P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility.The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates  correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls  had positive FRAC tests , whereas all of the subfertile bulls  had positive FRAC tests ( One out of three couples experiencing infertility have unexplained infertility5. It is expected that a significant portion of unexplained infertility is due to infertile men who, due to the limitation of current semen analysis methods, appear to have normal semen. Similarly, up to 5% of marketed, genetically elite Holstein bulls used in the artificial insemination (AI) industry have unexplained subfertility that is found only after being released to the market7. Because of this similarity and the similarity of cattle sperm centrioles' structure, composition, function, and inheritance pattern to human sperm centrioles, cattle subfertility is an important model for human infertility8.Unexplained infertility is a major problem in managing and treating reproductive health9. Currently, the markers used to diagnose bull subfertility explain ~ 3% of the total variance in artificial insemination field fertility deviations6. Bovine sperm is extensively analyzed prior to use and undiagnosed subfertility after analysis points to an unknown male factor and a gap in our understanding of sperm biology. One reason for this deficiency is the absence of a comprehensive list of independent biomarkers that identify defects in distinct properties of sperm, which are essential for selecting bulls with high fecundity10. Therefore, there is a need to identify novel biomarkers for male subfertility.Subfertility, a significantly reduced reproduction rate relative to average reproduction rate by most other bulls, is a major obstacle to the profitability of the U.S. meat and dairy industry15. Identifying biomarkers in bull sperm has the benefit of not only improving the selection of bulls, but it is also a good model for human sperm because they share a similar morphology, both having paddle or pear-like sperm heads with a canonical proximal centriole and an atypical distal centriole in the neck, whereas mice have sickle shaped sperm heads with no centrioles and numerous other embryonic developmental deviations reviewed in16. Robust fertility data provided by the bull artificial insemination industry, which examines over 300  insemination attempts per bull in the field, provide unprecedented reproductive statistics for determining normal biomarker ranges. Therefore, bulls are a valuable model to understanding human sperm biology, while also providing insight for the cattle industry. Yet, bull and human fertility are managed differently as bulls, unlike humans, are selected for fertility, creating challenges in using results from domestic animal testing for human reproductive assistance programs.One approach to identify new biomarkers is to study sperm structures that are implicated in sperm function and fertility but are not routinely used for diagnostics, such as the sperm centrioles17. A sperm component that functions in both processes are the centrioles. They form the sperm tail during spermatogenesis, are dynamic during sperm swimming, organize the microtubule in the zygote, and are essential for accurate cell division19. These multitude of functions imply that centriole quality could be an indicator of sperm fertility. Interestingly, despite most dividing cells requiring precisely two centrioles, there is only one canonical centriole, the proximal centriole (PC), in the sperm of humans, bovine, and other non-murine mammals; the second centriole is the remodeled atypical spermatozoa distal centriole (referred to as the DC in this paper)20. The role of the remodeling, or the centrioles in general, in sperm biology and male fertility is not well understood8. Since the centrioles are not examined during routine semen analysis, evaluating the centriole may be a useful independent biomarker for advancing semen analysis5. Recently, a high throughput method was developed to assess sperm centrioles, Fluorescence-Based Ratiometric Analysis of Sperm Centrioles (FRAC).Bull fecundity depends on the ability of sperm to travel through the female reproductive tract and the contributions of the sperm components in the early embryo21, as was suggested earlier by electron microscopy studies32. Also, FRAC identifies male factor infertility in couples with unexplained infertility33. FRAC is a sensitive method for identifying male infertility because it detects changes in relative protein distribution using fluorescent microscopy. Thus, FRAC could stratify centriole quality better than a low throughput assays like electron microscopy, but this requires futher evaluation.FRAC determines the intensity ratio of centriolar biomarkers at locations in the sperm neck and mid piece for each sperm . Then a mean ratio 95% confidence interval is calculated for each bull by analyzing over 100 sperm. The 95% confidence interval is compared to a reference population and, if it is outside, then the bull has a positive FRAC test indicating a possible centriole anomaly  and may be due to undiagnosed sperm defects. Similarly, lactating dairy cows, during the first week of gestation, lose 20–50% of pregnancies and during conceptus elongation (Days 8–27) an additional ~ 30% loss occurs. Some of these failures are lactation induced or due to heat stress reviewed in47. However, some of these failures may also be due to defects in the sperm centrioles that either lead to failure to properly polarize the pronuclei, or inaccurate cell division.After fertilization, the two centrioles organize the embryonic cytoskeleton and are the progenitors of centriole duplication within the embryo33. Tubulin is a building block of PC, DC, and Axoneme microtubules reviewed in48, and its labeling is more intense in the centrioles than in the axoneme of human sperm21. Acetylated tubulin is a posttranslational modification of tubulin that makes the microtubules more flexible and stable50. Its labeling has a similar intensity in the centrioles and the axoneme of bovine sperm51. POC1B and FAM161A are luminal centriole proteins52 that form the rods of the sperm centrioles but appear to function independently19. Their labeling is specific to centrioles and POC1B is essential for male fertility in humans and mice53.Centriolar biomarkers can be structural proteins or posttranslational modifications56. It is an outcome predictor based on more than 300 inseminations, sometimes from tens of thousands of inseminations. SCR is reported in deviation percentage units from average pregnancy rate within breed generated from more than 500 bulls. Average Holstein successful conception is approximately 34–35%, which is an SCR of 058. Using a bull with a significantly lower SCR is time-consuming and costly due to the need for increased rebreeding attempts, extended days open, widened calving intervals, and increased risk of culling when a threshold number of insemination attempts have been reached. Both farmers and the artificial insemination industry are interested in screening out such bulls.A measure of field fertility by artificial insemination in bulls  in the USA is Sire Conception Rate (SCR)59. They found that bulls with greater fertilization capacity have higher percentages of fast and nonlinear spermatozoa (P < 0.05). By examining 10 Holstein bulls with high or low SCR, Ortega and colleagues (2018) found that some low SCR bulls produced fewer day 8 blastocysts in IVF using slaughterhouse-sourced oocytes60. They also showed some low SCR bulls produced a lower percentage of fertilized oocytes or more degenerated embryos compared to high SCR bulls in super-ovulated heifers.Hidalgo and colleagues (2021) compared 12 bulls with pregnancy rates of 33–52% to 12 bulls with rates of 52–70% after fixed time artificial insemination in BrazilBecause the centriole functions during sperm swimming and embryo development, we hypothesized that centriolar anomalies act as biomarkers for bull subfertility. Therefore, we studied the correlation between sperm centriole protein distribution and SCR by studying fertile and subfertile bulls. We found that some subfertile bulls have abnormal centriole protein distribution that can be identified by FRAC. This finding is the first experimental evidence that bovine sperm centriole anomalies are associated with unexplained subfertility. Furthermore, the utilization of FRAC provides a path to improving artificial insemination efficiency in the short term by weeding out bulls with lower sperm quality and could be used to improve artificial insemination fertility in the long term by selecting bulls with higher sperm quality.33. No live animals were experimented on to generate these data. All experimental protocols were approved by UToledo Biosafety Committee (IBC). All methods were carried out in accordance with UToledo Biosafety Committee (IBC) guidelines. The ARRIVE guidelines  are not applicable as Animals were not used directly in the study.The methods utilized in this manuscript are similar to those published before in Jaiswal and colleagues (2022). A few changes were made, mostly to adapt the method from human semen to bovine semen6. Samples in straws were stored in liquid nitrogen until studied by FRAC. FRAC analysis was performed during 2020–2022 and ended once all 31 samples were analyzed. It should be noted that only semen samples collected by Select Sires were used to generate data presented herein.Semen samples from 31 bulls were obtained from Select Sires, Inc. in September of 2019 from available bulls with known, varying SCRs . Semen was collected by Select Sires as described in DeJarnette and colleagues and distributed to us cryopreserved in straws in liquid nitrogen tanks as regularly done in the dairy bull AI industryTo examine the sperm that are likely to participate in fertilization, semen samples were separated into less dense lower-quality and denser higher-quality sperm using differential gradient centrifugation following manufacturer’s instructions . Frozen samples and all necessary PureSperm media were brought to 37 °C in a water bath. Then, 1.0 mL of PureSperm 80%  was placed into a 15 mL conical tube labeled with the bull identifier and pellet. Next, 1.0 mL of PureSperm 40%  was carefully pipetted to create a layer on top of the lower phase. The contents of the sperm straw were emptied into a 1.5 mL tube by cutting both ends using scissors. The ~ 0.5 mL thawed semen sample was then layered on top of the upper phase using a pipette. The conical tube was centrifuged at room temperature for 20 min at 400 × g. Following centrifugation, the middle layer  was removed and placed into a second 15 mL conical tube, labeled with the bull identifier and interface. Remaining liquid was removed from the pellet tube without disturbing the pellet  and was discarded appropriately, leaving behind the high-quality pellet sperm. Both samples were washed with 2.0 mL PureSperm® Wash  media and were gently resuspended by pipette. Tubes were then centrifuged for 8 min at 250 × g. Enough supernatant was removed from both tubes without disturbing the pellets and discarded appropriately. Both pellets were resuspended with 100 μL of sterile water.The sperm was chemically fixed by adding 1 mL of – 20 °C methanol to the sperm solution  and the samples were immediately centrifuged for 2 min at 1000 × g. The methanol was pipetted off and 500 μL of sterile water was used to resuspend the sperm. 20 μL of high-quality or low-quality samples were pipetted onto gelatin coated 8 mm round coverslips and allowed to dry completely. Afterwards, the coverslips were stored at − 80 °C until needed. When needed for labeling, they were thawed at room temperature.Gelatin coated 8 mm round coverslips were generated by immersing in 0.1% v/v gelatin  for 10 min at room temperature. Next, the coverslips were placed onto a paper towel and allowed to dry at room temperature. Gelatin coated coverslips were either immediately used or stored at 4 °C.Coverslips containing sperm from each sample fraction/population were stained at least three independent times. Samples retrieved from − 80 °C storage were first permeabilized with 0.3% Triton X-100  in PBS (PBST) for 1 h and blocked with 1% BSA  in PBST for 30 min (PBSTB). Primary antibodies were diluted in PBSTB according to Table Slides were visualized using a Leica SP8 Confocal microscope in photon counting mode using a HC PL APO CS2 63 × 1.40 numerical aperture oil lens, 630× total magnification, format of 4096 × 4096 pixels (245 × 245 μm), 0.75 zoom factor, or with a format of 2048 × 2048 pixels (122.5 × 122.5 μm) 1.5 zoom factor, 2.0 frame accumulation, and three sequences:Sequence one produces two images, one of DNA and the other phase-like. To capture DNA labeling via Hoechst 33,342, we used 0.1% 405 nm (UV) laser. Absorption spectrum was set to cover 410–478 nm via HyD1detector, which was set to standard, and was assigned a blue color. To create a phase-like picture, the PMT Trans was set to ON with a gain of 300. Fluro turret was set to Scan-PH (Phase).Sequence two produces three images. To capture acetylated tubulin labeling via ALEXA 488, we activated a 488 nm laser set at 2.5% power. Absorption spectrum was set to cover 493–551 nm via HyD3 detector, which was set to counting, and was assigned a green color. To capture POC1B or FAM161A labeling via ALEXA 650, we activated a 633 nm laser set at 1% power. Absorption spectrum was set to cover 638–718 nm via HyD4 detector, which was set to counting, and was assigned a magenta color. This sequence produces a similar phase image to the first sequence. Sequence three produces two images. To capture tubulin labeling via ALEXA 555, we activated a 561 nm laser set at 0.75% power. Absorption spectrum was set to cover 566–623 nm via HyD3 detector, which was set to counting, and was assigned a red color. This sequence produces a similar phase image to the first sequence.We collected multiple Z sections (10–20) of 0.3 μm thickness from the top of the highest sperm to the bottom of the lowest sperm. Images were taken with the photon counting mode, a gain of 10, and a laser intensity (~ 1%) that prevented signal saturation of the centriole biomarkers so we could record both increases and decreases of signal.We generated a max projection of all the layers of each image using the LASX program. The labeling intensity of each marker in the acquired images was quantified in the PC, DC, and Ax of each sperm using a 1.0 × 0.75 μm rectangular region of interest (ROI) using the Draw Rectangle tool in the Las X program .Intensity quantification: We quantified the labeling intensity of each biomarker in the PC, DC, and Ax ROIs in each sperm  and location , an excel sheet compared the max intensity pixel sum at a specific location to the total max intensity pixel sum of the three locations for each sperm.Sperm population mean FRAC ratio : We calculated the mean and 95% confidence interval of each of these FRAC ratios per sperm population (> 100 sperm), Because we analyzed four biomarkers and looked at three sperm locations, we obtained 12 variables for each bull population. Because we analyzed 2 populations of sperm for each bull, we obtained 24 variables for each bull.Reference range: We determined the high-quality sperm population distribution range by calculating the mean FRAC ratio mean ± 2 standard deviations (SD) of the high-quality sperm of fertile bulls (having SCR above − 3). Sperm population mean ratio 95% confidence intervals that were partially to fully within the average mean FRAC ratio ± 2 SD were considered normal. 95% confidence interval mean FRAC ratios that were fully outside the boundaries of normal distributions were considered outliers and to have abnormal centriole labeling.FRAC test: A bull with normal centriole labeling in all 12 parameters in high-quality sperm populations is considered to have a negative FRAC test. A bull with one or more outliers in any of the 12 parameters in its high-quality sperm population is considered to have a positive FRAC test. Positive FRAC tests are more severe with increasing number of outliers and increasing distance of the mean FRAC ratio from the reference range as determined by number of standard deviations.Cohort FRAC tests: Cohorts were then compared to each other by comparing the ratio of outlier parameters out of total parameters in fertile and subfertile bulls (parameter-level FRAC test). Cohorts were also compared to each other by comparing the ratio of bulls with any outlier parameters out of the total number of bulls .We perform a FRAC test of individual bulls in five steps.To gain insight into the reproducibility of the quantification step, we tested reliability in two ways.61. Because we selected our raters from a large population of raters with similar characteristics (students with no prior experience), we used a “Two-Way Random-Effects” Model. This model allows us to generalize our reliability results to any raters who possess the same characteristics as the raters selected for the reliability study. As FRAC uses the mean ratio of multiple raters as the basis of measurement, we used the “Multiple Raters/Measurements” Type. Since we were concerned with different raters assigning the same score to the same subject, we used the “Absolute Agreement” Definition, culminating in ICC form  where 2 indicates a 2-way random effects model, and k indicates type: the mean of k raters. Then, we compared the raters to each other. Based on the 95% confidence interval of the ICC estimate, values less than 0.50 are indicative of poor reliability, values between 0.5 and 0.75 are indicative of moderate reliability, values between 0.75 and 0.90 are indicative of good reliability, and values greater than 0.90 are indicative of excellent reliability.First, we assessed rater performance by comparing the twelve mean FRAC ratios across four different pictures analyzed by the same three raters using intraclass correlation coefficient (ICC)61. These calculations treated the different samples as raters and the different biomarker locations as subjects. As there were variable numbers of labelings for the different bulls, the value of k  was not constant between the bulls.Second, sample to sample reliability of the FRAC analysis was also calculated by ICC mean FRAC ratios was determined after calculating the skewness and kurtosis by the functions SKEW and KURT in Excel. We also tested for normality  using https://www.gigacalculator.com/calculators/normality-test-calculator.php (used on date 5/27/2022)  of individual mean sample ratios were calculated using CONFIDENCE.T in Excel and were less than ± 0.04 from the mean in the high-quality sperm populations (Supplementary table 1) − ((p̂2))/SQRT((p̂)*(1 − (p̂))*(1/(n1) + 1/(n2)))),0,1,TRUE)))*2 in excel.T-Tests were performed using the T.TEST function in Excel. Z-tests for two population proportions were calculated using the formula . Pearson Correlation R and best fit were calculated by the function PEARSON and the automatic linear regression in Excel, respectively. The Pearson Correlation P-value was calculated using the function T.DIST.2T  in Excel. The Pearson Correlation R2 was calculated by the automatic linear regression in Excel. N was calculated using the functions COUNT and COUNTIF.The T statistic was calculated using the equation (R*SQRT(N − 2))/(SQRT(1 − R^2)), and the degrees of freedom was calculated by N − 2, in Excel.ICCs were calculated using the ICC function included in the Real Statistics Resource Pack for Excel.62.Odds ratios were calculated using the formula (number of true positive * number of true negative)/. The Haldane-Anscombe correction was used for any groups that were zeroBull sperm was purified as described above in the density gradient centrifugation, and the sperm suspension was added to a coverslip coated with 1 mg/mL poly-L-Lysine , and incubated 3–5 min to allow sperm adhesion. Samples were fixed in 1.5% formaldehyde at RT for 4 min and post-fixed in 100% methanol at − 20 °C for 4 min, followed by rehydration in PBS. Samples were blocked in immunofluorescence (IF) buffer (1% BSA and 0.05% Tween-20 in PBS) for 15 min at RT. Samples were incubated with anti-acetylated tubulin antibody  diluted 1:3000 in IF buffer for 24 h at 4 °C., followed by incubation in anti-mouse secondary antibody conjugated with STAR RED  diluted to 1:200 for 2 h at 37 °C. DNA was labeled with 0.5 µg/ml DAPI  in PBS for 2 min at RT. Samples were embedded in mounting medium and imaged.63. Coverslips containing immunolabeled sperm were postfixed in 4% formaldehyde in PBS at RT for 1 h and incubated at 40 °C for 16 h in a solution containing 30% acrylamide  and 4% formaldehyde in PBS. Following three 10 min washes with PBS, coverslips were cooled in an ice-water bath. Precooled gelling mixture , 0.04% bis-acrylamide , 0.5% ammonium persulfate , and 0.5% Tetramethylethylenediamine ) was pipetted to the coverslips and incubated on ice for 20 min and 1 h at RT. After polymerization of the gel, smaller samples were excised using a 4-mm biopsy puncher . And placed in an empty 50 ml conical tube and dry heated at > 90 °C for 10 min. Preheated SDS solution  was added to the punches, which were boiled for 1 h at > 90 °C. SDS solution with punches was cooled to RT. SDS was removed from punches by washing them extensively in PBS. To immunolabel acetylated tubulin, punches were blocked in IF buffer (1% BSA and 0.05% Tween-20 in PBS) for 1 h at RT and incubated with anti-acetylated tubulin antibody  diluted to 1:3000 in IF buffer for 48 h at 4 °C. Punches were washed in PBS for 1 h and incubated with anti-mouse secondary antibody conjugated with Abberior STAR RED  diluted to 1:50 in IF buffer for 24 h at 4 °C. Hoechst was added during immunolabeling to visualize DNA. Samples were expanded in deionized H2O and mounted in Rose chambers for imaging63.Samples were expanded as previously described2 inverted microscope (Nikon Inc.), using 100×, NA 1.45 Plan Apo objective. Avalanche photo detectors  were used to detect the signals. Browser-based control software (Abberior Instruments) was used to generate STED images. Images were acquired with a pinhole size of 32–65 µm and a pixel size of 10 nm. STAR RED was excited with 2–10% laser power and depleted with the STED laser at 97.88%. Signals were detected within a 7 ns gate.Imaging was performed with STEDYCON (Abberior Instruments) assembled on Eclipse TiSIM was performed on N-SIM, Nikon Inc., equipped with 405, 488, 561, and 640 nm excitation lasers, Apo TIRF 100 × NA 1.49 Plan Apo oil objective, and back-illuminated EMCCD camera . Images were reconstructed to generate a final image using Nikon NIS-Elements software.65. All 31 bulls passed a rigorous semen and sperm analysis before they were used for artificial insemination in the field6. Therefore, it was surprising that six of them (five Holsteins and one Jersey) had an SCR that is significantly lower than the average and in the subfertile range. For our study, we divided the 31 bulls to two categories: (1) the fertile category included 25 bulls with SCR deviations above − 3 and (2) the subfertile category included six bulls with SCR at or below − 3. This is a ratio of about four fertile bulls for each subfertile bull, which is preferred in small control case studies66. To determine bull sperm centriole quality, we used a quantitative immunofluorescence-based assay named FRAC, which was developed to compare labeling intensity ratios in the proximal centriole, distal centriole and the axoneme and, thereby, relative protein levels in human sperm21 . These bulls’ SCR scores ranged from a minimum of − 18.2 to a maximum of + 2.8  of 0.9900 with 95% confidence interval between 0.994 and 0.998. Therefore, we concluded that FRAC have “excellent” inter-rater reliability  distribution, which is needed to establish that 95% samples are expected to fall within two standard deviations of the meanIn contrast, the distributions of FRAC values of for fertile bulls’ individual sperm mostly show narrow distributions around one peak that rarely exhibit Gaussian distribution , and high for tubulin (0.19), acetylated tubulin (0.20), and FAM161A (0.20)  had mean ratio 95% confidence intervals outside of the high-quality sperm population’s reference range, making them outliers . When examining individual parameters, the low-quality sperm from fertile bulls had 22 parameters outside of the high-quality sperm population reference range, this rate difference (7/300 versus 22/300) is statistically significant (P = 0.004)  . This result suggests that subfertile bulls’ high-quality sperm population has inferior centrioles compared to fertile bulls’ high-quality sperm.We have analyzed five subfertile Holstein bulls and one subfertile Jersey bull Fig. C. All th08) Fig. E. These cutoff (aka threshold) of one FRAC outlier 95% confidence interval to indicate subfertility, the test is 100% sensitive, and 84% specific, which is a good diagnostic performance. A more stringent cutoff to indicate subfertility may be having two FRAC outlier 95% confidence intervals. Two outlier parameters were present in half of the subfertile bulls’ high-quality sperm populations . This rate is over ten times higher than high-quality sperm of fertile bulls  and is significantly different (P = 0.0025). Such a cutoff suggests a sensitivity of 50% and specificity of 96% for subfertility; these values must be considered carefully because of the small sample size.If we set a P = 0.62). We suspect that this is due to having a smaller group of subfertile individuals, and that, if the sample size were larger, we would see individuals with more than two outliers in their high-quality sperm population.None  of the subfertile bulls’ high-quality sperm populations had more than two outliers. However, one fertile bull did,  resulting in a larger proportion than in the subfertile population. This difference is not significant  , as expected from our hypothesis. Overall, the data suggests that abnormal centriole protein distribution is common in the unexplained subfertile bulls. Therefore, selecting a bull with a negative FRAC test, may improve predictions of successful breeding.P = 0.77) . Overall, the above data suggests that abnormal centriole protein distributions are common in subfertile bulls. Therefore, selecting a bull with high-quality centrioles may improve prediction of successful breeding.We analyzed the six subfertile bulls' low-quality sperm populations Fig. D. Indivi77) Fig. E. Half . Acetylated tubulin is a post translational modification of tubulin. Therefore, these markers allow us to examine which of these distinct functional aspects are more sensitive for identifying subfertility. To identify marker sensitivity, we counted the number of outlier parameters found in each of the four sperm populations Table . We founP = 0.0007) linear regression correlation between SCR and acetylated tubulin levels in the PCs of high-quality sperm . Based on this correlation, a cutoff of 0.31 in PC acetylated tubulin mean ratio identified five out of the six subfertile bulls and 0 of the 25 fertile bulls  and no correlation was found in Ax Acetylated tubulin (P = 0.90).Because we found that acetylated tubulin most effectively identifies lower-quality centrioles Table , we wonderm Fig. . This SCd R2 = 0.316. BaseInterestingly, the odds ratio (the strength of association between test-proposed fertility status and accepted fertility status) is much higher in the test based on the 0.31 cutoff generated from the linear regression analysis than tests based on bulls’ positive FRAC test results  super-resolution microscopy  Fig. A and fou19. Consistent with that, the acetylated tubulin immunolabeling central lines in the DC appeared continuous with Ax labeling in 96% of the analyzed sperm . The test also needs validation by clinicians and researchers to determine whether the new test results are meaningful in practice. A key point in the validation process is defining a threshold (cutoff) that would allow clinical use of the test with confidence. We developed FRAC testing and demonstrated that FRAC can detect differences between men with different infertility typesHere, we performed a small retrospective cohort-control study and found that FRAC can suggest the presence of centriole-based subfertility in bulls that previously would have been used for artificial insemination, only to find that they had lower rates of confirmed pregnancy. Future studies should include a more extensive retrospective cohort-control study that will estimate relative risk and a threshold, which would then be used for further validation in a prospective study. As artificial insemination centers might be collecting from thousands of bulls every year, promoting extensive use of FRAC requires developing automated labeling, imaging, and image quantification, which is currently underway. It would also be important to determine the stability of the centriolar phenotype, if it changes with bull age or external factors such as heat stress.An unexpected outcome of this study is that six out of the six subfertile bulls had positive FRAC tests, and five out of six subfertile bulls had high PC acylated tubulin—this is a very high proportion of anomaly for a single sperm structure, the centriole, out of many possible structures in sperm cells. There are two likely, non-mutually exclusive, explanations for this observation: (1) The standard semen analysis prescreen of bulls is effective at identifying most cases of subfertility before the semen is put on the market, but this analysis is unable to detect centriole abnormalities, thus enriching for centriole-based subfertility. Semen analysis in tandem with FRAC may reduce bulls coming onto the AI market only to be found to have unexplained subfertility. (2) FRAC testing is sensitive to sperm abnormalities beyond the centrioles and can report on causes that are not primarily in the centriole but do affect centriolar labeling. Centriole remodeling occurs during spermatogenesis; it is possible that if there are problems in spermatogenesis or sperm homeostasis, the centrioles might be reporting these problems.72. This low overall rate may be influenced by other factors such as DNA fragmentation73, oxidative stress74, and capacitation status75). If a bull is much better than average for any of these factors, the bull's centriole defect may be masked. Therefore, it would be essential to perform a multifactor analysis of the sperm in the future to test this hypothetical explanation. Finally, another explanation for these results is that FRAC assessment may vary within a bull across collections, and in this study, we have examined an individual ejaculate.One explanation for the presence of FRAC outliers in the fertile bull population is that their high SCR is due to having other fertility properties that are extraordinary and mask centriole impact. On average, a bull has about a 32% rate of impregnating a cow using AI  and histone deacetylase 6 (HDAC6)80, both of which are found in the sperm82. It is important to note that sperm are transcriptionally and translationally silent, and these modifications can allow sperm to respond to stimuli83 Reviewed in84.It was recently shown that protein acetylation protects sperm from spontaneous acrosome reactionsOverall, FRAC has the potential to be a method of identifying subfertile bulls before use in artificial insemination, saving time and money for both artificial insemination companies and cattle breeders. Furthermore, these results also support our previous findings using FRAC to evaluate human sperm centriole quality. An additional method of male fertility evaluation yields more information when couples are deciding whether or what assisted reproductive technologies to utilize and may open avenues for treatment.FRAC could help identify subfertile bulls, improving artificial insemination success rates, and increasing cattle industry reproductive efficiency.Supplementary Information.Supplementary Table 1.Supplementary Table 2.Supplementary Table 4.Supplementary Table 5.


Rousettus aegyptiacus) and their argasid tick ectoparasites, Ornithodoros (Reticulinasus) faini. Although tick salivary gland components have been shown to potentiate virus infection in vertebrate non-reservoirs , there is a lack of information on the effect of tick salivary gland components on viral infection and shedding in vertebrate reservoirs.The human-pathogenic Kasokero virus (KASV) circulates in an enzootic transmission cycle between Egyptian rousette bats  faini tick salivary gland extract (SGE).To determine the impact of tick salivary gland components on KASV infection and shedding in ERBs, KASV loads were quantified in blood, oral swab, rectal swab, and urine specimens collected daily through 18 days post inoculation from groups of ERBs intradermally inoculated with KASV or KASV + Bats inoculated with KASV + tick SGE had significantly lower peak and cumulative KASV viremias and rectal shedding loads compared to bats inoculated with KASV only.We report for the first time to our knowledge that tick salivary gland components dampen arbovirus infection and shedding in a vertebrate reservoir. This study advances our understanding of biological factors underlying arbovirus maintenance in nature.The online version contains supplementary material available at 10.1186/s13071-023-05853-7. Nairoviridae, genus Orthonairovirus) was first described by scientists at the Uganda Virus Research Institute (UVRI) following the isolation of infectious virus (2.7% [2/74] bats) and detection of virus-specific antibodies (67.6% [50/74] bats) in serum samples collected from Egyptian rousette bats  captured at Kasokero Cave, Uganda, in 1977 ). Anti-KASV IgG responses were measured using a previously described indirect enzyme linked immunosorbent assay ) protect the structural stability and functional activity of proteins in aqueous solutions subjected to high-dose gamma irradiation (5 megarads) [Previous studies investigating the effect of tick salivary gland components on virus infection in small animal models of human disease used SGE prepared from colonized ticks , 6. Due egarads) , 8.In conclusion, we report for the first time to our knowledge that tick salivary gland components dampen arbovirus infection and shedding in a vertebrate reservoir. This study advances the understanding of biological factors that influence arbovirus maintenance in nature.Additional file 1: Fig. S1. Anti-Kasokero virus (KASV) IgG responses. Whole blood for serology was collected at 0, 7, and 14 DPI and at the end of the study (18 or 20 DPI) from KASV- and KASV + tick salivary gland extract (SGE)-inoculated bats. Bat identification numbers correspond to numeric codes generated by scanning implanted passive integrated transponder tags.


Bangiomorpha and Proterocladus interpreted as crown-group eukaryotes are quite rare. Horodyskia is one of these few macrofossils, and it extends from the early Mesoproterozoic Era to the terminal Ediacaran Period. The biological interpretation of this enigmatic fossil, however, has been a matter of controversy since its discovery in 1982, largely because there was no evidence for the preservation of organic walls. Here we report new carbonaceous compressions of Horodyskia from the Tonian successions (~950–720 Ma) in North China. The macrofossils herein with bona fide organic walls reinforce the biogenicity of Horodyskia. Aided by the new material, we reconstruct Horodyskia as a colonial organism composed of a chain of organic-walled vesicles that likely represent multinucleated (coenocytic) cells of early eukaryotes. Two species of Horodyskia are differentiated on the basis of vesicle sizes, and their co-existence in the Tonian assemblage provides a link between the Mesoproterozoic (H. moniliformis) and the Ediacaran (H. minor) species. Our study thus provides evidence that eukaryotes have acquired macroscopic size through the combination of coenocytism and colonial multicellularity at least ~1.48 Ga, and highlights an exceptionally long range and morphological stasis of this Proterozoic macrofossils.Macrofossils with unambiguous biogenic origin and predating the one-billion-year-old multicellular fossils  Horodyskia from the Tonian successions reinforce the biogenicity of these macrofossils and indicate that they are among the oldest multicellular and coenocytic macro-organisms.Carbonaceous compressions of  However, fossils at the millimetric-centimetric scale are relatively scarce in successions older than the Neoproterozoic Era and their biogenicity and phylogenetic positions often remain contentious, hampering our ability to reconstruct the tempos and modes of early eukaryote evolution4. Horodyskia represents one of few examples with a fossil record extending from the early Mesoproterozoic Era (~1.48 Ga)6 to the terminal Ediacaran Period (~550 Ma)12. This genus is characterized by a string of beads with uniform size and spacing, and was first discovered as “enigmatic bedding-plane markings” from the ~1.48 Ga Appekunny Formation, Belt Supergroup in Montana, United States6. Subsequently reported occurrences extend the stratigraphic range of Horodyskia and Horodyskia-like fossils  from the Mesoproterozoic Era to the Ediacaran Period. These occurrences came from the 1.42–1.26 Ga Balfour Subgroup of the Rocky Cape Group in Tasmania14, the 1.17–1.07 Ga Backdoor and Stag Arrow formations of the Bangemall Supergroup in Western Australia17, the 550–539 Ma Zhengmuguan, Liuchapo, and Piyuancun formations in China12, and possibly the late Ediacaran Kauriyala Formation, Krol Group in India18. However, there had been no reports of the occurrence of Horodyskia from the Tonian Period (~1000–720 Ma).Proterozoic macroscopic fossils are crucial for our understanding of the early evolution of eukaryotes, particularly in terms of how and when early eukaryotes developed complex multicellular grades of organization and acquired body sizes that are visible to the naked eyesHorodyskia has a wide global distribution and long stratigraphic range, but its biological affinity remains controversial. Various opinions concerning about the nature of Horodyskia have been proposed, including dubiofossil5 or pseudofossil21, “enigmatic structure”5, brown alga15, Armstrongia-like sponge22, colonial eukaryote23, archaeal–bacterial consortium2, agglutinated foraminifer12, Geosiphon-like fungus24, testate amoeba9, and green alga12. One of the prominent features of Horodyskia in previously reported specimens is the cast-and-mold preservation in fine-grained siliciclastic rocks or in cherts (silicification), with no evidence for the preservation of organic walls25. This feature further impedes the biological and phylogenetic interpretation of Horodyskia.Horodyskia specimens from the Tonian Shiwangzhuang Formation (~850–720 Ma) in western Shandong and Jiuliqiao Formation (~950–720 Ma) in Huainan region, North China  with relatively constant size and spacing  of 0.76–0.84 .Other species—Horodyskia minor Dong et al.8.Emended diagnosis—Strings of sub-millimeter- to millimeter-sized bead-like  structures. Beads in the same string are uniform in diameter and are uniserially arranged, with a uniform gap in between. A halo may be present to envelop the beads. A stolon (or filament) may be present to connect adjacent beads.Remarks—Fossils with a string of beads were not treated taxonomically in early studies due to uncertainty about their biogenicity15. The genus Horodyskia and its type species H. moniliformis were established by Yochelson and Fedonkin6 to account for these fossils, and the type material from the Appekunny Formation in Montana were re-described in greater detail by Fedonkin and Yochelson23. The original diagnosis of Horodyskia was “presumed colonial organisms of small, vertically oriented, short wide cones, hemispherical on the upper surface, growing from a horizontal tube”6. Subsequently, the diagnosis was simplified as “Cones spaced along a horizontal tube”23. However, the reconstruction of the beads as “cones” has not been substantiated24. The beads were originally spherical, based on observations of Horodyskia specimens from the Shiwangzhuang and Jiuliqiao formations and other stratigraphic units25. The emended diagnosis recognizes the spherical morphology, as well as the sub-millimetric to millimetric size, of the beads.Horodyskia is the presence of a halo surrounding the beads. This feature has been described from Horodyskia specimens preserved in different taphonomic modes, including those preserved in Mesoproterozoic siliciclastic rocks from Montana23, Western Australia25, and Tasmania13, carbonaceous compression specimens from Tonian argillaceous limestone in North China (this paper), and silicified specimens from Ediacaran cherts in South China11. The halos have been interpreted as biological structures, such as agglutinated tests8, gelatinous sheaths13, or blade-like ribbons of seaweeds25. They have also been interpreted as abiotic structures resulting from current activity15 and organic matter degradation23.A defining feature of the genus 23. These features indicate that the halos were originally less rigid and more prone to deformation than the beads, and the eccentric offset may be caused by post-mortem shearing by water currents and sediment loading. The lack of a distinct boundary between the halos and the surrounding sediment matrix, as observed in light microscopy of polished slabs and thin sections cut perpendicular to the bedding surface  or silica12. These subtle geochemical differences are probably related to post-mortem mineral precipitation, preferentially within the halos, perhaps induced by the degradation of amorphous gelatinous material in the halos.Considering the consistent occurrence of the halos, we favor a biological interpretation and argue that the halos probably represent fossilized gelatinous matrix surrounding the organic-walled beads and were reinforced by post-mortem mineral precipitation. Contrary to the beads, the halos are not stable structures—they can be present in some specimens but absent in others from the same locality. The positions of the beads are not always located in the center of the halos. Eccentric offset of the bead centroids relative to the surrounding halos typically occurs toward the same direction along the string  have not been observed in the Shiwangzhuang and Jiuliqiao carbonaceous compression specimens, and rarely reported from specimens preserved in siliciclastic rocks23. Thus, either the stolons are not preserved in specimens from the Shiwangzhuang and Jiuliqiao formations, or the beads in these specimens were held together by a gelatinous halo rather than a stolon. If the latter case is correct, then the Tonian specimens may be different from the Liuchapo specimens of “Horodyskia minor” (or “Parahorodyskia minor”)12.Stolons are also present in some Horodyskia, including ridges surrounding the beads, linear chevron structures, top depressions or dimple structures, and radiate tubes25. These have either been considered as uncommon features or attributed to taphonomic variations23, and therefore are not included in the emended diagnosis. For example, ridges surrounding the beads are interpreted as an infilled scour mark23, linear chevron structures are related to current scouring structures15, top pits or dimple structures are identified as compression features25, and radiate tubes are considered as an unstable structure since they were not confirmed by subsequent study19.Numerous other features have been described in association with Parahorodyskia Liu and Dong in Liu et al.12 and Longbizuiella Yi et al.11 as junior synonyms of Horodyskia. Parahorodyskia and Longbizuiella were erected because of their smaller size and different preservational style from the Mesoproterozoic Horodyskia. But the diagnosis of Horodyskia6 does not exclude sub-millimetric beads for Horodyskia, and Horodyskia specimens with sub-millimetric beads can be preserved together with Horodyskia specimens with millimetric beads in the Shiwangzhuang Formation  were examined.Locality and horizon—Argillaceous limestone ~74 m below the top of the Tonian Shiwangzhuang Formation, Baishicun section, Anqiu region, western Shandong Province, North China.Emended diagnosis—A species of Horodyskia characterized by relatively large beads, typically 0.8–10 mm in average diameter.Description—Unbranched, uniseriate chains consisting of 3–29 bead-like structures , resembling a string of beads  of 0.82–3.79 mm . Generally, halos associated with adjacent beads are in contact or overlap with each other, but there are also a few exceptions where they are separated from each other by a short distance . The growth pattern established for H. moniliformis by Fedonkin and Yochelson23, i.e., some larger beads keep continuously growing while other intermediate smaller beads cease growing or have regressive development, has not been verified in subsequent studies24. Therefore, the absence of H. moniliformis-style growth pattern in Western Australian specimens, which was regarded as an important feature of H. williamsii, is not a solid taxonomic criterion either. The Shiwangzhuang specimens are similar to the Western Australian and Tasmania specimens in relatively small beads with a narrow range of bead size, when compared with H. moniliformis from the Appekunny Formation . Therefore, we propose that H. williamsii is a junior synonym for H. moniliformis, emend the diagnosis of H. moniliformis so that it can be objectively differentiated from H. minor by its larger bead size (0.8–10 mm in bead diameter), and assign the Shiwangzhuang specimens with bead diameter of 0.8–3.53 mm to H. moniliformis.ion Fig. . Moreoveion Fig. . HoweverHorodyskia minorDong et al., 2008, emend8Figures 2a, b, f; 5Horodyskia moniliformis? Yochelson and Fedonkin; Shen et al.10, p. 1401–1402, Fig. 4.9–4.12.2007 Horodyskia minor; Dong et al.8, p. 368–371, Fig. 3a, b, d–l.2008 Horodyskia cf. minor; Dong et al.7, Fig. 3.2012 Horodyskia sp. or Horodyskia specimens; Luo and Miao9, Figs. 3D, F; 5A, B; 6A .2020 Longbizuiella hunanensis Yi et al.11, p. 16, 17, Fig. 3.2022 Parahorodyskia disjuncta Liu and Dong in Liu et al.12, p. 5, 6, Fig. 5A–I .2022 Parahorodyskia or taxonomically unidentified specimens, Liu et al.12, Figs. 11A–D, F; 12A; 13A; 14; 15; 17.2022 Parahorodyskia minor  Liu et al.12, p. 6–7, Fig. 5J–L.non 2022 Referred material—More than 100 specimens (or strings) were examined.Locality and horizon—Argillaceous limestone ~74 m below the top of the Tonian Shiwangzhuang Formation, Baishicun section, Anqiu region, western Shandong Province, North China; argillaceous limestone ~14 m below the top of the Tonian Jiuliqiao Formation, Baiguashan section, Huainan region, northern Anhui Province, North China.Emended diagnosis—A species of Horodyskia characterized by relatively small beads, typically 0.1–0.8 mm in average diameter.Description.—Specimens from the Shiwangzhuang Formation are preserved on the top bedding surface as carbonaceous compressions. Unbranched string of 3–17 uniseriately arranged bead-like structures   is 0.07–0.89 mm . Paired beads  Figs. ; 5a–l. Sn = 24 beads from 6 strings), their spacing between 0.13 mm and 0.35 mm , and the inter-bead distance between 0.56 mm and 0.98 mm . Paired beads and halos are also found in some specimens . The bead sizes of the Jiuliqiao specimens fall well within the size range of the Shiwangzhuang specimens , and light-colored halos are 0.85–1.11 mm in outer diameter .Specimens from the Jiuliqiao Formation are also preserved on the bedding surface as carbonaceous compressions. Each string consists of 3–11 bead-like structures, and many strings can occur in aggregation Fig. . The beaens Fig. . The halens Fig. . Short sens Fig. , but theOccurrence—The Tonian Shiwangzhuang Formation, Tumen Group at the Baishicun section, Anqiu region, western Shandong Province, North China; the Tonian Jiuliqiao Formation, Feishui Group at the Baiguashan section, Huainan region, northern Anhui Province, North China; the late Ediacaran Zhengmuguan Formation, Ningxia Hui Autonomous Region, North China10; the late Ediacaran Liuchapo Formation, Guizhou and Hunan provinces, South China12; the late Ediacaran Piyuancun Formation, Anhui Province, South China7.Remarks—The genus placement of Horodyskia minor has been debated. Horodyskia minor was erected by Dong et al.8 on the basis of silicified material from the Liuchapo Formation in Guizhou Province of South China, and it is characterized by beads smaller than 0.7 mm in diameter. Silicified specimens of similar size and shape were subsequently reported from the Liuchapo Formation in Hunan Province (as Horodyskia sp.9) and the equivalent Piyuancun Formation in Anhui Province of South China (as Horodyskia cf. minor7). However, it is debated whether H. minor should be classified as a species of Horodyskia24. For example, Calver et al.13 excluded Horodyskia minor from the genus Horodyskia because the type species Horodyskia moniliformis has larger bead size (2.1–9.2 mm in diameter23) and lacks quartz in its halos.Horodyskia and assigned them to different genera. Yi et al.11 reported similar “string of beads” specimens from the Liuchapo Formation in Hunan Province but classified them as Longbizuiella hunanensis, based on their smaller size and different preservational style from the Mesoproterozoic Horodyskia. Yi et al.11 also re-assigned Horodyskia minor, Horodyskia cf. minor, and Horodyskia sp. fossils from South China9 to Longbizuiella hunanensis. However, Yi et al.11 synonymized H. minor, including its holotype, with L. hunanensis, effectively making L. hunanensis a junior synonym of H. minor. More recently, Liu et al.12 erected another new genus—Parahorodyskia, with the type species P. disjuncta characterized by the absence of connecting filaments, and the new combination P. minor diagnosed with the presence of connecting filaments. Liu et al.12 placed Liuchapo specimens without connecting filaments, including some specimens of Longbizuiella hunanensis in Yi et al.11, in P. disjuncta, but enigmatically, these authors placed the holotype of Horodyskia minor reported in Dong et al.8, which also lacks connecting filaments, in P. minor.Two recent studies have also removed the Liuchapo silicified specimens from the genus 6 nor the subsequent description of Horodyskia23 excludes sub-millimetric beads for Horodyskia, and different types of preservation have been found in the same stratigraphic unit . The Shiwangzhuang material also confirms the co-existence of “string of beads” with two different bead size classes on the same bedding surface of argillaceous limestone  are considered as H. minor. However, the specimens with larger beads in Luo and Miao9 include beads that contain smaller Nenoxites-like organisms . Whether these latter specimens can be classified as H. minor, H. moniliformis, or something else needs further examination.Based on compilation of published specimens of kia Fig. , we propParahorodyskia minor, which was diagnosed with the presence of connecting filaments but was typified by a specimen lacking connecting filaments . Considering that the connecting filaments between beads are likely genuine biological structures, we suggest that Liuchapo “string of beads” specimens with connecting filaments, including specimens in Fig. 3c in Dong et al.8 and specimens in Fig. 5J–L in Liu et al.12, may represent a new species of Horodyskia.21. Horodyskia fossils from the Shiwangzhuang and Jiuliqiao formations of North China provide evidence to illuminate their biogenicity. Their carbonaceous nature ; sensu ref. 30] . Horodyskia beads from other units also exhibit a bi-modal size distribution and a narrow A/SD ratio . Two groups of fossils with distinct bead sizes are observed on the same bedding plane in the Shiwangzhuang Formation  and long tubular cells of Ca. Thiomargarita magnifica  is two to three orders of magnitude less than the volume (≤2.3 × 1010 μm3) of the beads of Horodyskia. Importantly, no modern sulfur-oxidizing bacteria are known to have recalcitrant cell walls. Therefore, considering its extremely large cell size and inferred recalcitrant cell wall, Horodyskia is unlikely to be a prokaryotic organism, although we cannot entirely rule out the possibility that some extinct lineages of prokaryotes could have developed such a large cell size and cell-wall recalcitrance39.Morphological evidence indicates that Horodyskia indicates that it is not only a eukaryote, but also likely a multinucleated or coenocytic eukaryote. The sub-millimeter- to millimeter-sized cells of Horodyskia seem to require multiple nuclei to regulate the giant mass of cytoplasm, because a single nucleus, via the diffusion of messenger RNAs, can only control a limited volume of cytoplasm41. The cellular nature and the unconnected but occasionally dividing feature of Horodyskia beads, indicate that these fossils are unlikely to be complex multicellular organisms such as articulated brown alga15, branching sponge22, tissue-grade colonial metazoan23, or endocyanotic fungus24, but are more likely to be protists whose clonal cells forming simple and not fully integrated colonies . Multicellularity  occurs in a number of eukaryotic groups, including fungi, animals, choanoflagellates, slime molds , green algae, land plants, red algae, ciliates, oomycetes, diatoms, chrysophytes, xanthophytes, and brown algae44. However, protists with a construction of simple clonal coloniality, a sub-millimetric to millimetric cell size (or inferred coenocyte), and a recalcitrant cell wall, features that define Horodyskia, are limited to a smaller number of eukaryotic clades45. Here we will evaluate the following three most likely potential analogs: arcellinid testate amoebae46, foraminifers45, and some algal groups49, which are proposed in previous studies and show some, if not all, of the features of Horodyskia.The large cell size of Horodyskia9, although they are exclusively unicellular, not colonial44. Indeed, some arcellinid testate amoebae possess an organic-walled test45 that has the potential to be preserved as microfossil50. However, the lack of an aperture in the beads of Horodyskia argues against an affinity with arcellinid testate amoebae, which typically possess a characteristic aperture. The sizes of arcellinid testate amoebae, commonly between 50 μm and 200 μm in shell length51, are an order of magnitude smaller than H. moniliformis beads and also smaller than most of the H. minor beads. So far, vase-shape microfossils (VSMs) from the late Tonian Period are widely accepted as the earliest fossilized arcellinid amoebae51. Phylogeny of extant arcellinid amoebae and VSMs based on ancestral-state reconstructions put the divergence of major arcellinid lineages at ~759–734 Ma and molecular clock estimate using VSMs as a calibration places the divergence time at ~1000–730 Ma53, much younger than the oldest known Horodyskia fossils from the ~1.48 Ga Appekunny Formation23.Recently, arcellinid testate amoebae have been suggested as potential analogs of Horodyskia8, although rhizarians are also exclusively unicellular44. The halos, beads, and connecting filaments of Horodyskia minor (or “Parahorodyskia minor”) were interpreted as, respectively, agglutinated tests, cytoplasm-filled chambers, and cytoplastic passages of foraminifer cells12. Importantly, many modern foraminifers have an inner organic enveloping structure  that can be preserved as organic-walled microfossil54, and some foraminifers  even have an entirely organic test55, although with a limited fossil record56. The Xenophyophorea, a group of deep-sea foraminifers also belonging to the monothalamids, can reach a millimetric to centimetric size and are multinucleated and they are among the largest unicellular rhizarians57. Some xenophyophores  also possess a multi-chambered test resembling closely packed “string of beads”58. Thus, it is possible that the carbonaceous wall of Horodyskia beads may represent organic linings or organic tests of single-chambered monothalamid foraminifers, and hence the string of beads would represent either a multi-chambered foraminifer12 or, less likely, a colonial rhizarian. Still, the lack of an aperture in Horodyskia beads presents an obstacle to the foraminifer interpretation. In addition, the earliest unambiguous foraminifer fossils are of early Cambrian in age60, and a molecular clock study suggests that foraminifers diverged at ~920–650 Ma61, much younger than the oldest known Horodyskia fossils from the ~1.48 Ga Appekunny Formation23.Some rhizarians, particularly foraminifers, have also been proposed as modern analogs of Horodyskia may represent a giant-celled and colonial alga. Giant-sized cells, which are often coenocytic, occur in five extant algal clades, including Xanthophyceae, Rhodophyceae, Chlorophyceae, Charophyceae, and Ulvophyceae48. In the Xanthophyceae, Vaucheriaceae  is the only family that develops giant cells49. Vaucheria has a branching and tubular thallus with a holdfast, and Botrydium has large vesicles and branching rhizoidal extensions49. In the Rhodophyceae, Griffithsia is the only genus that develops giant cells, and it possesses a heavily branched thallus with a holdfast49. The Chlorophyceae, Charophyceae, and Ulvophyceae also contain giant-celled members62, and paleontological and molecular clock data do suggest a Mesoproterozoic origin for the “core Chlorophyta”64. Worth mentioning is the extant ulvophycean Valonia ventricosa (=Ventricaria ventricosa), which is characterized by spherical giant cells that are millimetric to centimetric in size and bear reduced rhizoids65. Individually, these giant algal cells resemble Horodyskia beads in size and shape, but they do not form uniseriate chains embedded in a gelatinous matrix. This difference, along with the lack of rhizoids in Horodyskia beads, makes the giant-celled algae yet an imperfect modern analog for Horodyskia. Nonetheless, it is also reasonable that we would not expect an evolutionary stasis experienced by eukaryotes for ca. 1.5 billion years and a parsimonious interpretation is that Horodyskia represents a total-group multicellular eukaryote that was composed of a string of giant-sized cells (probably coenocytic) and achieved a macroscopic size in the Proterozoic Eon.An alternative and perhaps better interpretation is that 66, and this has important evolutionary, ecological and geobiological implications. The Shiwangzhuang and Jiuliqiao materials indicate that Horodyskia acquired a large body size visible to the naked eyes probably through the combination of coenocytism and simple clonal coloniality. A coenocytic body plan has been proposed as the direct progenitor of some multicellular algal, animal, and fungal groups, through subsequent process similar to segregative cell division, whereas a colonial body plan is traditionally regarded as progenitor of multicellular organisms69. It is interesting to note that Horodyskia may have stood at an evolutionary crossroad of the “coenocytic-to-multicellular” and “unicellular-to-colonial-to-multicellular” transformation series69, and seems to represent a primitive condition for siphonocladous (multicellular multinucleate)68. Macroscopic body sizes can bring noticeable ecological advantages to eukaryotes, including increased speed and efficiency to occupy new adaptive niches or migration to more favorable environment, better protection against phagocytic predation, and increased possibility to capture larger preys72; larger eukaryotes can also act as a faster biological pump by accelerating the sinking flux and then enhancing the efficiency of organic carbon burial, and therefore facilitate marine ventilation74. Horodyskia is among the few examples of macrofossils reported from the early Mesoproterozoic23. Considering that the earliest Horodyskia fossils are from the ca. 1.48 Ga Appekunny Formation in Montana23, our study implies that macroscopic giant-celled (probably coenocytic) protists existed in the early Mesoproterozoic. Other examples of early Mesoproterozoic or earlier macrofossils include the helical fossils Grypania and Katnia76, the tomaculate fossil Tawuia75, and unnamed carbonaceous fossils from the 1.56 Ga Gaoyuzhuang Formation of North China77, all of which have been known as carbonaceous compressions. Together, these fossils provide paleontological evidence for the evolution of macroscopic size in the early-middle Proterozoic and it is worth evaluating the possibility that they may have also been coenocytic organisms73.Achieving a macroscopic body size occurred multiple times through multicellularity or coenocytism in different eukaryotic cladesValeria, Tappania, Shuiyousphaeridium, and Dictyosphaera79. Late Paleoproterozoic to early Mesoproterozoic macrofossils mentioned above, including Grypania, Katnia, Tawuia, and Chuaria, may also be eukaryotes80 . The interpretation of Horodyskia as a colonial giant-celled protist (probably coenocytic), considering its oldest occurrence dating back to ~1.48 Ga23, adds to a short but growing list of early eukaryote fossils existed in this critical time interval, and suggests that a macroscopic coenocytic body plan has probably been present since the early Mesoproterozoic.The late Paleoproterozoic to early Mesoproterozoic era witnessed the early diversification of eukaryotes, as represented by organic-walled microfossils with complex ornamentations, e.g., Sinosabellidites, Protoarenicola, and Pararenicola, the multicellular trichomes Anqiutrichoides and Eosolena, and the tomaculate fossil Tawuia84. Among them, Eosolena magna is somewhat similar to Horodyskia in its sub-millimetric to millimetric cells that are discoidal to isodiametric in shape. Together with the “string of beads” fossil Horodyskia herein and the siphonocladous Proterocladus from the early Tonian Nanfen Formation in North China63, these North China materials suggest that giant-celled protists may have been diverse in the Tonian Period when they became increasingly important in the paleoecology and geobiology during this time interval83.It is important to point out that the Shiwangzhuang and Jiuliqiao formations also preserve other giant-celled fossils that have been interpreted as possible macroscopic coenocytic algae, including the annulated tubular fossils Horodyskia is reconstructed as a benthic chain-like organism consisting of several to dozens of cells with a recalcitrant organic wall, and the cells are likely embedded in amorphous gelatinous matrix. Our study reinforces the biogenicity of Horodyskia through the detailed characterization of carbonaceous compression specimens from the Tonian successions in North China. The Tonian fossils herein also provide a valuable link, both chronologically and morphologically, between early Mesoproterozoic and late Ediacaran horodyskids insofar as two species with distinct bead sizes co-occur on the same bedding plane in the Shiwangzhuang Formation. The most plausible phylogenetic interpretation for Horodyskia is that it represent a multicellular and giant-celled protist, which has acquired a large body size and shares some similarities with living coenocytic algae and monothalamid foraminifers, although these two groups are phylogenetically distant and the latter are typically unicellular and therefore less likely. Our study reports evidence that Horodyskia, a genus of macroscopic fossils ranging from the early Mesoproterozoic Era to the terminal Ediacaran Period, may have attained its macroscopic size through the combination of coenocytism and simple clonal coloniality. Together with other possible early-middle Proterozoic coenocytic fossils73, Horodyskia provides an important temporal constraint on the origin of coenocytic eukaryotes.In conclusion, on the basis of the Shiwangzhuang and Jiuliqiao materials, Specimens were collected from an argillaceous limestone horizon ca. 74 m below the top of the Shiwangzhuang Formation (~850–720 Ma) at the Baishicun section , Anqiu region, western Shandong Province, North China Fig.  and an a25 and the spacing (or gap) between adjacent beads8 were measured to overcome the uncertainty of beads with fuzzy and irregular edges25.Specimens were examined and photographed using a Nikon D810 digital camera, an Olympus szx16 stereomicroscope attached with a DP74 digital camera, and a Zeiss Axioscope A1 microscope attached with an Axiocam 506 digital camera. Diameters of the bead and halo were averaged between the maximum and the minimum dimensions. Both the distance between the centroids of adjacent beads (inter-bead distance)Well-preserved specimens were selected for observation using both backscattered electron (BSE) and secondary electron (SE) detectors and elemental mapping using energy dispersive X-ray spectroscopy (EDS) on a TESCAN MAIA 3 GMU field emission scanning electron microscope (SEM) in Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (NIGPAS). The operating voltage in BSE-SEM was 20 kV in high vacuum conditions.–1 were acquired using the software LabSpec 6.0 with exposure time of 40 or 60 s and averaged after three times of acquisition85. At least thirteen spots were tested for each specimen. The spectral range of 1000–1750 cm–1 was used for baseline correction using the PeakFit 4.2 software and then normalized using the formula: normalized intensity = (X–Min)/(Max–Min), where the Min and Max represent, respectively, the minimum and maximum intensities of each spectrum85. The baseline-corrected and normalized Raman data were subsequently subjected to principle component analysis (PCA) in JMP13. The chemospace of carbonaceous material were defined by the first and second principal components from the PCA analysis85.Selected specimens were analyzed on a Horiba HR Evolution Jobin Yvon Raman microprobe in Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (NIGPAS). The Raman microprobe is equipped with a 532 nm argon laser source and a 600 grooves/mm grating. The laser beam was <2 μm in diameter and a 50× objective lens was used to collect backscattered radiation. Spectra in the range of 800–2000 cmFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Eriocheir sinensis), one of the most important aquatic commercial species, is still lacking. Here, we characterized the RNA viromes of asymptomatic, milky disease (MD)-affected, and hepatopancreatic necrosis syndrome (HPNS)-affected Chinese mitten crabs collected from 3 regions in China. In total, we identified 31 RNA viruses belonging to 11 orders, 22 of which were first reported here. By comparing viral composition between samples, we observed high variation in viral communities across regions, with most of the viral species being region-specific. We proposed to establish several novel viral families or genera based on the phylogenetic relationships and genome structures of viruses discovered in this study, expanding our knowledge of viral diversity in brachyuran crustaceans.Recent advances in viromics have led to the discovery of a great diversity of RNA viruses and the identification of a large number of viral pathogens. A systematic exploration of viruses in Chinese mitten crab (IMPORTANCE High-throughput sequencing and meta-transcriptomic analysis provide us with an efficient tool to discover unknown viruses and explore the composition of viral communities in specific species. In this study, we investigated viromes in asymptomatic and diseased Chinese mitten crabs collected from three distant locations. We observed high regional variation in the composition of viral species, highlighting the importance of multi-location sampling. In addition, we classified several novel and ICTV-unclassified viruses based on their genome structures and phylogenetic relationships, providing a new perspective on current viral taxa. Viruses are the most abundant and diverse biological entities on earth. The virome, the assemblage of viruses associated with a particular ecological niche or organism, is vast and heterogeneous . However2–9–Scylla serrata)  . The anaserrata) , 19–21. rata) 19–, 20, 22,Eriocheir sinensis H. Milne Edwards 1853, is one of the most important aquatic commercial species. The freshwater aquaculture production of Chinese mitten crab makes up 6.9% of global crustacean aquaculture production  genes, we used the original samples for reverse transcription-PCR (RT-PCR) and nested RT-PCR with primer sets designed based on the RdRp sequences of viral genomes . The products of RT-PCR were confirmed by Sanger sequencing. To ensure that our assembled viral genomes were not chimeric sequences, we aligned filtered Illumina reads to the genomes and calculated the genome coverage of reads as well as the heterozygosity of single-nucleotide polymorphisms (SNPs). The extremely high genome coverage (>99.5%) and low heterozygosity (<0.5%) indicate that our assemblies were not chimeric sequences and were high-quality (Table S2).There was large variation in the virus composition in Chinese mitten crab collected from the three sampling locations, which are separated from each other by at least 200 km  and B. MMartellivirales, Tolivirales, and Sobelivirales were abundant in MD-affected crabs, but showed a very low relative abundance in asymptomatic crabs  and Eriocheir sinensis reovirus isolate 816 (EsRV816) were both first reported in 2002  (Three double-stranded RNA (dsRNA) viruses found in this study belonged to the order  in 2002 –29. Erioin 2002 –. These ts (ICTV) . EsRV816s (ICTV) . Our phyAY542965) and EsRV2 (EF508130). We assembled a more complete genome of EsRV that consisted of 12 segments. The fifth segment of EsRV was not fully assembled, causing this segment to be divided into two fragments. EsRV was closely related to Callinectes sapidus reovirus 2 . A previous study proposed that the genome of EsRV2 had 10 segments, but only a complete segment encoding RdRp  was found in our assembled contigs , which was reported in 2019 and accepted by ICTV as a member of the family Cruliviridae in order Bunyavirales in 2020, was found in pools TS-MD and TS-Asym , except for members of the new family Tulasviridae which have a genome composed of a single large molecule of negative-sense RNA (~12 kb) encoding three open reading frames (ORFs) . EsBV shared 25.7% amino acid identity with Shahe bunya-like virus 3 (SBlV3) and 24.9% with Shahe bunya-like virus 4 (SBlV4). The unique genome organization of EsBV and its low sequence similarity with SBlV3 and SBlV4 suggested that EsBV is a novel virus. The phylogenetic result and distinct genome organization of EsBV indicated the possibility of establishing a novel family in the order Bunyavirales. However, there are some difficulties in the establishment of this novel family, mainly because EsBV and its relatives have no obvious common features, and further taxonomic research is needed in the future.Two negative-sense single-stranded RNA (ssRNA) viruses belonging to the order  TS-Asym , 34. A ns (ORFs) , 36. Hows (ORFs) , unlike Hepelivirales, Martellivirales, Amarillovirales, Nodamuvirales, Tolivirales, Timlovirales, Ourlivirales, Picornavirales, and Sobelivirales were identified in this study. Many of the viruses we found have different genome organizations compared to current ICTV-accepted members and independently clustered outside present genera or families in our phylogenetic trees. For these viruses, we preliminarily propose to establish novel genera or families mainly based on their phylogenetic results and genome organization.Twenty-six positive-sense ssRNA viruses belonging to the orders Hepelivirales.(i) Virus in the order  A viral sequence related to the family Alphatetraviridae in the order Hepelivirales, tentatively named Eriocheir sinensis alphatetra-like virus (EsAlV), was mainly discovered in the pool TS-Asym. EsAlV possessed a non-segmented genome with 10,673 nt encoding five ORFs . EsAlV shared 44.3% amino acid identity with Shahe hepe-like virus 2, which was the most closely related virus to EsAlV, indicating that EsAlV was a novel virus. The distinct genome structure of EsAlV and the long branch length of the clade containing EsAlV suggested that EsAlV may belong to a novel family in the order Hepelivirales. We propose to establish a novel family named “Pcahviridae” in Hepelivirales. This name was derived from the contraction of its characteristics, being a polycistronic, aquatic, hepe-like virus.ive ORFs , differeivirales –40. The Martellivirales.(ii) Viruses in the order  A kita-like viral sequence 12,933 bp in length, tentatively named Eriocheir sinensis kita-like virus (EsKlV), was discovered in this study. Current members of the family Kitaviridae have multiple segments (2 to 4 segments) and were considered to affect plants . EsKlV shared low amino acid identities with SASV1 (26.3%) and BAV1 (36.2%), suggesting that it may be a novel viral species in Kitaviridae. However, EsKlV was monopartite, and its genome contained 3 ORFs encoding 7 types of domains in total  Viruses in the order Flaviviridae is a family of positive-sense RNA viruses that have non-segmented genomes of approximately 9 to 13 kb and affect mammals  closely related to members of the family Flaviviridae, order Amarillovirales, was reported in 2016. Here, we found EsClV in almost all sampling pools except for pool SQ-Asym . The differences in genome length and host range between EsClV and current members of Flaviviridae indicate that EsClV and its relatives may represent a novel family in the order Amarillovirales. We propose to name this family “Megamonornaviridae.” This name was derived because these viruses have a large genome, are monopartite, and have a single-stranded RNA genome. Further study of the common characteristics of “Megamonornaviridae” needs to be performed to establish the novel family in the future. mammals . A parti SQ-Asym  and gene SQ-Asym . EsClV h SQ-Asym . PhylogeNodamuvirales.(iv) Viruses in the order  In our work, three novel viral sequences belonging to the order Nodamuvirales were identified, tentatively named Eriocheir sinensis nodavirus 1 (EsNV1), Eriocheir sinensis nodavirus 2 (EsNV2), and Eriocheir sinensis noda-like virus (EsNlV). Members of the family Nodaviridae have two genomic RNAs, encoding the RdRp (~3.1 kb) and capsid protein , respectively . The RdRp-encoded sequence of EsNV1 shared 58.1% amino acid identity with NoV and 66.2% with SIV11. Based on the ICTV report, definitive species demarcation within the genus Alphanodavirus requires the nucleotide sequence of the viral CP gene, as the two closest recognized species in this genus Black beetle virus and Flock house virus share 81% to 87% amino acid sequence identity in CP but are 99% identical in RdRp , and Shuangao insect virus 11 (SIV11) clustered together and fell within the genus  in RdRp . The limNodaviridae in the phylogenetic tree . EsNV2 shares limited similarity (47.8% amino acid identity) with Yunnan sediment noda-like virus 1, indicating that it was a novel virus and may represent a novel genus in Nodaviridae. The establishment of this novel genus requires more characteristics of EsNV2 and similar viruses, including the genomic RNAs encoding CP and other biological properties.EsNV2 formed a clade with Yunnan sediment noda-like virus 1 that is sister to current two genera of the family Sinhaliviridae, based on the phylogenetic tree . EsNlV shared 59.4% amino acid identity with Wenzhou crab virus 4 and 59.6% amino acid identity with Wenzhou tombus-like virus 18, suggesting EsNlV should be considered a new virus. The genome of EsNlV is 4,802 bp in length and has three ORFs that contain a methyltransferase domain, RdRp domain, and coat protein subunit, respectively  Viruses in the order Tombusviridae is a large family of viruses that infect monocotyledonous or dicotyledonous plants  that contained a partial RdRp domain, and renamed it Eriocheir sinensis tombusvirus 1 (EsTV1). We generated a nearly complete genome of EsTV1 of 4,861 nt in length that contained P33 protein, RdRp domain, and a coat protein subunit . EsTV2 shared limited similarity with Sanya tombus-like virus 2 (58.2% amino acid identity) and Forsythia suspensa tombusvirus (55.6% amino acid identity), suggesting that EsTV2 is a novel virus. The similarity of genome organization between EsTVs and the subfamily Procedovirinae and the phylogeny of EsTVs indicated that these viruses may represent novel genera of Procedovirinae in the family Tombusviridae. The phylogenetic result also showed that Procedovirinae was paraphyletic due to the introduction of EsTV1, EsTV2 and their relatives . The phylogeny of Procedovirinae needs to be confirmed using more reference sequences in the future.Phylogenetic analysis showed that EsTV1, EsTV2, and their related viruses formed a clade sister to the genus Timlovirales.(vi) Viruses in the order  The taxonomy of positive single-stranded RNA phages (+ssRNA phages) has been restructured due to the rapidly increasing number of known +ssRNA phages  with 5,266 nt and Eriocheir sinensis blumevirus 2 (EsBLV2) with 3,514 nt both had genomes containing 3 ORFs: their 3′ ORF encoded the RdRp, while the functions of the other ORFs were unknown (MW239254), but they did not cluster together in the phylogenetic tree. The criterion for establishing a novel genus in the class Leviviricetes is 50% pairwise amino acid identity of the RdRp  Viruses in the order  The family Botourmiaviridae in the order Ourlivirales includes viruses infecting plants and fungi; most of its genera, except for Ourmiavirus (tripartite), have a non-segmented genome (2.0 to 5.3 kb) containing one ORF that encodes the RdRp (Botourmiaviridae were identified in this study. Eriocheir sinensis botourmia-like virus 1 (EsBOlV1) with a length of 3,493 nt and Eriocheir sinensis botourmia-like virus 2 (EsBOlV2) with a length of 3,404 nt both had a genome containing only one ORF which encoded the RdRp , but the genome organizations of these viruses were more similar to those in other Botourmiaviridae genera than to those in Ourmiavirus. The distinct hosts of EsBOlVs and the long branch length of the clade containing EsBOlVs indicated that EsBOlV1 and EsBOlV2 may represent a novel genus in the family Botourmiaviridae. Here, we propose to establish a novel genus named “Crabovirus” for EsBOlVs. This name was derived from the crabs’ botourmia-like virus.the RdRp . Two novthe RdRp . EsBOlV1Picornavirales.(viii) Viruses in the order Picornavirales is a large order of positive-sense single-stranded RNA viruses and currently encompasses 8 families, 104 genera, and 344 species , showed high divergence and formed a clade far away from all other genera in the phylogenetic tree , was discovered. A previously reported virus, Shahe picorna-like virus 13 (SPlV13), with a length of 9,077 bp, was also found. These two viruses both had typical genome organizations of Picornaviridae: a long ORF containing a coat protein near the 5′ untranslated region (UTR) and a helicase, protease, and RdRp replication module (Hel-Pro-RdRp) near the 3′ UTR . Our phylogenetic tree also showed that the subfamily Paavivirinae of Picornaviridae clustered with the family Caliciviridae instead of with other subfamilies, indicating that the family Picornaviridae might be a paraphyletic group.tebrates . Previoutic tree . The lime 3′ UTR . PhylogeDicistroviridae is a family of RNA viruses with genomes containing two ORFs encoding nonstructural proteins (Hel-Pro-RdRp module) near the 5′ UTR and capsid proteins near the 3′ UTR, respectively , was 9,308 nt long. EsDlV shared 70.2% amino acid identity with Shahe arthropod virus 1 , which was also found in our study. EsDlV and SAV1 both had a typical genome organization of the family Dicistroviridae . The clade containing EsDlV and SAV1 was closer to Dicistroviridae than to other families. Due to the similar genome organization to Dicistroviridae and the phylogenetic results, we suggest that EsDlV and SAV1 may represent a new group of the family Dicistroviridae.ectively . Three noviridae . The phyDicistroviridae . Eriocheir sinensis dicistrovirus 1 (EsDV1), with a length of 7,577 nt, clustered with Changjiang picorna-like virus 11 and Riboviria sp. isolate 5cz-RDRP-2 and formed a clade that was sister to the genus Triatovirus. Eriocheir sinensis dicistrovirus 2 (EsDV2), with a length of 7,116 bp, clustered with Wenzhou shrimp virus 5 and Wenzhou shrimp virus 4 and formed a clade that was sister to the members of the genus Aparavirus isolated from crustaceans. According to the ICTV report, the species demarcation criterion for the family Dicistroviridae is 90% amino acid identity of the CP . The species demarcation criteria of Iflaviridae is 90% amino acid identity of the capsid proteins . EsMV4 appears sister to this clade, suggesting that these marnaviruses were potential members of Locarnavirus. The species demarcation criteria of the genus Locarnavirus are based on pairwise comparisons of the RdRP (90% identity) and capsid (75%) amino acid sequences (Locarnavirus. EsMV2 shared 65.3% amino acid identity of RdRp and 49.0% amino acid identity of capsid with Beihai picorna-like virus 47 (BPlV47), suggesting that EsMV2 may belong to a novel species in Locarnavirus. EsMV3 shared 40.8% amino acid identity of RdRp and 47.5% amino acid identity of capsid with Picornavirales sp. isolate HPLV-22 (GenBank accession no. OM622277), indicating that EsMV3 may also represent a novel species in Locarnavirus. EsMV4 shared 41.6% amino acid identity of RdRp sequence with mute swan feces-associated picorna-like virus 18 and shared <35.5% amino acid identity of capsid sequence with all similar viral sequences in the NCBI nonredundant protein (nr) database, suggesting that EsMV4 may also belong to a novel species.two ORFs . Some metebrates , 59. FouSobelivirales.(ix) Virus in the order  There are three families in the order Sobelivirales: Alvernaviridae, Barnaviridae, and Solemoviridae. Members of these families possess genomes longer than 4,000 nt  and Ixodes scapularis-associated virus 1 (38.7%). EsSlV2 shared 88.6% amino acid identity with Beihai sobemo-like virus 24. EsSlV1, EsSlV2, and their similar viruses formed a distinct clade and were distantly related to the current families of Sobelivirales in the phylogenetic tree constructed using the RdRp, indicating that EsSlVs may represent a novel group at the family level in Sobelivirales . We propose to establish a novel family for EsSlVs, named “Arthsoviridae,” which is derived from arthropod sobemo-like viruses.4,000 nt –62. Two ,000 nt –. CompareMany viromic studies have employed multi-location sampling to increase viral diversity and draw reliable conclusions. In our work, we observed high regional variation in viral species composition, with most of the viral species being region-specific, indicating the importance of multi-location sampling. For viromic research focused on uncovering novel viruses and expanding viral diversity, variation in viral species composition is an important component of viral diversity and should not be ignored. For viromic research focused on pathogen investigation, multi-location sampling can increase the reliability of analysis and exclude misleading regional differences.Metschnikowia bicuspidate  shared the highest similarity with two viruses from two traditional Chinese herbs, Leptochloa chinensis and Forsythia suspensa. A previous study reported that Macrobrachium rosenbergii virus 15, found in the giant freshwater prawn Macrobrachium rosenbergii, shared >99% amino acid identity with two viruses found in two traditional Chinese herbs, Trichosanthes kirilowii and F. suspensa  as the relative abundance. To assess the number of viral species in the sample and avoid false positive alignment, a species for which the number of mapped reads per million total reads (RPM) was greater than 10 was considered to be present in the sample. For higher viral taxa, we used RPM as the relative abundance because it is difficult to normalize the total amount of reads by length for multiple sequences from different viruses.Chinese mitten crabs with or without diseases were collected from farming ponds in three different regions in China . Specifig. The supernatant was collected and filtered through a Durapore PVDF (polyvinylidene difluoride) 0.45-μm-pore filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. The filtrates were subjected to ultracentrifugation  for 2 h to enrich the virus-like particles. After supernatant was carefully removed, the virus-like particles were resuspended for RNA extraction. RNA was extracted using a QIAamp Viral RNA minikit (Qiagen) in accordance with the manufacturer’s protocol. The quality and quantity of the RNA were measured on a Qubit 4.0 Fluorometer (Invitrogen) using the Qubit RNA HS assay kit.The hepatopancreas, branchia, and muscle tissues of 10 diseased or 10 healthy crabs collected from each region were pooled, resulting in 6 pools for sequencing library construction. The sample pools were homogenized with phosphate-buffered saline (PBS) (10% [mass/vol]) in a Lu kα sample freezing grinder for 3 min at 60 Hz and 4°C, frozen, and thawed three times on dry ice. The suspension of each pool was subjected to centrifugation for 10 min at 12,000 × The rRNA was removed for library construction by the NEBNext rRNA Depletion kit (New England Biolabs [NEB]). RNA sequencing libraries were constructed using a NEBNext Ultra II RNA Library Prep kit for Illumina (NEB) in accordance with the manufacturer’s protocol. The concentration of libraries was measured on a Qubit 4 Fluorometer (Invitrogen) using the Qubit dsDNA HS assay kit. Sequencing for the pair-end libraries were performed with 2 × 150 bp chemistry on the Illumina NovaSeq 6000 platform (Illumina).de novo using SPAdes (version 3.15.3)   and asse 3.15.3)  in rnavi 3.15.3) . Contigs 3.15.3) . Taxonom 3.15.3) , and con 3.15.3) .To confirm the assembly results and limit false positives, quality-controlled reads were mapped to viral contigs using Bowtie2 (version 2.4.2)  and SAMthttps://ictv.global/report/genome). The ORFs of viral genomes were identified using ORFfinder (https://www.chiplot.online/). The novel viruses found in this study were named according to the scientific name of the Chinese mitten crab and the phylogenetic analysis described below.Viral contigs were manually assigned to specific virus species by comparing their sequence similarity to each other and to the most similar sequences from nr database, based on the species demarcation criteria of specific taxa from the ICTV Report Chapters by Genome   using DiTo access the relative abundances of virus species in each library, quality-controlled reads were mapped to the whole genome of virus species using Bowtie2, and the FPKM of each virus species in each library was calculated using featureCounts (version 2.0.2) . The heaTo verify the presence of the RdRp genes, we used the original samples for RT-PCR and nested RT-PCR with primer sets designed based on the RdRp sequences of viral genomes. The PCR products were sequenced using Sanger sequencing and then compared with the assembled sequences. To calculate the genome coverage of reads and the heterozygosity of SNPs, raw sequencing reads were aligned to the assembled genomes using bwa (version 0.7.17) . Genome PRJNA921821. All viral sequences identified for the first time in this study have been deposited in GenBank under accession numbers OP019089 to OP019093, OP019095 to OP019108, and OP019111 to OP019132.Raw sequencing reads are accessible in NCBI under BioProject no.


In 2020, ASF occurred in wild boars throughout Latvia and Lithuania, and more than 21,500 animals were hunted and tested for the presence of the virus genome and antibodies in the framework of routine disease surveillance. The aim of our study was to re-examine hunted wild boars that tested positive for the antibodies and negative for the virus genome in the blood (n = 244) and to see if the virus genome can still be found in the bone marrow, as an indicator of virus persistence in the animal. Via this approach, we intended to answer the question of whether seropositive animals play a role in the spread of the disease. In total, 2 seropositive animals out of 244 were found to be positive for the ASF virus genome in the bone marrow. The results indicate that seropositive animals, which theoretically could also be virus shedders, can hardly be found in the field and thus do not play an epidemiological role regarding virus perpetuation, at least not in the wild boar populations we studied. When African swine fever (ASF) was introduced to the Baltic States and Poland in 2014, the epidemiological role of wild boars became evident and a new infection cycle was described: the wild boar–habitat cycle ,4. In thA large number of experimental studies have been conducted with domestic pigs to investigate the role of potential carriers and to explore their role in disease spread. In a detailed literature review , the autAccording to the strategic approach to the management of African swine fever for the European Union, all wild boars hunted in ASF-affected areas are subjected to ASF routine testing . Blood s2. However, these estimates should be considered rough figures of the populations under which the disease has spread. In Latvia, a total of 191 wild boar carcasses were found and tested, of which 99 were PCR-positive (51.8%). In Lithuania, 82 carcasses out of 177 were tested PCR-positive (46%). The geographical location of ASF cases is showed in In 2020, ASF was present all over the countries Latvia and Lithuania. The pre-reproductive wild boar population was estimated at about 15,300 wild boars in Latvia and 13,500 wild boars in Lithuania, which is around 0.2 wild boars per kmThe aim of our study was to take a closer look at seropositive wild boars that were shot in Latvia and Lithuania in the framework of routine surveillance in 2020.® GmbH for INDICAL BIOSCIENCE, Leipzig, Germany) for DNA manual extraction was used according to the manufacturer’s instructions. In total, we succeed in examining bone marrow samples from 244 seropositive wild boars, 139 from Latvia and 105 from Lithuania  for further virological examination. About 3 g of tissue or ligaments was extracted from the cancellous part of the bone with a bone chisel and was placed in 7 mL homogenization tubes with 2.5 mL PBS and 9–10 ceramic beads. The tubes were then placed in a homogenizer  and homogenized at 6000 rpm for 30 s in two cycles. For DNA extraction, the suspension was clarified via centrifugation for 2 min at 3000 rpm. The IndiSpin Pathogen Kit  = 8.9, p = 0.0029. On the contrary, the proportion of seropositive but PCR-negative animals  = 3.4, p = 0.066) did not differ significantly between the two countries. The prevalence of PCR-positive animals among the wild boars found dead did not differ either  = 0.9, p = 0.34). Only 2 (1.1%) of 244 re-examined seropositive animals but PCR-negative in the blood were found to be positive for the ASF virus genome in the bone marrow: a young male from Lithuania less than 12 months of age with a Ct value of 35 and a male from Latvia between one and two years old with a Ct value of 28. There was no significant difference between Lithuania and Latvia  = 0.27, p = 0.61).The test results of hunted and found dead wild boars in Latvia and Lithuania from May to December 2020 are presented in Four categories of animals can be distinguished in a population based on their virological and serological status in the blood: (i) animals that are neither seropositive nor virus/genome-positive and are thus fully susceptible; (ii) animals that are only virus/genome-positive and therefore potential virus shedders that may infect other animals; (iii) animals that are virus/genome-positive and seropositive at the same time, and could excrete the virus and contribute to pathogen perpetuation; and finally, (iv) animals that are only seropositive.The latter group of seropositive animals are suspected of being potential virus carriers, which can also excrete the virus and could thus play some role in the spread of the ASF virus. There are authors who support this hypothesis and others who see no epidemiological role of seropositive animals ,7,8,9,10The presented ratios of the proportions of seropositive and PCR-positive animals among hunted wild boars for Lithuania and Latvia for the period under consideration have also been described by other authors . Most poInterestingly, there are no significant differences between Lithuania and Latvia in the proportion of PCR-positive carcasses and the proportion of PCR-positive (bone marrow) and PCR-negative hunted wild boars. The latter could indicate common control and surveillance strategies in the infected areas of both countries.The results obtained in this study clearly indicate that seropositive animals, which theoretically could also be virus shedders, are hardly found in the field and thus do not play an epidemiological role, at least not in the wild boar populations we studied. Their prevalence is too low to explain the maintenance of an epidemic on the scale that is occurring in these two countries.Our results are also in line with the experimental studies in domestic pigs, where no convincing results for the epidemiological role of seropositive animals as virus shedders were found .Considering the results of our study, we conclude that seropositive animals do not play a significant role from the point of view of disease control. Therefore, ASF control measures must be focused on viremic animals and carcasses, as well as on biosecurity measures during and after hunting.