Philippa Kaur

This is an observational study with no intervention on flock and hatchery practices. None of the birds or eggs were exposed to experimental procedures. The study was based mainly on existing data provided by the hatchery company (DanHatch Denmark A/S) from five broiler breeder flocks in Denmark during the period from November 2018 to January 2019 when the breeders were 46 to 62 weeks of age, see details in Table 1. In addition, feed samples from the flock locations and eggs from grocery stores were acquired.Table 1 Flocks and production periods.Full size tableThe average age of breeders was 48–59 weeks (SD from 0.5 to 2.2) ranging from 46–50 weeks to 57–62 weeks (Table 1; Supplementary Fig. S1 online) with observation period ranging from 1.6 to 7.6 weeks in the five flocks. Average laying percent over observation days was 65% (SD = 5.4%) and average hatchability over deliveries was 79% (SD = 5.8%).Feed samplesTwenty-six feed samples were collected for analysis of glyphosate content, 3 to 10 feed samples per flock. The glyphosate concentration related to a given sampling date was assumed representative for the flock from this day and until next sampling. Average duration of the preceding samples were used as duration for the last sampling date within each flock. Glyphosate (N‐(phosphonomethyl) glycine) and the glyphosate degradation product, aminomethylphosphonic acid (AMPA) in feed samples were analysed by the method described by Nørskov et al.4.Production dataData on egg production and hatchability from periods following each feed sampling was obtained from the hatchery company. Daily information was available on laying percent (100% * number of eggs/number of breeders), breeder age (days) and egg weight. For the hatchability, this was calculated as the proportion of eggs placed in incubators from which a viable chicken hatched (but presented as a percentage, i.e. multiplied by 100%). Daily egg weight had been calculated as the average from approx. 30 randomly sampled eggs.Glyphosate concentration of the feed consumed by the breeders during the 10 days prior to laying was the explanatory variable of main interest. The weighted average of glyphosate concentrations across the 10 days of development from follicle to ovulation of egg was used with number of days each glyphosate sample is representative during these 10 days as weights. For hatchability, glyphosate concentrations were aggregated at the level of delivery by weighted averaging using number of hatch eggs as weights.Eggs from grocery storesNo eggs were obtained from the five flocks, however we acquired eight cartons of conventional as well as eight cartons of organic eggs from eight different grocery stores. Three eggs from each carton were selected and egg yolk were analysed for glyphosate by the microLC-MS/MS method as described by Nørskov et al.4 adjusted to the egg yolk matrix.Statistical analysisLaying percent and hatchability were analysed by linear mixed effects models, including a random effect of flock and a first order autoregressive correlation structure to account for the repeated measurements from each flock. Following two covariates were considered for both outcomes: average egg weight (g) and breeder age (decimal weeks). However, since egg weight and breeder age are highly correlated (Pearson’s correlation coefficient ranging from 0.73 to 0.95 in the five flocks; Supplementary Fig. S1 online), only breeder age was included in the models. An important reason for this choice being that average egg weight was missing for 24% and 43% of the days from flock 4 and 5, respectively. In the age range used for this study, laying percent decrease with breeder age (Supplementary Fig. S1 online) as substantiated by a correlation coefficient between − 0.38 and − 0.87. Hatchability also decrease with breeder age (Supplementary Fig. S1 online).In addition, storage time on farm until delivery (1 to 5 days) and storage time at hatchery until incubation starts (1 to 11 days) were included as covariates for hatchability. The incubation start date was determined as date of hatching minus 21 days. For hatchability, covariates obtained from flock production data were aggregated at the level of delivery by weighted averaging; using daily number of eggs as weights for the calculation of average egg weight, number of hatch eggs as weights for average storage time on farm, and current number of breeders as weights for average breeder age. Weighted average storage time on farm until delivery varied from 1.0 to 4.0 and was on average 2.1 days. For storage time at hatchery, deliveries had been split on one to four incubator start dates. Therefore, weighted average of storage days was calculated using number of delivered eggs as weights. Weighted average storage time at hatchery before incubation starts varied from 1.2 to 8.0 days and was on average 4.8 days.Final models were fitted with restricted maximum likelihood estimation using the lme function from the nlme package v. 3.1-152 in R version 4.0.45 and with a significance level of 0.05. Fixed effects were tested by χ2 likelihood ratio tests after maximum likelihood estimation. Model checking was carried out by examination of qq-plots for normality and scatter plots of residuals versus predicted values to look for uncovered trends and variance heterogeneity. More

We have analyzed the inbreeding-purging process in four captive populations of different ungulate species with effective sizes ranging 4–40 and with available pedigrees as well as survival and productivity records. This allows us to explore the role of inbreeding and purging in determining the evolution of fitness traits in a range of scenarios relevant in the context of conservation.In A. lervia (Ne ≈ 4), purging is expected only for the most severely deleterious alleles (those giving dNe  > 1, which implies d  > 0.25 as, for example, in completely recessive alleles with deleterious homozygous disadvantage s  > 0.5). Thus, it could be that purging has not been detected for this species because such severely deleterious alleles had been purged during the demographic decline in the wild, before the foundation of the captive population. This would be consistent with the low and non-significant inbreeding load estimated in this species. It is also possible that these estimates are non-significant due to the relatively small number of individuals available.G. cuvieri and N. dama have significant initial inbreeding loads that, adding up the direct and maternal components, is about 1.25 in both cases, which is on the order of other estimates published for captive populations (Ralls et al. 1988). Since in both species Ne  > 10, purging should be efficient against less severely deleterious alleles than in A. lervia (d  > 0.1). Purging is detected for both species with very low P values. This result is in agreement with Moreno et al. (2015), who suggested that purging had occurred in G. cuvieri as they found an increased juvenile survival parallel to an increased inbreeding coefficient. The relative contribution of severe and mild deleterious effects to the inbreeding load of populations is under a scientific debate with direct implications in conservation biology (Ralls et al. 2020, Kyriazis et al. 2021, Pérez-Pereira et al. 2021). The large d estimates obtained in our analysis indicate that a substantial fraction of the initial inbreeding load is being purged under modest effective population sizes, implying that such substantial fraction is due to relatively severe deleterious mutations in these two populations. As far as we are aware, these are the first estimates of this purging parameter obtained in managed, non-experimental populations. Previous estimates of d were obtained in D. melanogaster bottlenecked populations, first for egg-to-pupae viability in lines with Ne = 6 or 12 under noncompetitive conditions (d = 0.09, Bersabé and García-Dorado 2013), and second in lines with higher Ne ≈ 40–50 under more competitive conditions, giving a larger estimate of d, of the order of that estimated in these two ungulate endangered species (d ≈ 0.3, López-Cortegano et al. 2016).Regarding G. dorcas, given its larger population size, purging is expected even against alleles with mild recessive component of the deleterious effect (d  > 0.025). However, although a significant (if modest) inbreeding load was estimated, no significant purging was detected. Nevertheless, the number of equivalent complete generations by the end of the pedigree (EqG = 7) was smaller than our proposed minimum number of generations required to detect purging (tm = 10). This suggests that, due to the large size of this population, more generations are needed to detect purging.The results above support the use of tm to get an approximate idea about when a pedigree is too shallow for purging to be detected. Should the number of generations available be larger than tm, IP predictions could additionally be computed to search the d values that can be expected to produce detectable purging. Supplementary Fig. S3 shows that the true number of generations required to detect purging becomes increasingly larger than tm for alleles with smaller d values, as they suffer weaker purging each time they are exposed in homozygosis. The tm approach helps to understand the failure of many studies to detect purging. Such is the case of the extensive meta-analyses on 119 zoo populations by Boakes et al. (2007), where the median Ne value was 22.6 while the median number of generations was t = 3 meaning that, for most species, at least 5 more generations were needed before purging could be detectable. On the contrary, and in agreement with this tm approach, purging was experimentally detected in lines of D. melanogaster with Ne = 43 (i.e., tm ≈ 10) where, after an initial period of inbreeding depression, fitness experienced a substantial recovery beginning between generations 10 and 20 (López-Cortegano et al. 2016).A reason why detecting purging in captive populations is challenging is that a fitness rebound can also be due to adaptation to captive conditions or to environmental effects, such as those derived from improved husbandry (Clifford et al. 2007). In fact, this might have been the case in Speke’s gazelle breeding program, where the observed rebound of fitness was first ascribed to purging (Templeton and Read 1984, 1998), while Kalinowski et al. (2000) suggested that husbandry improvements could also be responsible for these findings. Our estimates of d and δ, however, are based on the association between the fitness trait and purged inbreeding at the individual level (Wi, gi) which, in our data, is mainly expressed within cohorts while average survival showed little variation through time. In addition, the analyses included temporal factors (YOB or POM) that should have removed confounding effects from adaptation to captivity or improved husbandry. Therefore, adaptive processes or time-dependent environmental factors are not expected to have biased our IP estimates.For productivity, the estimates of inbreeding load were high (overall inbreeding load ~5, P value  More

Insects and bamboosFive internodes (length: mean ± SD = 44.8 ± 1.1 cm, n = 5; diameter in the middle part of internodes: 21.4 ± 0.8 mm, n = 5) of five living mature culms of P. simonii bamboo were sampled at Kawaminami, Miyazaki Prefecture, Japan [32°9′ N, 131°29′ E] on 6 June, 2019. Per internode, four semi-cylindrical strips (ca. 15 × 2 cm) were made and stored at − 25 °C until use.To obtain fungus-free larvae of D. bucculenta, we sampled five beetle eggs from P. simonii bamboo obtained at Toyota, Aichi Prefecture, Japan [35°9′ N, 137°13′ E] on 9 May, 2019 in the laboratory from ovipositing females collected at Kawaminami on 10 and 11 April, 2019. The eggs were immersed in 99.5% ethanol for 10 s followed by 70% ethanol for 10 s for surface sterilization and then individually placed on potato dextrose agar (PDA) (Difco, Detroit, MI, USA) plates. The plates were incubated at 25 °C in the dark until 30 days after larval hatching to confirm the absence of the formation of yeast or other microbial colonies. Consequently, all five larvae hatched successfully and aseptically.The bamboo used in this study was morphologically identified using the literature29. This is native to the study areas and no other host bamboo species are distributed there29. Therefore, no voucher specimen of this bamboo has been deposited in a publicly available herbarium. No specific permits were required for the described field studies. The location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species. All applicable international, national, and/or institutional guidelines for the care and use of animals and plants were followed. This study is reported in accordance with ARRIVE guidelines.Component analyses of bamboo tissuesFor YP and LP, the yeast W. anomalus originating from D. bucculenta in Kawaminami (strain: DBL05Kawaminami) was cultured on a 9-cm PDA plate to obtain enough biomass for further experiments. Afterwards, yeast cells were suspended in ca. 10 mL of sterilized water, and were inoculated on the inner surface of the autoclaved internode strips using an autoclaved tissue paper immersed with the yeast suspension. For LP, additionally, the fungus-free 2nd instar larvae (weight: mean ± SD = 2.4 ± 0.4 mg, n = 5) were individually placed on the yeast-inoculated strips. Each of these yeast-inoculated and yeast-and-larva-inoculated strips was then put in a sterilized test tube (3.0 cm in diameter and 20 cm tall) with moistened cotton placed at the bottom. Each of the test tubes was covered with a sterilized polypropylene cap, sealed with Parafilm Sealing Film (Pechiney Plastic Packaging, Chicago, IL, USA) on which three small holes were made using a fire-sterilized insect pin to avoid oxygen shortage, and individually put in a plastic zipper bag. These yeasts and insects were incubated at 25 °C in the dark for 47 days for YP (n = 5), and 47 (n = 4) and 73 (n = 1) days until these larvae reached adulthood for LP (adult elytral length: mean ± SD = 9.2 ± 0.4 mm, n = 5). Microbial contamination was invisible to the naked eye.For FP, YP and LP, the inner surface (up to 0.3 mm in thickness, dry weight: 336 to 935 mg) of a strip was sampled using a small U-shaped gouge. In the case of FX, first, the pith of a strip was completely removed, and then xylem tissue (up to 0.5 mm in thickness, dry weight: 729 to 872 mg) was sampled using a small U-shaped gouge. These tissues were individually sampled from five strips derived from five different internodes for each tissue type.Samples were extracted by aqueous ethanol and hydrolyzed by sulfuric acid with reference to the literature30,31,32 as follows. Four types of samples were freeze-dried and pulverized using a rotor-speed mill (Fritsch, PULVERISETTE 14, 0.2 mm mesh). About 80 mg of powdered sample was extracted using 5-mL 80% ethanol aqueous solution (aq.) at 63 °C three times. The volume of the extracts was adjusted to 25 mL, filtered, and analyzed using ion exchange chromatography measurements (extractable sugar analysis). Their extracted residues were hydrolyzed using sulfuric acid as follows: 50-mg samples were immersed in 1.64-g 72% sulfuric acid aq. at 30 °C for 2 h, boiled in 39.4-g 3% sulfuric acid aq. for 4 h, and filtered to collect sulfuric acid residues as sulfuric acid lignin fractions. The volumes of the filtrates were fixed to 100 mL, passed through a sulfuric acid-removing filter (DIONEX OnGuard IIA), and submitted to ion exchange chromatography measurements (structural sugar analysis). For the uronic acid measurements, the sulfuric acid-removing filter was not used.Ion exchange chromatography measurements were conducted using a DIONEX ICS-3000 apparatus. The measurement conditions were as follows: column, CarboPac PA-1 (2.0 mm I.D. × 250 mm L, Dionex corp.); flow rate, 0.3 mL min−1; column temperature, 30 °C; injection volume, 25 µL; eluent, H2O (solvent A), 100 mM NaOHaq. (solvent B), aqueous solution containing 100 mM NaOH and 1.0 M CH3COONa (solvent C), and aqueous solution containing 100 mM NaOH and 150 mM CH3COONa (solvent D). The gradient conditions for monomers, dimers, and uronic acids were as follows: for monomers, with a gradient of B 0.5% C 0% 45 min, C 100% 10 min, B 100% 10 min, B 0.5% C 0% 20 min; for dimers, with a gradient of B 50% C 0% 50 min, C 100% 10 min, B 100% 10 min, B 50% C 0% 15 min; for uronic acids, with a gradient of D 100% 10 min. These extraction, hydrolysis, and measurement procedures were conducted using n = 5 samples. For the structural sugars, their yield was calculated as the dehydrated state. The values of other extractives % were calculated by the subtraction of total extractable sugars % from total extractives %.Elemental analysis (carbon, hydrogen, nitrogen) was conducted by 2400 CHNS Organic Elemental Analyzer (PerkinElmer Japan, Yokohama, Japan). About 1-mg dried samples were burned completely and the produced CO2, H2O, and N2 (after reduction of NOx species) gasses were quantified by a thermal conductivity detector.Means of components of bamboo tissues were compared among tissue types using the Steel–Dwass test after the Kruskal–Wallis test. Calculations were performed using R 3.5.133.Carbon assimilation testThe yeast W. anomalus (DBL05Kawaminami) was cultured aerobically in 20 mL of yeast nitrogen base (YNB) (Difco) containing 0.5% glucose at 25 °C in the dark for 2 days with shaking at 85 rpm. The culture media were centrifuged and cell pellets were suspended in sterile water, in which the OD600 was adjusted to 0.10. Fifty μL of the cell suspension was added into a tube (2 mL) with 1 mL of each of 14 different media containing YNB and one of the following carbon sources: d-glucose, d-galactose, d-mannose, d-xylose, l-arabinose, d-fructose, d-galacturonic acid, d-glucuronic acid, sucrose, cellobiose, starch from corn, xylan from corn, carboxymethyl cellulose, and no carbon source (n = 5 to 6). The concentration of each carbon source was 0.5 g L−1, except for xylan at 1.5 g L−1. The tubes were shaken at 85 rpm and incubated at 25 °C in the dark for 7 days. Afterwards, the presence of visible pellets of yeasts and OD600 were recorded to determine the growth of the strain. The degree of assimilation was scored according to the presence of the pellets and the difference in the turbidity increase (ΔOD600) between culture media containing no and a given carbon source as follows: no growth (without a pellet, ΔOD600  More

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Synergy between S. cerevisiae and R. etli in biofilm formationWhen S. cerevisiae Mat α Σ1278h and R.etli CE3 were grown in minimal medium with low glucose concentrations (0.1%), these species adhered to abiotic surfaces to form biofilms (Fig. 1). Interestingly, R. etli and S. cerevisiae formed a mixed biofilm whose biomass was ~ 3 times greater than that of either single-species biofilm (Fig. 1a). In addition, at 24 h, the number of colony-forming units (CFU)/cm2 of R. etli CE3 in the mixed biofilm was higher than that in the pure biofilm (Supplementary Fig. 1). Confocal laser scanning microscopy of biofilms stained with the Live/Dead Kit (propidium iodide and SYTO9) showed that in the mixed biofilm, the yeast cells formed patches, and the bacterial cells covered most of the surface (Fig. 1b). In contrast, monospecies biofilms of R. etli and S. cerevisiae had lower structural complexity and contained a greater (80%) number of dead cells, and their individual densities were lower than their populations in the mixed biofilm (Fig. 1b). These results suggest that in mixed biofilms, S. cerevisiae promotes bacterial growth.Figure 1The interaction between S. cerevisiae and Rhizobium etli CE3 results in the formation of a structurally complex and more productive biofilm in terms of biomass. (a) Biofilm formation of R. etli CE3 and S. cerevisiae Σ1278h Mat α and biofilm growth over time in minimal dextrose medium. The data are representative of 3 independent experiments +/− the S.D. values. (b) Top view and cross section of confocal micrographs of the S. cerevisiae-R. etli mixed biofilm and the single-species biofilms. Magnification 40 × . The images are representative of 3 independent experiments. Biofilms were developed on glass microscope slides and stained with a LIVE/DEAD viability kit. Red fluorescence indicates dead cells, and live cells are colored green. Images were acquired 24 h after inoculation.Full size image
S. cerevisiae secretes dicarboxylic acids that promote R. etli growth and biofilm formationWe found that the R. etli colonies that grew close to S. cerevisiae on solid glucose minimal medium were larger than those growing far from yeast colony (Fig. 2).Figure 2Yeast cells produce dicarboxylic acids that promote the growth of R. etli. (a) R. etli growth in coculture with S. cerevisiae BY4741 mutants (aco1Δ, fum1Δ, sdh1Δ and mdh1Δ) that accumulate dicarboxylic acids and a BY4741 strain with blockade of the aerobic respiratory chain (rho-). (b) Test on solid medium showing that S. cerevisiae BY4741 (*) secretes compounds that promote bacterial growth (  >). In contrast, BY4741 rho- cells (ρ), which do not produce dicarboxylic acids, do not promote the growth of R. etli CE3. R. etli CE3 cells were spread over MMD agar, and yeast cells were spotted in the center. (c) Top view of light micrographs of dual-species biofilms; S. cerevisiae (arrowhead) and R. etli (arrow). Biofilms were developed on glass microscope slides and stained with crystal violet. Magnification 20 × . The images are representative of 3 independent experiments. (d) Growth of R. etli strains in coculture with S. cerevisiae BY4741. The growth of the rhizobium strains was estimated at 24 h. R. etli CE3 strains: wild-type (wt), dctA- containing an empty expression plasmid (dctA-) and dctA- containing a plasmid expressing dctA (dctA-/dctA). The data are representative of 3 independent experiments +/− the S.D. values.Full size imageWe used a visual growth promotion assay on solid medium to screen for S. cerevisiae knockout strains (YKO library) that influenced bacterial growth. 159 yeast mutants were unable to promote R. etli CE3 growth (Supplementary Table 3). In general, these mutants were defective in mitochondrial function. Interestingly, we found that 5 strains with mutations in genes coding for enzymes involved in the TCA cycle showed an enhanced ability to promote bacterial growth compared to that of the wild-type strain (Fig. 2a).To determine how the S. cerevisiae mutants may affect the fungal-bacterial interaction, we analyzed factors that may be altered in mutants with mitochondrial function defects and a compromised TCA cycle.We compared the production of TCA intermediates between the wild-type and mutant yeast strains. Mutants defective in mitochondrial function (mef1Δ, gep5Δ, sdh2Δ, ppa2Δ, imp1Δ, cox7Δ, cyc1Δ and cyc2Δ) produced low amounts of tricarboxylic acids (Supplementary Fig. 2a). In contrast, the aconitase mutant (aco1Δ) produced 60% more citrate and succinate; the fumarase mutant (fum1Δ) resulted in fumarate accumulation; the succinate dehydrogenase mutants (sdh1Δ and sdh4Δ) produced 80% more succinate; and the mitochondrial malate dehydrogenase mutant (mdh1Δ) produced 60% more malate and succinate (Supplementary Fig. 2b). These results suggested that the large quantities of tricarboxylic acids secreted by the mutant yeast played a role in promoting bacterial growth in the cocultures.We analyzed the biomass of mixed biofilms formed by yeast cells defective in mitochondrial function (Σ1278B petit mutant). The ability of the wild-type and the petit mutant strains to form a monospecies biofilm was similar (Supplementary Fig. 3). In contrast, the mixed biofilm formed by yeast cells defective in mitochondrial function was significantly lower in biomass than that formed by the wild-type yeast strain (Fig. 2c). Also, Σ1278B petit mutant produced low amounts of tricarboxylic acids (Supplementary Fig. 2a).We next measured the biomass of the mixed biofilm formed by S. cerevisiae and a Rhizobium mutant unable to take up C4-dicarboxylic acids (dctA-). This evaluation revealed that C4-dicarboxylate uptake by R. etli is necessary to form mixed biofilms with high biomass (Fig. 2d).A symbiotic plasmid is involved in the phenotypic plasticity of R. etli.
The genome of Rhizobium etli CE3 is composed of a chromosome and 6 plasmids (pA, pB, pC, pD, PE and pF)11. To determine whether elements encoded by these replicons can participate in the establishment of commensalism, we evaluated the formation of biofilms by yeast and R. etli strains lacking these replicons12. We found that lack of pA, pB, pC or pF did not affect the ability of bacteria to coexist with yeast (Fig. 3a). Interestingly, a strain cured of plasmids pA-/pD- could not coexist with S. cerevisiae to form a mixed biofilm and obtain the benefits provided by the fungus (Fig. 3a).Figure 3Plasmids pA and pD encode proteins performing functions that are necessary for the coexistence of bacterial cells with yeast. Growth of R. etli strains in biofilms with S. cerevisiae S1278B. (a) Growth in mixed biofilms of R. etli strains lacking the plasmids; pA, pB, pC, pF and in one case of two plasmids, pA-/pD-. The growth of the rhizobia strains was assessed at 24 h. (b) Scheme of the genes contained in a cosmid that partially complements the growth of the pA-/pD- strain in mixed biofilms. Here, 3, 2 and only one gene was amplified to generate the plasmids AD1, AD2 and AD3, respectively, as indicated in the figure. (c) Growth of R. etli strains in mixed biofilms. Strains AD1 and AD2 are R. etli pA-/pD- cells that carried plasmids AD1 and AD2, respectively. The growth of rhizobium strains in mixed biofilms was estimated at 24 h. The data are representative of 3 independent experiments +/− the S.D. values.Full size imageTo determine the genetic elements from the symbiotic plasmid involved in the interaction with yeast, we complemented the R. etli pA-/pD- strain with a cosmid library containing fragments of partial digestion (EcoRI) of the R. etli CE3 genome13. We found that a cosmid containing 9 ORFs from plasmid pD (GenBank: U80928.5) partially restored the ability of R. etli pA-/pD- to form a mixed biofilm (Fig. 3b). This cosmid contains 7 insertion sequences (IS) and a predicted operon encoding a probable peptide pheromone/bacteriocin exporter (RHE_PD00332) and a probable bacteriocin/lantibiotic ABC transporter (RHE_PD00333) (Fig. 3b).The complete operon or only the ABC transporter gene, including its endogenous promoter and terminator regions, was cloned into plasmid pBBR1MCS-3, and the resultant plasmids were named AD1, AD2 and AD32, respectively (Supplementary table 1 and 2). We found that complementation with the complete operon (plasmid AD2) partially restored the ability of R. etli pA-/pD- to form a mixed biofilm with yeast (Fig. 3c). In contrast, complementing with the RHE_PD00332 gene (plasmid AD3) does not restore the phenotype. It is necessary to complement only with the RHE_PD00333 gene to determine if its product is involved in the phenotypic plasticity of R. etli. These results suggest that the ABC transporter gene (RHE_PD00333) is involved in the fungal-bacteria interaction.
S. cerevisiae produces a small molecule that affects R. etli growthTo determine how S. cerevisiae affects the growth of R. etli pA-/pD- (Fig. 4a), we evaluated the inhibitory activity of methanol extracts of S. cerevisiae culture supernatants.Figure 4S. cerevisiae s1278B produces a small molecule that only affects the growth of R. etli strains that do not harbor the symbiotic plasmid and plasmid A. (a) S. cerevisiae and R. etli strains were inoculated in close proximity onto MMD soft agar. R. etli pA-/pD- grew, forming a swarm far from the yeast colony. (b) Inhibition of R. etli pA-/pD- growth by 5 µg/mL of a purified compound from the yeast supernatant, which we named Sc2A. (c) Proposed molecular structure of Sc2A.Full size imageInterestingly, we found that the methanol extract inhibited R. etli pA-/pD- growth but had no activity against wild-type R. etli (Fig. 4b). We investigated the chemical constituents of the S. cerevisiae culture supernatants. After succesive organic solvent extractions, the methanolic extract was fractionated by HPLC and 8 fractions were obtained. Each fraction was tested for its determine its effect on the growth of R. etli pA-/pD-. Only a fraction with the ability to inhibit the growth of R. etli pA-/pD- was identified. This resulted in ~ 90% pure sophoroside, judging by its appearance as a dominant peak in the mass spectra obtained by Fast Atom Bombardment Mass Spectroscopy (FAB). As a result, a new sophoroside with bacteriostatic activity, named Sc2A, was isolated (Fig. 4c). The structure of Sc2A was elucidated by a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS.Sc2A was isolated as a crystalline powder with a positive optical rotation ([α]D25 + 13.7°, c0.58, H2O). The molecular formula of Sc2A was determined to be C30H50O24 from its positive-mode FAB data (m/z 794.26 [M + H]+), which was consistent with the 13C NMR data. RMN1H (CD3OD, 400 MHz) data for Sc2A: δ 5.1 d (J = 3.6 Hz), 4.4 d (J = 8 Hz), 4.23 dd (J = 9, 4.8 Hz), 3.79 t (J = 10.8, 14.4 Hz), 3.73 m, 3.67 m, 3.639 m, 3.63 dd (J = 8, 9.2 Hz), 3.53 dd (J = 5.6, 5.2 Hz), 3.36 dd (J = 3.6, 4 Hz), 3.31 dd (J = 8, 8 Hz), 3.10 dd (J = 8, 7.6 Hz), 2.77 dd (J = 4.4, 6.8 Hz), 2.61 m, 2.46 m, 2.33 m, 2.12 m. RMN13C-DEPT (CD3OD, 400 MHz) data for Sc2A: δ 181.2 (C), 175.9 (C), 98.1(CH), 93.8 (CH), 78.05 (CH), 78.02 (CH), 76.30 (CH), 74.92 (CH), 73.80 (CH), 73.11 (CH), 71.78 (CH), 71.72 (CH), 64.37 (CH2), 62.87 (CH2),62.72 (CH2), 57.24 (CH), 30.70 (CH2), 26.19 (CH2), 28.21 (CH2).The IR spectrum of Sc2A displayed characteristic absorptions of 3416.34 cm-1 (O–H), 1642.10 (C = O), 1405.44 (C–OH), 1242.93 (C–O–C), 1040.36 (C-H), and 598.48 (O-C-O).Sc2A possesses a sophorose linked by 2,5 hexanedione to another molecule of sophorose (Fig. 4c).Sc2A induces the expression of genes involved in symbiosisExpression from the nifH and fixA promoters was studied in R. etli monocultures and cocultures with yeast by monitoring GUS activity in living cells. Cells were grown on solid PY-D medium for 1 day, and monitoring of GUS expression showed that the nifH promoter was strongly induced when R. etli was grown with yeast in liquid medium and on solid medium (Fig. 5).Figure 5The expression of Rhizobium etli genes involved in symbiosis is induced in cocultures with yeast or by exposure to the small molecule Sc2A. (a) Activity of different R. etli promoters in monoculture (Re) or in coculture with yeast (+ Sc). Cells were cultured for 24 h in 1 ml of PY-D in 1.5-mL tubes. The tubes were kept closed to generate an environment with a low oxygen concentration. (b) Activity of the nifH promoter in R. etli cells grown alone (Re) or in coculture with yeast (+ Sc) on PY-D agar. (c) Effect of Sc2A on the expression of the nodA gene in R. etli cells grown in liquid culture. Cells stimulated with the flavonoid naringenin were included as a positive induction control. The data are representative of 3 independent experiments +/− the S.D. values.Full size imageAt the beginning of the symbiosis, the legume roots exude flavonoids, which induces in R. etli the expression of a group of genes (nod) involved in the synthesis of lipochitooligosaccharides, also called nodulation factors (NFs). Recognition of NFs by the host plant triggers both rhizobial infection and initiation of nodule organogenesis14. NodA protein is involved in N-acylation of the chitooligosaccharide backbone of NFs. Given the participation of nodA in the interaction of R. etli with a eukaryote, we decided to evaluate the expression of this gene in response to exposure to 5 µg/mL of Sc2A (this concentration is similar to that found in cocultures). We found that Sc2A induces the expression of nodA (Fig. 5c). However, the levels of induction of nodA were moderated compared to the values obtained upon naringenin induction (Fig. 5c). More

Morphology and light-emitting behavior of the undescribed Japanese Terebellidae wormIn 2016, some of the present authors were exploring shallow coastal waters (depth less than 1 m) to observe the ecological behaviors of marine animals in the Noto Peninsula, when they discovered unknown violet-light-emitting worms. At the sampling point, the worms were living in small holes (a few centimeters in diameter) or in cracks in rocks covered by sand at the shallow sea bottom (Supplementary Fig. S1). We successfully video-recorded their emission of violet light from the whole tentacle stretching into sea water when stimulated by air bubbling at night (Fig. 1A–C; Supplementary Videos S1 and S2). The violet-light emission consisted of rapid flashes with variable duration in the order of milliseconds (Supplementary Video S3), as observed for the worm P. perplexus in response to stimulation17. From our morphological observation, we identified the violet-light-emitting worm as a member of Polycirrus on the basis of the following characteristics18: (1) a sheetlike prostomium covering the upper lip; (2) avicular unicini on some neuropodia; (3) no branchiae. The specimens also have the following characteristics: (1) neurochaetae beginning on last notochaetigerous segment, chaetiger 14; (2) uncini with a long neck and concave base; (3) notopodial pre- and post-chaetal lobes both similar shape. These characters are also found in Polycirrus disjunctus Hutchings and Glasby18; however some of the characters in parapodial lobes and chaetae have differentiation. Thus, we concluded that this species should be treated as an undescribed species. Further comparative observation is needed to describe the species. At this time we treated the Polycirrus species observed in this study as Polycirrus sp. ISK. Application of an electric pulse also caused clear light emission from the tentacles of the living worm (Fig. 1D; Supplementary Video S3), and the luminescence spectrum showed that its λmax was 444 nm or slightly longer, depending on the individual (Fig. 2A). We also found that light emission was efficiently induced by the addition of KCl solution and observed the time course of light emission with rapid fluctuations with variable duration in the order of milliseconds for up to 30 s (Fig. 2B). The flash pattern was similar to that observed in a study of P. perplexus17. In the genus Polycirrus, P. medius and P. nervosus in Japan have been described18,19. However, the morphological features of the species in the present study differed from these species on the basis of our observations described above.Figure 1Photographs of Polycirrus sp. ISK. (A) Polycirrus sp. ISK in its natural habitat with bright-field illumination. (B) Bioluminescence of Polycirrus sp. ISK in its natural habitat without bright-field illumination. The worms were stimulated by air bubbling from SCUBA gear. (C) A single worm with stretched tentacles. Tentacles are indicated by white arrows. (D) The worm with light emission at the tentacles. This worm was stimulated by an electric shock. Scale bars = 100 mm for A and B, 10 mm for C and D. Each photograph was extracted from the videos recorded with the following settings: sensitivity, ISO 51200 or 11 lx; white balance, 4300 K or 5800 K; shutter speed, 1/30 s or 1/60 s; iris, F1.8-3.5; frame rate, 29.97 fps or 60 i; frame size, 1920 × 1080 pixels. Original high quality videos are available at https://youtu.be/KEsU0kWAEfg and https://youtu.be/24dxvPlBDB0Full size imageFigure 2Luminescence spectra and KCl-induced light emission of Polycirrus sp. ISK. (A) Spectrum analysis of Polycirrus sp. ISK using a living worm stimulated by an electric shock. The luminescence spectra were obtained from two different individuals. The λmax represented in closed circles and open circles were 444 nm and 446 nm, respectively. (B) Typical light-emission signal of a living worm soaked in 667 mM KCl. The black line indicates luminescence intensity after adding KCl solution, and the gray line indicates luminescence intensity before adding KCl solution.Full size imageJapanese Polycirrus spp. have not been described as luminous worms according to our review of the literature and web pages. In addition, the number of reports for new Polycirrus spp. from all over the world has been increasing, but a limited number of species are known to emit light13,17,18. Our finding of KCl-induced light emission from Polycirrus sp. ISK suggested that we can easily test the light-emitting ability of Polycirrus spp. by luminescence measurement just after adding KCl solution. A spectrum pattern has been reported for only one species, P. perplexus collected in California17, and it would be necessary for further understanding of these species to examine the light-emitting abilities and to compare light-emitting behaviors and spectrum patterns. The color of bioluminescence is often related to habitat, and light in the blue range is typical for pelagic species20. Thus, one of the points to be focused on is the ecological function of the violet-light emission of this worm inhabiting in a shallow coastal water environment. In P. perplexus, deterring predation is a possible function of luminescence based on that species’ habitat and its violet-light emission17,21. As shown in Supplementary Videos S1, S2, which are the first video records of in situ light emission of a Polycirrus species, the air bubble-stimulated luminescence of Polycirrus sp. ISK in its natural habitat also seemed to deter predation, but this explanation is still speculative.Differentially expressing genes between the tentacles and the rest of bodyA few years after discovering this worm, we found it difficult to collect enough of them to conduct common biochemical and chemical analyses because we did not find a place densely inhabited by hundreds of the worms whose wet weight was a few tens of milligrams (e.g. 16.5, 29.8, or 31.8 mg). Next, we conducted RNA-Seq analysis. In luminous animals with strong light emission, such as firefly or syllid polychaetes (Syllidae), luciferase expression is high especially at the luminous organ or in the whole body22,23. On the other hand, the light emission of Polycirrus sp. ISK was not so strong compared to that of fireflies, and the light-emitting area was limited to the tentacles. In addition, the genetic information related to the tentacles responsible for various ecological functions is still limited. Thus, in the present study we decided to purify RNA from the tentacles and the rest of body separately (Fig. 1C) and performed RNA-Seq analysis followed by a computational analysis using the MASER pipeline24. By de novo assembly, 110,775 contigs were predicted; 26.1% of them showed more than twice the expression level in the tentacles than in the rest of body, whereas 20.8% showed more than twice the expression in the rest of body than in the tentacles. When we performed a blastX search to the NCBI nr database for the contigs longer than 300 bp, 35.6% showed significant homology with registered genes with e-values of less than 1e−10. The average length for these contigs was 1384 bp, and half of them were in the range of 463–1863 bp (Supplementary Fig. S2). In the assembled sequence, we found the cytochrome oxidase subunit I (COI) gene and tried to construct a phylogenetic tree. However, the obtained phylogenetic tree was unreliable due to the low bootstrap values as shown in Supplementary Fig. S3.To focus on the tissue-specific genes, we first picked up genes with high expression levels based on high fpkm values (over 1000) and then ranked these genes based on the tissue-specificity judged by the comparison of fpkm values in tentacles and the rest of body. In tentacle-specific genes, we found that some genes coding for lectin(-like) domains were ranked in the top eight as shown in Supplementary Table S1. Of the top eight genes in the rest of body-specific genes (Supplementary Table S2), seven exhibited no similarity to any genes, and the remaining gene exhibited significant similarity to a hypothetical protein of Capitella teleta, which is a Polychaetes species with whole-genome information available25. Recently, TPM is preferably used to normalize expression level, and the value is used for statistical differential expression analysis26, and we also calculated TPM for tissue-specific genes (Supplementary Table S3).As we were unable to conduct statistical differential expression analysis due to no biological/technical replication resulted from difficulties in the sample collection, we simply compared TPM value between the tentacle and the rest of body samples. The ratio of TPM (tentacle/rest of body) was calculated, and then top 100 genes (Fig. 3A), which were highly expressing in the tentacle, were selected. Similarly, top 100 genes highly expressing in the rest of body were selected using the ratio of TPM (rest of body/tentacle) (Fig. 3B). These gene lists were annotated by gene ontology (GO) terms and analyzed using WEGO program27. WEGO results showed different expression patterns for the tentacle and the rest of body. In the tentacle, GO terms including cell adhesion, biological adhesion, small molecular binding, positive regulation of biological process, regulation of response to stimulus, carbohydrate binding, and immune response were significantly higher (Fig. 3C, D). In the rest of body, GO terms including hydrolase activity, catalytic activity, localization, and establishment of localization were significantly higher. In the top 100 genes highly expressing in the tentacle, we found 21 genes annotated as a gene coding for fucolectin by blast search (Supplementary Table S4). Fucolectin is a fucose-binding lectin involved in the innate immunity of diverse invertebrate species28. However, its function in invertebrates remains unclear, and no information is available for Terebellidae, including sequence information. Fucolectin was first identified in eel with mRNA distribution mainly in liver and gill28. In sea cucumber, expression of the fucolectin gene is confirmed in respiratory trees, muscle, and tentacle29. We were not able to see whether this gene was expressed in the respiratory organ of Polycirrus sp. ISK because a characteristic of the genus Polycirrus is the absence of branchiae18. Nevertheless, the tentacle-specific expression of fucolectin was consistent with the observation in sea cucumber, and the high expression of such proteins involved in innate immunity seemed reasonable because tentacles stretching out of their bodies can be damaged by attack of predators and thus are threatened by infectious bacteria and other pathogens11, as is the respiratory organ. In addition, localization of antimicrobial compounds in Terebellidae worms is suggested to be of antiseptic importance in damage by predation14. This study would provide indispensable information about the ecological meaning of Polycirrus sp. ISK’s life in future genetic studies.Figure 3WEGO analysis of highly expressing genes in the tentacle and the rest of body. (A) Box plot graph for the distribution of TPM value for top 100 genes highly expressing in the tentacle. Corresponding genes in each part are colored in the same gradation color according to the TPM value (red to blue form higher to lower value). (B) Box plot graph for the distribution of TPM value for top 100 genes highly expressing in the rest of body. Each gene is colored as in (A). (C) WEGO analysis of top 100 genes highly expressing in the tentacle (orange bar) and the rest of the body (blue bar). (D) P-values from Chi-square tests obtained by WEGO analysis. CC cellular component, MF molecular function, BP biological process.Full size imageTranscripts coding for luciferase-like genes in the wormTo find genes similar to the known luciferase, which is an enzyme oxidizing a specific compound called luciferin to emit light, from related species in polychaetes, we performed a blastX analysis against the Odontosyllis luciferase sequence using our RNA-Seq data. We found a gene coding for a protein that exhibited similarity to Odontosyllis luciferase, but the e-value was more than 1e−10 (Supplementary Fig. S4). In addition, the top hit for this gene analyzed by blastX was annotated to code an uncharacterized protein of Saccoglossus kowalevskii (Hemichordata), and its specific function was not predicted. Other hits were for genes from Chordata, Mollusca, and other phyla but there was no hit from Annelida. This result would suggest that the light-emission system of Polycirrus sp. ISK differs from that of the genus Odontosyllis, although further experiments using high purity Odontosyllis luciferase and the substrate will be necessary to confirm this. In further blastX analyses of representative luciferases, photoproteins, and a putative luciferase [luciferases from the ostracod Cypridina noctiluca (Accession number: BAD08210.1), the copepod Gaussia princeps (AAG54095.1), the deep-sea shrimp Oplophorus gracilirostris (BAB13775.1 and BAB13776.1), the firefly Photinus pyralis (AAA29795.1), the sea pansy Renilla reniformis (AAA29804.1); photoproteins from the hydrozoan jellyfish Aequorea victoria (AAA27720.1), the hydroid Clytia gregaria (CAA49754.1), the hydroid Obelia geniculate (AAL86372.1); a putative luciferase from the tunicate Pyrosoma atlanticum30 sequences using our RNA-Seq data], we found some tissue-nonspecific genes whose sequences exhibited similarity to firefly luciferase (FLuc) or Renilla luciferase-like protein (RLuc-like) sequences with an e-values of less than 1e−10 and percent identity of more than 50%. FLuc is a member of the acyl-adenylate-forming superfamily of enzymes responsible for firefly luciferin-dependent bioluminescence, which is found in terrestrial luminous beetles emitting light ranging from green to red31. Previously, a putative acyl-CoA synthetase protein was found in the luminous organ of firefly squid emitting blue light32, but there is no clear biochemical evidence that such protein is responsible for firefly squid’s bioluminescence. On the other hand, RLuc is responsible for coelenterazine-dependent bioluminescence, which is found in marine luminous organisms belonging to various taxa. An RLuc-like protein is found to be localized in luminous organs of the brittle star Amphiura filiformis, as revealed by taking advantage of the cross reactivity of anti-RLuc antibody to A. filiformis RLuc-like protein33. A recent study reported that recombinant RLuc-like protein found in P. atlanticum exhibited luciferase activity to coelenterazine30. However, an RLuc-like protein from sea urchin Strongylocentroutus purpuratus is confirmed to exhibit dehalogenase activity to various substrates but no luciferase activity to coelenterazine34. Therefore, it is suspected that Polycirrus sp. ISK possesses a luminescence system using an RLuc-like enzyme.Coelenterazine content in the wormTo investigate whether Polycirrus sp. ISK possesses not only a Renilla luciferase homologous gene but also coelenterazine, we analyzed an ethanolic extract of Polycirrus sp. ISK by UPLC with a UV–visible detector (Fig. 4). The obtained UPLC chromatogram did not show a peak corresponding to that of authentic coelenterazine. When further checking the chromatogram, we found the peak at a retention time similar to those of authentic coelenteramide and coelenteramine, which can be formed from coelenterazine. However, the absorption spectrum obtained by UPLC analysis and the mass spectrum obtained by MS/MS analysis were not identical to those of authentic coelenteramide or coelenteramine (Fig. 4 and Supplementary Figs. S5 and S6). In addition, when the worm extract was mixed with a recombinant RLuc, we did not detect luminescence using a luminometer. These results suggested that the luminescence system in the worm was independent of coelenterazine, although a RLuc homologous gene was found. Similarly, the existence of an RLuc homologous gene was reported in P. atlanticum, which has been suggested to use a coelenterazine-independent luminescence system relying on bacterial bioluminescent symbionts30,35. We also mixed the worm extract with a recombinant cypridinid luciferase, but we did not detect luminescence using a luminometer. This result was consistent with Harvey’s observation for P. caliendrum16. To examine whether the luminescence system is based on luciferin–luciferase reaction, which is found in various luminous animals including some syllid Odontosyllis spp.23,36,37,38,39, we prepared two different extracts of the worm using 100 mM HEPES–NaOH buffer (pH 7.4) and methanol, and subsequently subjected a mixture of the two to luminescent measurement. As a result, no light emission was detected from the mixture of the buffer and methanolic extracts of the worm. This result was also consistent with Harvey’s observation for P. caliendrum16. However, there is still a possibility that the light emission is based on luciferin–luciferase reaction, because luciferin–luciferase reaction found in fireflies or luminous mushrooms requires a cofactor such as ATP or NADPH, and we did not test all possible conditions due to the limitation of the number of collected specimens. In addition, extraction of luciferin and luciferase in the active form is sometimes difficult, as shown in previous studies37. Further studies using hundreds or more of the specimens must be performed to elucidate the mechanism underlying the violet-light emission.Figure 4Comparison of the ethanolic extract of Polycirrus sp. ISK with CTZ, CTMD, and CTM. (A) UPLC analysis of (a) the extract, (b) authentic CTZ, (c) authentic CTMD, and (d) authentic CTM using a multiwavelength detector. The black solid line indicates detection at 333 nm, and the blue solid line indicates detection at 435 nm. The compound between the red vertical dashed lines was collected for MS/MS analysis. (B) Absorption spectra of the compound from the extract, CTZ, CTMD, and CTM obtained at retention times of (a) 9.65, (b) 10.89, (c) 9.47, and (d) 9.27 shown in (A). CTZ coelenterazine, CTMD coelenteramide, CTM coelenteramine. These chemical structures are shown in Supplementary Fig. S5.Full size image More

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