Fusarium oxysporum f. sp. cubense 4 (Foc4), is regarded as one of the most devastating diseases worldwide. Cavendish cultivar ‘Yueyoukang 1’ was shown to have significantly lower disease severity and incidence compared with susceptible cultivar ‘Brazilian’ in greenhouse and field trials. De novo sequencing technology was previously performed to investigate defense mechanism in middle resistant ‘Nongke No 1’ banana, but not in highly resistant cultivar ‘Yueyoukang 1’. To gain more insights into the resistance mechanism in banana against Foc4, Illumina Solexa sequencing technology was utilized to perform transcriptome sequencing of ‘Yueyoukang 1’ and ‘Brazilian’ and characterize gene expression profile changes in the both two cultivars at days 0.5, 1, 3, 5 and 10 after infection with Foc4. The results showed that more massive transcriptional reprogramming occurs due to Foc4 treatment in ‘Yueyoukang 1’ than ‘Brazilian’, especially at the first three time points, which suggested that ‘Yueyoukang 1’ had much faster defense response against Foc4 infection than ‘Brazilian’. Expression patterns of genes involved in ‘Plant-pathogen interaction’ and ‘Plant hormone signal transduction’ pathways were analyzed and compared between the two cultivars. Defense genes associated with CEBiP, BAK1, NB-LRR proteins, PR proteins, transcription factor and cell wall lignification were expressed stronger in ‘Yueyoukang 1’ than ‘Brazilian’, indicating that these genes play important roles in banana against Foc4 infection. However, genes related to hypersensitive reaction (HR) and senescence were up-regulated in ‘Brazilian’ but down-regulated in ‘Yueyoukang 1’, which suggested that HR and senescence may contribute to Foc4 infection. In addition, the resistance mechanism in highly resistant ‘Yueyoukang 1’ was found to differ from that in middle resistant ‘Nongke No 1’ banana. These results explain the resistance in the highly resistant cultivar and provide more insights in understanding the compatible and incompatible interactions between banana and Foc4.Banana wilt disease, caused by the fungal pathogen  Musa spp. (bananas and plantains) is one of the most important fruit crops in the world and their global annual production account to more than 120 Mt Musa crop not only has the prominence as a dessert fruit, but also provides a vital source of food to many inhabitants of the humid tropics. Banana cultivation, like that of all other crop species, is affected by distinct constraints, among which Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is considered to be one of the most important threats Fusarium oxysporum, Foc4 is a typical necrotrophic fungus As de novo sequencing technology has been successfully used for molecular mechanism investigation of plants after pathogen infection, such as grape Currently genomics and disease-resistance genomics resources of banana that are available online are very limited. The lack of massive genomics resources is one of the major constraints in banana wilt-resistant breeding. Recently the rapid, low-costing next generation sequencing (high-throughput deep sequencing) technology has become a powerful tool for discovery of novel gene, expression profiling, comparative genomics study and evolutionary genomics research To obtain more transcriptome information of banana and further understand the molecular mechanism of the banana resistance against wilt disease in highly resistant cultivar, we performed transcriptome sequencing of the highly resistant ‘Yueyoukang 1’ banana using Illumina technology. A total of 87,845 unigenes were obtained and subsequently served as reference database for gene expression profile analysis. KEGG annotation revealed that ‘Plant-pathogen interaction’ and ‘plant hormone signal transduction’ pathway respectively included 2579 and 2696 genes. We further monitored gene expression profile changes in both ‘Yueyoukang 1’ and ‘Brazilian’ at time points 0.5, 1, 3, 5 and 10 days post Foc4 infection. The results showed that the number of differentially expressed genes in highly resistant ‘Yueyoukang 1’ was far more than that in susceptible ‘Brazilian’ at the first three infection time points. It was interesting that the expression patterns of many defense genes involved in PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), regulation of ion influx and cell wall reinforcement were different between ‘Yueyoukang 1’ and ‘Brazilian’ challenged with Foc4. The study first investigates the resistance mechanism in highly resistant banana against Foc4 using the Illumina Solexa sequencing technology and provides more insights into the understanding of banana-Foc interaction.6 condia/ml for 30 minutes and then were planted back the pots. The entire root system was harvested at time points 0.5, 1, 3, 5 and 10 days post infection (DPI), flash frozen in liquid nitrogen and stored at −70°C. Nine plants were used for each time point and all collection roots of the nine plants were pooled together. The roots harvested from the uninfected banana plants at 0 day as described above were served as a control.‘Brazilian’  is a cultivar susceptible to Foc4, and ‘Yueyoukang 1’  is a cultivar highly resistant to Foc4 Total RNA was extracted from roots of cv. ‘Brazilian’ and cv. ‘Yueyoukang 1’ using the QIAGEN RNeasy plant mini kit , respectively, and treated with RNase-free DNAse I . RNA integrity was confirmed using the 2100 Bioanalyzer (Agilent Technologies) with a minimum RNA integrated number value of 8. The mixture of equal amounts of the two cultivar root RNA samples for transcriptome analysis were prepared using Illumina's kit following manufacturer's recommendations. Briefly, mRNA was purified from 20 µg of total RNA using Sera-mag Magnetic Oligo (dT) Beads (Illumina) and then was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were used for double-stranded cDNA synthesis using double-stranded cDNA Synthesis kit  with random hexamer (N6) primers (Illumina). These cDNA fragments then went through an end repair process, phosphorylation, and ligation of adapters. Then these products were purified and enriched with PCR to create the final cDNA library.The cDNA library prepared was sequenced on the Illumina HiSeq™ 2000. And both ends of the cDNA were sequenced. The 90 bp pair-end raw reads were generated by the Illumina Genome Analyzer II system. Image deconvolution and quality value calculations were performed using the Illumina GA pipeline 1.3. Clean reads were obtained by moving the empty reads, the adaptor sequences, and the low-quality sequences (reads with unknown base pairs ‘N’). The clean reads were then assembled into contigs and scaffolds based on pair-end information using SOAPdenovo. −5, KEGG and COG. The proteins from NCBI nr database with the highest sequence similarity to the unigenes were used to assign functional annotations to the unigenes Annotation of the unigenes was performed by running our assembly against protein databases including NCBI nr with a cut-off E-value of 10Total RNA was respectively extracted from roots of cv. ‘Brazilian’ and cv. ‘Yueyoukang 1’ after TR4 infection 0, 0.5, 1, 3, 5, and 10 DPI using the QIAGEN RNeasy plant mini kit . DGE libraries were prepared using the Illumina gene expression sample prep kit. Briefly, mRNA was purified from 6 µg of total RNA using oligo(dT) magnetic beads. Double-stranded cDNA were directly synthesized on the RNA-bound beads and was subsequently digested with NlaIII. The fragmentized cDNAs which contained 3′ ends were washed away the magnetic beads, and then the Illumina adaptor 1 with an additional restriction site of Mme I was ligated to the 5′ ends of these cDNA fragments. After digestion with Mme I and CATG site, 21–22 bp tags containing the adaptor 1 sequence were released. Then the Illumina adaptor 2 was added to the 3′ end of the tags to produce a tag library. After 15 cycles of PCR amplification, 85 bp strips were purified from 6% PAGE gels. The cDNAs were digested, and the single-stranded molecules were attached to the Illumina sequencing chip for sequencing.Low quality tags (tags with unknown nucleotide “N”), empty tags (no tag sequence between the adaptors) and tags with only one copy number (which might result from sequencing errors) were removed from the DGE tags to generate clean tags. In order to annotate the tags, the clean tags containing CATG and 21 bp tag sequences were mapped to our transcriptome reference database, allowing no more than one nucleotide mismatch. All the tags mapped to reference sequences from multiple genes were filtered and the remaining tags were designated as unambiguous tags. For gene expression analysis, the number of expressed tags was calculated and normalized to the number of transcripts per million (TPM) tags. The differentially expressed tags were used for further mapping and annotation.−ΔΔCt method.Total RNA was extracted as described for the transcriptome library preparation and sequencing. RNA was reverse transcribed in a 20 µl reaction system using the PrimeScript™ RT Master Mix Kit (TaKaRa). Gene-specific primers were designed base on the gene sequences using Primer 5.0 software and the primer sequences are listed in The differentially expressed genes (DEGs) of cv. ‘Brazilian’ and cv. ‘Yueyoukang 1’ in different TR4 infection stages  were identified using an algorithm developed according to “The significance of digital gene expression profiles” described by Audic et al A cDNA library for transcriptome analysis was prepared from the equally mixed RNA of roots of the susceptible cultivar ‘Brazilian’ and the resistant cultivar ‘Yueyoukang 1’. A total of 56.79 million 90-pair end raw reads (SRA accession number SRP026137) were generated through Illumina sequencing. To facilitate sequence assembly, repetitive, low-complexity and low-quality reads were filtered out. Reliable reads were assembled into 193,809 contigs with a mean length of 293 bp. After clustering the contigs together, we finally obtained 87,845 distinct sequences that cannot be extended on either end. Such sequences were defined as unigenes, including 33,532 clusters and 54,313 singletons, with a mean length of 755 bp. The size of each unigene was not less than 300 bp. And between 200 bp and 1000 bp, there were 67412 distinct sequences. The size distribution of these unigenes is shown in −5. A total of 59,340 sequences had good comparability with known gene sequences in existing species, which represented about 67.54% of the total unigenes. The species distribution of the best match result for each sequence is shown in Vitis vinifera, followed by Oryza sativa Japonica Group (11.1%), Sorghum bicolor (8.4%), Brachypodium distachyon (8.2%), Ricinus communis (6.9%), Populus trichocarpa (6.2%), Oryza sativa Indica Group (5.7%). The proteins from NCBI nr database with the highest sequence similarity to the unigenes were used to assign functional annotations to the unigenes.The annotation of 87,845 unigenes was based on the sequence similarity search in protein databases using BLAST algorithm. We first searched the reference sequences using BLASTX against the non-redundant (nr) NCBI protein database with a cut-off E-value of 10Gene Ontology (GO) terms were assigned to banana unigenes based on their sequence matches to known proteins in the nr databases. A total of 43,305 unigenes, which were assigned with at least one GO term, can be categorized into 59 functional groups . Within Using the COG databases to annotate the banana unigenes, we found 20,713 distinct sequences having a COG classification. Among the 25 COG categories, the largest group was ‘General function prediction’ , followed by ‘Transcription’ (6053), ‘Replication, recombination and repair’ (4506), and ‘Signal transduction mechanisms’ (4223). The smallest groups were ‘Nuclear structure’ (5) and ‘Extracellular structures’ (17) .KEGG is a bioinformatics resource for linking genomes to life and the environment. The PATHWAY database records networks of molecular interactions in the cells, and variants of them specific to particular organisms. To identify the biological pathways that are active in the banana, we mapped the 59,340 unigene sequences to the reference canonical pathways in Kyoto Encyclopedia of Genes and Genomes. A total of 35,313 unigenes could be annotated in KEGG database and assigned to 127 KEGG pathways. The ‘metabolic pathway’ was the biggest term and contained 8747 unigenes occupying 24.77%, following by ‘biosynthesis of secondary metabolites’ , ‘plant hormone signal transduction’ , ‘endocytosis’ , ‘Plant-pathogen interaction’  .Total RNA was respectively extracted from roots of cv. ‘Brazilian’ and cv. ‘Yueyoukang 1’ after TR4 infection 0, 0.5, 1, 3, 5, and 10 DPI. Twelve DGE libraries (SRA accession number SRP026137) were sequenced and named BX, BX1, BX2, BX3, BX4, BX5 (from ‘Brazilian’), and YY, YY1, YY2, YY3, YY4, YY5 (from ‘Yueyoukang 1’), respectively. Each library generated about five million raw tags. After removing the low quality reads, the total number of clean tags in each library ranged between 4.3 and 4.8 million, occupying from 92.67 to 97.23%. All clean tags were mapped to our transcriptome reference database which was mentioned above. The percentage of these clean tags that could be mapped to the reference database ranged from 65.16 to 76.98%.The differentially expressed genes (DEGs) of cv. ‘Brazilian’ and cv. ‘Yueyoukang 1’ at different TR4 infection stages were identified using an algorithm developed according to the method described by Audic et al Metabolic pathways’ ‘Biosynthesis of secondary metabolites’ ‘Phenylpropanoid biosynthesis’ and ‘RNA transport’ were significantly enriched at all of the five infection stages. There were 13, 22, 8, 10 and 12 significantly enriched pathways for TR4 infection at 0.5, 1, 3, 5 and 10 DPI, respectively. The number of down-regulated DEGs was far more than up-regulated DEGs in almost all significantly enriched pathways at the first three time points. The remaining two time points contained most of significantly enriched pathways in which up-regulated DEGs were more than down-regulated DEGs. In susceptible cv. ‘Brazilian’, there were 13 significantly enriched pathways. Among these pathways, 3 common pathways including ‘Metabolic pathways’ ‘Phenylpropanoid biosynthesis’ and ‘Phenylalanine metabolism’ were found at all of the five infection stages. 0.5, 1, 3, 5 and 10 DPI respectively contained 5, 4, 9, 9 and 9 significantly enriched pathways. It was different from ‘Yueyoukang 1’ that the proportion of up-regulated DEGs in significantly enriched pathways was high in the first three time points of ‘Brazilian’. The time points of 0.5, 1 and 3 DPI respectively included 4, 2 and 3 significantly enriched pathways that had more up-regulated DEGs than down-regulated DEGs. And all the significantly enriched pathways of the following two time points contained more up-regulated DEGs than down-regulated DEGs. The summary of significantly enriched KEGG pathways influenced by Foc4 infection was shown in We evaluated the TR4-affected biological pathways of susceptible and resistant banana by significantly enrichment analysis of DEGs. Pathways with Q value≤0.05 were regarded as significantly enrichment. 25 KEGG pathways showed significantly enrichment in resistant cv. ‘Yueyoukang 1’, of which 4 common pathways, namely ‘Plant-pathogen interaction’ ‘Ribosome’ ‘Purine metabolism’ and ‘Phenylpropanoid biosynthesis’. ‘Plant-pathogen interaction’ with DEGs from 46 to 61 was involved in 1, 3, 5 and 10 DPI. ‘Ribosome’ with DEGs from 47 to 49 was involved in 0.5 and 3 DPI. ‘Purine metabolism’ with DEGs from 47 to 51 was involved in 0.5 and 1 DPI. ‘Phenylpropanoid biosynthesis’ with DEGs from 31 to 37 was involved in 5 and 10 DPI. ‘Brazilian’ had seven common top enriched pathways at all the five infection stages, including ‘Metabolic pathways’ ‘Biosynthesis of secondary metabolites’ ‘RNA transport’ ‘Endocytosis’ ‘Ether lipid metabolism’ ‘Glycerophospholipid metabolism’ and ‘mRNA surveillance pathway’. The total number of DEGs in each common top enriched pathway ranged from 23 to 342. Other top enriched pathways contained ‘Phenylpropanoid biosynthesis’ ‘Plant-pathogen interaction’ ‘Purine metabolism’ ‘Ribosome’ ‘Protein processing in endoplasmic reticulum’ and ‘Plant hormone signal transduction’. ‘Phenylpropanoid biosynthesis’ with DEGs from 15 to 45 was involved in 0.5, 3 and 10 DPI; ‘Plant-pathogen interaction’ with DEGs from 14 to 57 was involved in 0.5, 1, 5 and 10 DPI; ‘Purine metabolism’ with 13 DEGs was involved in 0.5 DPI; ‘Ribosome’ with DEGs from 25 to 34 was involved in 1, 3 and 5 DPI; ‘Protein processing in endoplasmic reticulum’ with 24 DEGs was involved in 1 DPI; ‘Plant hormone signal transduction’ with DEGs from 25 to 54 was involved in 3, 5 and 10 DPI  are one of the most important fruit crops in the world. Recently, banana draft genome sequencing was completed but the genome sequences are still not released −5. Functional annotation analysis revealed that 43,305 and 20,713 unigenes could be annotated in GO and COG databases, respectively. KEGG annotation analysis showed a total of 35,313 unigenes could be annotated in KEGG database and assigned to 127 KEGG pathways. The top 20 enriched KEGG pathways were shown as Bananas and plantains  libraries for each cultivar during different TR4 infection stages . DGE technology provides a quantitative measure of transcript abundance and has a higher precision than microarrays Pattern recognition receptors (PRRs) detect the conserved pathogen-associated molecular patterns (PAMPs) and trigger plant immunity to protect host from pathogen infection. Therefore PRRs play a fundamental role in PAMP-triggered immunity(PTI) Arabidopsis showed extreme susceptibility to necrotrophic fungi Brassinsteroid insensitive 1-associated receptor kinase 1 (BAK1) is another significant pattern recognition receptor in plant. It was required for PTI triggered by bacterial flagellin and elongation factor Tu. Flagellin-sensitive 2 (FLS2), a leucine-rich repeat receptor kinases, was characterized as PRR for the bacterial flagellin. FLS2 and BAK1 form a complex to initiate innate immunity in plant 2+ is utilized as an important secondary messenger in signaling cascades by plant cell and plays an essential role for production of hypersensitive reaction in plant response to biotic stress 2+ influx changed in banana response to Foc4 infection. CNGC4 was induced in response to pathogen attack and might participate in signaling pathways leading to HR CaPseudomonas syringae strains expressing avrRpm1 or avrB effector Pseudomonas syringae effector AvrPphB and the cleavage activates RPS5 to initiate ETI immunity in ArabidopsisThe majority of R proteins in plant containing cytoplasmic nucleotide-binding site (NB) and leucine-rich repeat (LRR) domains confer resistance to diverse pathogens such as bacteria, fungi, viruses, nematodes and insects ely. However, no any WRKY22 gene showed differently expressed in ‘Brazilian’ until 5 DPI. The number of up-regulated WRKY33 genes was more and the down-regulated was less in ‘Yueyoukang 1’ than ‘Brazilian’ at almost all the five time points. And up-regulated WRKY33 exhibited higher increase level in ‘Yueyoukang 1’ than ‘Brazilian’ at the first two time points. DREB protein was mainly up-regulated at 3, 5 and 10 day after inoculation and its increase level was very higher in ‘Yueyoukang 1’ than ‘Brazilian’. They have been documented to play an important role in resistance response to pathogen attack Plant transcription factors participated in regulation of the expression of defense-related genes against different types of microbial pathogen. In our research, three transcription factors including WRKY transcription factor 22, 33 and DREB protein showed different expression pattern between ‘Yueyoukang 1’ and ‘Brazilian’. In ‘Yueyoukang 1’, two WRKY22 genes were up-regulated and one was down-regulated at both first two time points; three, one and three WRKY22 genes showed up-regulated at 3, 5 and 10 DPI, respectivOne of the features of plant defense response is production of pathogenesis-related (PR) proteins that have been widely studied in plant pathogen resistance. In our research, the constitutive expression of glucanases and chitinases was significantly higher in cv. ‘Yueyoukang 1’ compared with the susceptible cv. ‘Brazilian’. When inoculated with the FOC4, gene expression of pathogenesis-related (PR)-1, glucanases and chitinases were affected in both cultivars. PR-1 was mainly up-regulated in the resistant ‘Yueyoukang 1’ and down-regulated or changeless in the susceptible ‘Brazilian’ at the first three time points. However, PR-1 mainly showed down-regulated in ‘Yueyoukang 1’ but up-regulated in ‘Brazilian’ at the last two time points, suggesting that PR-1 may be involved in the incompatible interaction at the early infection stages. The expression of glucanase changed dramatically over time in both cultivars. Glucanase genes were highly expressed in the resistant cv. ‘Yueyoukang 1’ at all time points compared with those in the susceptible cv. ‘Brazilian’. Chitinase gene was up-regulated starting at 1 DPI and came to the highest level at 5 DPI in ‘Yueyoukang 1’, while it showed up-regulated only at 1 and 10 DPI in the susceptible cv. ‘Brazilian’. The induction and greater accumulation of mRNA of PR-1, glucanases and chitinases in ‘Yueyoukang 1’ after TR4 infection suggested that these genes were associated with plant defense in banana roots. This was consistent with the previous study which reported that PR-1, glucanases and chitinases might serve to resist Foc in banana Arabidopsis, and treatment of Arabidopsis with JA induces COI1-dependent senescence Fusarium oxysporum in ArabidopsisFusarium oxysporum hijacks COI-mediated JA-signaling pathway to cause wilt-disease symptoms that lead to plant death in ArabidopsisIt has been reported that salicylic acid, jasmonic acid and ethylene play important roles in signaling networks and regulation developmental processes in plant response to biotic stress Fusarium oxysporum f. sp. NiveumPlant cell wall forms the first plant barriers to pathogen infection. On the one hand, plant cell wall acts as physical barrier to protect the whole cell. On the other hand, plants may perceive pathogen attack by sensing cell wall integrity In this study, we performed transcriptome and expression profiles sequencing of the highly resistant ‘Yueyoukang 1’ and the susceptible ‘Brazilian’ banana using Illumina technology, and compared expression profile differences between the two cultivars infected by Foc TR4. The results showed that genes related to CEBiP, BAK1, NB-LRR proteins, PR proteins, transcription factor and cell wall lignification were induced in ‘Yueyoukang 1’ banana, suggesting that these genes play important roles in the incompatible interactions between banana and Foc TR4. Meanwhile those genes controlling hypersensitive reaction and senescence, such as CNGC4, Rboh and genes in jasmonic acid pathway, were suppressed by TR4 in ‘Yueyoukang 1’ but were induced by TR4 in ‘Brazilian’, suggesting that TR4 may induce this response as part of its infection strategy. These genes initially identified by the method of comparative transcriptome analyses still require further research to determine their functions and dissect the complex interacting signaling networks. Some of defense-related genes show different expression patterns between highly resistant ‘Yueyoukang 1’ and middle resistant ‘Nongke No 1’ challenged with Foc TR4, suggesting that the two cultivars had different pathogen-resistance mechanism. In conclusion, our study expands the transcript information of banana, explains the resistance in the highly resistant cv. ‘Yueyoukang 1’ and gains more insights into banana-necrotrophic pathogen interactions. However, many genes involved in the incompatible interactions between banana and Foc4 still remain unknown. In the future, more work will be required to decipher the true nature of banana wilt resistance and successfully control banana wilt disease.Table S1Primers used in qRT-PCR for validation of differentially expressed genes.(XLS)Click here for additional data file.Table S2Significantly enriched KEGG pathways in ‘Yueyoukang 1’ and ‘Brazilian’.(XLS)Click here for additional data file.Table S3The first ten top enriched pathways in ‘Yueyoukang 1’.(XLS)Click here for additional data file.Table S4The first ten top enriched pathways in ‘Brazilian’.(XLS)Click here for additional data file.Table S5Verification of DGE analysis results by qRT-PCR.(XLS)Click here for additional data file.Table S6Differentially expressed genes related to plant resistance.(XLS)Click here for additional data file.


Puma concolor) cubs. We examined cub body mass, survival, and age of natal dispersal in relation to specific timing of birth. We also investigated the role of maternal age relative to timing of births. We captured mountain lion cubs while in the natal den to determine birth date, which allowed for precise estimates of the population birth pulse and age of natal dispersal. A birth pulse occurred during June–August. Body mass of cubs was related to litter size and timing of birth; heaviest cubs occurred in litters of 2, and those born after 1 July. Cubs born within pulse months exhibited similar survival to those born out of the pulse. We found that cubs born April–June dispersed at younger ages than those born after 1 July. There was less variation in birth timing for 1st litters of females than older females. We hypothesize that cubs born after the peak in births of neonate prey are advantaged by the abundance of vulnerable prey and those cubs and mothers realize an evolutionary advantage.We investigated potential advantages in birth timing for mountain lion ( Puma concolor) is better characterized as a pulse over several months Pattern of births for mountain lions . Months with higher than expected births included the period June–August; thus, we defined our birth pulse as occurring from 1 June to 31 August. We further delineated the year as spring (April–May), early pulse (June), middle pulse (July), late pulse (August), autumn (September–October) and winter , so we censored those animals from analyses regarding cub weights. We found that timing of birth  and litter size  influenced body mass of cubs at 1 week of age, whereas sex  had no effect. Mean body mass for male (n = 34) and female (n = 40) cubs at 1 week of age was 1.162 kg±0.112 (SE) and 1.102 kg±0.095, respectively.We used data for 74  cubs originally captured in natal dens, at ages ranging from 1–4 weeks, to estimate influence of birth timing on weight at 1 week of age. We found few cubs born during winter .There was a declining trend in body mass as litter size increased. Mean body mass of cubs in 2-, 3-, and 4-cub litters were 1.114 kg±0.135, 1.053 kg±0.091, and 0.912 kg±0.089, respectively. Cubs born in litters of 2 were 22% larger at 1 week of age than cubs in litters of 4 ; however, a trend existed, with cubs born in spring lightest and body mass of cubs increasing through autumn . Cubs born during spring and early pulse averaged 230 g±72 (SE) lighter in mass than cubs born during the middle pulse through autumn .After accounting for sex and litter size, mean mass of cubs born within the birth pulse (June–August) was similar to cubs born outside the birth pulse . Cubs born before the ungulate birth peak (n = 10) weighed 869 g (mean)±96 (SE) whereas cubs born coincident with ungulate birth peaks and 30 days following (n = 20) weighed 934±109 g. Those cubs born 30–60 days after the ungulate birth peak (n = 31) weighed 1,174±100 g, whereas cubs born >60 days after the ungulate birth peak (n = 13) weighed 1172±102 g.When we reevaluated birth timing relative to ungulate birth periods, we found a strong relationship to timing of birth and weight at 1-week of age  = 0.55, SE = 0.08; Female, n = 36, S(i) = 0.58, SE = 0.08; 2-sample t76 = 0.264, 2-sided P>0.50); therefore, we pooled sexes in subsequent analyses. We found no support  for our hypothesis that survival was greater for cubs born within the birth pulse  = 0.55, 95% C.I. = 0.41–0.69) compared to those born outside the pulse  = 0.58, 95% C.I. = 0.41–0.74). However, we found an increasing trend in survival relative to prey abundance and vulnerability  = 0.50, SE = 0.09) had mean survival rates lower than those born 30–60 days after the ungulate birth peak, but before winter  = 0.53, SE = 0.09; Oct–Nov, n = 12, S(i) = 0.67, SE = 0.14).No difference was documented in sex-specific survival . There was evidence that cubs born during the birth pulse dispersed when 2.5 months younger (±1.3 SE) than those born outside the pulse . However, there was stronger evidence  that cubs born during spring, early-, and mid-pulse  dispersed 4±1.3 months younger than cubs born in the last month of the birth pulse and winter . When we excluded cubs born in July , there was strong evidence of a mean 4±1.5 month difference in dispersal ages .We obtained dispersal ages for 22 juvenile mountain lions . Male mountain lions dispersed at a mean age of 14.7 months±0.84 (SE) and females dispersed at 15.3±1.15 months. While accounting for variation in timing of birth, we found no sex-related differences in natal dispersal age (All cubs that were born in early or mid-pulse dispersed during the summer (June–August) or autumn (September–November) after attaining an age of 1-year. All cubs born late in the birth pulse dispersed during winter (December–February) or spring (March–May) after becoming 1-year of age.n = 11) and occurred mostly in July (n = 6), and few occurred in June or August  were dispersed throughout the year, while still exhibiting a pulse in June–August , mule deer (O. hemionus), and elk (Cervus elaphus) exhibit a scent-less hiding strategy for 2–4 weeks, presumably to avoid predators until they are mobile enough to flee More cubs were born during July and August  than any other 2-month period, which occurred >30 days after the births of ungulate neonates in our study area. Neonates of white-tailed deer  are presumably heavier and more experienced, than those dispersing at 13 months (born before the switch). Some of the advantages might influence survival through the subadult life-stage, which translates to an evolutionary advantage for both mother and offspring. Thus, births of mountain lions should trend toward a narrower as opposed to wider birthing period.st time mothers than for older mothers. These data are in accordance with the prediction that birth timing is related to prey availability Because of the stochastic nature of most cub mortalities However, breadth of the population birth pulse is maintained by variation in reproductive maturity, birth intervals, and the vagaries of mortality. Logan and Sweanor Ovis canadensis], guanaco [Llama guanacoe]), yet birth peaks are predictable , that first litters of mountain lions will be closely timed to the birth peak of those primary prey species. We predict that in environments where prey are not born in predictable peaks or the sum of births in multiple primary prey species provide abundant neonates throughout the year , that mountain lions will not exhibit a birth pulse or the pulse will be broader than 3–4 months, characteristic of populations studied in North America We offer a hypothesis and predictions regarding the birth pulse in mountain lions and recommend testing with data from other populations. We hypothesize that mountain lion births are adaptively timed closely to the availability of primary prey in environments, where primary prey exhibit predictable birth peaks, but that reproductive biology and random mortality events after the initial litter result in a broadened birth pulse that adheres less strongly to the timing of prey availability. We predict that in environments where the primary prey for mountain lions exhibit “hider" predator-avoidance strategies  and where timing of ungulate birth peaks is predictable  that first litters of mountain lions will be closely timed to follow the switch from “hider" to “fleer" by neonates. We also predict that in environments where primary prey species do not exhibit a “hiding" anti-predator strategy as neonates are capable of fleeing shortly after birth  forests, but also contains spruce (Picea glauca), aspen (Populus tremuloides), birch (Betula spp.), and oak (Quercus macrocarpa) trees Odocoileus virginianus), mule deer (O. hemionus), elk, American bison (Bison bison), mountain goat (Oreamnos americanus), and bighorn sheep as well as domestic livestock species. No other large carnivores were present in this system. Bobcat (Lynx rufus) and coyote (Canis latrans) are mesocarnivores that occur sympatrically with mountain lions.We studied mountain lions in the Black Hills ecoregion  of South Dakota, USA between 2006 and 2009. The Black Hills are a complex of forested ridges, valleys, and steep canyons Field Methods.—We used foot-snares When cubs were approximately 9 months old, we recaptured them by using the same techniques as those used for adult mountain lions and refitted cubs with larger radiocollars  or global positioning system (GPS) collars . We used fixed-wing aircraft or satellite-telemetry to relocate subadults and mothers. When a juvenile mountain lion left the natal area, we recorded the mid-date between the last location in the natal area and the first location outside the natal area. In some cases, dispersing mountain lions made exploratory movements and returned to the natal area and subsequently left again or established a home area at least partly overlapping the natal area . We recorded dispersal age as the mid-date the juvenile made the initial exploratory movement, regardless of subsequent return to the natal area. If an animal was not located outside the natal area until the animal became 3 years old, we censored the animal from analyses. These animals were censored because exploratory movements can be of short duration st one-third), middle , late , and outside pulse. In addition, we delineated cub births as before ungulate birth peak (before 1 June), before neonate ungulates switch predator avoidance strategies (hiding vs. fleeing unpublished data). The birth peak for mule deer was 7–14 June unpublished data).We plotted all birth dates and defined the birth pulse as the period where ≥40% of births occurred and number of births were higher than expected F-tests within a MANOVA framework to test for differences in birth timing, while accounting for differences in sex, litter size, and age at capture. We used a known-fates model in Program MARK t-test to determine if differences in survival occurred between male and female cubs, despite timing of birth. We ran Program MARK analyses with and without sex as a covariate to determine if sex was related to survival. We analyzed cub survival in 2 ways. First, we placed cubs in categories; within or outside of the birth pulse to replicate the analysis conducted by Laundré and Hernández We used JMP 8.0  statistical software to perform statistical analyses. We used chi-squared test for independence to determine if the monthly distribution of litters was uniform through the year st litter, >1st litter). We visually compared birth month frequencies by mother's age and litter sequence.To investigate the role of mother's age on birth timing, we calculated the monthly frequency distribution of litters born by mother's age . We also calculated the monthly frequency distribution of litters born by litter sequence (i.e., 1


Photoactivatable fluorescent probes developed specifically for single molecule detection extend advantages of single molecule imaging to high probe density regions of cells and tissues. They perform in the native biomolecule environment and have been used to detect both probe position and orientation.2 minimization.Fluorescence emission from a single photoactivated probe captured in an oil immersion, high numerical aperture objective, produces a spatial pattern on the detector that is a linear combination of 6 independent and distinct spatial basis patterns with weighting coefficients specifying emission dipole orientation. Basis patterns are tabulated for single photoactivated probes labeling myosin cross-bridges in a permeabilized muscle fiber undergoing total internal reflection illumination. Emitter proximity to the glass/aqueous interface at the coverslip implies the dipole near-field and dipole power normalization are significant affecters of the basis patterns. Other characteristics of the basis patterns are contributed by field polarization rotation with transmission through the microscope optics and refraction by the filter set. Pattern recognition utilized the generalized linear model, maximum likelihood fitting, for Poisson distributed uncertainties. This fitting method is more appropriate for treating low signal level photon counting data than χResults indicate that emission dipole orientation is measurable from the intensity image except for the ambiguity under dipole inversion. The advantage over an alternative method comparing two measured polarized emission intensities using an analyzing polarizer is that information in the intensity spatial distribution provides more constraints on fitted parameters and a single image provides all the information needed. Axial distance dependence in the emission pattern is also exploited to measure relative probe position near focus. Single molecule images from axial scanning fitted simultaneously boost orientation and axial resolution in simulation. Single molecule detection characterizes individual states of a system providing the “bottom-up” description that can be uniquely formulated and tested without the ambiguities inherent in ensemble derived observations Light is emitted by a probe in the aqueous medium but near the glass/aqueous interface formed by the coverslip of a high numerical aperture (NA) oil immersion microscope objective. The interface substantially affects the emitted light it transmits The lateral PSF readily undergoes the 6 basis pattern devolution in 2-D because the CCD camera records the lateral photon distribution in the 2-D pixel array. The 2-D spatial pattern defines the dipole orientation and contains information specifying the axial position of the probe. Basis patterns depend on the axial dimension conferring sensitivity to the image for the axial position of the probe because a changing sample axial position alters pattern shape. We calibrated the axial dimension of image space to exploit the position sensitivity of the patterns using an axial translating camera. The setup records the axial photon distribution using the translating CCD detector. It provides <10 nm precision (super-resolution) of the peak position of a point object and calibrates the axial image space against the real axial dimension in sample space. When calibrated, we assign relative sample space axial distances to emission patterns. We also show that an axial scan data set consisting of three images from a translating objective on a single emitter provides superior resolution for all quantities investigated.We tested the basis pattern analysis method for probe dipole orientation detection on photoactivated green fluorescent protein (PA-GFP) tagged myosin regulatory light chain (RLC) exchanged, permeabilized skeletal muscle fibers, in rigor. Orthogonal polarized photoactivation laser pulses were applied to the PA-GFP tagged myosin cross-bridges in separate fiber fields to photoselect contrasted oriented sub-populations of probes that are intrinsically oriented by fiber structure. Evidence for both orientation selecting processes seen in the data validates the approach.Our protocol for obtaining rabbit tissue is approved by the Mayo Institutional Animal Care and Use Committee, Protocol A4208.ATP, dithiothreitol (DTT), leupeptin, and phenylmethanesulfonyl fluoride (PMSF) were from Sigma Chemical . All chemicals were analytical-grade or Ultra-Pure if available. Carboxylate-modified fluorescent microspheres were purchased from Molecular Probes .Rigor solution contains 10 mM imidazole, 2.5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 2.2 mM Mg acetate, 130 mM potassium propionate, 0.2 mM PMSF, 0.8 µg/mL leupeptin, and 1 mM DTT.Psoas muscle fibers were prepared from rabbit and the native myosin regulatory light chain (RLC) exchanged with PA-GFP tagged human cardiac RLC (HCRLC-PAGFP) as described 4 fold dilution from stock into distilled water giving sphere concentration of 1.4×1011 spheres/mL.Red-orange carboxylate-modified fluorescent spheres had 40 nm diameter and excitation/emission maxima at 565/580. Sphere concentrations were computed using the formula from the manufacturer and diluted in water. We used a 10All experiments were conducted at room temperature.Clean #1 glass coverslips were sonicated for 10 min in ethanol then plasma cleaned  for 15–30 min. Coverslips were placed on a 1×3-inch brass slide with a large hole cut out, permitting the objective from the inverted microscope to contact the coverslip through immersion oil. A water tight chamber was constructed on top of the coverslip as described Through-the-objective total internal reflection (TIR) occurs at the coverslip/aqueous interface where a HCRLC-PAGFP exchanged muscle fiber in aqueous buffer solution contacts the coverslip and is illuminated by the evanescent field. Diluted nanospheres were flowed into the sample chamber and allowed to dry. After drying, some spheres were strongly attached to the substrate surface in a sparse random spatial distribution that remained intact when distilled water refills the sample chamber. Fluorescent spheres were TIR or epi-illuminated depending on the experiment.Overall computer control of the microscope is exercised through a custom written Labview  routine and drivers supplied by the manufacturers. The Labview software coordinates image capture by the camera with movement of the various translating elements in or around the microscope. Translating elements were controlled via a RS232 port (LEP stage) or a USB interface (MCL nanopositioners). Wasabi! software  captures images following triggering by the Labview program via a counter output TTL pulse (NI6602).For TIRF microscopy, laser excitation is focused on the back focal plane of the objective and incident from the glass side of a glass/aqueous interface at angles greater than critical angle for TIR. Although light is totally reflected, an evanescent field created in the water medium and decaying exponentially with distance from the interface, excites fluorophores within ∼100 nm of the surface A HCRLC-PAGFP exchanged muscle fiber is illuminated by the evanescent field. Sparse PA-GFP photoactivation was accomplished under TIR using 10–20 sec exposure to 488 nm light from the argon ion laser. Laser intensity was ∼10–100 fold higher during photoactivation compared to that used during fluorescence excitation of photoactivated PA-GFPs. A larger field of the muscle fiber was shown previously showing the sparse photoactivation of the PA-GFP under these conditions B→NA, where total molecules N is the sum of un-photoactivated (NB) and photoactivated (NA) species, describes fluorescence photoactivation. Solving for NA(t),A is the activation rate and tA is the activating light pulse duration. Integrated absorption cross-section, kA ∝ i, the normalized probability for its photoactivation, γA,i, is, A,i << 1. In simulation a random number, ξ, between 0 and 1 is compared to γA,i. When ξ < γA,i, molecule i is photoactivated. This procedure selects with higher probability molecules having Irreversible isomerization, Ni,e,ν, th molecule of the photoactivated species (wavelength band near 490 nm), ||  =  / will contain dependence on For comparison with results reporting polarized emission intensity ratios, we compute the photoactivated single molecule polarized fluorescence, FSimulation of the photoselected set of activatable probes allows separate orientations for rger see .O is the objective effective focal length, fT is the tube lens focal length, and L is tube length. In all measurements, ε≤1 µm, while other quantities in eq. 5 are 3–180 mm hence δ and ε are linearly related with axial magnification, Ma, given by the relationship nMa ≈ −(fT/fO)2 for n the sample space refractive index. The negative sign in eq. 5 indicates that when the sample moves away from the objective the image moves towards the tube lens. Parameters appropriate for the Olympus IX71 and TIRF objective  have fT = 180 mm, fO = 3.0 mm, and L ∼150 mm indicating nMa ≈ −3600. Projective transformation has nMa = −ML2 where ML is the lateral magnification likewise suggesting nMa = −3600 The linear relationship between object and image dimensions in lateral coordinates is the objective magnification. The analogous relationship for the axial dimension was formulated from the lens equations. 2, that is the sum of variances from camera and signal noises, σc2 and σs2, such that,d the dark current in electrons/(pixel-sec), p the number of pixels in the array containing the in-focus point source image, Δt the collection time interval for a single image capture, R the rms read out noise, s above-background signal in electrons/sec, q the CCD quantum efficiency, and g the CCD gain. SNR is s/σ. ORCA CCD camera specifications have id  = 0.1 electrons/(pixel-sec), R = 6, and q = 0.7 for light at 550 nm wavelength. We utilized p = 4 or 9, Δt = 500 ms, and g = 1. A camera scan pathway shown in The axial PSF observed from fluorescent sphere point source at various distances from the objective are compared to the theoretical PSF to estimate the mean sphere axial position. We describe how to compute the theoretical PSF in panels A and B show measured emission axial intensity profile from a fluorescent point source fixed on the coverslip under epi-illumination. Experiments were conducted by focusing the objective on a fluorescent sphere specimen then translating the CCD camera axially through the saw-tooth pattern indicated. Axial translation sweeps the camera through the point source image space recording the axial PSF. Total photon count at each axial position is the sum of counts in the lateral pixels occupied by the focused point object image. Panel A shows the camera position saw-tooth pattern (solid line) and the fluorescence intensity observed from the point source (▪). The left hand side abscissa scale applies to the saw-tooth curve. The right hand side abscissa scale applies to intensity (▪). In Panel B, the camera axial position (independent variable) vs fluorescence intensity (dependent variable) includes only the middle portion of the saw-tooth pattern where camera position changes monotonically. We computed the fitted curve in Panel B (solid line) as described in Axial camera scanning was usually slightly off axis such that the image from the point-object moved laterally in the camera field of view. Slightly off-axis camera scanning does not affect calibration because lateral movement is negligible compared to axial movement. Images were aligned by requiring maximal intensity overlap between the in-focus point-object image frame and the other frames in the scan. Best results were obtained when several point-objects covered the field of view and multiple sources were optimally aligned simultaneously. a = −2644±42, in agreement with the lens equation estimate giving Ma ≈ −3600/1.334 = 2699 for n = 1.334 (refractive index of water in sample space). Experiments were conducted as in Precision, indicated in The electric field at the camera image plane was computed for the TIRF microscope described above using a method taken from Richards and Wolf 1 = Eu for,1The electric field approximately conserves its polarization relative to the meridional plane after refraction in the objective x(π).A1x.such that p and ts. We assume the DM contains a dielectric thin film interface with multiple interfering reflections to give the DM the ability to reflect and transmit the desired light wavelengths. We adjusted film thickness parameters in tp and ts to give relative intensities consistent with a correction factor measured from a sample of rapidly tumbling chromophore emitters in solution. The rapidly tumbling chromophores emit unpolarized light that is linearly polarized before the DM by introduction of an analyzer. The tumbling probe and analyzer provide linearly polarized light of uniform intensity for any direction selected by the analyzer orientation. Polarized light intensity collected with analyzer along the x-axis compared to that collected with analyzer along the y-axis gives the ratio |tp|2/|ts|2 ≈ 1.4. Transmission coefficients multiply the incident field with the x- and z- polarization components multiplied by tp and the y-component by ts given in matrix form by,g is the refractive index of glass, field amplitudes 2 = Eu, rotates the electric field polarization due to refraction at the tube lens giving the electric field at the tube lens focus, 21 while,T from θ where an is the refractive index of air, NAT(A) the numerical aperture of the tube (objective) lens, and γ the ratio of aperture radii for the tube and objective lenses. Constant η for our microscope is ∼0.01 hence θT ranges over a smaller interval than θ implying the correction from A2 is much less significant than that from A1.Time-averaged fluorescence intensity observed from individual molecules,b is background, F0 the signal amplitude, ‖,⊥ the total power emitted by a dipole oriented parallel (with μz = 0) or perpendicular (with μz = 1) to the dielectric interface, and μi the single chromophore dipole components in lab-coordinates. In our application, h ∼ 0.12. Re-expressing eq. 13 in real quantities,pCosφp,SinθpSinφp,Cosθp) into eq. 14 and rearranging terms to find,p–dependent vectors 2 minimization p and φp are sampled over the interval {0,π} since dipole inversion symmetry implies it covers all of sample space. We sampled axial distance ε over the {−250,250} nm interval since it extends beyond the ∼100 nm evanescent illumination depth used in experiments on muscle fibers. With each choice of φp, GLM fitting finds the coefficients for p we choose the maximum of all the maximum likelihood fits to select the best φp and coefficients of b, signal intensity, F0, and angle θp. The fluorescence pattern determines  up to the emission dipole inversion symmetry .Substituting p,φp). Several combinations of normally distributed angles and various axial dipole positions were investigated. We report here on normally distributed angles covering a 15 degree width with average values   =   degrees and with dipoles positioned axially at −50 nm corresponding to a dipole in the aqueous phase and 50 nm above the glass/aqueous interface. Signal fluorescence, F0, and background, Fb, (eqs. 6 and 14) from background light and camera noise are 143 and 35 and similar to typical muscle fiber data. Quantities were substituted into eq. 16 to generate an ideal pattern then each pixel intensity was Poisson distributed. p,φp)  =   with Poisson distributed noise , the fitted pattern , and the residual of the two patterns normalized to fill the 8-bit dynamic range . The fit gave   =   and the axial position ε = −50 nm. 4, c4. The c4 sets the sign forp. The wrong sign converts the correct θp to its complement π−θp as can be seen in 8 cases out of the 70 shown in p ≈ 0, 90, or 180 degrees when c4 is close to zero and parameter standard error (determined from the covariance matrix) is > |c4|. Rising signal-to-noise , however, the mean axial position of the dipole is −60 nm. The results probably reflect different sensitivities of basis patterns for axial position. Dipole orientations containing larger contributions from I4. A different approach was tested in simulation that shows promise. Simulated images from an axial scan of the single molecule emitters produced data like that used above plus images from above and below the nominal focal plane. The axial scanned images were fitted simultaneously to constrain the dipole orientation degrees of freedom. The scan consisted of three images of the emitter at −100, −50, and 0 nm replacing the single image at −50 nm discussed above. Scanning does not change dipole distance from the glass/aqueous interface but the objective is moved these distances in the axial dimension  and the parallel polarization photoactivated subset . The middle panels are the fit to the data (fit) and the bottom panels the residual normalized to fill the 8-bit dynamic range (res). The perpendicular polarization photoactivated pattern has the characteristic donut shape of a dipole (the emission dipole of the photoactivated species or μe[A]) perpendicular to the coverslip/aqueous interface. In Lab-coordinates where pCosφp,SinθpSinφp,Cosθp) this particular dipole has   =   in degrees. The residual shows that the fit is less sprawling than data suggesting the image is somewhat unfocused. The parallel polarization photoactivated pattern has the filled in and more compact shape of a dipole parallel to the coverslip/aqueous interface. In Lab-coordinates it has   =  . Perpendicular and parallel polarization photoactivation tends to select cross-bridges perpendicular and parallel to the fiber axis. Transforming  to the Fiber-coordinates azimuthal and polar angles , that specify the Euler angles for the GFP emission dipole, we find  and  for perpendicular and parallel photoactivation.Single molecule data from PA-GFP tagged myosin cross-bridges in permeabilized muscle fibers was collected from several fiber samples over several days. PA-GFP photoactivation with light polarized parallel or perpendicular to the fiber axis photo-induced an ordered subset of single probes within a differently ordered set of cross-bridges. Myosin cross-bridges are intrinsically orientationally ordered due to the fiber structure. 79 and 70 patterns like those in B)i specify B from B from fluorescence polarization anisotropy with B δ24 degrees . The correction factor is handled differently in the two methods. In the past it changed intensity of half the data sets uniformly while in the pattern recognition method it influences a fit applied independently to each single molecule intensity pattern. In the present study fluorescence patterns from fewer single molecules were quantified due to the more extensive analysis needed for each pattern.The orientation distribution of μThe emission dipole axial position is also quantified in the pattern fitting method. This is demonstrated in a linearly calibrating the image to the sample space axial dimension.Point objects viewed in a microscope have the objective PSF defining the narrowest photon distribution in 3-dimensions detectable by the instrument. The objective PSF correlated with the measured sample photon distribution identifies the distribution center with precision determined by S/N ratio rather than the PSF distribution width PSF shape also depends on light polarization linking it to the emitting probe dipole orientation. Probe orientation linkage to fluorescence spatial distribution has been demonstrated for back focal plane images p,φp) and . We tested the method on simulated data then treated data from myosin cross-bridges in permeabilized muscle fibers to deduce single myosin lever arm exchanged RLC tagged PA-GFP orientation in rigor.After propagation through the microscope optics, we devolve the point image spatial pattern or PSF into the 6 basis patterns shown in p,φp) angles for the dipole moment 50 nm from the glass/aqueous interface (in the aqueous phase) and with Poisson distributed noise from signal and two background sources. One example of simulated data is shown in Muscle fiber data is summarized in a[B], that disorients the photoactivated molecules. The PA-GFP chromophore is locally more flexible than GFP because it is highly accessible to quenchers a[B] is asymmetrical in the photoactivation of myosin cross-bridges  because the disordering retains the mostly perpendicular μa[B] orientation distribution enabling perpendicular photoactivation and de-enabling parallel photoactivation. This effect is captured qualitatively by the model distribution shown in Orthogonal polarized photoactivation laser pulses were applied to the PA-GFP tagged myosin cross-bridges in separate fiber samples to photoselect contrasted oriented sub-populations of probes that are intrinsically oriented by fiber structure. Evidence for both orientation selecting processes was seen in the data. Cross-bridge orientation distribution in the fiber azimuthal degree of freedom, α, is uniform while the parallel polarized photoactivated sample must photoselect a uniform distribution in α due to symmetry. In contrast, the perpendicular polarized photoactivated sample should have a non-uniform distribution in α despite the fiber myosin symmetry in this degree of freedom. These data are contrasted in Probe axial position detection is an added benefit of intensity pattern fitting that has not been exploited experimentally. Simulations summarized in In conclusion, the lateral and axial PSF for a dipole emitter is sensitized to the emission polarization and dipole axial position providing links between the PSF pattern and dipole orientation and axial position. A general expression for the 3-dimensional PSF shows it is composed of 6 basis patterns that in linear combination can specify any single molecule emission pattern. Given the basis patterns, we invert an observed image to deduce their coefficients by using maximum likelihood fitting for Poisson distributed uncertainties. The coefficients for the basis patterns depend algebraically on the dipole orientation. We tested the method on simulated data then treated data from myosin cross-bridges in permeabilized muscle fibers to deduce single myosin lever arm exchanged RLC tagged PA-GFP orientation in rigor. Orthogonal polarized photoactivation laser pulses were applied to the PA-GFP tagged myosin cross-bridges in separate fiber fields to photoselect contrasted oriented sub-populations of probes that are intrinsically ordered by fiber structure. Evidence for both orientation selecting processes was detected in the data and quantified by dipole orientation distributions. Axial probe position dependence in the PSF was quantified in simulation but evidence of it from real data was inconclusive. A method utilizing axial scanning was indicated that boosts axial and orientational resolution in simulation.


Pathological brain states that result in [K+]o and pH dysregulation have been shown to cause astrocyte swelling. However, whether astrocyte volume changes occur under physiological conditions is not known. In this study we used two-photon imaging to visualize real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were observed to swell by 19.0±0.9% in response to a small physiological increase in the concentration of [K+]o (3 mM). Astrocyte swelling was mediated by the influx of bicarbonate (HCO3−) ions as swelling was significantly decreased when the influx of HCO3− was reduced. We found: 1) in HCO3− free extracellular solution astrocytes swelled by 5.4±0.7%, 2) when the activity of the sodium-bicarbonate cotransporter (NBC) was blocked the astrocytes swelled by 8.3±0.7%, and 3) in the presence of an extracellular carbonic anhydrase (CA) inhibitor astrocytes swelled by 11.4±0.6%. Because a significant HCO3− efflux is known to occur through the γ-amino-butyric acid (GABA) channel, we performed a series of experiments to determine if astrocytes were capable of HCO3− mediated volume shrinkage with GABA channel activation. Astrocytes were found to shrink −7.7±0.5% of control in response to the GABAA channel agonist muscimol. Astrocyte shrinkage from GABAA channel activation was significantly decreased to −5.0±0.6% of control in the presence of the membrane-permeant CA inhibitor acetazolamide (ACTZ). These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.Astrocytes, the major type of non-neuronal cells in the brain, play an important functional role in extracellular potassium ([K Astrocytes play an essential role in maintaining optimal physiological brain functioning by tightly regulating the concentrations of K+ and pH in the ECS, as alterations can affect a wide range of cellular processes that include neuronal excitability, synaptic transmission, metabolism, and the activity of many enzymes, channels, and transporters + buffering +, well outside physiological range. As a result, the currently available literature reflects only what might be occurring during pathological states Neuronal firing of action potentials leads to the release of potassium ions , and can be blocked with the removal of extracellular HCO3−, or by inhibition of the main astrocyte HCO3− transporter, the sodium-bicarbonate cotransporter (NBC) Under physiological conditions even moderate amounts of neuronal activity can lead to a build up of extracellular K+]o, and to shrink in response to GABAA receptor activation. Both the astrocyte swelling and shrinking were mediated by the flux of HCO3−. These dynamic astrocyte volume changes may represent a previously unappreciated yet fundamental mechanism by which astrocytes regulate physiological brain functioning.In the present study we performed two-photon imaging in the brain slice preparation to directly examine real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were found to undergo robust swelling in response to a physiological increase in o from an initial control ACSF containing 2.5 mM [K+]o. +]o ACSF (A green) and after maximal swelling has occurred with the 3 mM increase of [K+]o (B red). The overlayed image  = 22.094, p<0.05] -1-piperazineethanesulfonic acid (HEPES) buffered ACSF. HCO3− free ACSF significantly decreased the +3 mM [K+]o –induced astrocyte swelling compared to the swelling observed under HCO3− buffered ACSF, conditions; 5.4±0.7%, n = 31 astrocytes from 7 slices, p<0.05 (3− was reduced using a membrane impermeant extracellular carbonic anhydrase (CA) inhibitor to decrease the production of extracellular HCO3−. The membrane impermeant CA inhibitor 1-(4-sulfamoylphenylethyl)-2,4,6-trimethyl-pyridinium perchlorate (STPP) (100 µM) was used to selectively inhibit the formation HCO3− in the extracellular solution. +3 mM [K+]o –induced astrocyte swelling was significantly reduced in the presence of STPP; 11.4±0.6%, n = 50 cells from 7 slices, p<0.05 (3− influx through the sodium-bicarbonate cotransporter (NBC) cotransporter. Inhibition of NBC activity with the NBC inhibitor DIDS (500 µM) significantly decreased the +3 mM [K+]o –induced astrocyte swelling; 8.3± 0.7%, n = 40 astrocyte from 6 slices, p<0.05  was found to cause significant astrocyte shrinking; −7.7± 0.5% of control, n = 42 astrocyte from 8 slices, p<0.05)  (100 µM) was used to block the formation of intracellular HCO3− during muscimol applications. ACTZ significantly decreased the amount of GABAA channel mediated astrocyte shrinkage by muscimol; −5.0±0.6% of control, n = 34 astrocytes from 7 slices, p<0.05 (+]o for 40 minutes and astrocytes that were incubated with ACSF containing ACTZ . A one-way ANOVA confirmed significance between the control, muscimol, and muscimol with ACTZ groups  = 46.28, p<0.05).The selective GABA p<0.05) . To dete, p<0.05 . A Stude+]o, a concentration that is well within physiological range, and to shrink in response to GABAA receptor activation. These dynamic volume changes were regulated in a HCO3− dependent manner.In the present study, two-photon imaging was used to directly examine real-time astrocyte volume changes in the stratum radiatum of the hippocampus CA1 region. Astrocytes were found to swell in response to a 3 mM increase in [K+]o caused a 19% increase and GABAA channel activation an 8% decrease for the same time period. A number of recent elegant studies have used comparable high-resolution imaging approaches to investigate dynamic astrocyte volume changes in the brain slice preparation, and thus offer the opportunity for quantitative comparisons to be made between the magnitude of the astrocyte volume changes we report here and those that resulted from pathological conditions, such as osmotic stress, ischemia, and cortical spreading depression (SD) events et al. (2010) induced spreading depression in the brain slice preparation by perfusing ACSF containing a +40 mM increase in [K+]o and found that astrocytes swelled 40% +]o, which suggests that astrocytes are not the primary contributor to SD propagation. As in the Zhou et al. (2010) study, Risher et al. (2009) found that brain slices perfused with ACSF containing high [K+]o (+26 mM) also caused a doubling in the magnitude of astrocyte swelling compared to our findings, showing that astrocytes swelled 38.9% +]o -induced astrocyte swelling was greater than the astrocyte swelling that resulted from −40mOsm osmotic challenge (17%) and oxygen/glucose deprivation (29%) et al. found that astrocytes swelled to a maximum of 40% in vivo to examine osmotic- and ischemia-induced volume changes in astrocytes. Astrocyte somas were shown to swell by 21% as a result of hypoosmotic stress induced by IP distilled water injection and 33% from induced global ischemia induced from cardiac arrest et al. (2012) showed that astrocyte somas in the metabolically compromised ischemic penumbra-like area swelled by 31% as a result from the initial induced spreading depolarizations and by 30% as a result of spreading depolarization induced by a transient bilateral common carotid artery occlusion model of global ischemia To the best of our knowledge this is the first study to report on astrocyte volume changes in the brain slice preparation that resulted from non-pathological induction methods. We were able to achieve high image resolution and stable measurements of astrocyte area during long-term imaging experiments and determined that astrocyte area changed less than 1% over 40 minutes in control ACSF, while a modest +3 mM increase in [K+]o and muscimol once applied, were perfused for the remainder of imaging experiments. While this model allowed us to very precisely quantify astrocyte volume changes and unmask a previously underestimated contribution of HCO3− flux, and certainly does comment on the astonishing propensity for astrocytes to change their volumes, it in no way predicts the likely magnitude of astrocyte volume changes that are to occur during normal physiological brain functioning. Indeed, currently available in vivo data indicates that [K+]o changes in a fluctuating manner in the ECS and that modest astrocyte volume changes may occur in this manner as well under physiological brain functioning, such as during slow (∼1 Hz) sleep oscillations The robust magnitude of the astrocyte volume changes found in the present study that occurred from the modest induction methods compared to the studies above, likely result from the experimental paradigm used here; that is, the +3 mM [K+]o that occur as a result of neural activity must be tightly regulated. Astrocytes are thought to play a key role in this regulation by taking up excess K+ from the ECS +]o astrocytes are known to rapidly undergo an intracellular alkalinization 3− in [K+]o -induced astrocyte swelling under physiological conditions was only recently speculated +]o and that the removal of HCO3− from the extracellular solution, inhibition of the extracellular CA activity, and inhibition of the NBC activity all resulted in significant decreases in the magnitude of the [K+]o -induced swelling.For normal neuronal functioning to be maintained, the local changes in o as a result of neuronal activity causes a net driving force for K+ influx into astrocytes, while at the same time causes a depolarization of the astrocyte membrane. This depolarization is believed to activate the uptake of Na+ and HCO3− through the NBC, which is highly expressed in astrocytes +, Na+, and HCO3− in the astrocyte cytoplasm would then generate an osmotic gradient and drive water influx through aquaporin 4 water channels (AQP4) that are highly expressed in astrocytes +, and the metabolic bi-product, CO2, of neuronal activity from the ECS at the same time. Recent work from the Nagelhus group suggests that activity-dependent astrocyte water uptake may be partly offset by an AQP4-mediated water efflux that may serve to moderate the activity-dependent astrocyte volume changes and offset the resulting ECS shrinkage Our findings support a mechanism for K3− from the extracellular solution, inhibition of the extracellular CA activity, and inhibition of the NBC activity all resulted in significant decreases in the magnitude of the [K+]o -induced swelling, but each of the individual conditions did not fully prevent the astrocyte swelling. A wide variety of other transporters are known to contribute to the regulation of astrocyte volume. These include the sodium-potassium chloride cotransporter (NKCC), the K+-Cl- cotransporter (KCC), the Cl–HCO3− anion exchanger (AE), the Na+ dependent Cl–HCO3− exchanger (NDCBE) and the Na+-H+ exchangers (NHE)  used moderate astrocyte swelling to test for VRAC activity under conditions resembling those to which astrocytes may be exposed to upon physiological stimulation and elegantly demonstrated Ca2+-dependent VRAC glutamate release Finally, in an attempt to counteract swelling and to recover normal volume, astrocytes initiate volume regulatory mechanisms by modifying their concentration of osmotically active solutes through a process termed regulatory volume decrease (RVD). Volume-regulated anion channels (VRACs) are activated by increased cell volume and are an important conduit through which glutamate release from astrocytes occurs during pathological brain states The results of this study demonstrate the existence of dynamic astrocyte volume changes that may be an intrinsic phenomenon that occur under physiological conditions. These volume changes may play a fundamental role in the control of extracellular brain signaling and in the regulation of synaptic plasticity.Movie S1This file contains a movie showing astrocyte swelling and recovery in a series of two-photon z-stack projection images of SR101 labeled astrocytes in the CA1 stratum radiatum region of the hippocampus. Images were acquired every 10 minutes over the course of 100 minutes. At time zero the slice was switched from 2.5 mM [K+]o ACSF to 5.5 mM [K+]o ACSF. After the 40-minute time point the perfusate was switched back to 2.5 mM [K+]o.(MOV)Click here for additional data file.Movie S2A agonist muscimol.This file contains a movie showing astrocyte shrinking from the selective GABA At time zero muscimol was added to the ACSF and images were acquired every 10 minutes for 40 minutes.(MOV)Click here for additional data file.


The diet of free-ranging carnivores is an important part of their ecology. It is often determined from prey remains in scats. In many cases, scat analyses are the most efficient method but they require correction for potential biases. When the diet is expressed as proportions of consumed mass of each prey species, the consumed prey mass to excrete one scat needs to be determined and corrected for prey body mass because the proportion of digestible to indigestible matter increases with prey body mass. Prey body mass can be corrected for by conducting feeding experiments using prey of various body masses and fitting a regression between consumed prey mass to excrete one scat and prey body mass (correction factor 1). When the diet is expressed as proportions of consumed individuals of each prey species and includes prey animals not completely consumed, the actual mass of each prey consumed by the carnivore needs to be controlled for (correction factor 2). No previous study controlled for this second bias.Acinonyx jubatus), to establish both correction factors. In contrast to previous studies which fitted a linear regression for correction factor 1, we fitted a biologically more meaningful exponential regression model where the consumed prey mass to excrete one scat reaches an asymptote at large prey sizes. Using our protocol, we also derive correction factor 1 and 2 for other carnivore species and apply them to published studies. We show that the new method increases the number and proportion of consumed individuals in the diet for large prey animals compared to the conventional method.Here we use an extended series of feeding experiments on a large carnivore, the cheetah (Our results have important implications for the interpretation of scat-based studies in feeding ecology and the resolution of human-wildlife conflicts for the conservation of large carnivores. Diet is an important part of carnivore ecology and conservation. Information on diet composition is needed when predator-prey relationships are studied The conversion of prey body mass into a number of scats and their weights excreted by an individual carnivore after prey consumption is a crucial step that diet studies based on scats ought to infer accurately. We use seven quantities to evaluate the steps required to establish the consumed prey mass and the number of consumed prey individuals derived from carnivore scats collected in the field . By appl1Q) on the consumed prey mass to excrete one scat (5Q), previous studies established a correction factor  using feeding experiments (wolves (Canis lupus): Felis concolor): Acinonyx jubatus): y = ax + b) with x being the provided prey body mass (1Q) and y being the consumed prey mass to excrete one scat (5Q). The diet in terms of the proportions of consumed mass per species is then determined by multiplying the frequency of occurrence of prey species i identified in n collected scats (in) by y for prey species i ( = 5iQ) using the (reasonable) assumption of prey body mass x = 1iQi by a carnivore can be described as in×5iQ. Other studies determined CF1 for a given prey species by calculating the ratio of consumed mass to dry mass of prey remains in scats and determined the diet in terms of the proportion of consumed mass per species using this CF1 (wolves: Lynx lynx): To control for the influence of prey body mass , side-striped jackals (Canis adustus) and red fox (Vulpes vulpes), we suggest that the specific conversion factors from feeding experiments with these food items should be used We evaluated this possibility in the cheetah, a species for which a previous study used a linear relationship between i is commonly established by dividing the consumed mass of prey species i, which is in×5iQ, by the entire body mass of the consumed prey species i, i.e. (in×5iQ)/1iQ. Such calculations, however, are only appropriate when the carnivore consumes the prey animal entirely, but not when it consumes only part of it, as this leads to an underestimate of the numbers of large prey animals consumed. To our knowledge this issue has not been considered in previous studies 3Q) as suggested by i is then equivalent to (in×5iQ)/3iQ for solitary carnivores and ((in×5iQ)/3iQ)/2Q for carnivores feeding in groups with a mean size of 2Q. Because 5iQ = 3i/Q4iQ, 5iQ can be substituted by 3i/Q4iQ. The number of consumed individuals per prey species i is then simply i/Q4in for solitary carnivores and (i/Q4in)/2Q for carnivores feeding in groups with a mean size of 2Q. 4iQ is the number of scats excreted after consuming prey species i and can be derived from the feeding experiments using a regression between the number of excreted scats (4Q) and body mass (1Q). We termed this regression correction factor 2 (CF2).Studies may not only be interested in the biomass of prey consumed by a carnivore but also, or primarily, in the number of prey individuals consumed These calculations are suggested if the prey species is large, the consumption of one individual (one feeding event) is likely to produce more than one scat and scats are collected in a defined area at regular intervals  so that all detected scats excreted in the last couple of days or weeks are collected. Under the assumption that the likelihood of finding scats with remains of one feeding event of a given prey species is similar to the likelihood of finding scats with remains of different feeding events of the same prey species, scats containing remains of this prey species from one sampling interval can be pooled. Then, these scats should be treated as dependent samples and several scats assumed to represent one feeding event.4Q) to account for the missed scats of the carnivore individual. The number of consumed individuals per prey species is then equivalent to ((in×4iQ)×5iQ)/3iQ for solitary carnivores and for carnivores feeding in groups since only one group member was sampled. Because 5iQ = 3i/Q4iQ, 5iQ can be substituted by 3i/Q4iQ and the number of consumed individuals per prey species for scats collected independently from each other is simply in. It is important to be aware of whether scats collected in the field arise from one feeding event (dependent objects) or from independent feeding events, because the results and therefore the interpretation differ substantially.In contrast, if single carnivores of known identity are followed and observed to defecate or are immobilised and directly sampled, and if the sampling interval is longer than the time period during which a carnivore excretes scats after having fed on one prey animal, the scats are independent samples and cannot originate from one feeding event. In this case each scat containing remains of a particular prey species has to be multiplied by the mean number of scats a carnivore excreted after consuming from such a prey animal . AfriCat Foundation is registered by the Namibian Ministry of Environment and Tourism (MET) as a large non-profit carnivore captive facility since 1993 (permit office 2004/11) and runs a rehabilitation centre for free-ranging carnivores, mainly cheetahs. AfriCat is equipped with high standard veterinary facilities and qualified staff caring for the animals.Lepus sp.) that weigh about 2 kg to adult zebra (Equus burchelli) that weigh up to 270 kg Cheetahs are capable of killing a wide range of prey species. Reports range from hares (Tragelaphus strepsiceros), gemsbok (Oryx gazella), hartebeest (Alcelaphus buselaphus), springbok , warthog (Phacochoerus africanus), goat (Capris sp.), springhare (Pedetes capensis), ground squirrel (Xerus inauris), Namaqua rock mouse (Aethomys namaquensis) and hairy-footed gerbil (Gerbillurus paeba) as prey species. All large carcasses were animals killed by trophy hunters who booked their hunting with a registered professional Namibian hunting guide who had a hunting permit from the Ministry of Environment and Tourism (MET) in Windhoek, Namibia. Once the animal was shot in the field according to the hunting regulations from the NAPHA  and the trophy, i.e. head and horns, was removed, we retrieved the carcass. The smaller animals were trapped at the farms where our research stations are based. Commercial mouse traps of different sizes were used and once an animal was trapped it was quickly killed. Our study was approved by the MET (permit number 1089/2006), the scientific advisory board of the AfriCat Foundation and the leadership and Ethical Committee of the Leibniz Institute for Zoo and Wildlife Research in Berlin.For the feeding experiments we used kudu . During the experiments, the cheetahs were kept in observation enclosures of 20 m×10 m provided with water  of food . In thes of food , with inn = 13). Approximately ten minutes after the last cheetah of the group stopped feeding, we collected and weighed the remains of the carcass(es). In two feeding experiments with a goat and a springhare the cheetahs were still feeding after 3 hours 25 minutes and 2 hours 30 minutes, respectively, when darkness set in at 19:00 h. In these cases the carcass was left over night and we collected and weighed all remains the next morning. During period 3, we again collected all scats during each day until the cheetahs excreted no more scats or only scats containing grass or cheetah hairs. We distinguished between ‘collectable’ (hard to soft) and ‘non-collectable’ (viscous) scats according to their consistency and included only collectable scats in the determination of correction factors to provide correction factors suitable for field studies Following 5Q) for each feeding experiment ( = 3Q/4Q) and fitting an exponential function of this parameter on mean prey body mass provided per feeding experiment (1Q) which is a representation of an asymptotic process , not those that were rejected and left untouched . We determined CF2 by fitting a non-linear regression of the number of excreted collectable scats per cheetah and prey animal (4Q) on mean prey body mass provided per feeding experiment (1Q). We initially expected this to also be an asymptotic process, best represented by an exponential function. Alternatively, if at large prey sizes either the behaviour of the predator or the ratio of digestible to indigestible matter changes, then it is possible that the number of excreted collectable scats might decline again. Such a process is usefully represented by a peak logarithmic function. For our study the peak logarithmic function runs best through the data; in the supporting information we also present the exponential function (Canis lupus papllipes) we also compare the fit of the exponential function with that of the peak logarithmic function , steenbok (Raphicerus campestris), springhare and squirrel The application of the correction factors to scats collected in the field and the subsequent determination of the diet were based on weights of prey animals consumed by cheetahs. Since identified prey remains in scats rarely provide information on their age and thus approximate weight of the consumed prey individual, age categories of the prey species were estimated and corresponding weights applied 3Q– 6Q) * 100/3Q. This digestibility is termed ‘apparent’ because the scats contain also metabolic components from the animal and the fresh scat might also include fluid originating from drinking by the cheetahs We determined the apparent digestibility for different prey sizes as (mean fresh prey mass consumed per cheetah – mean fresh collectable scat mass per cheetah)*100/mean fresh prey mass consumed per cheetah P = 0.37, n = 85).One hundred and twenty (92%) of the 131 scats were collectable scats . The rat5Q) increased with prey body mass (1Q) and levelled out at certain weight of prey body mass. This relationship, the CF1, was described by the exponential function y = 2.358(1-exp(−0.075x)) with 2.358 kg being the consumed prey mass per collectable scat at which the curve reaches an asymptote  (y = 2.821(1-exp(−0.057x)), 2R = 0.493, P = ns, n = 15).The amount of food from one prey animal a cheetah consumed to excrete one collectable scat ( n = 14) . The war n = 14)  was excly = 7Q) increased with increasing mean prey body mass per feeding experiment (x = 1Q) and levelled out at 94.2% (y = 94.192(1-exp(−17.222x)), 2R = 0.541, P<0.001, n = 15).Apparent digestibility (n). When applying the empirically determined CF1 to calculate the consumed prey mass per collectable scat (5Q), the consumed prey mass per species was lowest for small prey species (n * 5Q).The hypothetical example consisted of 100 scats. In these scats, 10 prey species were chosen, each of which was represented in 10 scats. The relative frequency of occurrence for each prey species was 10% (y = 4Q) increased with mean prey body mass (x = 1Q) and then decreased again (y = 3.094exp(-0.5((ln(x/16.370))/2.584)2), 2R = 0.626, n = 14, y = 6Q) similarly first increased with the provided mean prey body mass (x = 1Q) and then decreased again (y = 0.310exp(−0.5((ln(x/11.600))/2.022)2), 2R = 0.696, n = 14), with a peak mean collectable scat weight of 0.31 kg per cheetah and prey animal at 11.60 kg of mean prey body mass provided. The outlier of the warthog with 1 excreted collectable scat per cheetah . This contrasts with the results of the conventional method (dividing consumed mean mass per prey species by the entire prey weight 1Q) where large prey species showed the lowest number in the diet (n * 5/Q1Q). The numbers of the three prey species with the highest mass in the example, juvenile kudu, juvenile hartebeest and adult goat, were 17.5 times, 10.8 times and 6.6 times higher when using CF2 than the conventional method  for solitary cheetahs revealed the highest number of individuals consumed for small prey animals, the lowest number for medium-sized prey animals and a medium number for large prey  was 0.337 kg (x = 1Q). Cheetah scats with remains of prey with a body mass below this threshold will therefore consist of remains from two consumed individuals of this particular species. Thus, the number of consumed individuals for such prey should be multiplied by a factor of 2.For very small prey species a particular problem arises. Our results indicate that for such prey animals the consumption of one prey individual will not produce an entire scat . The smaLynx rufus) containing deer remains experienced a larger mass loss than scats containing mice and rats or rabbits when left in the field for three weeks, reducing the likelihood for scats containing deer to be reliably recognised as bobcat scats Scats containing remains of different prey species are likely to change their shape and consistency at different rates, which bias the likelihood of collecting scats containing remains of particular prey species and thus bias diet composition. For example, scats from bobcats  + (10×5BQ×0.5). Similarly, to calculate the consumed number of individuals per prey species we suggest to allocate equal 4Q contributions of different prey species to the scats. Using the same example, the consumed number of individuals per prey species would be (10/4AQ×0.5) + (10/4BQ×0.5).Our hypothetical example is based on scats containing remains of one prey species only. However, scats sometimes contain more than one prey species. Studies that determined the dry mass or volume of all prey remains in the scats use the proportions of dry masses or volumes of the different species in the scats, sum these proportions up and apply CF1 to determine the consumed mass per prey species applied y = 1.798(1-exp(−0.008x); in Europe: y = 0.621(1-exp(−0.012x); in India: y = 1.382(1-exp(-0.020x); lynx: y = 1.045(1-exp(−0.145x)). We also fitted an exponential function through the data from the four studies to derive CF2 (wolf in North America: y = 37.311(1-exp(−0.021x); in Europe: y = 39.473(1-exp(−0.061x); in India: y = 13.940(1-exp(−0.980x); Eurasian lynx: y = 6.995(1-exp(−0.201x)), however, the data from the study on wolves in India were best described by a peak logarithmic normal function with three parameters (y = 21.792exp(−0.5((ln(x/6.444))/1.273)2)), similar to our cheetah study , leopard (Panthera pardus) or dhole  in India. We determined the diet of these carnivore species in terms of consumed prey mass and number of prey individuals (Bos gaurus) and sambar (Cervus unicolor) was between 1.7 and 2.2 times higher, and chital (Axis axis) was the main prey only for leopard and dhole, overriding previous assessment of niche overlap and predator-prey relationships  and 37.3 (North America) and 39.5 (Europe) ; similarCF1 and CF2 determined in this study are only applicable to carnivores feeding chiefly on mammalian prey species and on species that are not completely consumed. For carnivore species mainly feeding on fruits, invertebrates, birds and small mammals such as black-backed jackals, side-striped jackals and red fox, the diet should be determined using the specific conversion factors for these food items The design of the feeding experiments will influence the outcome in terms of the non-linear functions for CF1 and CF2. How much and which parts of a prey animal a carnivore consumes depends on factors such as its state of hunger, the prey mass provided and competition with conspecifics Figure S1orrection factor 2 (CF2) described as exponential function from our data set in . Mean number of collectable scats excreted per cheetah and prey animal (4Q) as a function of mean prey body mass (kg) provided per feeding experiment (1Q). CF2 follows the exponential function y = 2.654(1-exp(−0.960x)), 2R = 0.705, P<0.05, n = 14. For details and comparison with CF2 described as a peak logarithmic function see C(TIF)Click here for additional data file.Table S1Determination of correction factors 1 (CF1) and 2 (CF2) from four published feeding experiments with the new method. We used the studies on wolves from North America (DOC)Click here for additional data file.Table S2Application of correction factors 1 (CF1) and 2 (CF2) to four published studies on carnivore diet following the new method. We applied CF1 and CF2 derived from our feeding experiments with cheetahs (Acinonyx jubatus) to a cheetah study in Namibia Panthera tigris) and leopard (P. pardus) study in India Cuon alpinus) study in India (DOC)Click here for additional data file.Table S3Guidelines for feeding experiments to ensure the experiments simulate as closely as possible natural feeding situations.(DOC)Click here for additional data file.