Ecology

Climatic conditions and phenologyThe growth and development of T. durum plants was moderately differentiated by weather conditions in the analyzed years (Table 1).Table 1 The duration of growing seasons (Days), sum of temperatures (Temp.) and sum of precipitation (Prec.) during the growth and development of T. durum Desf. in the analyzed years.Full size tableThe growing seasons of 2015, 2016 and 2017 lasted 136, 132 and 145 days, respectively; the sum of temperatures was determined at 2011.3, 1895.6 and 2069.9 °C, respectively, and the sum of precipitation was determined at 366.7, 360.1 and 350.5 mm, respectively. However, a comparison of cumulative temperatures and precipitation in the phenological phases of T. durum in each year of the study indicates that temperature and precipitation could have influenced the duration of the examined phases and plant growth indicators (Fig. 1). Weather conditions were generally favorable for the growth and development of T. durum in 2015 and 2016. Cumulative temperatures and precipitation were quite similar in 2015 and 2016 up to the booting stage, but precipitation levels in successive stages were higher in 2016 than in 2015. The growing season was shortest in 2016 and longest in 2017, mainly due to low temperatures during sowing and seed germination, and high precipitation during tillering, grain formation and ripening.Figure 1Cumulative temperatures and precipitation in the phenological phases of T. durum in 2015–2017.Full size imageBiophysical parameters: LAI and SPADThe LAI denotes the area of photosynthetic tissue per unit ground surface area (m2 m−2). The LAI is directly associated with plant canopy, and it is an indicator of net primary production, water and nutrient use, and the carbon balance. SPAD is a measure of leaf greenness that is directly associated with chlorophyll content and nitrogen sufficiency.The main effects of LAI and SPAD were analyzed separately in the framework of the Zadoks scale to reveal the significant effects of years, nitrogen rates and sowing density, and an absence of significant effects associated with the application of the growth regulator (see Tables 1.1–1.6 in the Supplementary Information). In the analyzed years, LAI and SPAD were similar in the 2nd node detectable stage (Z32), but they differed in the stem elongation stage (Z45) and the ear emergence and heading stage (Z59), when LAI values were higher and leaf greenness values were lower in 2016 and 2017 than in 2015. These findings can be attributed to moderate temperatures and precipitation in 2015, and high precipitation in the critical growth stages in the remaining years. The general trend associated with the nitrogen rate was similar across the examined growth stages, i.e. a significant increase in LAI and SPAD values with nearly identical effects were noted in treatments with nitrogen rates of 80 and 120 kg ha−1. A similar trend was observed in sowing density. In treatments with a sowing density of 450 and 550 germinating seeds per m2, LAI values continued to increase, whereas SPAD values were below those noted in the treatment with a sowing density of 350 germinating seeds per m2. The only significant interaction was observed between years and nitrogen rates.The average values of LAI continued to increase in successive growth stages and were determined at 1.30 at Z32, 1.75 at Z45, and 1.99 at Z59. In turn, leaf greenness was significantly lower in the stem elongation stage (Z45) than in the preceding (Z32) and subsequent (Z59) stages.The significant effect of the years × growth stages interaction for LAI and SPAD values resulted from similar means in stage Z32 in all years, as well as higher LAI values and lower SPAD values in subsequent growth stages in 2016 and 2017 than in 2015. In 2015, the increase in the nitrogen rate induced only a rising trend in LAI and SPAD values, whereas significant differences were observed in 2016 and 2017. To summarize, it should be noted that in successive Zadoks growth stages, the interactions between years, nitrogen rates and sowing density exerted significant effects on LAI and SPAD values, whereas the effects of year × nitrogen rate interactions were significant only in selected growth stages.Contribution of different sources of variation to physiological and biophysical parameters of plant growthThe calculated eta-squares η2 provide information about the contribution of different sources of variation to physiological variables (Table 2). The experimental years and agronomic factors (33.1% and 38.6%), growth stages, and interactions with other factors (32.5% and 39.3%) and random factors (34.4% and 22.1%) made similar contributions to the variation in the LAI and chlorophyll content. The variation in the net photosynthetic rate was related mostly to variations across years (32.6%) and the interactions between growth stages and other factors (24.3%). The variation in the transpiration rate was attributed mostly to the main effects of growth stages (45.8%) and the year × growth stage interaction (16.1%). Instantaneous WUE was strongly determined by variation in agronomic factors and growth stages (22.3% and 21.1%, respectively).Table 2 Eta-square (η2) values for the sources of variation in the leaf area index (LAI), chlorophyll content (SPAD), net photosynthetic ratio (Pn), transpiration rate (E) and instantaneous water use efficiency (WUE).Full size tableIt is worth noting that the variation in agronomic factors made a considerable contribution to the total variation in LAI (22.3%) and SPAD (11.8%), but only a marginal contribution to the net photosynthetic rate (0.4%) and transpiration (2.0%).Photosynthetic indicators— net photosynthetic rate, transpiration rate, and instantaneous water use efficiencyThe effects of the net photosynthetic rate (Pn), transpiration rate (E) and instantaneous WUE were highly differentiated in successive growth stages, and relatively small differences were noted for agronomic factors (see Tables 2.1–2.9 in the Supplementary Information). At the same time, the analyzed photosynthetic indicators differed in successive stages of growth. The net photosynthetic rate was similar in the 2nd node detectable stage (Z32) and the stem elongation stage (Z45) at 29.7 μmol CO2 m–2 s–1, and it was 15% higher at the end of the heading stage (Z59) than in the preceding stages. The transpiration rate continued to increase by 60% on average in successive stages of growth and development, from 1.59 H2O m–2 s–1 in stage Z32, to 2.52 mmol H2O m–2 s–1 in stage Z45, and 4.06 mmol H2O m–2 s–1 in stage Z59.An analysis of the results noted in different growth stages across years revealed significant year × growth stage and growth regulator × growth stage interactions (Fig. 2).Figure 2Mean values and standard error of photosynthesis indicators for year × growth stage (upper) and growth regulator × growth stage interactions (GR 0—without growth regulator, GR 1—with growth regulator).Full size imageThe year × growth stage interaction resulted from differences in the rates of photosynthesis and transpiration in the analyzed growth stages across years. In 2015, the net photosynthetic rate was similar in the first two growth stages, and it increased by around 30% at the end of the heading stage (Z59). In 2016, the photosynthetic rate continued to increase in successive growth stages. In 2017, the net photosynthetic rate was around 10% higher in the 2nd node detectable stage (Z32) than in the stem elongation stage (Z45) and at the end of the heading stage (Z59). The transpiration rate increased significantly in successive stages of plant growth and development, and the only exception was noted in 2015, when the analyzed parameter was similar in stages Z32 and Z45. The WUE index was highest in stage Z32, and a significant interaction was noted due to the correlation between the net photosynthetic rate and the transpiration rate in the remaining stages. Water use efficiency was similar in stages Z32 and Z45 in 2015, and in stages Z45 and Z59 in 2016, whereas significantly lower values in successive stages of plant growth were noted in 2017.The growth regulator was the only agronomic factor that induced significant differences in the net photosynthetic rate across the examined growth stages. Photosynthesis indicators were similar regardless of the application of the growth regulator, and significant interactions resulted mainly from varied disproportions between the end of heading and the stem elongation stage in treatments with and without the application of the growth regulator.It should be noted that the interactions between growth stages and nitrogen rates and sowing density were not significant, which implies that the effects of the interactions between increasing nitrogen rates and sowing density on photosynthetic indicators in successive growth stages were similar to the average values of photosynthetic indicators in the corresponding growth stages (Supplementary Information).Agronomic traitsThe means for yield components and yield are presented in Tables 3.1–3.8 of the Supplementary Information. Stem length was differentiated by the nitrogen rate and nitrogen rate × year interaction. Nitrogen rates of 80 and 120 kg N per ha increased stem length by 11% and 13%, respectively, relative to the unfertilized control. The significant year × nitrogen rate interaction resulted from the fact that the nitrogen-induced increase in stem length was smaller in 2015 (0.07 cm per 1 kg of nitrogen) than in 2016 and 2017 (0.09 cm per 1 kg of nitrogen). In 2015, ear length was similar to that noted in the remaining years, and only in 2017, ear length was 7% higher than in 2016. Ear length and the number of kernels per ear increased with a rise in nitrogen rate and decreased with a rise in sowing density.Grain weight per ear and 1000 kernel weight were highest in 2015 and significantly lower in the following years. Grain weight per ear increased only in response to the nitrogen rate of 120 kg ha−1, but 1000 kernel weight was not affected. Both traits decreased with a rise in sowing density. The significant year × nitrogen rate and year × sowing density interactions for both traits can be largely attributed to the magnitude of differences between years, rather than an increase or a decrease in this trend.The biological yield (grain and straw) differed across years and nitrogen rates. In 2016, the biological yield was similar to that noted in 2015 and significantly higher (by 30%) than that noted in 2017. The biological yield increased by 28% and 35% in response to nitrogen rates of 80 and 120 kg ha−1, respectively, relative to the unfertilized control. The significant year × nitrogen rate interaction was associated with variations in nitrogen use efficiency, and the difference between maximal biological yield was determined at 0.5 t ha−1 in 2015, 2.3 t ha−1 in 2016, and 2.8 t ha−1 in 2017.Grain yield was similar in 2015 (4.94 t ha−1) and 2016 (5.38 t ha−1), and it was significantly lowest in 2017 (3.87 t ha−1). Straw yield was highest in 2016 (2.86 t ha−1), and it exceeded the values noted in the remaining years by 16%. The harvest index was similar in 2015 and 2016, and it was 9% lower in 2017. Grain yield increased by 30% and 36%, whereas straw yield increased by 20% and 35% in response to the nitrogen rates of 80 and 120 kg ha−1, respectively. A minor increase in grain yield (3%) was observed in treatments with a sowing density of 550 seeds m−2 relative to the remaining sowing densities.Path modellingA simple correlation analysis of manifest variables in all phenological stages revealed significant correlations between the LAI and leaf greenness (SPAD) only in stage Z32, as well as a very strong correlation between the net photosynthetic rate and the transpiration rate, which was positive in stages Z32 and Z45 and negative in stage Z59. No simple correlations were noted between the indicators of physiological processes (Pn, E, WUE) and biophysical parameters (LAI, SPAD). AAll correlations between the manifest variables of yield components and biological yield were statistically significant, excluding the correlation between stem length and ear length (Supplementary information).All correlations between the manifest variables of yield components and biological yield were statistically significant, excluding the correlation between stem length and ear length (Supplementary Information). The outer and inner PLS-PM models well fit the data, and their goodness of fit was determined at 0.973 and 0.786, respectively. The outer weights provide information about the relative importance of a manifest variable for the corresponding latent variable (for details please see the Supplementary Information). Outer weights that exceed 0.3 are considered meaningful. By the same token, loading estimates represent the correlations between a latent variable and the corresponding manifest variables. Loadings higher than 0.7 capture more than 50% of the variability contributed by a latent variable to the corresponding manifest variable. In general, both indicators in the outer model, i.e. outer weights and loadings, exceeded the thresholds, which indicates that manifest variables were strongly related with latent variables. Growth regulators (({w}_{GR}) = − 0.007) and the length of the growing season (({w}_{DAYS})=0.197) provided the only evidence for the low explanatory value of latent variable A (agronomic factors).In the inner model, all equations that regressed latent variables well fit the data and were statistically significant (Table 3). The latent variables expressed by the value of R2 increased in successive stages of T. durum growth and development, from 0.218 in physiological processes in stage Z32 (Table 3, Eq. 1) to 0.698 and 0.708 in yield components and Biological Yield, respectively (Table 3, Eqs. 7 and 8). It is worth noting that in successive stages of growth, the value of physiological processes was relatively lower in comparison with biophysical parameters.Table 3 Parameters of regression models for latent variables.Full size tableThe analysis of path coefficients (βi) revealed that agronomic factors (A) and climate conditions (CC) in stages Z32, Z45 and Z59 exerted a specific influence on physiological processes (PP) and biophysical parameters (BP) of T. durum plants. Agronomic factors directly determined physiological processes in all stages and biophysical parameters in stages Z32 and Z59. At the same time, climate conditions did not exert a direct influence on physiological processes in any stage, but directly affected biophysical parameters in all stages. All of the modeled parameters, i.e. agronomic factors, climate conditions and physiological processes, significantly influenced biophysical parameters in stages Z32 and Z59, but not Z45. Consequently, it can be stated that agronomic factors were the main determinant of variability in physiological processes (photosynthesis, transpiration) in a model evaluating the impact of agricultural practices on yield and the manifest variables associated with T. durum growth and development. At the same time, physiological processes made a significant but negative contribution to biophysical parameters. A one unit increase in photosynthesis processes with constant values of agronomic factors and climate conditions implies a decrease of − 0.382, − 0.065 and − 0.395 in biophysical parameters in stages Z32, Z45 and Z59, respectively.The performance of every preceding latent variable in terms of its total impact on the target latent variable, i.e. the biological yield of T. durum (IPMA – Importance-Performance Map Analysis), was analyzed to highlight latent variables associated with agricultural practices that improve biological yield. The total effect (importance) of preceding latent variables (A, CC32, PP32, BP32, CC45, PP45, BP45, CC59, PP59, BP59, YC and CC) on the anticipated performance of the specific target (Biological Yield) is presented in Fig. 3.Figure 3Importance-Performance Map Analysis presenting the impact of latent variables on biological yield (A—agronomic factors, YC—yield components, CC32, CC45, CC59—climate conditions in growth stages, PP32, PP45, PP59—physiological processes, BP32, BP45, BP59—biophysical parameters in the phenological stages of plant growth and development Z32, Z45 and Z59, CC—climate conditions for the entire growing season).Full size imageThe importance and performance of latent variables that influenced the biological yield of T. durum varied. The biological yield of T. durum was affected mostly by agronomic factors (A), followed by yield components (YC) and biophysical parameters (BP) in growth stages Z59 (BP59) and Z32 (BP32), climate conditions in stage Z59 (CC59), and climate conditions in stage Z32 (CC32). A one unit increase in the above latent variables led to an increase of 0.575, 0.422, 0.234, 0.203 and 0.109 units in biological yield, respectively. At the same time, the performance scores of these latent variables were determined at 53.1, 53.5, 67.1, 59.6 and 61.8, respectively (scores closer to 100 denote higher performance). The remaining latent variables, in particular climate conditions for the entire growing season and physiological processes in stage Z32, were characterized by low importance and exerted a relatively small effect on biological yield performance.The results of the importance-performance analysis clearly indicate that latent variables have considerable potential to optimize the agricultural conditions for the growth and development of T. durum plants. More

Direct nucleotide sequencingReadable electropherograms were obtained from both direction in COI fragments of all three individuals of the Japanese spiny lobster. COI sequences determined by direct nucleotide sequencing ranged from 807 to 864 bp and have been deposited in International Nucleotide Sequence Database Collection (INSDC) under accession numbers of LC571524‒LC571526. No stop codon was observed in these sequences (designated by PJK1-direct, PJK2-direct, and PJK3-direct). No indel was observed between these sequences. All nucleotide substitutions at 19 variable sites observed between these sequences were transition at the 3rd position of a codon, and all substitutions were synonymous. The mean Kimura two parameter (K2P) distance between these three haplotypes was 1.510 ± 0.352% SE and that between these sequences and a reference sequence of P. japonicus (NC_004251) was 1.087 ± 0.270%, which were all well within the range reported for Japanese spiny lobster samples collected in Japan and Taiwan9,10.Electropherograms obtained by forward primer for 12S fragments were not readable, while those by reverse primer were readable in all individuals. 12S sequences determined by direct nucleotide sequencing using reverse primer alone ranged from 551 to 570 bp and have been deposited in INSDC under accession numbers of LC605705‒LC605707. Of nine variable sites, eight were transition and one was indel. The mean K2P distance between these three haplotypes (designated by PJK1-12Sdirect, PJK2-12Sdirect, and PJK3-12Sdirect) was 0.970 ± 0.338%, and that between these sequences and a reference sequence of P. japonicus was 0.835 ± 0.282%.Electropherograms obtained by both primers for Dloop fragments were readable only in one individual (PJK2). This Dloop sequence determined by direct nucleotide sequencing was 762 bp and deposited in INSDC under accession number of LC605749. K2P distance between this haplotype (designated by PJK2-Dloopdirect) and a reference sequence of P. japonicus was 3.666%. No indel was observed between the two sequences, and 25 of 27 variable sites were transition.Phylogenetic analysis of clones, heteroplasmy and NUMTsAmong the 36–42 positive COI clones examined per individual, sequences (809–892 bp) of 22–31 clones per individual (75 clones in total) were successfully determined. After alignment, both ends of all sequences were trimmed to fit the shortest sequence obtained by direct nucleotide sequencing, yielding 774–810 bp sequences. Eleven clones of PJK1 were identical to PJK1-direct, as well as seven of PJK2 to PJK2-direct and three of PJK3 to PJK3-direct. These dominant haplotypes (807 bp) were determined to be genuine COI haplotypes of each individual, and representative sequences of these three genuine haplotypes were deposited in INSDC (LC 571527, LC571533 and LC571538). Nucleotide sequences of the remaining 54 clones were all different one another, in which 20 haplotypes were observed in PJK1, 14 in PJK2, and 20 in PJK3 (LC571541–LC571577, OK429332–OK429343, LC654683-LC654687).Phylogenetic tree constructed using three genuine COI haplotypes, 57 unique haplotypes and eight sequences of reference lobster species is shown in Fig. 1. Haplotypes detected from P. japonicus were segregated into four groups (designated by A, B, C and D). Among the outgroup species used, Australian rock lobster (P. cygnus) that morphologically and genetically belongs to the P. japonicus group11,12, appeared to be the closest kin to all haplotypes detected from P. japonicus. All haplotypes in group A were of the same length (807 bp), and no indel was observed. Three distinct clades (designated by c-I to c-III) were observed in group A, in which 14 haplotypes from PJK1, 11 from PJK2 and 11 from PJK3 were cohesively clustered together with their corresponding genuine haplotypes (bold italic). PJK1-C25 was outlier, having 10 nucleotide differences from the genuine COI sequence. The numbers of variable nucleotide sites between haplotypes within c-I, c-II and c-III were 20, 15 and 26, respectively, of which nonsynonymous nucleotide substitutions were observed at 11, 13 and 10 sites. Stop codon was observed only in one haplotype (PJK3-C1). The mean K2P distance between different haplotypes within these clades ranged from 0.320 ± 0.075 to 0.561 ± 0.103%. The mean K2P distances between three clades ranged from 1.343 ± 0.339 to 2.178 ± 0.464%. Although group A must be composed of sequences containing those caused by Taq polymerase error or true heteroplasmic sequences as well as genuine haplotypes, it is difficult to determine the former two categories. All of the non-genuine haplotypes in group A had singleton difference one another, supporting the occurrence of Taq polymerase error. We determined haplotypes (marked with dagger in Fig. 1) differed by less than two substitutions from the genuine haplotype to be due to Taq polymerase error. This criterion may be reasonable, since Taq polymerase-mediated errors were estimated to occur approximately at a frequency of 7.2 × 10−5 per bp per cycle13 to one mutation per 10,000 nucleotides per cycle14. When Taq polymerase error is taken into account, these K2P distances within and between clades and number of haplotypes are likely to be somewhat overestimated. PJK1-C25, two (PJK1-C5 and PJK1-C60) in c-I clade, one (PJK2-C26) in c-II, and five (PJK3-C1, PJK3-C5, PJK3-C26, PJK3-C31, PJK3-C34) in c-III differed by 3 to 10 nucleotides from their genuine haplotypes, which were determined to be heteroplasmic haplotypes.Figure 1Neighbor-joining phylogenetic (NJ) tree showing relationships among 57 different haplotypes of cytochrome oxidase subunit I (COI) or COI-like sequences obtained from the Japanese spiny lobster (Panulirus japonicus), and COI sequences of eight congeneric species derived from the GenBank database. Haplotypes detected from the same individual of the Japanese spiny lobster share the same color. Genuine mtDNA haplotype is shown in bold italic and number of clones examined is shown in parenthesis. Stop codons were observed in haplotypes carrying asterisk. Haplotypes carrying dagger differ from the corresponding genuine mtDNA haplotype by less than two nucleotides (including indel). The bootstrap values greater than 60% (out of 1000 replicates) are shown at the nodes.Full size imageSequence size of haplotypes in groups B to D ranged from 774 to 810 bp. K2P distance between haplotypes of groups A and B ranged from 7.169 to 8.177% with a mean of 7.754 ± 0.973%, that between A and C ranged from 12.073 to 17.392% with a mean of 14.521 ± 1.151%, and that between A and D ranged from 17.472 to 23.880% with a mean of 21.042 ± 1.600%. Multiple stop codons were observed in a haplotype of group B, in five of eight haplotypes of group C, and all haplotypes of group D. Three haplotypes in group C had no stop codon but differed in four to 10 deduced amino acids from the genuine haplotypes. BLAST homology search revealed no identical sequence for haplotypes in groups B to D but indicated that the closest species were P. japonicus or P. cygnus with moderate similarity (83–89% homology). Therefore, all haplotypes of groups B to D (LC571565–LC571570, LC571572–LC571577, LC654683-LC654687) were determined to be NUMTs.Among the 30–35 positive 12S clones examined per individual, sequences (772–806 bp) of 25–27 clones per individual (77 clones in total) were successfully determined. After alignment, primer sequences were trimmed, yielding 731–765 bp sequences. Thirteen clones of PJK1 were identical one another, as well as 12 of PJK2 and three of PJK3, and these were identical to PJK1-12Sdirect, PJK2-12Sdirect and PJK3-12Sdirect, respectively. These dominant haplotypes ranging from 761 to 762 bp in size were determined to be genuine 12S haplotypes of the individual, and representative sequences of these three genuine haplotypes were deposited in INSDC (LC605708‒LC605710). Nucleotide sequences of the remaining 49 clones were all different one another, in which 12 haplotypes were observed in PJK1, 23 in PJK2, and 14 in PJK3 (LC605711‒LC605748, OK429126–OK429131, LC654678-LC654682).Since incorporation of all eight Panulirus species sequences made sequence alignment ambiguous because of multiple indels, reference sequences of P. japonicus and of closely related P. cygnus were used for constructing phylogenetic tree (Fig. 2). Haplotypes detected from P. japonicus were segregated into three groups (designated by A to C). Sequence size of haplotypes in group A ranged from 760 to 762 bp. Three distinct clades (s-I to s-III) were observed in group A, in which 12 haplotypes each from PJK1, PJK2 and PJK3 were cohesively clustered together with their corresponding genuine haplotypes (bold italic). The numbers of variable nucleotide sites between haplotypes within s-I, s-II and s-III were 24, 17 and 16, respectively. Of these variable sites, transversion was observed at five, one and three sites, and indel was observed at one, zero and one sites, respectively. The mean K2P distances between different haplotypes within these clades ranged from 0.345 ± 0.081 to 0.519 ± 0.101%. The mean K2P distances between three clades ranged from 0.936 ± 0.275 to 1.371 ± 0.359%. Haplotypes differed by less than two substitutions (including indel) from the genuine haplotypes are marked with dagger. Five haplotypes in s-I clade and two haplotypes in s-III clade differed by three to six nucleotides from their genuine haplotypes, which were determined to be heteroplasmic copies.Figure 2Neighbor-joining phylogenetic (NJ) tree showing relationships among 52 different haplotypes of clones of 12S rDNA (12S) or 12S-like sequences obtained from the Japanese spiny lobster (Panulirus japonicus), and 12S rDNA sequences of P. japonicus and P. cygnus derived from the GenBank database. Haplotypes detected from the same lobster individual share the same color. Genuine mtDNA haplotype is shown in bold italic and number of clones examined is shown in parenthesis. Haplotypes carrying dagger differ from corresponding genuine mtDNA haplotype by less than two nucleotides (including indel). The bootstrap values greater than 60% (out of 1000 replicates) are shown at the nodes.Full size imageSequence size of haplotypes in group B varied from 731 to 762 bp. K2P distance between groups A and B ranged from 1.336 to 7.445% with a mean of 3.449 ± 0.398%, and those between a reference sequence of P. japonicus and groups A and B were 0.864 ± 0.236% and 3.189 ± 0.410%, respectively. Sequence size of haplotypes in group C varied from 744 to 765 bp. K2P distance between groups A and C ranged from 3.104 to 22.434% with a mean of 12.049 ± 0.901%, and those between a reference sequence of P. japonicus and group C ranged from 3.951 to 21.287% with a mean of 11.764 ± 0.901%. BLAST homology search indicated that the closest species for haplotypes in groups B and C was P. japonicus or P. cygnus with moderate to high similarity (84–98% homology). Therefore, all 13 haplotypes (LC605741‒LC605748, LC654678-LC654682) in groups B and C were determined to be NUMTs.Among the 36–49 positive Dloop clones examined per individual, sequences (777–893 bp) of 26–38 clones per individual (92 clones in total) were successfully determined. After alignment, primer sequences were trimmed, yielding 736–853 bp sequences. Three clones (821 bp) of PJK1 were identical one another and determined to be genuine haplotype of this individual. Nine clones (813 bp) of PJK2 were identical to PJK2-Dloopdirect and determined to be genuine haplotype of this individual. Three clones (821 bp) of PJK3 were identical one another and determined to be genuine haplotype of this individual. Representative sequences of these three genuine haplotypes were deposited in INSDC (LC605750‒LC605752). Nucleotide sequences of the remaining 78 clones were all different one another, in which 25 haplotypes were observed in PJK1, 17 in PJK2, and 36 in PJK3 (LC605753‒LC605815, LC654419-LC654430, LC654675-LC654677).Incorporation of all eight Panulirus species sequences made sequence alignment considerably unreliable because of multiple indels, reference sequences of P. japonicus and of closely related P. cygnus were used for constructing phylogenetic tree (Fig. 3). Haplotypes detected from P. japonicus were segregated into four groups (designated by A to D). Sequence size of haplotypes in group A ranged from 812 to 822 bp. Three distinct clades (d-I to d-III) were observed in group A, in which 17 haplotypes from PJK1, 13 from PJK2 and 15 from PJK3 were cohesively clustered together with their corresponding genuine haplotypes (bold italic). The numbers of variable nucleotide sites between haplotypes within d-I, d-II and d-III were 27, 61 and 28, respectively, of which indels were observed at five, two and four sites and transversion was observed at 0, six and six sites. The mean K2P distance between different haplotypes within these clades ranged from 0.340 ± 0.067 to 1.097 ± 0.139%. The mean K2P distance between these three clades ranged from 7.577 ± 0.951 to 8.770 ± 0.984%. Haplotypes differed by less than two substitutions (including indel) from the genuine haplotypes are marked with dagger. Eight haplotypes in d-I clade, three in d-II clade, and four in d-III clade differed by three to five nucleotides from the genuine haplotype were determined to be heteroplasmic copies.Figure 3Neighbor-joining phylogenetic (NJ) tree showing relationships among 80 different haplotypes of control region (Dloop) or Dloop-like sequences obtained from the Japanese spiny lobster (Panulirus japonicus), and control region sequences of P. japonicus and P. cygnus derived from the GenBank database. Haplotypes detected from the same lobster individual share the same color. Genuine mtDNA haplotype is shown in bold italic and number of clones examined is shown in parenthesis. Haplotypes carrying dagger differ from corresponding genuine mtDNA haplotype by less than two nucleotides (including indel). The bootstrap values greater than 60% (out of 1000 replicates) are shown at the nodes.Full size imageSequence size of haplotypes in groups B to D largely varied from 736 to 853 bp. K2P distances between group A and others ranged from 14.748 ± 1.030% (A vs B) to 61.619 ± 3.045% (A vs D), whereas that between haplotypes of group A and a reference sequence of P. japonicus was much smaller (6.333 ± 0.663%). BLAST homology search revealed no identical sequence for haplotypes in groups B to D and indicated that the closest species for haplotypes in groups B and C was P. japonicus with low to moderate similarity (74–88% homology). On the other hand, no significantly similar sequence was found for haplotypes in group D. Therefore, all 31 haplotypes (LC605788‒LC605815, LC654675-LC654677) in groups B to D were determined to be NUMTs.Impact of heteroplasmy and NUMTs for direct nucleotide sequencingPartial electropherogram obtained by direct nucleotide sequencing for COI amplicon of PJK3 is shown in Fig. 4 (top). Peak signals of this electropherogram are readable, but there are a number of sites where two (asterisk) or three (dagger) signals overlap. Alignment of a genuin haplotype (PJK-C7) and nine NUMTs sequences, corresponding to this partial electropherogram, is shown in Fig. 4 (bottom). At the sites where plural peaks overlap, different NUMT haplotypes were observed to share the same nucleotide different from the PJK3-direct. Heteroplasmic copies in COI determined in this study may have little negative impact on direct nucleotide sequencing, since nucleotides different from the genuine haplotypes were all unique to each heteroplasmic haplotype. Thus, the plural peaks at a site were composed of signals from genuine plus NUMT haplotypes, and the intensity of each peak was positively related to the copy numbers of these haplotypes. Frequent failure to obtain readable electropherograms in 12S and Dloop regions by direct sequencing may be due to extensive indels observed in the NUMT haplotypes.Figure 4A part of electropherogram obtained by direct nucleotide sequencing for COI region of PJK3 (top), and corresponding sequences from genuine haplotype (PJK3-C7) and nine NUMT haplotypes (see Fig. 1) are aligned (bottom). Apparent double (asterisk) and triple (dagger) peaks are observed at seven and five sites, respectively, which are comprised of signals from genuine and NUMT haplotypes.Full size image More

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Field trialsField trials were conducted in three raised beds (1 × 2 × 0.6 m) on the Penn State University campus from July to August 2020. The raised beds were separated by at least 8 m to avoid treatment cross-contamination. Faba bean (Vicia faba L.) seeds were planted at a density of 20 seeds/ m2 (50 plants per bed), and each bed was caged using a metal-framed tent. “Noseeum” nylon mesh (Outdoor Wilderness Fabric s, Inc., Caldwell, ID) was draped over the frame and the edges buried in the soil of the bed. The sides of the cages were fastened closed with zippers to allow access.InsectsAphid and predator beetle colonies were raised separately on faba bean plants in cages (BugDorm 20 cm × 40 cm × 20 cm, BioQuip Products, Inc., Rancho Dominguez, CA) in the field. Larvae and adults of predator beetles were fed with a combination of A. pisum and Rhopalosiphum padi every other day (Supplementary information Fig. S1). Trials involving plants, insects, and entomopathogenic fungi were conducted according to institutional, national, and international guidelines and legislation.Fungal inoculations (Beavueria bassiana)We released first instar aphid nymphs on each faba bean plant on the raised beds (~ 1100 aphids) by gently shaking plastic containers with groups of 20 nymphs and placing them on the plants using a paintbrush. They were allowed to grow and reproduce for fifteen days. During the night, we sprayed spore suspension of the Beauveria strain GHA (BotaniGard ®, MT, USA) at 1.4 × 106 and 1.4 × 1012 spore ha−1, low and high load respectively. Two days after inoculation, we collected adult aphids (~ 4–5 days old) from the experimental plots and measured physiological parameters (see details below). Next, we released 300 adult beetles inside each aphid–fungal inoculated cage, allowed them to feed for 2–3 days in our experimental cages, and then collected beetles for physiological measurement.Identification of critical thermal limits (CTMax and CTMin) of healthy and infected insectsTo determine critical thermal maximum for locomotion (CTMax) of healthy and infected individuals of each species, we employed a protocol modified from Ribeiro et al.25, using a hotplate with a programmable heating rate controlled by a computer interface (Sable Systems, LV, USA). The temperature was monitored by independent thermocouple channels connected to a Hobo 4-channel data logger. One thermocouple was attached to the surface of the hotplate, and the other sensor was attached inside the glass tube plugged by a cotton ball in which we placed an individual insect. This equipment was located inside an automated thermal chamber (interior dimensions: width 40.5 cm × 35 cm length × 40 cm height). We transferred an adult aphid (4-day-old) into the glass tube and exposed it to increasing temperatures at a rate of 0.3 °C min−1 until its locomotion stopped. CTMax was recorded when the insect turned upside down and could no longer return to the upright position within 5 s. The insect was returned to a faba bean leaf for recovery (n = 10 individuals per treatment).To measure the critical thermal minimum for locomotion (CTMin) of healthy and infected individuals of each species (n = 10 individuals per treatment), we used an insulated incubator where the temperature was monitored by independent thermocouple channels connected to a Hobo 4-channel data logger. The sensors were attached inside three glass tubes, each tube with an adult (3 to 4-day-old), and plugged by a cotton ball. The glass tube was exposed to decreasing temperature at a rate of 0.3 °C min−1 until its locomotion stopped. CTMin was recorded when no movement was recorded within 5 s. The insect was returned to an aphid-infested faba bean leaf for recovery. Data were only considered valid if the insect displayed normal activity 2 h after a CTMax or CTMin test.Impacts of infection on voluntary exposure aphids and predator beetles to extreme thermal zonesTo examine how voluntary exposure to ETZ was affected by fungal infection, we collected aphids and predator beetles (3 to 5 day-old) from our field plots and transferred them to a dark plastic bottle. Next, a bottle containing the insects was attached to a choice test arena following a modified protocol from Navas et al.24. This experimental arena allows insects to freely move across extreme temperatures to access food in containers located at each end of the device. To reach food, individuals had to cross an ETZ, either warm or cold. The location of each insect was recorded after 60 min, and it was classified as: exploration for individuals that left the initial black bottle, warm or cold ETZ crossings. The experiment was replicated ten times for each species and treatment condition [aphid: healthy, infected (low and high spore load); predator beetle: healthy, infected (low and high spore load)].Effects of fungal infection and thermal conditions (critical thermal limits and voluntary exposure to ETZs) on longevity of aphids and predator beetlesTo examine whether fungal infection and thermal conditions alter longevity in aphids and beetles, we isolated three individuals from each factor combination (low, high fungal load, CTMin, CTMax, behavior: crosses to ETZ cold, warm, and no cross) from previous experiments, and counted the number of days the adults survived after the exposure to the thermal condition (n = 3 factor combination).Energetic cost associated with fungal infection of aphid and predator beetles under critical thermal limits and voluntary exposure to ETIntracellular ATP content was determined in neutralized perchloric acid extracts and by a spectrophotometric coupled enzyme assay, based on modified protocol from Churchill and Storey26 content (n = 3 per treatment condition). An insect was ground to powder using a mortar and pestle cooled in liquid nitrogen, and then weighed into 1.5 mL microcentrifuge tubes (Eppendorf). Powder was dissolved with 0.1 mL ice-cold TE buffer (50 mM Tris–HCl, pH 7.5 plus 1 mM EGTA) and homogenized by sonication (15 s, three times), using a Q500 Sonicator system (QSonica, Newtown, CT, USA). An aliquot (10 µL) of the well-mixed homogenate was removed for protein determination. Cells were lysed by adding 6% (v/v) ice-cold perchloric acid, strongly vortexed for 2 min and incubated at 4 °C for 10 min. Next, the cell homogenate was centrifuged at 14,462 rpm and 4 °C for 5 min. The resulting supernatant was neutralized by adding KOH/Tris (3 M/0.1 M) and centrifuged again to discard the perchlorate salts. Extracts were kept at 4 °C for their immediate utilization. ATP content was determined spectrophotometrically by following the production of NADPH at 340 nm (ε = 6.22 mM−1 cm−1) and using CARY WinUV-Vis Spectrophotometer (Agilent, Santa Clara, CA, USA). The following reagents were used for the spectrophotometric coupled enzyme assay: 5 U Hexokinase, 10 U Glucose 6-phosphate dehydrogenase, 1 mM NADP + , 5 mM MgCl2 and 10 mM Glucose in HE buffer (100 mM Hepes-HCl plus 1 mM EGTA, pH 7.0) at 25 °C. Chemicals were purchased from Roche (Manheim, Germany) and Sigma (St Louis, MO, USA).Infection statusWe used two different protocols to confirm fungal infection: (1) placing each individual in wet towel paper inside a Ziploc bag to observe hyphal growth27. (2) For insects used in ATP measurements, we followed a modified protocol from Wraight and Ramos28 and Castrillo et al.29. Insect were washed using a serial dilution technique, vortexed for 10 s, and mounted in a drop of lactophenol blue, diluted with distilled water. We then preserved insect body parts (i.e., legs and abdomen terga) at − 80 °C for 12 months and placed in Petri dishes containing potato dextrose agar (PDA HiMedia-GM096) medium (pH 6.8), and incubated for ten days. To confirm infection by B. bassiana, we observed plates every 3 days, identified fungal growth (dense white mycelia), then randomly chose three samples, collected mycelia, and DNA was extracted using PureLink Genomic DNA Kit (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer’s protocol. Next, we used PCR essays (25 µL) contained 1 × Q5 Hot Start High-Fidelity Master Mix (New England BioLabs), following a protocol modified from Castrillo et al.29 using primers GHTqF1 (5′-TTTTCATCGAAAGGTTGTTTCTCG) and GHTq R1 (5′-CTGTGCTGGGTACTGACGTG) amplified a 96-bp region of the SCAR fragment. The PCR protocol was initial denaturation at 98 °C, followed by 30 cycles at 98 °C for 1 min, annealing at 58 °C for 1 min; and extension at 72 °C for 1 min. PCR products were visualized in a 1.0% (wt/vol) agarose gel stained with ethidium bromide.Data analysisAll data were tested for statistical test assumptions using a qqplot, Levene’s homogeneity test and the Shapiro–Wilk normality test at alpha = 0.05 significance level. For critical thermal limits (CTMax and CTMin) experiments, the data sets were non-normal and transformation did not normalize the residuals, so we used nonparametric ANOVAs (Kruskal–Wallis) followed by post-hoc nonparametric multiple comparisons. For voluntary exposure to ETZs, we used a generalized linear model with treatment (healthy, low and high spore load) with Poisson distribution, followed by comparisons within each treatment group. For healthy insects, we used a t-test to compare crosses between warm or cold ETZs; for infected insects, we conducted ANOVAS for comparisons among 23 °C, warm or cold ETZs.ATP data: Data for CTMax of A. pisum were non-normal, and transformation did not normalize the residuals, nonparametric ANOVAs (Kruskal–Wallis) were then used and followed by post-hoc nonparametric pairwise comparisons with Wilcoxon tests. ATP data sets from voluntary exposure to ETZs were analyzed following the same protocol as described previously for in crosses analysis of ETZ experiment. Longevity was analyzed using a two-way ANOVA with fungal load and thermal condition (critical temperature and behavior) as factors. Analyses were performed in the R programming environment (v. 3.4.3., CRAN project)30 and JMP-Pro version 15 (SAS Institute 2020). More

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Here we show, for the first time, that the toxicity of a pesticide formulation to bees is caused exclusively by a co-formulant (alcohol ethoxylates), rather than the active ingredient. A 0.8 µL acute oral dose of the agricultural fungicide formulation Amistar® caused a range of damage to bees: both lethal, with 23% mortality, and sublethal, with 45% reduced sucrose consumption, 3.8% drop in body weight (whereas the negative control gained 4.8%), and a 302% increase in gut melanisation. For all metrics tested, the Amistar® and alcohol ethoxylates treatments were not statistically different, demonstrating conclusively that the toxicity of the formulation, Amistar®, to bumble bees is driven by the alcohol ethoxylates. These results demonstrate gaps in the regulatory system and highlight the need for a greater research focus on co-formulants.The mortality in the Amistar® treatment, and treatments containing alcohol ethoxylates reached 32% at its highest, which is substantial given that bees are likely to have a high level of exposure to Amistar® and alcohol ethoxylates. The mechanism by which the alcohol ethoxylates cause mortality has not been explicitly isolated, but our results suggest two potential, possibly related, causes. We recorded a 302% increase in the melanised area of bee midguts in the alcohol ethoxylates treatment. A similar effect was observed in Melipona scutellaris exposed to the pure fungicide active ingredient pyraclostrobin alongside a similar reduction in survival37. We suggest that the alcohol ethoxylates are disrupting the structure of the midgut, which the bee immune system is reacting to with melanisation44 (see Fig. 5). In parallel with this gut damage, alcohol ethoxylate treatment drove a 54% reduction in sugar consumption, which persisted throughout the experiment. Supplementary Fig. S3 shows a plot comparing sugar consumption against gut melanisation, with increasing gut melanisation correlated to reduced sugar consumption in the Amistar®, co-formulant mixture and alcohol ethoxylates treatments. Consequently, we propose that mortality was driven by energy depletion due to reduced consumption, which in turn may have been driven by damage to the gut.Figure 5(Left) Bumble bee midgut in the negative control treatment. (Right) Bumble bee midgut in the co-formulant mixture treatment, which contains alcohol ethoxylates. The dark brown patches are areas of melanisation, indicative of damage to the gut. Both bees survived the full 120 h.Full size imageLikely as a consequence of the reduced consumption of sucrose, bumble bees in the alcohol ethoxylates treatment lost 8.4% of their original weight, in stark contrast to the negative control where bees gained 4.8% over the five-day period. This indicates the alcohol ethoxylate treated bees were expending more energy than they were consuming, and thus exhibiting a negative energy balance. This weight loss, while considerable as a percentage of the bee’s total body mass, is also similar in scale to the weight of the sucrose bees consume in one sitting (EA Straw pers. obs.), for which rigorous data do not exist. As such it is possible that a portion of the weight loss is attributable to the reduced sucrose consumption of the bees, meaning they would have less sucrose in their guts at the time of weighing. Sucrose consumption does not, however, explain the failure of alcohol ethoxylate treated bees to gain weight, which was observed in the control treatment. The weight loss, and lack of weight gain, are concerning because they are likely to indicate a reduction in fat reserves, although this has not been experimentally confirmed. Bee fat reserves are important physiologically, in particular in responding to immune threats45,46. Fat reserves allow bees the energetic resources to buffer against challenges, and thus their depletion could expose bees to greater risk from future threats47.The reduced appetite and negative energy balance in alcohol ethoxylates treated bees could have broader effects in the natural environment. Bees pollinate flowers as they forage for nectar and pollen, so a reduction in their appetite could subsequently have effects on ecosystem services. In our experiment, bumble bee appetite was reduced immediately after ingesting a single dose of alcohol ethoxylates or Amistar®. This effect persisted for five days after exposure, indicating a persistent change in consumption behaviour. While nectar-foraging in bumble bees is driven by the needs of the colony48, a reduction in appetite would reduce overall colony nectar consumption, and thus the number of foraging trips made for nectar. Fewer visits to flowers for nectar may lead to reduced pollination, which would be detrimental to crop yields and farm profits. Further studies of how the impacts we have found map onto foraging and pollination are clearly needed. Importantly, the reduction in appetite recorded in our experiment is a sublethal effect, which standard lower tier testing would not detect. When Amistar® is tested on bumble bees for the 2025 renewal of azoxystrobin, this sublethal effect will be missed by regulatory testing, despite the impact it may have on the pollination services such testing is designed to protect. We suggest that a simple modification to the regulatory protocol OECD 247 would be to weigh the sucrose syringes at the start and end of the trials to calculate sucrose consumption, which would allow measurement of this sublethal effect with minimal additional workload.Our results show a slightly, but not significantly, higher level of mortality in the alcohol ethoxylates treatment (30%) than the Amistar® treatment (23%). If this is a real biological difference, one explanation might be that the concentration of alcohol ethoxylates in the Amistar® formulation was lower than that used in the alcohol ethoxylates treatment solution. This is possible because the Amistar® material safety data sheet lists concentrations as a range (10–20% for alcohol ethoxylates), and here we used the upper end of the range. The co-formulant mixture treatment in all metrics was statistically indistinguishable from the alcohol ethoxylates treatment, showing that the toxicity of alcohol ethoxylates is not a result of synergism with other co-formulants.We believe that the implications of our results are not limited to a laboratory setting and a single species, as other published and unpublished research supports our findings. Semi-field flight cage experiments, where Amistar® was applied to a crop, found effects on full bumble bee colonies (Bombus terrestris). Amistar® caused a reduction in average bee weight and a reduction in foraging activity, as our results predict49. This demonstrates that the effects observed in our laboratory testing scale up to effects at a field realistic level. Additionally, in honeybees (Apis mellifera) Amistar® has been found to cause mortality in laboratory experiments at a range of doses50,51, demonstrating the mortality effect found in our experiment is not species specific. However, no mortality was seen in trials on the red mason bee Osmia bicornis (Hellström and Paxton, unpublished data). Additionally, a similar compound, C11 and lower alcohol ethoxylates, has been found in small scale laboratory testing to cause 100% mortality after contact exposure in honeybees31.To measure the exposure of bees to PPP’s, the EU mandates trials that measure chemical residues in pollen and nectar after crops have been sprayed with either active ingredients or formulations34. However, these residue analysis studies only measure active ingredient concentrations, not the co-formulants. As such, we have no systematic data on the exposure of bees to co-formulants7,8,9. This dearth of data means that the exposure of bees to co-formulants is very poorly characterised. To estimate exposure to alcohol ethoxylates, residue data for Amistar®’s active ingredient azoxystrobin could be used as a proxy18,52. However, the chemical properties of alcohol ethoxylates, specifically their surfactant action, make it unlikely that they have an equivalent environmental fate to azoxystrobin, so this would not be appropriate.While we have very little data to quantify bee exposure to alcohol ethoxylates, we know Amistar® can be applied to crops, such as strawberries, during flowering while bees are foraging on them. The Environmental Information Sheet for Amistar® states “[For bees] no risk management is necessary. Amistar® is of low risk to honey bees”53,54,55. In addition, we would note that exposure of bees to alcohol ethoxylates, and related substances, is not exclusively from Amistar®. For example, a cursory search of the Syngenta website56 immediately identified alcohol ethoxylates in five other Syngenta products. Worryingly, the chemical group alcohol ethoxylates sit in, alkoxylated alcohols, are also widely used in adjuvants, which are products which can be added to tank mixtures to modify the action of the agrichemical6. 89 adjuvant products licenced in the UK contain alkoxylated alcohols as the primary ingredient15. To our knowledge, these adjuvants have never been toxicity tested on bees and have no bee exposure mitigation measures in place whatsoever.To complement measures to promote academic research, moving regulatory research beyond its mortality and active ingredient-centric approach to toxicity testing would better reflect the risks pesticides, as used in the field, pose. For regulatory systems to accurately characterise risk they need to estimate the scale of sublethal effects, regardless of initial mortality results33. The results presented here demonstrate that even substances assessed by regulators as ‘bee safe’ can pose a serious hazard to bee health. To reflect potential sublethal differences caused by co-formulation composition, all formulations could undergo a much more rigorous set of lower tier testing or be automatically entered for higher tier testing.In the face of declining bee populations we advocate that a precautionary approach minimising the exposure of bees to potential stressors, where possible, would be prudent. The current legislation allowing application of PPPs directly onto bees and flowering plants does not align with the emerging evidence that co-formulants, adjuvants, herbicides and fungicides can be hazardous to bees8,57. The wealth of untested and undisclosed co-formulants used abundantly in agriculture is a serious and pressing concern for the health of pollinators worldwide. More

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