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Preparation of positive controls for molecular testingZIKV strains MR766, PRVABC59, and DakAR41525 were separately propagated on Vero cells (ATCC CCL-81). Cell supernatant was harvested 72 hpi, and RNA extraction was performed using Trizol. Due to undetectable RNA concentration, the maximum input volume of 11 µL was used for cDNA generation using the SuperScript IV First-Strand Synthesis System with random hexamers (Thermo Fisher Scientific, Waltham, MA, United States). A ten-fold dilution series of RNA was generated for each strain to validate detection of phylogenetically divergent strains of ZIKV using our primer set. For all molecular assays, 3 µL of 10−3 of MR766 was used experimentally as the positive control. Propagation of ZIKV was conducted under CSU biosafety protocol 17-059B.Infection protocol, RNA Extraction, and cDNA synthesis for A129 mice and Jamaican fruit batsAll animal studies were carried out in accordance with ARRIVE guidelines and all procedures approved by and carried out under the Colorado State University Institutional Animal Care and Use Committee (protocol 15-6677AA). Three sub-adult male A129 mice and three female Jamaican fruit bats (Artibeus jamaicensis) were obtained from their respective breeding colonies at Colorado State University. Mice were subcutaneously inoculated with 1 × 103 PFU supernatant from PRVABC59-infected Vero cells, and bats were subcutaneously inoculated with 7.5 × 105 PFU supernatant from Vero cells infected with one of three strains (either PRVABC59, MR766, or DakAR41525; one strain per individual). Mice were euthanized at 7 days post-infection (dpi). The bat infected with ZIKV strain MR766 was euthanized at 28 dpi, while the two bats infected with strains PRVABC59 and DakAR41525 were euthanized at 45 dpi to provide a broader of time window in which to characterize sfRNA persistence. Organs and blood were harvested and placed into DMEM supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, United States) and 10% FBS (Atlas Biologicals, Fort Collins, CO, United States) and stored at − 80 °C until RNA extraction using the Mag-Bind Viral DNA/RNA 96 kit (Omega Bio-Tek Inc., Norcross, GA, United States) on the KingFisher Flex Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA, United States). RNA was eluted in 30 µL nuclease-free water.Droplet digital PCR (ddPCR) to detect ZIKV sfRNATo detect ZIKV sfRNA, primers were designed to target the 3′ UTR of multiple strains of ZIKV according to recommended ddPCR primer design guidelines, resulting in an amplicon 123 bp in length (F: TTCCCCACCCTTYAATCTGG and R: TGGTCTTTCCCAGCGTCAAT). Each reaction consisted of 50 ng cDNA, 125 nM foward primer, 125 nM reverse primer, and 10 µL QX200 ddPCR EvaGreen Supermix (Bio-Rad Laboratories, Hercules, CA, United States). Following reaction preparation, 20 µL of reaction and 60 µL of QX200 Droplet Generation Oil for EvaGreen (Bio-Rad Laboratories, Hercules, CA, United States) were loaded into a DG8 Cartridge for droplet generation in the QX200 Droplet Generator (Bio-Rad Laboratories, Hercules, CA, United States). Following droplet generation, plates were sealed in the PX1 PCR Plate Sealer (Bio-Rad Laboratories, Hercules, CA, United States). PCR was performed on a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, United States), using the following cycling parameters: 95 °C for 5 min, 40 cycles of 95 °C for 30 s followed by 57.5 °C for 1 min, 4 °C for 5 min, 90 °C for 5 min, and held at 4 °C until reading the plate. Plates were read on the QX200 Droplet Reader (Bio-Rad Laboratories, Hercules, CA, United States). Analysis was performed by two individuals using QuantaSoft Software (Bio-Rad Laboratories, Hercules, CA, United States) to determine results.Gradient PCR was performed to identify the optimal annealing temperature, resulting in selection of 57.5 °C (Fig. S1). At this annealing temperature, the ddPCR reaction using the 3′ UTR primers successfully amplified ZIKV strains MR766, DakAR41525, and PRVABC59 (Fig. S2). As an additional and more biologically relevant sample type, 50 ng cDNA from the organs of A129 mice experimentally infected with ZIKV PRVABC59 were tested using this same assay; successful ZIKV sfRNA amplification was obtained from mouse kidney and spleen (Fig. S2). Blood and tissue samples from the three female Jamaican fruit bats were tested in duplicate on the QX200 Droplet Digital (ddPCR) System (Bio-Rad Laboratories, Hercules, CA, United States) using the ZIKV sfRNA primers as described above.Testing of archived samples from free-ranging Ugandan batsThis study utilized archived tissue samples from bats previously captured in Uganda from 2009 to 201318,26 (Table 1). Bats were captured using harp traps or mist nets, identified using a field guide specific to East African bats, and placed in holding bags prior to anesthesia via halothane and euthanasia by cervical dislocation27. This study used historic archived samples from a previous study, in which all bat captures and sampling were conducted under the approval of CDC IACUC protocols 1731AMMULX and 010-015 and carried out according to ARRIVE guidelines. RNA was extracted from frozen tissue homogenates (spleen, and in some cases both spleen and liver separately) using the MagMax 96 total RNA isolation kit (Applied Biosystems, Foster City, CA, United States), and cDNA generation was performed as above. To confirm RNA integrity via amplification of a housekeeping gene, we used previously published primers demonstrated to amplify GAPDH from two Old World bat species (black flying fox and Egyptian rousette bat) and one New World bat species (common vampire bat) (F: GTCGCCATCAATGACCCCTTC and R: TTCAAGTGAGCCCCAGCC)31. For samples with undetectable RNA concentration on the Qubit RNA HS assay, 6 µL cDNA was used as input. ddPCR was performed as above, except that an annealing temperature of 60˚C was used. Plates were read as above, and only samples deemed ‘suspect’ or ‘positive’ for GAPDH amplification were subjected to ddPCR testing with ZIKV sfRNA (3′ UTR). For these samples, the same volume of input cDNA was used to test for the presence of ZIKV sfRNA in duplicate; results were analyzed by two individuals.Table 1 All bat species and trap sites collected from 2009 to 201318,26.Full size tableSequence confirmationTo confirm specific amplification of GAPDH sequence for each of the 8 Old World species, the same primers were used in a conventional PCR assay using GoTaq HotStart Polymerase (Promega corporation, Madison, WI, United States). Cycling parameters were as follows: 95 °C for 2 min; 35 cycles of 95 °C for 1 min, 57.5 °C for 1 min, and 72 °C for 30 s; followed by 72 °C for 5 min and samples were held at 4 °C until being analyzed for the presence of a 248-bp amplicon via gel electrophoresis. Amplicons were verified by Sanger sequencing (GENEWIZ, Inc., South Plainfield NJ, United States). Results obtained from Sanger sequencing were subjected to quality analysis prior to aligning forward and reverse reads, and the consensus read was subjected to a BLAST search.Confirmation of ZIKV sfRNA ddPCR results in Ugandan bat samples using conventional PCR and sequencingSamples deemed ‘suspect’ via screening on the ddPCR system with ZIKV 3′ UTR primers were subjected to additional PCR and Sanger sequencing using the same primer set targeting the 3′ UTR of ZIKV. ZIKV strain MR766 was used as a positive control in these assays. Samples were considered ‘suspect’ if (1) the automatically-defined threshold yielded ≥ 1 positive droplet in the same 1D amplitude as the positive control cDNA (ZIKV MR766) or (2) the negative droplet populations existed in the same 1D amplitude region of positive control droplets and thus, precluded the ability to differentiate positive and negative populations. The cDNA from these samples was amplified using the GoTaq HotStart system (Promega corporation, Madison, WI, United States), with each reaction consisting of 50 ng cDNA, 25 µL GoTaq HotStart Master Mix, 400 nM forward primer, 400 nM reverse primer, and 1 M Betaine. Cycling parameters were as follows: 95 °C for 2 min; 35 cycles of 95 °C for 1 min, 57.5 °C for 1 min, and 72 °C for 30 s; followed by 72 °C for 5 min and samples were held at 4 °C until being analyzed for the presence of a 123-bp amplicon via gel electrophoresis. Positive samples were verified by Sanger sequencing (GENEWIZ, Inc., South Plainfield NJ, United States). Results obtained from Sanger sequencing were subjected to quality analysis prior to BLAST search and subsequent alignment of forward and reverse reads with the 3′ UTR of ZIKV MR766 in Geneious v11.1.5 (www.geneious.com).Comparison of detection sensitivity between sfRNA and NS5 in field-caught samplesThe four samples from which ZIKV sfRNA was amplified were subjected to cPCR amplification with GoTaq HotStart MasterMix as described above and primers designed for this study targeting NS5 from MR766, PRVABC59, and DakAR41525 in order to compare detection sensitivity (F: TGC CGC CAC CAA GAT GAA CT, R: CAT TCT CCC TTT CCA TGG ATT GAC C). Cycling parameters were as follows: 95 °C for 2 min; 35 cycles of 95 °C for 1 min, 57.5 °C for 1 min, and 72 °C for 30 s; followed by 72 °C for 5 min and samples were held at 4 °C. cDNA from ZIKV MR766 was used as a positive control. Results were sent for Sanger sequencing if a band was present. All methods in this study were carried out in accordance with relevant guidelines and regulations. More

Fossils, such as this skeleton of a T. rex on display in the Netherlands, may be even rarer than scientists realised. Credit: Marten van Dijl/AFP via Getty

Ever wondered how many Tyrannosaurus rex ever roamed the Earth? The answer is 2.5 billion over the two million or so years for which the species existed, according to a calculation published today in Science1. The figure has allowed researchers to estimate just how exceedingly rare it is for animals to fossilize.Palaeontologists led by Charles Marshall at the University of California, Berkeley, used a method employed by ecologists studying contemporary creatures to estimate the population density of T. rex during the late Cretaceous period. “You hold a fossil in your hand and you know it’s rare. The question is, how rare?” says Marshall. “To know that, you need to know how many of them existed.”To do that, he and his co-authors turned to a method used to estimate the population density of living animals from their body mass and the geographic ranges that they occupy. Damuth’s Law stipulates that the average population density of a species decreases in a predictable way as body mass increases; for example, there are fewer elephants than mice in a given area.Chances of being fossilized vanishingly smallThe team used their estimates of the total range of T. rex across modern North America, combined with their estimates of the dinosaur’s body mass, to calculate that, at any one time, around 20,000 T. rex would have been alive on the planet. That translates to around 3,800 T. rex in an area the size of California, or just two T. rex patrolling Washington DC. Calculating that T. rex survived for about 127,000 generations before becoming extinct, the researchers came up with a figure of 2.5 billion individuals over the species’ entire existence. Only 32 adult T. rex have been discovered as fossils, so the fossil record accounts for just 1 in about every 80 million T. rex. This means that the chances of being fossilized — even for one of the largest-ever carnivores — were vanishingly small.These numbers suggest that fossils in general are exceedingly rare, and hint that many species that were much less widespread than T. rex were probably never preserved, says Marshall, who adds: “The fossil record is our only direct knowledge of these completely unimaginable past histories of our planet.”Thomas Holtz, a vertebrate palaeontologist at the University of Maryland in College Park, calls the calculation an “interesting speculation”, adding that “we always knew that the chance of any individual becoming a fossil was exceedingly rare, but we lacked the calculation to figure out how rare”.But he says it would be good “to see someone ground-truth these kinds of estimations against living species to get a better sense of accuracy”. He’d also like to see comparable studies made on extinct species with more abundant fossils, such as woolly mammoths, Neanderthals and dire wolves, which might allow us to better understand historic ecosystems. More

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The BNNs demonstrated that the average yields of cacao farmer groups, in Sulawesi over distinct time periods, are closely associated with the ENSO OI patterns 9 to 25 months before harvest. The ENSO OI short term pattern explained slightly less (69%) of the variation in the average yield than the long term pattern (77%). We consider both these levels of prediction to be high, however, the short term pattern level was simpler and was used for further analyis. The linear regression between predicted and actual yields indicates that the model will tend to underestimate cacao productivity at high yields (e.g. in excess of 100 kg ha−1 month−1).The predictions made by the BNNs indicated that cacao yields are substantially impacted by ENSO conditions, which accords with prior observations21. The fertilizer response varied according to the ENSO profile: the greatest predicted response was in the Neutral ENSO profile with a smaller response under the MinCent ENSO profile, especially when unfertilized yields were low, and essentially no response under the MaxCent ENSO profile. Hence, the analysis provides insights into the appropriate fertilizer regime for distinct ENSO OI patterns in the period 9 months before harvest. We also note that recent methods to improve prediction of future ENSO OI patterns make it possible to predict them with reasonable accuracy for up to 1 year3. Thus, it is possible to relate average cacao crop performance and management practices directly to ENSO patterns in a given region without the need for weather data when the following conditions are met: (1) data exist on crop performance in any given site over time with distinct management practices; and (2) the weather patterns are driven by ENSO OI. We have used cacao as proof-of-principle, and suggest that this principle can readily be applied to other crops.A great advantage that Bayesian methods have over other machine learning approaches is that they can utilise variance based probability distributions to predict the likelihood of any given outcome. The model was used to predict the most likely monthly yield and expected standard deviation from each farm group under a specific ENSO profile when either fertilized or unfertilized. The standard deviations attained across all predicted responses was remarkably low, typically less than 1 kg ha−1 per month. Both the construction of the model and the subsequent predictions were based upon the mean yield data from 10 farms in each group at each monthly harvest under a single management type. As a result, all variations in yield across those 10 farms would have been excluded from the network constructed. As a consequence, while the predictions returned by the model might precisely reflect the mean response from each group, the limited input data will mean that the range of possible outcomes under any predicted scenario is likely to be underestimated. Up to now we have established proof-of-principle stage, the next stage will be first to improve the assessment of the predicted probability distributions and then to develop channels for communicating the results of the analysis to farmers followed by appraisal of their opinions and use of the information provided. Options for improving estimates of the probability distribution include both incorporating all observations from within each group, to ensure that farm-to-farm variance is adequately captured, and to extend the observations across more seasons to ensure that the variability of response to contrasting ENSO profiles is better represented.The analysis presented here is based on the average yields for each group of farmers. However, previous analysis indicates much variation in yield within the farmers groups20. Furthermore, those farmers with higher average yields tended to maintain their yield advantage relative to those with lower yields, even when conditions were adverse. This supports the view that the differences in yield between the high average yield and the low average yield farmers are due to management skills, rather than more favorable soils and weather conditions20. This suggests that if the average yields of individual farmers relative to the mean of all farmers are known, then the ENSO predictions can be used to predict their yield levels, and also their response to fertilizer applications.The demonstration that on farm yields and response to one management variable, fertilizer, can be linked directly to ENSO OI data supports the view that, in the future, with cacao or other crops, data on farm yields obtained with distinct management practices can be coupled with ENSO OI data to both determine probable crop yields and also to define differential crop response to management at specific sites under distinct ENSO OI patterns without the need for accurate weather data. The ENSO OI data exists, what is often lacking is data on yield with distinct management practices. To obtain this type of information in heterogeneous growing environments using traditional Randomized Control Trials is simply not possible. However, we suggest that schemes, such as those to collect the cacao data we have here with distinct management treatments superimposed on farmers fields20, can be used. Furthermore, even without superimposing management practices, simply monitoring crop performance, weather and the variation in management practices of farmers can be used to relate yield to variation in weather patterns and management28,29,30. However, this is only effective if the data of a large number of cropping events is brought together for analysis, which requires social organization and the willingness to share data28. Our experience with cacao indicates that small farmers are willing to share data, but an external agency is required to manage the overall process of data collection and compilation20. Similar experiences with CropCheck and in Australia and Chile support this point of view31,32. The value of shared information through formation of farmer groups is well established33,34 and we suggest that the methodology described here could be implemented through farmer groups. Hence, through monitoring of crop performance and management coupled with Bayesian based machine learning tools and currently available ENSO OI information and predictions, farmers and agronomists can adjust management practices, in this case fertilizer applications, according to ENSO profiles. This will require social organization and support for the collection, compilation and analysis of the data; however, we believe it offers a route to provide farmers with an improved and cost effective knowledge base, derived from sparse data resources, to better manage their crops.Social organization is not only required for the collection of data to be analysed, but also for the disemination to farmers of the knowledge generated though its interpretation. Current tendencies of providing farmers with the basis to make better decisions recognise the restrictions of the linear model for extension and tend towards active farmer participation in the interpretation of data through such mechanisms as farmers field schools35, formation of farmers groups (see for example Montaner 200434) and innnovation networks (see for example Klerkx et al. 201036, Wood et al. 201437, World Bank, 200838). Further development of farmers´organizations and innovation networks will be required to effectively deploy the concepts presented in this paper.The principles developed here could be applied to other crops, such as coffee, olive and oil palm, and this type of analysis could be extended to other regions, such as Africa where data on crop response to management and weather variation is sparse. At the same time, we note that additional information on, inter alia, crop management, topography and soil types could substantially improve the predictive power of the networks. Furthermore, these machine learning techniques can be used to mine existing big data sets collected by large commercial interests, to discover relationships between environment, management and crop production, and thereby supplement, at low cost, the findings generated by formal controlled scientific experiments. In the case of small farmers, social organization and external support will be required.There are several caveats on the use of this proposed methodology. First, the relationship between the ENSO phenomenon and the weather patterns will be specific to each location or recommendation domain. Hence, models and inferences for management cannot be readily transferred from one recommendation domain to another. Furthermore, the definition of the area that comprises a recommendation domain is not simple. Thus, whilst we consider the principles developed here to be universal, the models themselves will be specific to each recommendation domain, which are currently still difficult to define but new approaches are becoming increasingly available to do so (e.g. Rubiano et. al. 201618; Rattalino Edreira et al. 201817).A further complication of the suggested approach is the lack of understanding of the underlying mechanisms that establish the associations. This deficiency limits the ability to identify the specific causes of different crop productivities, and thus limits our ability to resolve these unidentified problems.Growers decisions on how much to invest in their crop production practices depends on the expected prices of the commodities they produce: when prices are expected to be high, they will invest more, and when prices are low they may even abandon their crops. It has not escaped our notice that the predictive power of the machine learning resources would also provide the cacao industry as a whole with insights into the fluctuations in future cacao supply and hence prices. This would allow farmers and others in the cacao supply chain to minimize uncertainty and better manage the overall industry. The experiences strongly support the idea that machine learning is a useful tool in our armoury opening the opportunity to utilize information from on farm performance coupled with publicly available data to improve agricultural management. More