to sixth lactations). At parturition, cows included in the present study had an average BW of 576 ± 76.9 kg, an average BCS of 2.57 ± 0.572, and an average parity of 2.4 ± 1.40. The relatively narrow range of BCS values reflected management practices adopted on the experimental farm. Pedigree records were available for the majority of the cows (n = 7), milk samples were taken in proportion to yield during six consecutive milkings (either weekly or fortnightly), and a single bulked sample analyzed. However, in later experiments (n = 20) samples were taken during two consecutive milkings  and each individual sample analyzed, and a weighted composition for the 24 h sampling period, subsequently, determined. Samples in all experiments were analyzed for fat, protein, and lactose concentrations , while milk somatic cell count (SCC) was determined monthly using a Fossomatic 360 . Fat-to-protein ratio in milk was calculated as milk fat content (g/kg) divided by milk protein content (g/kg). A number of animal measurement protocols changed over the 20-year period during which the 27 experiments were undertaken, while others remained largely unchanged. The feed intake of each individual cow was recorded daily using feed-boxes mounted on weigh cells, access to which was controlled by a Calan Gate feeding system  linked to an electronic cow identification system. All diets were offered ad libitum. In all experiments, cows were milked twice daily, with milk yields recorded automatically at each milking, and a total daily milk yield for each cow determined for each 24 h period. In early experiments  was calculated assuming the GE content of 1 kg ‘standard milk’ to be 3.1 MJ/kg , BW was recorded weekly, immediately after pm milking. However, in later studies, BW was recorded twice daily (immediately after each milking) using an automated weighbridge, and an average BW calculated for each week. Body condition scores were recorded weekly or fortnightly through each lactation, with BCS assessed by an experienced trained operator on a 1 to 5 scale [Milk energy output (MJ/cow/day) was calculated by multiplying the GE content of milk (Equation (1)) by the daily milk yield. Feed efficiency was calculated by dividing ECM yield (kg/day) by the total DMI (kg/day). In early studies -based swards, and were produced from primary growth and primary, secondary, and tertiary regrown herbages. Herbages were normally wilted prior to ensiling  and treated with a bacterial inoculant at harvesting. While silage offered in most studies was ensiled in clamp silos, a number of studies involved silage made in big bales (n = 4). In a number of studies (n = 16), grass silage was partially replaced with corn silage . In addition, in one study, a small quantity of chopped wheat straw (0.3 kg/cow/day) was included in the diet. In all studies, the forage component of the diet was offered ad libitum . Although diets offered in these studies were predominantly based on grass silage and concentrates, diets changed over time, including concentrate feed levels and the ingredient composition of the concentrates offered. Grass silages offered were produced from perennial ryegrass (® and molasses) were included in some concentrates, while most concentrates contained a mineral/vitamin supplement. The concentrate component of the diets was offered either mixed with the forages , separate from the forages (via in-parlor or out-of-parlor feeders), or via a combination of these practices. The mean forage: concentrate DM ratio across the 27 studies was 49:51.A wide range of concentrate types, feeding levels, feeding strategies, and feeding methodologies were adopted within and between studies, according to the objectives of each individual study. The concentrate supplements consisted, principally, of cereal grains , protein supplements , and by-products from the food industry . Additional energy sources  analysis, with fresh samples normally analyzed weekly for nitrogen (N), gross energy (GE) and fermentation products. Silage ODM contents were, subsequently, corrected for volatile losses during drying, with all intakes presented on a volatile corrected DM basis. Samples of dried silage, composited for each 2–4-week period, were, subsequently, analyzed for fiber and ash concentrations. n = 8), the digestible OM in total DM content  of silages was determined by offering the silages to sheep confined in ‘digestibility crates’ at maintenance level . The metabolizable energy (ME) content of these silages was then estimated by multiplying the DOMD by 0.16 (assuming that one percentage point of DOMD equates to 0.16 MJ/kg DM of ME) [n = 20), the ME value of the forages offered was derived using NIRS as described by Park et al. [®, SRUC, Edinburgh, UK).In early experiments (M of ME) . The calM of ME) ,33. In lk et al. . In two k et al.  (Equatio®). The mean calculated ME content of the concentrate offered was 12.9 ± 0.25 MJ/kg DM. Total ME intakes were determined as the sum of the DM intake of each diet component multiplied by the ME content of that component. Further details of analytical methods used to determine the chemical composition of the feedstuffs and fermentation quality of the silages are presented within the individual studies, which are listed in Concentrates offered were normally sampled weekly, and composite samples analyzed for each 2–4 wk. period. The ME content of each concentrate was calculated using the ME content of each individual ingredient, based on values reported in UK feed composition tables , measured values were applied to each day during the 3-day period pre and post the day of measurement (in the case of weekly measurements), or to the 7-day period pre, and to the 6-day period post the day of measurement (in the case of fortnightly measurements). The mean ME content of all individual silage samples taken from each silo was applied to all days during which that silage was offered.maint+milk: MJ/kg of BW0.75) was determined using Equation (3).mk, with the efficiency of utilization of ME for walking assumed to be the same as that for maintenance (mk = 0.35 × ME/gross energy + 0.503) [The daily estimated EB  of each individual cow was calculated using equations contained within ‘Feed into Milk’ (FiM), the current UK dairy cow rationing system, as the difference between the cow’s total ME requirements  and total ME intake . The sumw et al.  (shown i+ 0.503) . This asi is the ME intake (MJ/cow per day). Mean weekly EB values were, subsequently, calculated for each week post-calving (up to a maximum of 20 WIM), with calving date considered as day 1 of the first WIM.The term METhe full dataset comprised 25,605 cow-week observations, with the individual cow lactation considered as the experimental unit. Individual cows included in the dataset were not fully nested within a study since individual cows were often used in more than one study. The dataset was subdivided by lactation number as follows: lactation 1, lactation 2, lactation 3, and lactation ≥ 4, the latter comprising cows in lactations 4, 5, and 6. Least mean squares evaluating the interaction between lactation number  and WIM were calculated to obtain general trends for DMI, ECM, EB, BW and BCS, milk composition parameters, and blood metabolites (BHB and NEFA). Both study and cow were included as random crossed effects in the model, and differences between least square means tested with the Tukey–Kramer test when relevant. Partial correlations between FPR and EB were determined using the MANOVA option of PROC GLM of SAS  to obtain partial correlations controlling for the effect of individual study within the dataset, at five 4 wk. intervals, namely, 1–4, 5–8, 9–12, 13–16, and 17–20 WIM. Univariate linear mixed model regressions were built to evaluate FPR as a single predictor of EB at the same time intervals. The best fit to the equation was chosen based on the lowest root mean square error (RMSE). All regressions analyses were performed within the MIXED procedure of SAS , and theijk is a dependent variable, μ is the mean for all observations, Fi is the effect of FPR score i, Sj is the effect of study j, Ck is the effect of cow k, and Eijk ~N represents the residual error. Both study and cow were included as random crossed effects in the model and differences between least square means tested by pairwise comparisons with the Tukey–Kramer test when relevant. Denominator degrees of freedom were calculated by the Kenward–Roger adjustment [p ≤ 0.05.Three FPR score categories  were adopted, and the PROC FREQ procedure of SAS used to determine the percentage occurrence of each FPR score during each four wk. interval from calving until 20 WIM. Prior to analysis, a natural logarithm transformation was applied to NEFA (lnNEFA) and BHB (lnBHB) concentrations to achieve normality. Animal performance , body reserves (BW change), log-transformed blood metabolites (lnBHB and lnNEFA), and daily EB associated with each FPR scores were assessed at selected four-week intervals using the following statistical model:justment . For allp < 0.001; milk fat content p < 0.006) and with the stage of lactation (p < 0.001), while there was a significant lactation × WIM interaction for all parameters , with the exception of milk fat content (p > 0.10). As expected, peaks in the ECM yield across lactations occurred earlier compared to responses in DMI (p < 0.001). No significant difference was detected for the nadir point between lactations three and ≥ four. Primiparous and second lactation cows reached positive EB earlier (≈10.5 WIM) compared to third and ≥ fourth lactations (≈12.5 WIM), respectively. Dry matter intakes reached a plateau (95% of maximum observed intake) at 10 and 6 WIM in primiparous and multiparous cows, respectively, while the peak ECM (maximum production measured as weekly least square means) occurred at 6 WIM in all lactations .In general, trends across lactations for the main parameters  were as s in DMI A,B. Irres in DMI C, and beConcentrations of milk fat declined from the beginning of lactation until, approximately, 8 WIM, remaining relatively constant thereafter A. In conp > 0.001). With all parities, the cows tended to lose BW during the first few WIM, gaining BW thereafter, while the BCS decreased throughout most of the first 20 WIM in primiparous cows, while, with multiparous cows, BCS decreased until 6–14 WIM (depending on lactation number), with cows tending to slowly gain BCS thereafter. Multiparous cows had higher BHB concentrations in early lactation than primiparous cows, with concentrations decreasing until approximately 8 WIM, remaining relatively constant thereafter. Non-esterified fatty acids concentrations tended to decrease until, approximately, 12–14 WIM, remaining relatively constant thereafter, with second lactation cows tending to have lower NEFA concentrations until, approximately, 7 WIM. Maximum plasma concentrations of both BHB and NEFA were observed during the first 2 WIM.Changes in body tissues (BW and BCS) and blood metabolites (BHB and NEFA) are presented in p < 0.001) with EB , while within each of lactations 1–4, partial correlations between these two variables tended to become less negative with an increased stage of lactation (p < 0.001), with FPR consistently negatively related to EB. The data used to develop each of the models in When examined across 1–20 WIM, FPR was moderate and negatively correlated . Similarly, across all parities and lactation intervals, increasing FPR scores were associated with increasing milk fat contents and decreasing milk protein contents (p < 0.009). Increased FPR was often associated with a greater BW loss or smaller BW gain, and increasing lnBHB and lnNEFA concentrations, with these effects mostly significant during 1–4 and 5–8 WIM post calving. Finally, ‘High’ FPR scores were always associated with a decrease in EB, irrespective of parity and the stage of lactation (p < 0.001).When examined during each four-week interval during each lactation, the incidence (%) of ‘High’ FPR scores (>1.5) tended to decrease as each lactation progressed, whereas, in general, the incidence of ‘Low’ scores (<1.0) tended to increase until 9–12 WIM, remaining relatively constant thereafter . Least sData analyzed within this paper were derived from a single farm. This reflected the fact that the determination of daily EB values relied on the use of individual cow intake data, which are generally only recorded on research farms. However, a wide range of diet types and qualities were examined within the studies used in the analysis. Furthermore, while the genetic potential of cows in the research herd changed over time, this genetic variability within the dataset made the results highly applicable to commercial farms where genetic variation both within herds and between herds can be substantial.In addition to characterizing the relationships between FPR and EB during the first 20 WIM, this paper sought to examine how the main drivers of EB (DMI and ECM) and other commonly used EB indicators  might help explain FPR–EB relationships across the stage of lactation and parity in Holstein-Friesian dairy cows. The mean forage-to-concentrate proportion in diets offered across the 27 experiments was 49:51 (on a DM basis), with this ratio varying little across lactations; thus, minimizing possible confounding effects arising from differences in energy supply. This concentrate proportion in the diet is typical of that in diets offered to moderate–high-yielding dairy herds within grass silage-based systems in the UK.An examination of weekly trends in DMI, milk production, milk composition, EB, BW, BCS, and blood metabolites allowed changes in FPR to be placed within a wider context. The trend for DMI and ECM to increase from lactations one to three within the lactation profiles reflected the increasing size (reflected in increasing BW) of the cows with increasing parity, their correspondingly greater intake and milk production potential, and their increased capacity for tissue mobilization in support of lactation, resulting in an increased duration of NEB . In suppWhile the EB curves in the present study were based on weekly least square means, these curves were in agreement with modelled EB curves  for Holstein cows offered grass silage-based diets ,40. AlthThe milk fat and milk protein content followed normal lactation trends , but with subtle differences between lactations and over time, which influenced FPR. The FPR ‘curves’ in the present study, which decreased from maximum values in early lactation and ‘plateaued’ around 10–13 WIM, were broadly similar to curves observed with Holstein cows in Germany , Canada Nevertheless, across published studies, there is much variation in the ratio at which the curves plateaued, with curves plateauing at, approximately, 1.20 to 1.25 in the present study, between 1.05 (first and second lactation) and 1.15 (third and further lactation) in the study by Buttchereit et al. , and 1.1That cows in all lactations started to gain BW from approximately 4 WIM onwards, long before reaching positive EB, has been observed in earlier studies ,48, withThe decreasing concentrations of BHB and NEFA in blood plasma, both of which are known to be indicators of body fat mobilization ,16,53, wUniquely, the present study allowed the relationships between FPR and EB to be examined at four wk. intervals during the first 20 WIM, and within each of lactations one, two, three, and > four. Irrespective of the lactation stage and parity, the partial correlations demonstrated that FPR was negatively related to EB, with the range of correlations observed broadly in agreement with those reported by Buttchereit et al.  for primTo the best of our knowledge, no previous study has examined FPR–EB relationships using a Linear Mixed Model approach evaluating between and within lactation responses as shown in the present study. The slopes of these regression models provide an estimate of the rate of decrease in EB per unit of increase in FPR. In the present study, when data from all 20 WIM were combined (slopes for the entire 20 WIM period for each lactation), the slope of the regression in each of lactations one to ≥ four was considerably more negative than for any of the individual four WIM periods within that lactation. An examination of the When data were compared across time points within any lactation, the The data presented in While a ‘baseline’ FPR score between 1.0 and 1.5 is considered to represent normal physiological conditions in Holstein cows during early lactation , an FPR The results presented in In agreement with the differences in EB between FPR scores, cows with an FPR score > 1.5 had a consistently greater BW loss, or lower BW gain, than cows with an FPR score < 1.0 throughout the first 12 WIM in all parities (except for 9–12 WIM in lactation ≥ four). In contrast, there were few differences in actual BCS between FPR score groups, and while this does not imply that changes in BCS did not follow a similar pattern for BW changes, it does suggest that cows with a different BCS were not more likely to be in one FPR score band than in another. This is perhaps surprising as there is ample evidence that cows with a high BCS are likely to have lower DMI and, subsequently, greater tissue loss in early lactation . HoweverThe increasing concentrations of both BHB and NEFA (ln transformed) in blood metabolites at higher FPR scores in early lactation reflected the greater NEB and greater BW loss with higher FPR scores. These metabolites, produced from the mobilization of body tissue reserves to produce glycerol for use as an energy resource, are normally associated with high milk fat concentrations and, as such, ‘High’ FPR scores (>1.5) . GreaterWhile this paper established clear relationships between FPR and EB, it is important to examine the utility of FPR as a prediction tool of EB for individual cows. Partial correlations and linear regression models highlighted strong relationships between EB and FPR, while data presented in Results obtained in the present study provide a better understanding of the EB responses per unit of FPR in milk during early–mid lactation (1 to 20 WIM) in Holstein-Friesian cows offered grass silage-based diets. While FPR in milk may provide a useful indication of EB at a herd level, it does not provide a robust indicator of EB at an individual cow level. The use of ‘High’ FPR scores in conjunction with additional indicators of EB appears to be helpful when describing physiological changes encompassing EB profiles at specific stages of lactation, until cows achieve positive EB. Caution should be exercised when extrapolating results of the present study, since the value of FPR in milk as an indicator of EB during early lactation may differ considerably depending on experimental conditions.


Vespula pensylvanica, is native to western North America and has become naturalized in Hawaii, where it exerts pressures on native arthropod communities as a competitor and predator. As invasive species may alter the microbial and disease ecology of their introduced ranges, there is a need to understand the microbiomes and virology of social wasps. We used 16S rRNA gene sequencing to characterize the microbiome of V. pensylvanica samples pooled by colony across two geographically distinct ranges and found that wasps generally associate with taxa within the bacterial genera Fructobacillus, Fructilactobacillus, Lactococcus, Leuconostoc, and Zymobacter, and likely associate with environmentally-acquired bacteria. Furthermore, V. pensylvanica harbors—and in some cases were dominated by—many endosymbionts including Wolbachia, Sodalis, Arsenophonus, and Rickettsia, and were found to contain bee-associated taxa, likely due to scavenging on or predation upon honey bees. Next, we used reverse-transcriptase quantitative PCR to assay colony-level infection intensity for Moku virus (family: Iflaviridae), a recently-described disease that is known to infect multiple Hymenopteran species. While Moku virus was prevalent and in high titer, it did not associate with microbial diversity, indicating that the microbiome may not directly interact with Moku virus in V. pensylvanica in meaningful ways. Collectively, our results suggest that the invasive social wasp V. pensylvanica associates with a simple microbiome, may be infected with putative endosymbionts, likely acquires bacterial taxa from the environment and diet, and is often infected with Moku virus. Our results suggest that V. pensylvanica, like other invasive social insects, has the potential to act as a reservoir for bacteria pathogenic to other pollinators, though this requires experimental demonstration.Invasive species present a worldwide concern as competition and pathogen reservoirs for native species. Specifically, the invasive social wasp,  Vespula are particularly problematic in this respect [th century, members of the genus Vespula have become invasive in South America, New Zealand, Australia, South Africa, and a variety of islands where native arthropods have not coevolved with predatory vespines [Vespula pensylvanica became widespread in the 1970s, introduced from its native range of western North America [V. pensylvanica typically exhibits the annual life cycle common to most temperate social wasps and bees: colonies are founded by solitary queens in the spring, grow through the summer, and produce many dispersing males and daughter queens in the fall before dying [V. pensylvanica and porous colony boundaries, apparent in both the native and introduced range [Social insects present some of the most widespread and damaging examples of invasive species worldwide . Invasiv respect . During vespines . Given t America . Throughre dying  and morere dying , coloniere dying . A prerere dying . Foreigned range . UnderstCephalotes spp.) and wasps (Vespa and Vespula spp.) seem to have simple core microbiomes, although it is unknown how ubiquitous the taxa are between colonies [Rickettsia, Arsenophonus, and Wolbachia [Sodalis [V. pensylvanica is native to the Western United States and is invasive to Hawaii [V. pensylvanica represents an excellent model species to understand the microbial ecology of isolated populations of invasive social wasps and possible mechanisms of microbial transmission.Invasive species potentially alter microbes present in their introduced environment , or the colonies –28. Convcolonies , 29–31, colonies , 33. Furolbachia , 34–36, [Sodalis , 38. Giv[Sodalis , and thao Hawaii , V. pensVespula spp. For example, honey bees harbor several debilitating viral diseases such as Deformed Wing Virus (DWV), Slow Bee Paralysis Virus (SBPV), Sacbrood Virus (SBV), Black Queen Cell Virus (BQCV), Kashmir Bee Virus (KBV), and others known to contribute to colony loss and impaired fitness  originally described in wasp colonies in Hawaii [Vespula spp. from several geographical locations, and has been found in honey bees and their associated Varroa mites [Vespula spp. are the natural reservoirs of this disease [V. pensylvanica suggests that Moku virus loads are strongly bimodal at the colony level, that colonies with high loads have reduced longevity in some years, and colonies that are at higher densities have higher Moku loads [Viral infections are widespread concerns for social Hymenoptera, and have been studied extensively in honey bees and iewed in ). While iewed in , 40–44. ic hosts , 40, 44.n Hawaii . Aside foa mites , 45, 46.ku loads .V. pensylvanica are largely unknown, we conducted an exploratory study using 16S rRNA gene sequencing and RT-qPCR on these invasive social wasps at the colony level. We investigated three lines of inquiry to better understand the microbial communities of our study organism: First, does V. pensylvanica associate with a defined microbiome, is there any evidence of environmental transmission of microbes, and are their microbiomes similar in the native and invasive range? Second, does this wasp species associate with endosymbiotic bacteria? Third, are there associations between Moku virus and the wasp microbiome?As the microbial ecology and effects of Moku virus on V. pensylvanica workers from nest entrances using a net or a portable vacuum. While collecting, we attempted not to disturb the colony and thus primarily collected foragers, though in some cases we likely collected guard wasps as well. We flash-froze the collected adults alive in a liquid nitrogen dry shipper, and then transferred to a -80°C freezer upon return to the lab until processing. Thirty-seven colonies sampled in Hawaii are included in a companion study on colony longevity in V. pensylvanica [Fieldwork was conducted under a permit from the National Park Service (HAVO-2016-SCI-0050). We collected adult worker wasps from the entrances of colonies found along Hilina Pali Road and at Kipuka Kahali’i in Hawaii Volcanoes National Park (HAVO) on the Big Island of Hawaii and the campus of the University of California, Riverside (UCR) in Riverside, CA. Colonies were discovered by following foragers back to the nest site, or by noticing characteristic foraging traffic at the colony entrance. We collected between 20 and 40 adult worker wasps from each of 40 colonies at HAVO between August 25–27, 2017, as well as from 13 colonies at UCR between October 6–26, 2017 (see ylvanica . Three oWe extracted DNA by first pooling 20 wasps per colony (N = 53 colonies). We homogenized the samples by adding still-frozen insects to 15 mL tubes with grinding balls using a Geno/Grinder , with the block pre-chilled with liquid nitrogen. We then quickly transferred the homogenate to new sample tubes and re-froze at -80°C until DNA or RNA extraction. Ultimately, we extracted DNA from a ~20 mg aliquot of thawed homogenate using a Qiagen DNeasy kit  by following the manufacturer’s protocol plus an overnight incubation at 55°C.CMGGATTAGATACCCKGG and 1115R: AGGGTTGCGCTCGTTG) chosen to avoid plant plastid contamination [We prepared paired-end 16S rRNA gene libraries for samples N = 53) using a protocol based on Engel et al. , Rothmanmination , 53, a u using a Vespula eIF3 [moku-CqeIF3) for each sample, resulting in an index with higher values corresponding to greater viral titer on a log scale. Because colony virus titer was strongly bimodal in a larger dataset including two years of data [We used data reported in another study on Moku Virus and colony longevity  and repeula eIF3  as a ref of data , we usedhttp://bioinformatics.psb.ugent.be), and plotted heatmaps with “pheatmap” [We processed the 16S rRNA gene libraries with the QIIME2 pipeline (v2019.7)  by firstheatmap” . We usedheatmap”  to test We obtained 1,033,410 quality-filtered  16S rRNA gene sequences with an average of 19,498 reads per sample from two separate populations  that clustered into 2,943 unique ASVs . ThroughPseudomonas spp, Shewanella spp., Halomonas spp., Micrococcus spp., Cutibacterium acnes, Streptococcus oralis, and Rhizobia), the top 10 most abundant bacterial families and their relative abundances in our samples were as follows: Leuconostocaceae (32.8%), Enterobacteriaceae (17.1%), Microbacteriaceae (9.9%), Streptococcaceae (3.6%), Lactobacillaceae (3.6%), Halomonadacae (3.1%), Rhizobiaceae (2.9%), Acetobacteraceae (2.6%), Rickettsiaceae (2.4%), and Moraxellaceae (1.9%), while all other families combined comprised an average of 20.2% of the proportional abundance of taxa (Frigoribacterium faeni ASV (9.7%), four Fructobacillus ASVs , an Arsenophonus sp. ASV (6.3%), a Leuconostoc sp. ASV (3.7%), an ASV of Lactococcus lactis (2.9%), a Rhizobium sp. ASV (2.7%), and a Zymobacter palmae ASV (2.5%), which cumulatively comprise 51.6% of the wasp microbial communities  in our samples, and did not find any significantly differentially abundant ASVs between high-titer and low-titer colonies. We also ran mantel tests using Spearman correlations to test relationships between wasps’ alpha (Shannon diversity index) and beta (Bray-Curtis dissimilarities) microbial diversity with Moku virus titer, but Moku virus titer was not significantly correlated with microbial alpha or beta diversity in our samples .We compared the microbiomes of wasp colonies with low Moku virus titer (N = 7) or high Moku virus titer (N = 26) in our Hawaiian samples (we did not detect Moku virus in any Riverside samples). We found no significant effect of viral titer on either the alpha  or beta diversity  was correlated with the proximity of wasp colonies to honey bee hives at the Hawaii field site [Wasps in our study harbored a variety of putatively endosymbiotic bacteria—especially those collected in Hawaii. These endosymbionts included bacteria in the genera  species , 68, 69,emiology . Additiodynamics . Suggesteld site , which cV. pensylvanica and honey bees, and has been previously detected in Hawaii, the United Kingdom, and Belgium [Vespula colony longevity in 2016 at our Hawaii field site, but not in 2017 when the samples in this study were collected, suggesting a variable role of this virus in wasp colony dynamics [Neither Moku virus presence nor titer affected the wasps’ microbiomes. Moku virus is a recently-discovered virus that is known to infect  Belgium , 46—althdynamics . Moku vidynamics . Many otdynamics , 73, anddynamics . Even thdynamics , 77.V. pensylvanica possesses a simple microbiome mainly composed of lactic acid bacteria and putatively environmentally-acquired taxa along with several species of endosymbionts. Furthermore, we show that these invasive wasps likely maintain some of these core bacteria across geographically distinct regions, indicating potential symbioses. We also found that wasp colony microbiomes are not affected by Moku virus presence, despite high titers, which supports the hypothesis of V. pensylvanica being a natural reservoir for this disease, along with a potential vector to other insects. We suggest that future studies examine the physiological effects of Moku virus on wasps and other Hymenoptera, and the potential for invasive wasps to change the microbial and viral ecology of their introduced ranges. We are also interested in assaying the contributions of wasps’ microbes to colony fitness and nutrition and subsequent work should be conducted to understand the microbial ecology of social wasps.In summary, our data suggest that the social wasp S1 FigCurves saturate before a sequencing depth of 2,232 reads, which we used for all diversity analyses.(DOCX)Click here for additional data file.S1 File(TXT)Click here for additional data file.S1 Table“R” denotes “Riverside” and “H” denotes Hawaii sampling locations.(DOCX)Click here for additional data file.


Nonalcoholic fatty liver disease (NAFLD) and gallstone disease (GSD) often coexist in the general population owing to shared risk factors. This study explored the relationship between NAFLD and GSD in patients with type 2 diabetes.We conducted a retrospective cross-sectional analysis of 4325 patients with type 2 diabetes. GSD and NAFLD were confirmed using ultrasonography. GSD was defined as either asymptomatic gallstones or previous cholecystectomy, and each was analyzed separately.P = 0.15). After case–control matching (1:1) of baseline data such as age, sex, duration of diabetes, and HbA1c between patients with and without NAFLD, there was still no significant difference in the prevalence of GSD . The prevalence of NAFLD in patients with asymptomatic gallstones was lower than that of patients without GSD , whereas the prevalence in those who had undergone cholecystectomy was much higher . The ratio of cholecystectomy to asymptomatic gallstone in patients with or without NAFLD was 1.97 and 0.79, respectively. The rate of cholecystectomy was higher in the patients with NAFLD than in those without NAFLD , consistent with the result after case–control matching . Multivariate logistic regression analysis, after adjusting for numerous potential confounding factors, revealed that GSD  and cholecystectomy  were both strongly associated with NAFLD. However, asymptomatic gallstone  seemed to be negatively correlated with NAFLD.There was no significant difference in the prevalence of GSD between patients with and without NAFLD (23.8% vs. 21.2%, The prevalence of GSD was similar in patients with type 2 diabetes with and without NAFLD. The higher proportion of cholecystectomy and lower proportion of asymptomatic gallstones in patients with NAFLD suggests that NAFLD may increase the risk of complications of GSD. The liver is an essential organ in systemic metabolism, contributing substantially to the development of insulin resistance and type 2 diabetes mellitus (T2DM). Nonalcoholic fatty liver disease (NAFLD) refers to histological liver abnormalities ranging from isolated steatosis to nonalcoholic steatohepatitis (NASH), which may result in cirrhosis and is related to being overweight or obese and having metabolic disorders, such as insulin resistance, T2DM, and dyslipidemia . NAFLD aGallstone disease (GSD) is another common condition that exhibits a concomitant increase in prevalence as the incidence of obesity increases . GSD is This study included inpatients with T2DM at the Department of Endocrinology and Metabolism of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital between January 2011 and December 2014. Participants suffering from the following conditions were excluded: diabetic ketosis, hyperglycemic hyperosmolar syndrome, and other acute complications of diabetes, hyperthyroidism, acute pancreatitis, acute hepatitis, intrahepatic and intrahepatic bile duct stones, gallbladder polyps, cirrhosis, malignant tumors, and pregnancy. A total of 4325 patients with T2DM (2411 men and 1914 women) were enrolled.GSD was confirmed with abdominal ultrasonographic examination, which was performed using a 3.5-MHz ultrasound transducer; the presence of gallstones or the absence of the gallbladder ascribed to previous cholecystectomy was determined. The presence of intraluminal echoes dependent on gravity or attenuation of ultrasonic transmission (acoustic shadowing) was considered diagnostic for gallstones.NAFLD was defined based on the revised definition proposed by the Chinese Hepatology Association in 2010 : ① No hiAll these data were retrieved from patients’ medical records, including body mass index (BMI), waist circumference (WC), blood pressure (BP) and other clinical characteristics.The general biochemical parameters, including serum levels of fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), glycosylated hemoglobin (HbA1c), glycosylated albumin (GA), gamma-glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were determined using an automated biochemical detector . Blood samples were collected through venipuncture for laboratory testing from each study participant after overnight fasting. All patients underwent ultrasonographic examination.t test, or paired t test after a 1:1 case control. Chi-square test was constructed to compare differences for categorical variables. Logistic regression analysis was performed to assess the association between GSD and NAFLD. A P-value < 0.05 indicates a statistically significant difference.SPSS v22.0  was used for statistical analysis. Continuous variables are presented as mean ± standard deviation, and binary variables as numbers (percentages). The Kolmogorov-Smirnov test was taken to assess the normal distribution of the continuous variables. All continuous variables between cases and controls were compared by the Student’s n = 2241), those with NAFLD (n = 2084) were younger (P < 0.001) and had higher WC (P < 0.001), BMI (P < 0.001), and TG (P < 0.001). There was no significant difference in the prevalence of GSD between patients with and without NAFLD . However, the prevalence of gallstones in persons without NAFLD (11.9%) was nearly 1.5 times that in persons with NAFLD . Meanwhile, the prevalence of cholecystectomy was significantly higher in patients with NAFLD . However, patients with NAFLD were characterized by higher level of ALT, AST, Gamma-GT as well as TG. Meanwhile, the proportion of patients with NAFLD who underwent cholecystectomy was approximately 1.5 times that of those without NAFLD . Conversely, the proportion of patients with NAFLD who had asymptomatic gallstones was much lower than those without NAFLD and concomitant asymptomatic gallstones , which is consistent with the original results , cholecystectomy , and without GSD . The prevalence of GSD was 22.45% (971/4325). Compared with patients without GSD, patients with GSD had significantly higher age, female sex, longer diabetes duration, higher BMI, larger WC, higher blood pressure, and higher GGT levels. Regardless of the presence or absence of GSD, the prevalence of NAFLD was similar , and cholecystectomy  were significantly associated with the prevalence of NAFLD in the univariate analysis, and asymptomatic gallstone  was likely to be negatively associated with NAFLD. After adjustment for age, sex, BMI, blood pressure, diabetic duration, HbAlc, TC, TG and LDL-C, GSD  and cholecystectomy  were still associated with increased risk of NAFLD, while asymptomatic gallstone  was just the opposite (Table Univariate and multivariate logistic regression analyses were conducted to further investigate the association between GSD and NAFLD. GSD (OR = 1.16, 95%CI: 1.007–1.340, NAFLD and GSD are common comorbid conditions that are associated with the same risk factors, including insulin resistance, obesity, metabolic syndrome, and T2DM –18. InsuIn this study, we analyzed the prevalence of asymptomatic gallstones and cholecystectomy. The relationship between asymptomatic gallstones and NAFLD remains controversial . HoweverTill now, direct evidence proving the chronological sequence of GSD and NAFLD or cholecystectomy and NAFLD is lacked. Meanwhile, the exact mechanism underlying the association between NAFLD and GSD or cholecystectomy still remains unknown. Hepatic insulin resistance may create an inner link between GSD and NAFLD, which is widely accepted as an independent risk factor for both conditions –31. The This study had several limitations. First, the lack of a nondiabetic control group may limit the generalizability of our results. Second, our findings are limited to Chinese population; therefore, this conclusion may not be generalizable to other populations in other geographical locations. Third, the cross-sectional design precluded the determination of a causative relationship. Longitudinal studies with long-term follow-up and larger sample sizes are required. Finally, both the inner mechanism of NAFLD and the underlying pathophysiology of the association between NAFLD and GSD are so complex since a lot of co-variates or mediation factors could not be excluded, distinguishing causes from effects is very difficult –42.In our cohort of patients with T2DM, the prevalence of GSD was not associated with NAFLD, and the higher incidence of complications of GSD was likely related to NAFLD, which is reflected in the higher proportion of cholecystectomy in NAFLD patients. NAFLD may induce the complications of GSD, leading to a higher prevalence of cholecystectomy.


Drosophila melanogaster testis, a new class of small regulatory RNAs initially named repeat-associated small interfering RNA (rasiRNAs). rasiRNAs were first described as regulating a protein-coding gene. They are derived from the Suppressor of Stellate [Su(Ste)] locus located on the Y chromosome and were shown to target the X-linked Stellate repeated genes by sequence complementarity. Stellate genes encode proteins with homology to the regulatory β subunit of the protein kinase CK2. Stellate repression occurs during male gametogenesis and is essential for male fertility. These small RNAs were renamed as PIWI-interacting RNAs (piRNAs); they are 23 to 29 nucleotides (nt) long and bind to proteins of the PIWI family. Accordingly, piRNA pathway mutants are sterile, and they contain crystalline aggregates of Stellate-coded protein [In the early 2000s, Aravin and colleagues discovered, in the  protein .D. melanogaster, both sexes depend on a functional piRNA pathway for their fertility. Our understanding of piRNA biogenesis and function comes predominantly from studies of the female Drosophila germline, but the male’s piRNA pathway remains poorly understood. Previously, studies reported that many proteins involved in the female piRNA pathway are also required for male fertility and Stellate silencing in the testis, supporting the conservation of piRNA pathway machinery in both sexes [Su(Ste) genes and AT-chX [D. melanogaster spermatogenesis and show piRNAs adaptation, dependent on sex-specific expression of transposons [Rhino mutants was not compromised and stellate crystals were absent, RDC function in spermatogenesis was neglected [piRNAs were then discovered in mammals and in most other animal germ cells . Nowadayth sexes . While Ath sexes –6 . Furthermore, Moonshiner  then links the RDC to RNA polymerase II to initiate a promoterless noncanonical transcription.In this issue of germline . UnexpecSu(Ste) dual-strand cluster is Rhino independent, and Stellate genes are properly silenced in Rhino mutants [Su(Ste) locus may be canonically transcribed by RNA polymerase II, with initiation occurring from convergent promoters. Sense transcripts of Su(Ste) are spliced and polyadenylated [Su(Ste) locus lacking RDC, which would actually suppress splicing and polyadenylation if present. Importantly, when Su(Ste) repeats start to be expressed in spermatocytes during the mitosis-to-meiosis transition, the general transcription machinery is replaced by testis-specific machinery, like testis-specific TBP-associated factor (tTAF), testis-specific meiotic arrest complex (tMAC), and the coactivator mediator [Su(Ste) expression.One striking result from Chen and colleagues is that transcription of the  mutants . Similarenylated . This ismediator . It remaAT-chX locus [AT-chX locus not colocalize with pericentromeric D1? (ii) How does AT-chX become highly expressed at this stage? One possibility might be that AT-chX loops out to be excluded from the down-regulated X chromosome, similarly to the Y chromosome. Also, we cannot exclude that the single Rhino foci results from the concentration of multiple piRNA clusters, similarly to rDNA that cluster in the nucleolus to be transcribed by RNA-pol-I in many organisms [AT-chX and other piRNA cluster expression and nuclear organization during spermatogenesis.In this new study, Chen and colleagues showed that RDC first forms multiple nuclear foci in GSCs and spermatogonia due to its association with multiple dual-strand piRNA clusters and then concentrates in spermatocytes in a single dot corresponding to a unique piRNA cluster, the X-linked hX locus . ImportahX locus . (2) ThehX locus ,16. (3) hX locus . Thus, 2rganisms . Future AT-chX is the second most expressed piRNA cluster in the testis after Su(Ste), its function still remains poorly understood. One possibility is that AT-chX could be a master locus required for the piNG-body formation, as flamenco is necessary for the Yb body formation in ovarian somatic cells [AT-ChX piRNAs could participate together with the Su(Ste) system to assist the emergence of reproductive isolation and speciation of D. melanogaster during evolution (D. melanogaster has Su(Ste) and AT-chX repeats [D. melanogaster females mate with other Drosophila species males, Stellate genes cannot be repressed, and the hybrids flies are sterile. Intriguingly, AT-chX piRNAs have 76% homology to vasa transcripts within D. melanogaster but do not have the ability to repress vasa in the testis or ovaries. However, vasa sequences from related species that diverged from a Drosophila common ancestor several million years ago have more than 90% complementarity with piRNAs from AT-chX repeats of D. melanogaster. AT-chX piRNAs are more similar to “alien vasa” than endogenous vasa, suggesting that those piRNAs could help to preserve reproductive isolation and speciation through the repression of “alien vasa” when crossed to other species, since vasa is crucial for the piRNA pathway, germline formation, and fertility.Finally, even if ic cells . Indeed,ic cells . It has volution . Indeed, repeats ,21. WhenThanks to their discovery, Chen and colleagues open the possibility to reconsider many other proteins of the piRNA pathway thought to be futile in males, which will help to better understand the global piRNA mechanisms and piRNA sexual dimorphism.


Neocaridina palmata. Therefore, organisms were exposed to 20–20,000 particles L−1 of either polyethylene (PE) beads (41 μm and 87 μm) or polyvinyl chloride (PVC) fragments (<63 μm). Moreover, shrimps were exposed to 20,000 particles L−1 of either 41 μm PE and 11 μm polystyrene (PS) beads or the PVC fragments for 24 h, followed by a post-exposure period of 4 h to analyze the excretion of particles. To simulate natural conditions, an additional fragment ingestion study was performed in the presence of food. After each treatment, the shrimps were analyzed for retained or excreted particles. Our results demonstrate that the ingestion of beads and fragments were concentration-dependent. Shrimps egested 59% of beads and 18% of fragments within 4 h. Particle shape did not significantly affect MP ingestion or egestion, but size was a relevant factor. Medium- and small-sized beads were frequently ingested. Furthermore, fragment uptake decreased slightly when co-exposed to food, but was not significantly different to the treatments without food. Finally, the investigations highlight that the assessment of ingestion and egestion rates can help to clarify whether MPs remain in specific organisms and, thereby, become a potential health threat.The ingestion of microplastics (MPs) is well documented for various animals and spherical MPs (beads) in many studies. However, the retention time and egestion of MPs have been examined less, especially for irregular MPs (fragments) which are predominantly found in the environment. Furthermore, the accumulation of such particles in the gastrointestinal tract is likely to determine whether adverse effects are induced. To address this, we investigated if the ingestion and egestion of beads are different to those of fragments in the freshwater shrimp The online version contains supplementary material available at 10.1007/s11356-021-15068-x. The ingestion of microplastics (MPs) has been previously described for more than 70 freshwater organisms . This shrimp is characterized by a transparent exoskeleton and, therefore, eggs in breeding females, and food uptake is easy to detect. The genus is native to Asia  of global rivers  was purchased and cultured in 20 L glass aquaria at Goethe University (Department Aquatic Ecotoxicology). Individuals were acclimatized at least for 1 week and were kept under constant conditions at 23 ± 2 °C and a 16:8 h light/dark cycle (460 lux). Reconstituted water based on the OECD guideline 242: Potamopyrgus antipodarum Reproduction Test  and continuous aeration. Twice a week, the medium was partially renewed, and the shrimps were fed ad libitum with CrustaGran and Shrimp King Mineral (Dennerle GmbH). The number of individuals in the culturing aquaria varied greatly, depending on the reproduction rate of the individuals at the time.est OECD  was usedSpherical MPs and fluorescent polyethylene (PE) beads  of two different size ranges (UVPMS-BG-1.035g/cc 38–45 μm and UVPMS-BG-1.025g/cc 75–90 μm) were purchased from Cospheric LLC©  and Fluoresbrite® YG 10 μm polystyrene (PS) beads in a 2.5% aqueous suspension  from Polysciences Europe GmbH . Irregular MPs (fragments) were prepared from a fluorescent  polyvinyl chloride (PVC) cord ; the PVC cord was cut into small pieces (<1 cm) and milled cryogenically for 1–2 min at 30 Hz . The grinding steps were repeated until a fine powder was formed, which was sieved (<63 μm) using the Vibratory Sieve Shaker AS 200 basic . Since there were no data available for the specific density of the PVC cord, the density was determined based on the weight and volume of one PVC cord piece  to avoid the agglomeration of beads  for 24 h  of Tween®20 as we dispersed the beads with this solution. To elucidate whether the shrimps feed preferentially within a specific size range, the experiments with beads were conducted with mixtures (1:1) of 75–90 μm and 38–45 μm PE beads in the ingestion study and 38–45 μm PE and 10 μm PS beads in the egestion study, respectively  for 24 h but with added 4–5 mg of CrustaGran per test vessel; this food quantity corresponds approximately to 10% of the shrimps’ wet weight  were determined for each individual using an Olympus SZ40 stereo microscope  . Lysates were then vacuum-filtered onto cellulose nitrate membrane filters and analyzed for ingested particles using the fluorescence microscope. The data were corrected for the negative control of the beads that served as a blank and for the airborne control that was necessary during the microscopical fragment analysis  and the sex  of a PE-PS beads mixture and PVC fragments, respectively. Half of the individuals were then transferred into particle-free vessels with food (10 mg CrustaGran), which was added once to the vessels. A higher food amount than in the fragment ingestion study with food was chosen to increase the encounter rate for natural particles and, thereby, enhance the excretion. A post-exposure period of 4 h (t = 4 h) for the egestion of particles was chosen since preliminary tests revealed that <4 h is sufficient for the shrimps to egest more than 50% of beads. The other half of the individuals not intended for excretion analysis were removed from the test after particle exposure to serve as a reference for particle uptake (t = 0 h). After the egestion period, the shrimps were cleaned and lysed under the same conditions as previously described. Lysates and excretions were vacuum-filtered and analyzed microscopically next to the shrimps that had no egestion period (t = 0 h). Here, the negative control of the beads study served as a blank, while another filter accounted for the introduction of airborne fragment-like particles during microscopy. A further blank accounted for potential fragment-like particles introduced by the food source during post-exposure .Data were analyzed with GraphPad Prism® (5.00 and 9.00) . The data were tested for normal distribution. If the data were not normally distributed or in cases of variance inhomogeneity, the Kruskal-Wallis test followed by Dunn’s post hoc test was conducted; otherwise a one-way ANOVA with Dunnett’s post hoc test was performed. Statistical comparisons were made between the control group without MPs and the exposure treatments. Relationships between the body length and ingested or egested particles were analyzed using the Pearson or Spearman correlations, depending on whether the data met the parametric criteria. In order to test if the particle type, sex, and added food influenced the ingestion or egestion, a two-way ANOVA with Bonferroni’s post hoc test was performed. The significance level was defined with α = 0.05  with rising exposure concentrations   and 20,000 (p <0.0001) beads L−1. During exposure to 2000 beads L−1, one individual out of eight individuals died. Regarding the PVC particles, the mean number of ingested particles ranged from 6.13 to 204 fragments individual−1 after exposure to 20–20,000 fragments L−1. Once again, significant increases were observed for 200 fragments L−1 (p <0.01) as well as for 2000 and 20,000 fragments L−1 (p <0.0001) when compared to the control group. A significant influence of the differently shaped MPs in relation to the uptake was not detected. Moreover, neither significant differences for the MP ingestion between males and females nor a correlation between MP ingestion and body length were observed  Table S, whereas−1. In another treatment, individuals had an additional post-exposure time (t = 4 h) in particle-free medium to allow the measurement of egested particles. Here, the shrimps’ excretions as well as lysates were examined to elucidate whether particles remained in the digestive system. On average, the shrimps contained 243 beads individual−1 (t = 0 h), i.e., 146 of 11 μm beads and 96.4 of 41 μm beads, and egested 143 beads individual−1 after 4 h ; the latter was corrected for two beads found in the corresponding negative control (Table S−1 remained in the shrimp lysates but were still significantly different to the reference treatment (p <0.05)  from 371 in the reference (t = 0 h) to 14.0 in the lysate within 4 h of post-exposure. One individual died in the egestion treatment (t = 4 h)  compared to the 75–90 μm PE beads), the organisms were further exposed to a PE-PS beads mixture of an even smaller size range in the egestion experiment Table . For the5 μm comp) Table S. Neocari) Table S. No sign−1 and, therefore, included more particles than the exposure treatment with 20 fragments L−1. Here, an average of 4.75 PVC particles was detected per shrimp .Finally, we investigated whether food availability influenced the ingestion of fragments Fig. . The ingNeocaridina palmata. In addition, we used MP concentrations measured in the sediment of global freshwaters  fragments than of 30–165 μm PE and PS beads (Gray and Weinstein Mysis relicta ingested high rates of acrylonitrile butadiene styrene (ABS) fragments, but not polyethylene terephthalate (PET) fragments, when compared to PS beads. This could be attributed to the individual’s ingestible size range since ABS fragments were smaller than the PET. Moreover, diverse MP properties  could contribute to bioavailability issues  and detected high loads of natural particles. This indicated active particle uptake enabling food to be mechanically fragmented. Based on this observation, they concluded that shrimps may be less selective in their search for food and therefore less susceptible towards MP contamination in their environment. Our findings are plausible in that food probably reduces the animals’ encounter rate for MPs due to dilution effects  within 4 h of post-exposure. The shrimps only partially egested the particles within this specific excretion period as 123 beads and 14 fragments remained in the gastrointestinal tract , we observed a comparably higher excretion rate for beads (85%), while only a small fraction was found in the digestive systems, and the rest could not be detected due to methodological reasons. Saborowski et al.  is very limited, and solely the analysis of sampled animals would elucidate true accumulation rates of MPs  for PE beads and powdered PVC particles by Stock et al.