Farewell to the Lose–Lose Reality of Policing Plant Imports

In an age of free international shipments of mail-ordered seeds and plants, more policing will not stop the global migration of hitchhiking pests. The solution is in a preemptive response based on an internationally coordinated genomic deployment of global biodiversity in the largest breeding project since the “Garden of Eden.” This plan will enrich the narrow genetic basis of annual and perennial plants with adaptations to changing environments and resistances to the pests of the future.

“Plan for what is difficult while it is easy; do what is great while it is small.”—Sun Tzu, The Art of War

When 182 countries become party to a common cause, it is reason to rejoice. Such an opportunity was provided when the Food and Agriculture Organization of the United Nations (FAO) approved the International Plant Protection Convention (IPPC) on December 6, 1951, with the objective of developing and implementing international phytosanitary standards to reduce the risks associated with the spread of plant pests to agriculture and natural ecosystems [1]. Over the years, the IPPC has been amended to enforce safer trade of plants by preventing the entry and spread of new pests. This led to the establishment of dedicated government agencies, usually associated with the ministries of agriculture, which are responsible for inspecting and policing against the entry of pests. These agencies have grown tremendously over the years because their noble mission is simply to explain to the “want to do good” elected officials who are responsible for the allocation of funds. However, the IPPC is currently implementing a losing defensive strategy, for which a scientific alternative based on a broad view of our interconnected global reality is presented below.     

Agricultural biosecurity

The prevention and control of new pest and disease introductions is an agricultural challenge which is attracting growing public interest. This interest is in part driven by an impression that the threat is increasing, but there has been little analysis of the changing rates of biosecurity threat, and existing evidence is equivocal. Traditional biosecurity systems for animals and plants differ substantially but are beginning to converge. Bio-economic modelling of risk will be a valuable tool in guiding the allocation of limited resources for biosecurity. The future of prevention and management systems will be strongly influenced by new technology and the growing role of the private sector. Overall, today''s biosecurity systems are challenged by changing national priorities regarding trade, by new concerns about environmental effects of biological invasions and by the question ‘who pays?’. Tomorrow''s systems may need to be quite different to be effective. We suggest three changes: an integration of plant and animal biosecurity around a common, proactive, risk-based approach; a greater focus on international cooperation to deal with threats at source; and a commitment to refocus biosecurity on building resilience to invasion into agroecosystems rather than building walls around them.     

Integrative Analysis of the Physical Transport Network into Australia

Effective biosecurity is necessary to protect nations and their citizens from a variety of threats, including emerging infectious diseases, agricultural or environmental pests and pathogens, and illegal wildlife trade. The physical pathways by which these threats are transported internationally, predominantly shipping and air traffic, have undergone significant growth and changes in spatial distributions in recent decades. An understanding of the specific pathways and donor-traffic hotspots created by this integrated physical transport network is vital for the development of effective biosecurity strategies into the future. In this study, we analysed the physical transport network into Australia over the period 1999–2012. Seaborne and air traffic were weighted to calculate a “weighted cumulative impact” score for each source region worldwide, each year. High risk source regions, and those source regions that underwent substantial changes in risk over the study period, were determined. An overall risk ranking was calculated by integrating across all possible weighting combinations. The source regions having greatest overall physical connectedness with Australia were Singapore, which is a global transport hub, and the North Island of New Zealand, a close regional trading partner with Australia. Both those regions with large amounts of traffic across multiple vectors (e.g., Hong Kong), and those with high levels of traffic of only one type (e.g., Bali, Indonesia with respect to passenger flights), were represented among high risk source regions. These data provide a baseline model for the transport of individuals and commodities against which the effectiveness of biosecurity controls may be assessed, and are a valuable tool in the development of future biosecurity policy.     

The viral era: New biotechnologies give humans an unprecedented control over Nature and require appropriate safeguards

New biotechnologies such as gene drives and engineered viruses herald a viral era that would give humans exceptional power over any organism at the level of the genotype. Subject Categories: Synthetic Biology & Biotechnology, S&S: Economics & Business, Ecology

We are entering a new phase in our relationship with nature: after mechanization, automation and digitalization, a new era of autonomous technical objects is dawning. The most advanced of these technologies are characterized by viral behaviour. The COVID‐19 pandemic has again aptly demonstrated the power of viral systems: not only because of the SARS‐CoV‐2 virus'' ability to jump into and rapidly spread among the human population while wreaking havoc with human societies, but also because some of the vaccines developed against the virus are themselves based on viruses. Both developments give us some ideas of the possible impact of new biotechnologies that aim to create artefacts with viral behaviour in order to shape and control our natural environment. In this essay, the focus is on the use of genetically engineered organisms and the genetic manipulation of wild species. This change has a more direct relationship to our natural environment than autonomous software artefacts such as computer apps or digital viruses that “live” in their artificial “ecosystems” of information‐processing devices. The development of artificial biological systems will therefore require new methods for monitoring and intervention given their potential to autonomously spread within natural ecosystems.     

Modelling biosecurity change: Ecology, economics, technology and social values

The threats and responses to biosecurity are constantly changing, creating decision problems for policy makers setting priorities for future biosecurity systems. In the United Kingdom during 2003–04, the Department for Environment, Food and Rural Affairs (DEFRA) commissioned a Horizon Scanning project to predict the future (20–30 years) of biosecurity needs in the United Kingdom. This project created an integrated model of key ecological, economic and technological processes involved in the development and control of invasive species, across a range of taxa, and also sought views on social values that could limit response options and affect the economic and political importance of introduced species. The model demonstrates the ability to make useful probability- based estimates of economic impact given practical assumptions on ecological, economic and technological inputs. Sensitivity analyses show where improved data could reduce uncertainty. The model establishes a framework that has been used to identify major drivers of biosecurity change affecting the next generation: increased and more diverse trade and travel increasing the entry of new species; climate change affecting establishment and spread of pests introduced from new zones that could approximate Britain’s climate; social values affecting attitudes to control measures such as animal culling and greater concern for environmental and amenity resources rather than agriculture; and technological improvements in pest detection. An important economic issue affecting the value of the impact caused by invasions is the time scale over which the impact is felt, ranging from immediate in the case of many livestock diseases through to the long-delayed recognition of loss of environmental quality from competition or diseases affecting native plants. New pest detection technology offers substantial opportunity to improve eradication of introduced species and could affect the prevention versus cure paradigm for many species for which general exclusion systems are presently adopted. An integrated modelling framework allows some quantification of these drivers and offers a tool to guide biosecurity planning.     

BeeDoctor,a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.     

Recent non‐native invertebrate plant pest establishments in Great Britain: origins,pathways, and trends

1 An appraisal of non‐native invertebrate plant pest establishments in Great Britain, between 1970 and 2004, was carried out to improve our understanding of current invasion processes by non‐native plant pests, and to assist national strategies in managing the risks they pose. 2 A total of 164 establishments, comprising 50 natural colonists and 114 human‐assisted introductions, were recorded across 13 major taxonomic groups. 3 The mean rate of establishment was 22.1 species per 5‐year period: 19.1 and 3.0 species outside and inside protected cultivation, respectively. Despite the continuing rapid growth in international trade and a general perception that rates of pest invasions are accelerating, no significant temporal trends in the rate of establishments in Great Britain were detected, either for natural colonists or human‐assisted introductions, or for pests of plants grown indoors or outside. 4 The plant trade, particularly in ornamental plants, accounted for nearly 90% of human‐assisted introductions; apiculture, biological control, timber imports, transport stowaways and intentional releases each contributed less than 5%. Only eight (4.9%) of the establishments could be considered as having no direct potential economic impact because all other species have been recorded as feeding on cultivated plants. A greater proportion of establishments by both natural colonists and human‐assisted introductions occurred on non‐native, woody plants. 5 The present study confirms previous work in other European countries that highlight the predominant role of the ornamental plant trade in introducing new plant pests to the European continent, mainly from Asia and North America.     

Deathly Drool: Evolutionary and Ecological Basis of Septic Bacteria in Komodo Dragon Mouths

Komodo dragons, the world''s largest lizard, dispatch their large ungulate prey by biting and tearing flesh. If a prey escapes, oral bacteria inoculated into the wound reputedly induce a sepsis that augments later prey capture by the same or other lizards. However, the ecological and evolutionary basis of sepsis in Komodo prey acquisition is controversial. Two models have been proposed. The “bacteria as venom” model postulates that the oral flora directly benefits the lizard in prey capture irrespective of any benefit to the bacteria. The “passive acquisition” model is that the oral flora of lizards reflects the bacteria found in carrion and sick prey, with no relevance to the ability to induce sepsis in subsequent prey. A third model is proposed and analyzed here, the “lizard-lizard epidemic” model. In this model, bacteria are spread indirectly from one lizard mouth to another. Prey escaping an initial attack act as vectors in infecting new lizards. This model requires specific life history characteristics and ways to refute the model based on these characteristics are proposed and tested. Dragon life histories (some details of which are reported here) prove remarkably consistent with the model, especially that multiple, unrelated lizards feed communally on large carcasses and that escaping, wounded prey are ultimately fed on by other lizards. The identities and evolutionary histories of bacteria in the oral flora may yield the most useful additional insights for further testing the epidemic model and can now be obtained with new technologies.     

Two Different Macaviruses,ovine herpesvirus-2 and caprine herpesvirus-2, Behave Differently in Water Buffaloes than in Cattle or in Their Respective Reservoir Species

The ongoing global spread of “exotic” farm animals, such as water buffaloes, which carry their native sets of viruses, may bear unknown risks for the animals, into whose ecological niches the former are introduced and vice versa. Here, we report on the occurrence of malignant catarrhal fever (MCF) on Swiss farms, where “exotic” water buffaloes were kept together with “native” animals, i.e. cattle, sheep, and goats. In the first farm with 56 water buffaloes, eight cases of MCF due to ovine herpesvirus-2 (OvHV-2) were noted, whereas additional ten water buffaloes were subclinically infected with either OvHV-2 or caprine herpesvirus-2 (CpHV-2). On the second farm, 13 water buffaloes were infected with CpHV-2 and two of those succumbed to MCF. In neither farm, any of the two viruses were detected in cattle, but the Macaviruses were present at high prevalence among their original host species, sheep and goats, respectively. On the third farm, sheep were kept well separated from water buffaloes and OvHV-2 was not transmitted to the buffaloes, despite of high prevalence of the virus among the sheep. Macavirus DNA was frequently detected in the nasal secretions of virus-positive animals and in one instance OvHV-2 was transmitted vertically to an unborn water buffalo calf. Thus, water buffaloes seem to be more susceptible than cattle to infection with either Macavirus; however, MCF did not develop as frequently. Therefore, water buffaloes seem to represent an interesting intermediate-type host for Macaviruses. Consequently, water buffaloes in their native, tropic environments may be vulnerable and endangered to viruses that originate from seemingly healthy, imported sheep and goats.     

How accurately can we assess zoonotic risk?

Identifying the animal reservoirs from which zoonotic viruses will likely emerge is central to understanding the determinants of disease emergence. Accordingly, there has been an increase in studies attempting zoonotic “risk assessment.” Herein, we demonstrate that the virological data on which these analyses are conducted are incomplete, biased, and rapidly changing with ongoing virus discovery. Together, these shortcomings suggest that attempts to assess zoonotic risk using available virological data are likely to be inaccurate and largely only identify those host taxa that have been studied most extensively. We suggest that virus surveillance at the human–animal interface may be more productive.

Determining which organisms harbour viruses that could potentially infect humans is of great topical interest. This Essay demonstrates that the data on which such zoonotic risk assessments are conducted are incomplete, biased, and rapidly changing with ongoing virus discovery.     

Scent of a killer: How could killer yeast boost its dispersal?

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.     

Temperate Bacterial Viruses as Double-Edged Swords in Bacterial Warfare

It has been argued that bacterial cells may use their temperate viruses as biological weapons. For instance, a few bacterial cells among a population of lysogenic cells could release the virus and kill susceptible non-lysogenic competitors, while their clone mates would be immune. Because viruses replicate inside their victims upon infection, this process would amplify their number in the arena. Sometimes, however, temperate viruses spare recipient cells from death by establishing themselves in a dormant state inside cells. This phenomenon is called lysogenization and, for some viruses such as the λ virus, the probability of lysogenization increases with the multiplicity of infection. Therefore, the amplification of viruses leads to conflicting predictions about the efficacy of temperate viruses as biological weapons: amplification can increase the relative advantage of clone mates of lysogens but also the likelihood of saving susceptible cells from death, because the probability of lysogenization is higher. To test the usefulness of viruses as biological weapons, we performed competition experiments between lysogenic Escherichia coli cells carrying the λ virus and susceptible λ-free E. coli cells, either in a structured or unstructured habitat. In structured and sometimes in unstructured habitats, the λ virus qualitatively behaved as a “replicating toxin”. However, such toxic effect of λ viruses ceased after a few days of competition. This was due to the fact that many of initially susceptible cells became lysogenic. Massive lysogenization of susceptible cells occurred precisely under the conditions where the amplification of the virus was substantial. From then on, these cells and their descendants became immune to the λ virus. In conclusion, if at short term bacterial cells may use temperate viruses as biological weapons, after a few days only the classical view of temperate bacterial viruses as parasitic agents prevails.     

Surveillance of acute respiratory virus infections in general practice

The ordinary postal system provides a simple method for the transportation of specimens for virus isolation to the laboratory. This was used to carry out an epidemiological survey, in general practice, of respiratory viruses in patients with acute respiratory disease. Three distinct outbreaks of myxovirus infections were recognized and most other types of respiratory viruses were isolated sporadically. After the initial “pilot” phase of the study an isolation rate of 24% was obtained over the months from October 1968 to June 1969 (20% isolations from adults'' specimens and 29% from those of children).     

Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) “COM,” (ii) “LYM” or “MYE,” (iii) “UNI,” and (iv) “UND.” The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.     

Molecular Basis for the Generation in Pigs of Influenza A Viruses with Pandemic Potential

Genetic and biologic observations suggest that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.     

Brevipalpus mites Donnadieu (Prostigmata: Tenuipalpidae) associated with ornamental plants in Distrito Federal, Brazil

Brevipalpus mites colonize a great number of fruit and ornamental plants. Mite species belonging to this genus have been associated with many plant viruses. Citrus leprosis (CiLV) is the most economically important virus transmitted by B. phoenicis mites. It has recently been shown that ornamental plant species can be alternative hosts of this virus. The high volume of trade and frequent movement of live ornamental plants make them efficient pest disseminators. Because of this, it is desirable to expand knowledge of potential pests aiming to guide the adoption of quarantine measures. This work reports ornamental plant hosts of Brevipalpus mites in the Distrito Federal (DF), as well the occurrence of symptoms consistent with Brevipalpus-borne plant viruses in these same hosts. Between July and September of 2005, five surveys were carried out in 14 localities within DF. Leaves and branches of fifty-five ornamental plant species were sampled. The species Pithecellobium avaremotemo Mart. is for the first time reported as a host for B. phoenicis (Geijskes), B. californicus Banks and B. obovatus Donnadieu species. Additionally, seven new species are reported as hosts for Brevipalpus within South America. New hosts are also listed for individual mite species. Typical symptoms of Brevipalpus-borne viruses were observed in Ligustrum sinense Lour., Pelargonium hortorum L.H. Bailey, Hibiscus rosa-sinensis L. and orchids (Dendrobium and Oncidium). The results of this work emphasize the potential role of the ornamental plants as vehicles for dissemination of Brevipalpus mites.     

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn’t recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3–6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3–6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3–6 hairpin than the wild-type hairpin. The SΔ1Δ3–6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30–37 days, while mice infected with SΔ1Δ3–6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV “flip and flop” telomeres are arranged.     

Down on the farm: Farm coronaviruses are a large reservoir for new spillover events into humans

Until COVID‐19, coronaviruses were largely overlooked by virologists. Yet, their abundance in mammalian species could cause new spillover events into humans. Subject Categories: Ecology, Microbiology, Virology & Host Pathogen Interaction

By now, coronaviruses have become notorious. Yet, before the SARS coronaviruses started wreaking havoc with human society, coronaviruses were largely overlooked, even amongst virologists. “When I told another virologist at a meeting what I was working on”, recalled Peter Rottier at Utrecht University, the Netherlands, who began his coronavirus research in 1979, “that person said they''d never heard of coronaviruses”. With plenty of other viruses such as influenza and HIV demanding attention, funders saw no reason to support research on the endemic human coronaviruses in the 1980s and 1990s since they caused mostly common cold.

With plenty of other viruses such as influenza and HIV demanding attention, funders saw no reason to support research on the endemic human coronaviruses in the 1980s and 90s…

Clinicians and virologists began taking much notice around 2003 and 2004 after the SARS coronavirus inflicted about 800 deaths before the outbreak receded, but the potential of this virus family to spillover into new species and mutate to more virulent strains had long been recognized by veterinarians, because coronaviruses inflict significant diseases in poultry, cattle and especially pigs. Virologists now are taking a closer look at some of these diseases to better understand coronaviruses and why they may be relevant for potential future pandemics.     

Plant history and soil history jointly influence the selection environment for plant species in a long‐term grassland biodiversity experiment

Long‐term biodiversity experiments have shown increasing strengths of biodiversity effects on plant productivity over time. However, little is known about rapid evolutionary processes in response to plant community diversity, which could contribute to explaining the strengthening positive relationship. To address this issue, we performed a transplant experiment with offspring of seeds collected from four grass species in a 14‐year‐old biodiversity experiment (Jena Experiment). We used two‐ and six‐species communities and removed the vegetation of the study plots to exclude plant–plant interactions. In a reciprocal design, we transplanted five “home” phytometers (same origin and actual environment), five “away‐same” phytometers (same species richness of origin and actual environment, but different plant composition), and five “away‐different” phytometers (different species richness of origin and actual environment) of the same species in the study plots. In the establishment year, plants transplanted in home soil produced more shoots than plants in away soil indicating that plant populations at low and high diversity developed differently over time depending on their associated soil community and/or conditions. In the second year, offspring of individuals selected at high diversity generally had a higher performance (biomass production and fitness) than offspring of individuals selected at low diversity, regardless of the transplant environment. This suggests that plants at low and high diversity showed rapid evolutionary responses measurable in their phenotype. Our findings provide first empirical evidence that loss of productivity at low diversity is not only caused by changes in abiotic and biotic conditions but also that plants respond to this by a change in their micro‐evolution. Thus, we conclude that eco‐evolutionary feedbacks of plants at low and high diversity are critical to fully understand why the positive influence of diversity on plant productivity is strengthening through time.