Lifespan estimation in marine turtles using genomic promoter CpG density.

Maximum lifespan for most animal species is difficult to define. This is challenging for wildlife management as it is critical for estimating important aspects of population biology such as mortality rate, population viability, and period of reproductive potential. Recently, it has been shown cytosine-phosphate-guanine (CpG) density is predictive of maximum lifespan in vertebrates. This has made it possible to predict lifespan in long-lived species, which are generally the most intractable. In this study, we use gene promoter CpG density to predict the lifespan of five marine turtle species. Marine turtles are a particularly difficult group for lifespan estimation because of their migratory behaviour, longevity and high juvenile mortality rates, which all restrict individual tracking over their lifespan. Sanger sequencing was used to determine the CpG density in selected promoters. We predicted the lifespans for marine turtle species ranged from 50.4 years (flatback turtle, Natator depressus) to 90.4 years (leatherback turtle, Dermochelys coriacea). These lifespan predictions have broad applications in marine turtle research such as better understanding life cycles and determining population viability.

Introduction

Marine turtles are slow growing, long-lived, and migrate vast distances in the ocean [1]. This makes it difficult to determine demographic characteristics of wild populations. Although mark-recapture studies of marine turtles can determine certain features of populations such as survival probabilities, it is difficult to determine the full extent of life cycles [2]. Consequently, making broader predictions relating to the risk of extinction, population growth, and viability due to limited age and longevity data is challenging [3]. Although marine turtles are known to be long-lived, the true longevity of each species is unknown [4]. Marine turtles epitomise the difficulties in generating lifespan or longevity information in many wild animal species.

Lifespan is difficult to define for most species of animals, especially in long-lived species, which may outlive a generation of researchers. Lifespan is commonly regarded as being the highest recorded age of an individual or the age at death within a selected population [5]. Lifespan is an essential characteristic for any species and has implications for wildlife management. Lifespan is associated with life-history traits such as reproductive capacity and the probability of mortality [6]. Currently, of the seven marine turtle species that occur globally, only the Green sea turtle (Chelonia mydas) has a reliable lifespan value in the Animal Ageing & Longevity Database (An Age; 75 years) [7-9]. Lifespan predictions for the remaining species is typically based on a small number of ad hoc and opportunistic records, often for animals held in captivity [10]. This limits the number of analyses relating to population growth and viability that can be performed for the other marine turtles as they require longevity data [3].

Previous research has found the frequency of cytosine-phosphate-guanine (CpG) sites in selected gene promoters can be predictive of lifespan [11, 12]. This provides an alternative method to predicting lifespan in long-lived species. Here, we predict the lifespan of marine turtle species that occur in Australian waters using CpG density in gene promoters. The molecular lifespan predictions provided in this study have broad application in the wildlife management of marine turtles.

Materials and methods

Animal ethics

Animal ethics for the collection of tissue was approved by the Department of Biodiversity, Conservation and Attractions (FO25000245).

Tissue collection and DNA extraction

Tissue was collected from one individual of each species (Table 1). Flipper biopsies from marine turtles were stored in 70% ethanol. DNA was extracted from tissue using the DNeasy Blood & Tissue Kit (QIAGEN) following the manufacture’s protocol. DNA was quantified using a QIAxpert (QIAGEN).

Table 1

Locations of sea turtles where tissue was collected for DNA extraction.

Species	Location	Latitude	Longitude
Leatherback sea turtle (Dermochelys coriacea)	Albany, Western Australia	-30.505	115.066
Loggerhead sea turtle (Caretta caretta)	South Muiron Islands, Western Australia	-25.498	112.987
Olive Ridley sea turtle (Lepidochelys olivacea)	Roebuck Bay, Western Australia	-18.019	122.237
Hawksbill sea turtle (Eretmochelys imbricata)	Delambre Island, Western Australia	-20.451	117.076
Flatback sea turtle (Natator depressus)	Roebuck Bay, Western Australia	-18.036	122.284
Green sea turtle (Chelonia mydas)	South Muiron Islands, Western Australia	-20.425	115.591

PCR design and sanger sequencing

Since the five marine turtles of interest do not have published genomes, we used the green sea turtle genome (CheMyd 1.0) as a reference genome [13]. The green sea turtle is the only marine turtle with a reference genome available. The lifespan promoters were identified using Basic Local Alignment Search Tool (BLAST) v2.2.31 (S1 Appendix) [14]. Primers were designed using Primer3 v0.4.0 for an optimal primer length of 20bp and temperature of 60°C [15]. A temperature gradient (45–60°C) was used for each primer pair to determine the optimal annealing temperature in each species (S2 Appendix). PCR reactions that produced single band visualised on an agarose gel were used for Sanger sequencing (Australian Genome Research Facility). Promoter CpG density was determined by calculating the CpG frequency within the BLAST hit based on the Green Sea Turtle genome and dividing it by the BLAST hit length (bp).

Predicting lifespan

Lifespan prediction was determined using the model developed previously that is exclusive to five vertebrate classes [12]. Lifespan prediction is conducted by determining the CpG density within selected genomic promoters. Genomic promoters predictive of lifespan were identified by comparing the sequences of 29,598 promoters to a database of known animal lifespans [7, 16]. An elastic net regression model was used to regress the lifespans of 252 species against the CpG densities of the genomic promoters. The model returned a total of 42 genomic promoters and coefficients that can be used to predict lifespan. The model returns the most informative genomic promoters but does allow redundancy as not all species will contain all 42 genomic promoters. The model was found to have an error range of 5.9%. The model returns a single lifespan prediction, but the 5.9% error is given in ± years. CpG densities were calculated for each promoter that received a BLAST hit to the Green Sea Turtle genome. A significant BLAST hit in the green sea turtle genome was considered with an identity > 70%.

Average mass and length data

To determine whether basic morphological traits correlated with predicted lifespans, physical features including the average carapace length (mm) and mass (g) for each species was obtained from the Animal Diversity Web (ADW) database [17]. Olive ridley sea turtles did not have data available in the ADW database and were removed from the analysis. Pearson correlations between physical features and maximum lifespan were natural log (ln) transformed to determine if there was a linear relationship. All analyses were performed in R v3.5.1 [18].

Results

Lifespan prediction

Promoter CpG density used for lifespan prediction is provided in S3 Appendix. The lifespan prediction for five marine turtle species are detailed in Table 2. The green sea turtle was excluded from the analysis as it has a known lifespan [7-9]. Leatherback sea turtles were found to have the longest lifespan prediction at 90.4 ± 5.3 years and flatback sea turtles with the shortest at 50.4 ± 2.9 years.

Table 2

Lifespan prediction of marine turtle species using promoter CpG density.

Species	Prediction (- 5.9% Error)	Prediction	Prediction (+ 5.9% Error)
Leatherback sea turtle (Dermochelys coriacea)	85.1	90.4	95.7
Loggerhead sea turtle (Caretta caretta)	59.1	62.8	66.5
Olive Ridley sea turtle (Lepidochelys olivacea)	51.1	54.3	57.5
Hawksbill sea turtle (Eretmochelys imbricata)	50.1	53.2	56.4
Flatback sea turtle (Natator depressus)	47.4	50.4	53.4

Lifespan and physical features

We found a strong positive correlation between both the average length (cor = 0.95, p-value = 0.012) and mass (cor = 0.98, p-value = 0.0038) with the lifespan prediction from CpG densities in marine turtles (Fig 1). Positive correlations were also observed in untransformed data for both length (cor = 0.91, p-value = 0.030) and mass (cor = 0.96, p-value = 0.010).

Fig 1

Increasing a. carapace length and b. mass of marine turtle species with lifespan. Each dot represents a species. Average length and mass data was obtained from the Animal Diversity Web database [17].

Discussion

Marine turtles globally face many anthropogenic threats [19]. However, as with other long-lived organisms their lifespan is difficult to determine and data on this key life-history attribute is sparse. This may partly be attributed to the fact that they may out-live research projects or researchers themselves. Age-estimates for marine turtles do exist but are based on much weaker data than typically is available for short-lived species. A lifespan prediction, provides an immediate value thereby providing useful demographic parameter regarding marine turtle ecology. In this study, we have used a molecular approach to confirm marine turtles as being long-lived animals. We found the leatherback sea turtle to have the longest lifespan and the flatback sea turtle with the shortest, with a difference of 40 years. This suggests a high variance and specific lifespan between species. The lifespan predictions provide a fundamental parameter used in determining mortality rates [20]. This can be used in the wildlife management of marine turtles and determine if specific populations are at risk of extinction.

Reliable lifespan values for long-lived species are difficult to find within the literature, although some do exist for selected individuals. Leatherback sea turtles were found to have the longest lifespan at 90 years. They have been reported to live at least 30 years in the wild with informal evidence suggesting a longevity of 70–80 years [21]. Loggerhead sea turtles have also been reported to have a lifespan of at least 30 years and up to 60 years in the wild [22, 23]. Similarly, the Olive Ridley, Hawksbill, and Flatback sea turtles have had reported lifespans of at least 30 years and up to 50 years in the wild [24, 25]. These reported lifespan values are supportive of the molecular predictions. A limitation of these studies is the low samples size as they only followed selected individuals. The longevity of marine turtle’s life cycles makes it challenging to study and determine the maximum lifespan.

Age estimates of wild animals can provide insight into age at sexual maturation and longevity [26, 27]. Skeletochronology is used to determine the age of stranded deceased marine turtles [28, 29]. Previous studies have found, depending on the species, that the age at sexual maturity ranges from as early as 6 years (Kemp’s ridley sea turtle) to 35 years (leatherback sea turtle) [30-33]. Other studies researching the same species, but different populations have recorded different ages at sexual maturity. For example with loggerhead sea turtles age at sexual maturation can range from 20 years of age in North American populations to 35 years in Australia [34-36]. Although longevity can be determined from age at sexual maturation, it can range greatly between different populations. Older age at sexual maturation ranges suggest marine turtles are long-lived animals. Other studies have found wild sea turtles of at least 40 years of age [28, 37]. Many of these age estimates are on the lower end of the lifespan predictions presented in this paper. Older individuals may exist in the wild and are not recorded since skeletochronology can only be carried out on deceased individuals. Therefore, the lifespan predictions provide a useful but potentially conservative values. However, it is important to note that older sea turtles are known to exist, primarily in captivity. It is well known animals, including reptiles, which are kept in captivity generally live longer than their wild counterparts [38, 39]. The lifespan predictions presented here suggest they may be on the upper end of what can be achieved in the wild but may be considered low to what can occur in captivity.

We found two morphological metrics of turtle size to strongly correlate with increasing lifespan (Fig 1). When more data becomes available the loggerhead and Kemp’s ridley sea turtle morphological and lifespan data can be added to the analyses. As a life-history strategy this may reflect a lower death rate in larger animals from extrinsic causes such as predation [40]. This may be the case with marine turtles since except for humans and large sharks, adults have few predators [41]. This correlation between size and longevity is well established in other taxa, supporting our findings in marine turtles [42]. To our knowledge this is the first time that this has been demonstrated in marine turtles, an ancient vertebrate group [42].

The main limitation of using a molecular method to predict lifespan is the generalisation of the species. A single molecular prediction does not account for population differences. Environmental pressures differ between populations which may reduce life expectancies. Without factoring environmental pressures, the molecular method cannot be used to make predictions for specific populations or individuals. Rather, it represents a potential maximum lifespan for the species. A maximum lifespan can be used as a reference tool see if individuals within a population are reaching their natural limit. If their life expectancy is low compared to their maximum lifespan it may indicate a potential environmental factor that may be limiting their longevity. Another limitation is the lack of known age data. Skeletochronology is used to determine the age of turtles but by having a non-invasive method, older aged turtles can be determined. This can then be used to determine if some turtles are either approaching or exceeding the lifespan predictions in this paper. A limitation of the method used in this study to predict lifespan is the dependency on an assembled genome. Reference genomes are in different stages of assembly such as contigs, scaffolds, or at the chromosome level. This can introduce artefacts and may result in inaccurate CpG densities. In this study, sanger sequencing was used to determine CpG density thereby removing the possibility of a lack of coverage. Lifespan prediction from DNA has shown to be highly predictive across most speciose vertebrate classes, including reptilia [12]. In the absence of robust observational information on the lifespans of wild marine turtles, molecular predictions represent useful consistently derived foundation values for this iconic and vulnerable group of marine animals.

Green sea turtle genomic coordinates and primer sequences used to amplify promoter sequences.

(XLSX)

Click here for additional data file.

Species specific annealing temperatures for each primer pair.

(XLSX)

Click here for additional data file.

Promoter CpG density from sanger sequencing used to predict marine turtle lifespan.

(XLSX)

Click here for additional data file.

30 Jun 2020

PONE-D-20-15932

Lifespan estimation in marine turtles using genomic promoter CpG density

PLOS ONE

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Reviewer #1: For long-lived species, it is difficult or impossible for human observers to accurately estimate maximum lifespan through direct observation, leaving ecologists & other biologists with an important missing datum for their species of interest. In this manuscript, Mayne et al take advantage of a published phenomenon for vertebrates – the correlation between CpG density in a select set of gene promoters and observed maximal lifespan – to predict the lifespans for several species of long-lived marine turtles. This manuscript develops no method nor tests a hypothesis; instead, it provides the only source of maximum-lifespan data currently available for these species. And due to the impracticality of direct observation, it is unlikely that an alternative, direct observation will ever be produced. This manuscript will likely therefore be a valuable reference for those who study marine turtles.

The main problem with this manuscript would be easily remedied with text edits: the authors generally describe the results of their analysis as “lifespans” or “estimated lifespans”. This diction inaccurately reflects the nature of the manuscript: “estimate” suggests an imprecise but nonetheless observation-based measurement. There are NO observed lifespans in this manuscript, just predictions. As I’ve said above, that’s fine, and there is great value in these predictions. But it is important that the authors make that abundantly clear throughout, so that it will be obvious to even the most casual reader. In their prior publication, where they established this correlative phenomenon to hold across vertebrates (listed as ref. 12), these authors were very good about using the diction of “predictied lifespan”. They should maintain that diction in this manuscript.

A more specific example of my comment above: the y-axis labels in Fig 1 (and descriptive text on lines 90-3). The figure represents the correlation between actual data collected in this paper versus literature values for length & weight. It would be more appropriate to label the Y-axis in terms of the observed CpG density values (the actual data!) than in terms of a value inferred based on those densities.

There are a few places where this manuscript is overly vague, and some additional clarification should be added. In Table 2, the statistical nature of the “lower bound” and “upper bound” intervals is unclear and should be specified. Ditto for the “+/-“ qualifiers on line 88 of the Results. Are these all based on the 5.9% error for the correlation in reference 12? Or are statistics for these particular measurements of CpG density also taken into account? Secondly, the methods for “Lifespan estimation” should be somewhat expanded. I appreciate that published methods should only require brief summaries, but this is too brief given how central this computational method is to the manuscript. The authors cite reference 12, which in turn cites reference 11. The summary following “Briefly, as described previously…” in reference 12 should serve as a guide to the authors as to the level of detail that is needed here.

Reviewer #2: The authors present lifespan estimates for five sea turtle species which are noted for their longevity and with life history traits making experimental determination of lifespan intractable. The method used is based solely on CpG density and is applied here in non-model organisms for which only one species has a fully sequenced genome. Therefore, this work demonstrates a number of novel and useful discoveries, 1) lifespan can be estimated for wild populations that are unable to be tracked individually, 2) a subset of promoter sequences can be sequenced to estimate lifespan without full genome sequencing, 3) lifespan can be calculated in marine vertebrates which are not closely related to other species with known lifespans, and 4) only small tissue samples from individuals at any age are necessary to calculate lifespan, negating the need to keep animals in captivity or sacrificial tissue collection. While the authors had previous experience in generating lifespan data for species with whole genomes available, this is the first report of species lifespan estimates being generated using de novo Sanger sequencing from tissue in non-sequenced animals. The authors also report the first link between size and longevity in marine turtles, a feature seen in many terrestrial species but never before confirmed in marine turtles. These lifespan estimates will be particularly useful, the authors note, in ecological studies of population age structure to determine whether ecological pressures are limiting populations from reaching their natural lifespans.

I note that the lifespan estimates do not uniformly correspond to phylogeny. For example, Lepidochelys olivacea and Caretta caretta are most closely related, and have similar estimates (62 and 54 years), while Natator depressus is most closely related to the green sea turtle Chelonia mydas, yet their lifespans are quite divergent, 50 and 75 years respectively. These differences suggest that these species have probably been under diverging selection pressures, at least regarding lifespans, although a 20-30 million range since speciation is a long time to collect differences.

Overall the authors did an outstanding job and provided novel, useful age estimates for a vulnerable keystone species with previously unreliable age estimates. This paper is concise, well-presented, and serves as a guide for others to report future molecular based lifespan estimates.

Minor concerns:

Though I recommend “Accept”, the authors should still briefly summarize the model used for the prediction and how it was used with the density data determined here in the methods section lines 72-73. It’s a bit too brief in this manuscript.

Calculating CpG density by dividing CpG frequency by the BLAST hit length is acceptable when using a single reference genome with uniform coverage and relatively closely related species. However, there is a risk of artefacts if this method is expanded using multiple reference genomes that may have disparate quality or coverage, leading to variable length BLAST hits for similar promoter regions. This concern is not applicable here due to study design but may be an issue that could be addressed by determining a consistent length or algorithm for CpG density measures in genomic regions in future studies.

Reference 6 has a URL attached that does not seem correct.

In the results, line 87, the green sea turtle lifespan refers to ref #6 and should probably be ref #7-9 or another.

**********

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Reviewer #2: Yes: Christopher Faulk

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13 Jul 2020

Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: For long-lived species, it is difficult or impossible for human observers to accurately estimate maximum lifespan through direct observation, leaving ecologists & other biologists with an important missing datum for their species of interest. In this manuscript, Mayne et al take advantage of a published phenomenon for vertebrates – the correlation between CpG density in a select set of gene promoters and observed maximal lifespan – to predict the lifespans for several species of long-lived marine turtles. This manuscript develops no method nor tests a hypothesis; instead, it provides the only source of maximum-lifespan data currently available for these species. And due to the impracticality of direct observation, it is unlikely that an alternative, direct observation will ever be produced. This manuscript will likely therefore be a valuable reference for those who study marine turtles.

The main problem with this manuscript would be easily remedied with text edits: the authors generally describe the results of their analysis as “lifespans” or “estimated lifespans”. This diction inaccurately reflects the nature of the manuscript: “estimate” suggests an imprecise but nonetheless observation-based measurement. There are NO observed lifespans in this manuscript, just predictions. As I’ve said above, that’s fine, and there is great value in these predictions. But it is important that the authors make that abundantly clear throughout, so that it will be obvious to even the most casual reader. In their prior publication, where they established this correlative phenomenon to hold across vertebrates (listed as ref. 12), these authors were very good about using the diction of “predictied lifespan”. They should maintain that diction in this manuscript.

Response: As the reviewer points out there are no observed lifespans for marine turtles and therefore the use of estimated lifespan in the manuscript is inaccurate and can cause confusion. To amend the problem the term estimated lifespan has been replaced with predicted lifespan.

A more specific example of my comment above: the y-axis labels in Fig 1 (and descriptive text on lines 90-3). The figure represents the correlation between actual data collected in this paper versus literature values for length & weight. It would be more appropriate to label the Y-axis in terms of the observed CpG density values (the actual data!) than in terms of a value inferred based on those densities.

Response: The reviewer makes an important point in regards to the Y-axis labels in Figure 1. We have now modified the Y-axis labels from “Maximum Lifespan” to “Lifespan Prediction from CpG density”. The text on lines 90-3 have also been updated to reflect the Y-axis label changes.

There are a few places where this manuscript is overly vague, and some additional clarification should be added. In Table 2, the statistical nature of the “lower bound” and “upper bound” intervals is unclear and should be specified. Ditto for the “+/-“ qualifiers on line 88 of the Results. Are these all based on the 5.9% error for the correlation in reference 12? Or are statistics for these particular measurements of CpG density also taken into account? Secondly, the methods for “Lifespan estimation” should be somewhat expanded. I appreciate that published methods should only require brief summaries, but this is too brief given how central this computational method is to the manuscript. The authors cite reference 12, which in turn cites reference 11. The summary following “Briefly, as described previously…” in reference 12 should serve as a guide to the authors as to the level of detail that is needed here.

Response: The reviewer makes an important point regarding being too brief in the methods and the model.

In table 2 we have changed the headings to Prediction ± 5.9% and on lines 72-81 we have provided more detail on the model that was used for lifespan prediction. We now explain that the ± qualifiers are representing the 5.9% error in years. We also provide a brief discussion in the methods on how the model was initially concepted, build and how it can be applied on other species.

Reviewer #2: The authors present lifespan estimates for five sea turtle species which are noted for their longevity and with life history traits making experimental determination of lifespan intractable. The method used is based solely on CpG density and is applied here in non-model organisms for which only one species has a fully sequenced genome. Therefore, this work demonstrates a number of novel and useful discoveries, 1) lifespan can be estimated for wild populations that are unable to be tracked individually, 2) a subset of promoter sequences can be sequenced to estimate lifespan without full genome sequencing, 3) lifespan can be calculated in marine vertebrates which are not closely related to other species with known lifespans, and 4) only small tissue samples from individuals at any age are necessary to calculate lifespan, negating the need to keep animals in captivity or sacrificial tissue collection. While the authors had previous experience in generating lifespan data for species with whole genomes available, this is the first report of species lifespan estimates being generated using de novo Sanger sequencing from tissue in non-sequenced animals. The authors also report the first link between size and longevity in marine turtles, a feature seen in many terrestrial species but never before confirmed in marine turtles. These lifespan estimates will be particularly useful, the authors note, in ecological studies of population age structure to determine whether ecological pressures are limiting populations from reaching their natural lifespans.

I note that the lifespan estimates do not uniformly correspond to phylogeny. For example, Lepidochelys olivacea and Caretta caretta are most closely related, and have similar estimates (62 and 54 years), while Natator depressus is most closely related to the green sea turtle Chelonia mydas, yet their lifespans are quite divergent, 50 and 75 years respectively. These differences suggest that these species have probably been under diverging selection pressures, at least regarding lifespans, although a 20-30 million range since speciation is a long time to collect differences.

Overall the authors did an outstanding job and provided novel, useful age estimates for a vulnerable keystone species with previously unreliable age estimates. This paper is concise, well-presented, and serves as a guide for others to report future molecular based lifespan estimates.

Minor concerns:

Though I recommend “Accept”, the authors should still briefly summarize the model used for the prediction and how it was used with the density data determined here in the methods section lines 72-73. It’s a bit too brief in this manuscript.

Response: Reviewer 2 shares the same concern as reviewer 1 regarding the methods section as being to brief. The model is described in more detail on lines 72-81.

Calculating CpG density by dividing CpG frequency by the BLAST hit length is acceptable when using a single reference genome with uniform coverage and relatively closely related species. However, there is a risk of artefacts if this method is expanded using multiple reference genomes that may have disparate quality or coverage, leading to variable length BLAST hits for similar promoter regions. This concern is not applicable here due to study design but may be an issue that could be addressed by determining a consistent length or algorithm for CpG density measures in genomic regions in future studies.

Response: The reviewer makes an important point regarding reference genomes with disparate quality or coverage. This would be difficult to incorporate into a model as genome coverage would be factor of both genome size and sequencing depth. It would be ideal to only use the model to predict lifespan using fully assembled genomes. We have now provided this discussion on lines 168 -172, to urge the reader that the method is ideal to fully assembled genomes.

Reference 6 has a URL attached that does not seem correct.

In the results, line 87, the green sea turtle lifespan refers to ref #6 and should probably be ref #7-9 or another.

Response: Ref 6 was incorrect and has been replaced with correct refs 7-9 as the reviewer has pointed out.

16 Jul 2020

Lifespan estimation in marine turtles using genomic promoter CpG density

PONE-D-20-15932R1

Dear Dr. Mayne,

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PLOS ONE

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Reviewers' comments:

21 Jul 2020

PONE-D-20-15932R1

Lifespan estimation in marine turtles using genomic promoter CpG density

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