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22 pages, 25162 KiB  
Article
Antibody-Mediated Protein Knockdown Reveals Distal-less Functions for Eyespots and Parafocal Elements in Butterfly Wing Color Pattern Development
by Yugo Nakazato and Joji M. Otaki
Cells 2024, 13(17), 1476; https://doi.org/10.3390/cells13171476 - 2 Sep 2024
Viewed by 391
Abstract
One of the important genes for eyespot development in butterfly wings is Distal-less. Its function has been evaluated via several methods, including CRISPR/Cas9 genome editing. However, functional inhibition may be performed at the right time at the right place using a different [...] Read more.
One of the important genes for eyespot development in butterfly wings is Distal-less. Its function has been evaluated via several methods, including CRISPR/Cas9 genome editing. However, functional inhibition may be performed at the right time at the right place using a different method. Here, we used a novel protein delivery method for pupal wing tissues in vivo to inactivate a target protein, Distal-less, with a polyclonal anti-Distal-less antibody using the blue pansy butterfly Junonia orithya. We first demonstrated that various antibodies including the anti-Distal-less antibody were delivered to wing epithelial cells in vivo in this species. Treatment with the anti-Distal-less antibody reduced eyespot size, confirming the positive role of Distal-less in eyespot development. The treatment eliminated or deformed a parafocal element, suggesting a positive role of Distal-less in the development of the parafocal element. This result also suggested the integrity of an eyespot and its corresponding parafocal element as the border symmetry system. Taken together, these findings demonstrate that the antibody-mediated protein knockdown method is a useful tool for functional assays of proteins, such as Distal-less, expressed in pupal wing tissues, and that Distal-less functions for eyespots and parafocal elements in butterfly wing color pattern development. Full article
(This article belongs to the Section Reproductive Cells and Development)
Show Figures

Figure 1

Figure 1
<p>Wing color patterns of <span class="html-italic">J. orithya</span>, a sexually dimorphic species. The dorsal hindwings of males (<b>left</b>) and females (<b>right</b>) have blue and brown background colors, respectively. The present study focused on the anterior eyespot and its surroundings on the dorsal hindwing. These butterfly samples were obtained from Ishigaki-jima Island, Okinawa, Japan.</p>
Full article ">Figure 2
<p>Molecular structure of the <span class="html-italic">J. coenia</span> Dll protein as predicted by AlphaFold. (<b>A</b>) Predicted Dll protein structure. The colors indicate model confidence levels: cyan: very high, blue: confident, yellow: low, and red: very low. The homeodomain structure (the core helical structure) was predicted with high confidence. (<b>B</b>) The same image as (<b>A</b>) but with the green highlight for the 18-aa sequence of the synthetic peptide used for immunization. Its sequence is shown at the bottom. The first G (glycine) and the last R (arginine) of the peptide sequence are indicated adjacent to the structure. (<b>C</b>,<b>D</b>) Predicted <span class="html-italic">J. coenia</span> Dll protein structure from different angles. Locations of the first G and the last R of the peptide sequence are indicated.</p>
Full article ">Figure 3
<p>Antibody delivery to butterfly pupal wing tissue (dorsal hindwing) via the sandwich method. Fluorescent colors are indicated by colors of letters on the right. (<b>A</b>) Anti-<span class="html-italic">Drosophila</span> axons antibody conjugated with Alexa 488. (<b>B</b>) The same visual field as in (<b>A</b>) but with only fluorescent signals from the anti-<span class="html-italic">Drosophila</span> axons antibody. Most green signals are colocalized with red signals. (<b>C</b>,<b>D</b>) Anti-tubulin antibody conjugated with DyLight 550. (<b>E</b>,<b>F</b>) Anti-histone H3 antibody conjugated with DyLight 550. These two images are from different individuals. There are purple signals from nuclei (arrows), indicating the localization of the anti-histone H3 antibody in the nucleus.</p>
Full article ">Figure 4
<p>Liquid distribution on the surface of the dorsal hindwing after the anti-tubulin antibody conjugated with DyLight 550 was sandwiched. (<b>A</b>) A pupa before eclosion. The forewing was removed, and the dorsal hindwing was exposed. Anterior and posterior eyespots are clearly visible. (<b>B</b>) Magnification of the eyespots in (<b>A</b>). (<b>C</b>) A pupa with the forewing lifted and the hindwing covered with a piece of plastic film under visible white light. The anti-tubulin antibody is sandwiched between the hindwing and plastic film. (<b>D</b>) Magnification of (<b>C</b>). The dorsal hindwing has wing veins (tracheae). (<b>E</b>) The same visual field as (<b>C</b>) under green excitation light. (<b>F</b>) Magnification of (<b>E</b>). There are numerous red-fluorescing liquid droplets.</p>
Full article ">Figure 5
<p>Antibody delivery to butterfly pupal wing tissue (dorsal hindwing) via the injection method. Fluorescent colors are indicated by colors of letters on the right. A pair of panels was obtained from different individuals. (<b>A</b>,<b>B</b>) Anti-<span class="html-italic">Drosophila</span> axons antibody conjugated with Alexa 488. Most green signals are colocalized with red signals in (<b>A</b>), producing orangish or yellowish signals when three channels of fluorescent colors are observed simultaneously, as shown in the left panel of (<b>A</b>). A single channel image of the anti-<span class="html-italic">Drosophila</span> axons antibody conjugated with Alexa 488 is shown in the right panel of (<b>A</b>). (<b>C</b>,<b>D</b>) Anti-tubulin antibody conjugated with DyLight 550. (<b>E</b>,<b>F</b>) Anti-histone H3 antibody conjugated with DyLight 550. There are many purple signals from the nuclei (arrows), indicating the localization of the anti-histone H3 antibody in the nucleus.</p>
Full article ">Figure 6
<p>Delivery of the anti-Dll antibody to butterfly pupal wing tissue (dorsal hindwing) via the sandwich and injection methods. Fluorescent colors are indicated by colors of letters on the right. There are many blue–green signals from the nuclei (arrows), indicating the localization of the anti-Dll antibody in the nucleus. A pair of panels was obtained from different individuals. (<b>A</b>,<b>B</b>) Sandwich method. (<b>C</b>,<b>D</b>) Injection method.</p>
Full article ">Figure 7
<p>Immunohistochemical analysis of Dll in the pupal wing tissue (dorsal hindwing) immediately after pupation. (<b>A</b>) Two wing compartments with and without a prospective eyespot focus. The prospective anterior eyespot focal area shows positive staining (black arrow). The prospective PFE/SMB also shows positive staining (white arrow). (<b>B</b>) Annotations of the positive areas shown in (<b>A</b>). (<b>C</b>) Enlargement of the prospective anterior eyespot focal area shown in (<b>A</b>), indicated by a broken circle. (<b>D</b>) Prospective anterior eyespot focal area of an individual different from (<b>A</b>–<b>C</b>), showing a ring-like pattern. (<b>E</b>,<b>F</b>) Enlargement of (<b>D</b>). The prospective anterior eyespot focal area is indicated by a broken circle in (<b>F</b>).</p>
Full article ">Figure 8
<p>Effect of anti-Dll antibody delivery on eyespot size. (<b>A</b>) Representative male (top) and female (bottom) hindwings treated with an anti-Dll antibody. The anterior eyespot (red arrows) in the left hindwing was compared with the untreated anterior eyespot in the right hindwing within a single individual. (<b>B</b>) Two examples of an automatic processing for the eyespot area values from digital images. (<b>C</b>) Comparison of the eyespot area values in males (two-sided paired <span class="html-italic">t</span> test). **: <span class="html-italic">p</span> &lt; 0.01. (<b>D</b>) Comparison of the eyespot area values in females (two-sided paired <span class="html-italic">t</span> test). (<b>E</b>) Left/right ratio comparisons between the anti-Dll and anti-spike antibodies in males (left) and females (right) (two-sided unpaired <span class="html-italic">t</span> test). *: <span class="html-italic">p</span> &lt; 0.05.</p>
Full article ">Figure 9
<p>Color pattern modifications induced by the application of the anti-Dll antibody via the sandwich method. In a pair of panels, an area indicated by an arrow or arrows in the left panel is magnified in the right panel. Individual numbers are indicated in the first panel of an individual (<a href="#app1-cells-13-01476" class="html-app">Supplementary Files S1 and S2</a>). (<b>A</b>,<b>B</b>) A female individual with invagination of parafocal element (PFE). The PFE is curved along two adjacent eyespots, and the submarginal band (SMB) is expanded proximally. Additionally, there may be a small ectopic eyespot (arrow). (<b>C</b>,<b>D</b>) A female individual with a phenotype similar to (<b>A</b>,<b>B</b>). (<b>E</b>,<b>F</b>) A female individual with possible knockdown of eyespot-less areas, including the midline and the imaginary focal areas. The PFE is curved, although less intense than in the previous cases, and black spots (arrows) emerge. (<b>G</b>,<b>H</b>) A female individual with an ectopic black and orange pattern between the two eyespots. The PFE is not deformed. (<b>I</b>,<b>J</b>) A female individual with small ectopic black and orange patterns (arrows) between the two eyespots. (<b>K</b>,<b>L</b>) A female individual with PFE deletion (arrow) and a thickened SMB. (<b>M</b>,<b>N</b>) A female individual showing a partial loss of PFE (arrow) and PFE invagination (arrow) toward the eyespot focus. (<b>O</b>,<b>P</b>) A female individual showing PFE invaginations (arrows) toward eyespot foci. (<b>Q</b>,<b>R</b>) A male individual with two ectopic black spots (arrows) in a single compartment. (<b>S</b>,<b>T</b>) A male individual with ectopic black areas (arrows) in two compartments.</p>
Full article ">Figure 10
<p>Color pattern modifications induced by the application of the anti-spike antibody via the sandwich method. In a pair of panels, an area indicated by an arrow or arrows in the left panel is magnified in the right panel. Individual numbers are indicated in the first panel of an individual (<a href="#app1-cells-13-01476" class="html-app">Supplementary Files S3 and S4</a>). (<b>A</b>,<b>B</b>) A female individual with an ectopic color pattern (arrow) in the eyespot-less imaginary focal area. (<b>C</b>,<b>D</b>) A female individual with an ectopic color pattern, similar to (<b>A</b>,<b>B</b>), but outside the imaginary focal area. (<b>E</b>,<b>F</b>) A female individual with a possible ectopic color pattern (arrow). (<b>G</b>,<b>H</b>) A female individual with an ectopic color pattern (arrow) near the imaginary focal area. (<b>I</b>,<b>J</b>) A male individual with two ectopic black areas (arrows). Some scales appear to be removed. (<b>K</b>,<b>L</b>) A male individual with two ectopic black areas (arrows). They are likely associated with the removal of scales.</p>
Full article ">Figure 11
<p>Color pattern modifications induced by the application of the anti-Dll antibody via the injection method. Three panels in the same row indicate a serial magnification of the first panel. Individual numbers are indicated on the left (<a href="#app1-cells-13-01476" class="html-app">Supplementary Files S5 and S6</a>). (<b>A</b>–<b>C</b>) A female individual with a remnant eyespot (arrow) on the left hindwing. Note scarcity of scales. (<b>D</b>–<b>F</b>) Same female individual with an eyespot size reduction on the right hindwing. (<b>G</b>–<b>I</b>) A female individual with a potentially affected eyespot (arrow) and impaired scale development in the background. (<b>J</b>–<b>L</b>) A female individual with a reduced eyespot (arrow) on the forewing.</p>
Full article ">Figure 12
<p>Interpretations of the antibody-mediated color pattern modifications in the present study. (<b>A</b>) An illustration of a hindwing with elemental nomenclature. The wing compartment indicated by red lines has both the anterior eyespot and parafocal element (PFE) in the adult wing. Their organizer is located at the prospective eyespot focus in the pupal wing tissue. The adjacent compartment indicated by the yellow lines does not have an eyespot but has a PFE, suggesting that the eyespot-less imaginary focal area is an organizing center for PFE. (<b>B</b>) Time series of color pattern determination proposed by the induction model. Green arrows indicate the directions of signal movement from the eyespot organizer (pink area). (<b>C</b>) Color pattern configuration of the normal wing (no treatment). Pink areas indicate Dll expression at the pupal stage. Additionally, the PFE and peri-wing veins may have their own Dll expression, but this is not shown in this illustration. (<b>D</b>–<b>G</b>) Interpretations of the knockdown results. The numbers of individuals that supported these interpretations via the sandwich method are indicated. For the sandwich method, the total number of individuals was 67 including both sexes. These individuals can be identified in <a href="#app1-cells-13-01476" class="html-app">Supplementary Materials</a>. The crosses indicate the knockdown areas. (<b>D</b>) Eyespot focal knockdown (KD). In addition to the number of individuals via the sandwich method, the number of individuals supporting this interpretation via the injection method is indicated (Inj). For the injection method, the total number of individuals was 10 including both sexes. (<b>E</b>) Eyespot-less midline KD. (<b>F</b>) Parafocal element KD. (<b>G</b>) Background KD.</p>
Full article ">Figure A1
<p>ELISA results of serial dilutions of antibodies before and after purification. (<b>A</b>) Anti-Dll antibody. (<b>B</b>) Anti-spike antibody.</p>
Full article ">
14 pages, 3217 KiB  
Article
Preparation and Application of Multi-Walled Carbon Nanotube-Supported Metconazole Suspension Concentrate for Seed Coating to Control Wheat Sharp Eyespot
by Xuexiang Ren, Dongdong Qi, Zhao Li, Yu Chi, Xianyan Su, Kaixin Gu, Zhenghe Ye, Shun He and Li Chen
Agronomy 2024, 14(9), 1985; https://doi.org/10.3390/agronomy14091985 - 1 Sep 2024
Viewed by 459
Abstract
Wheat sharp eyespot is a prevalent soil-borne disease that causes substantial economic losses in agriculture. Metconazole, a new triazole broad-spectrum fungicide, has demonstrated effective control of soil-borne diseases. Multi-walled carbon nanotubes (MWCNTs) are an innovative adsorbent material known for their large surface area [...] Read more.
Wheat sharp eyespot is a prevalent soil-borne disease that causes substantial economic losses in agriculture. Metconazole, a new triazole broad-spectrum fungicide, has demonstrated effective control of soil-borne diseases. Multi-walled carbon nanotubes (MWCNTs) are an innovative adsorbent material known for their large surface area and high absorptive capacity. This study identifies MWCNTs as the optimal adsorption material for metconazole, achieving an adsorption rate of 85.27% under optimal conditions (stirring time of 30 min and feeding ratio of 6:1). The optimized formula consists of 1.5% dispersant sodium wood, 1% emulsifier BY-112, 2% AEO-15, 3% glycol, 3% filmogen, and 4% red dyes. A 0.5% MWCNT–metconazole suspension concentrate for seed coating (FSC) significantly enhances the inhibitory effect of metconazole on wheat growth and promotes root development. At the tillering stage, a coating ratio of 1:100 shows a marked impact on wheat growth, and MWCNTs can improve the control effect of metconazole to Rhizoctonia cerealis. This work offers a novel approach for applying metconazole in a wheat suspension concentrate for seed coating. Full article
(This article belongs to the Section Pest and Disease Management)
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Figure 1

Figure 1
<p>Electron microscope pictures of MWCNTs.</p>
Full article ">Figure 2
<p>Establishment of standard curve for metconazole. (<b>A</b>) Ultraviolet absorption spectrum of metconazole; (<b>B</b>) standard curve for metconazole.</p>
Full article ">Figure 2 Cont.
<p>Establishment of standard curve for metconazole. (<b>A</b>) Ultraviolet absorption spectrum of metconazole; (<b>B</b>) standard curve for metconazole.</p>
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<p>Adsorption curves for different MWCNT inputs over time. The abscissa reflects time, and the ordinate indicates the adsorption rate. The gray, red, blue, green, and purple lines represent ratios of 1:1, 2:1, 4:1, 6:1, and 8:1, respectively.</p>
Full article ">Figure 4
<p>The field effect of 0.5% MWCNT–metconazole FSC (DM) and 0.5% metconazole FSC (M) at the seedling stage (<b>top</b>) and tillering stage (<b>bottom</b>). CK: the blank control.</p>
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<p>Plant height (<b>A</b>), root length (<b>B</b>), fresh weight (<b>C</b>), dry weight (<b>D</b>), SPAD (<b>E</b>), and nitrogen content (<b>F</b>) of 0.5% MWCNT–metconazole FSC (DM) and 0.5% metconazole FSC (M) at seedling stage. Significant differences are marked by letters (Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 6
<p>Plant height (<b>A</b>), number of tillers (<b>B</b>), fresh weight (<b>C</b>), dry weight (<b>D</b>), SPAD (<b>E</b>), and nitrogen content (<b>F</b>) of 0.5% MWCNT–metconazole FSC (DM) and 0.5% metconazole FSC (M) at tillering stage. Significant differences are marked by letters (Student’s <span class="html-italic">t</span>-test, <span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">
20 pages, 11837 KiB  
Article
Comparative Genomic and Functional Analysis of c-di-GMP Metabolism and Regulatory Proteins in Bacillus velezensis LQ-3
by Rong Li, Panlei Yang, Hongjuan Zhang, Chunjing Wang, Fang Zhao, Jiehui Liu, Yanbin Wang, Yan Liang, Ting Sun and Xiansheng Xie
Microorganisms 2024, 12(8), 1724; https://doi.org/10.3390/microorganisms12081724 - 21 Aug 2024
Viewed by 467
Abstract
Bacillus velezensis is a promising candidate for biocontrol applications. A common second messenger molecule, bis-(3,5)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has the ability to regulate a range of physiological functions that impact the effectiveness of biocontrol. However, the status of the c-di-GMP signaling pathway in biocontrol [...] Read more.
Bacillus velezensis is a promising candidate for biocontrol applications. A common second messenger molecule, bis-(3,5)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has the ability to regulate a range of physiological functions that impact the effectiveness of biocontrol. However, the status of the c-di-GMP signaling pathway in biocontrol strain LQ-3 remains unknown. Strain LQ-3, which was isolated from wheat rhizosphere soil, has shown effective control of wheat sharp eyespot and has been identified as B. velezensis through whole-genome sequencing analyses. In this study, we investigated the intracellular c-di-GMP signaling pathway of LQ-3 and further performed a comparative genomic analysis of LQ-3 and 29 other B. velezensis strains. The results revealed the presence of four proteins containing the GGDEF domain, which is the conserved domain for c-di-GMP synthesis enzymes. Additionally, two proteins were identified with the EAL domain, which represents the conserved domain for c-di-GMP degradation enzymes. Furthermore, one protein was found to possess a PilZ domain, indicative of the conserved domain for c-di-GMP receptors in LQ-3. These proteins are called DgcK, DgcP, YybT, YdaK, PdeH, YkuI, and DgrA, respectively; they are distributed in a similar manner across the strains and belong to the signal transduction family. We selected five genes from the aforementioned seven genes for further study, excluding YybT and DgrA. They all play a role in regulating the motility, biofilm formation, and colonization of LQ-3. This study reveals the c-di-GMP signaling pathway associated with biocontrol features in B. velezensis LQ-3, providing guidance for the prevention and control of wheat sharp eyespot by LQ-3. Full article
(This article belongs to the Section Microbial Biotechnology)
Show Figures

Figure 1

Figure 1
<p>Strain LQ-3 could serve as a potential biocontrol agent against <span class="html-italic">R. cerealis</span>. (<b>A</b>,<b>B</b>) The antagonistic activity of both the strain LQ-3 (<b>A</b>) and its fermentation broth (<b>B</b>) toward <span class="html-italic">R. cerealis</span>. (<b>C</b>,<b>D</b>) The biocontrol strain LQ-3 (<b>C</b>) and fermentation broth (<b>D</b>) significantly inhibited the growth of mycelia. (<b>E</b>) The biocontrol effect of LQ-3 toward <span class="html-italic">R. cerealis</span> under different concentrations (10<sup>6</sup>, 10<sup>7</sup>, and 10<sup>8</sup> CFU/mL) on the plate. (<b>F</b>) The disease index of control and LQ-3 (10<sup>6</sup>, 10<sup>7</sup>, and 10<sup>8</sup> CFU/mL) in plate experiments. (<b>G</b>) The biocontrol effect of LQ-3 (10<sup>7</sup> CFU/mL) toward <span class="html-italic">R. cerealis</span> in a greenhouse experiment. (<b>H</b>) The disease index of control and LQ-3 (1 × 10<sup>7</sup> CFU/mL) in greenhouse experiments. Error bars indicate ± SD of three replicates. The statistical analysis was performed using GraphPad Prism 8 software by <span class="html-italic">t</span>-test and one-way ANOVA (<span class="html-italic">p</span> &lt; 0.05). ****, <span class="html-italic">p</span> &lt; 0.0001, and **, <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Phylogenetic analysis of <span class="html-italic">B. velezensis</span> LQ-3. (<b>A</b>) The ML tree of different <span class="html-italic">Bacillus</span> strains was generated based on 806 single-copy core genes using RAxML 8.2.10. <span class="html-italic">P. polymyxa</span> M1 was used as the out-group. Percent bootstrap values (from 100 replicates) are indicated at the nodes. (<b>B</b>) Heat map of ANI values among different <span class="html-italic">Bacillus</span> strains. The numbers represent the size of ANI values.</p>
Full article ">Figure 3
<p>Pan-genomes of <span class="html-italic">B. velezensis</span> strains. (<b>A</b>) The number of specific genes for each strain of <span class="html-italic">B. velezensis</span>. The inner circle displays the core genomes shared between all strains. The unique genes for each strain are displayed in each of the outer circles. The number below the strain name represents the CDS of each strain. (<b>B</b>) The curves for <span class="html-italic">B. velezensis</span> pan-genomes and core genomes. The blue plots represent the pan-genome size of <span class="html-italic">B. velezensis</span> for each genome comparison, while the green plots represent the core genome size of <span class="html-italic">B. velezensis</span> for each genome comparison. The median values are linked to display the relationship between the number of genomes. (<b>C</b>) COG distribution of core, specific, and accessory genes present in all 30 analyzed <span class="html-italic">B. velezensis</span> strains.</p>
Full article ">Figure 3 Cont.
<p>Pan-genomes of <span class="html-italic">B. velezensis</span> strains. (<b>A</b>) The number of specific genes for each strain of <span class="html-italic">B. velezensis</span>. The inner circle displays the core genomes shared between all strains. The unique genes for each strain are displayed in each of the outer circles. The number below the strain name represents the CDS of each strain. (<b>B</b>) The curves for <span class="html-italic">B. velezensis</span> pan-genomes and core genomes. The blue plots represent the pan-genome size of <span class="html-italic">B. velezensis</span> for each genome comparison, while the green plots represent the core genome size of <span class="html-italic">B. velezensis</span> for each genome comparison. The median values are linked to display the relationship between the number of genomes. (<b>C</b>) COG distribution of core, specific, and accessory genes present in all 30 analyzed <span class="html-italic">B. velezensis</span> strains.</p>
Full article ">Figure 4
<p>The domain analysis of seven potential c-di-GMP-related proteins in <span class="html-italic">B. velezensis</span> LQ-3. (<b>A</b>) Distribution of DgcK, DgcP, YdaK, YybT, PdeH, YkuI, and DgrA in <span class="html-italic">B. velezensis</span> group. The yellow box represents the presence of a gene within a genome, and the green box indicates the absence of a gene within a genome. (<b>B</b>) Domain composition and organization of seven c-di-GMP-related proteins in <span class="html-italic">B. velezensis</span> LQ-3. The orange box indicates the conserved domain of diguanylate cyclases (DGCs), the GGDEF domain. The blue box indicates the conserved domain of phosphodiesterases (PDEs), the EAL domain.</p>
Full article ">Figure 5
<p>Analysis of conserved amino acid residues in the GGDEF, EAL, and PilZ domains. (<b>A</b>) Conservation analysis of amino acid residues in the GGDEF domain. The I site and catalytic site are highlighted with blue boxes. (<b>B</b>) Conservation analysis of amino acid residues in the EAL domain. The c-di-GMP binding site and metal binding site are marked with red boxes. (<b>C</b>) Conservation analysis of amino acid residues in the PilZ domain. The c-di-GMP binding site is identified by an orange box. The letters in the figure represent the abbreviations of amino acids.</p>
Full article ">Figure 6
<p>Structural features of the GGDEF, EAL, and PilZ domains in LQ-3 and their comparison with the corresponding templates. (<b>A</b>) The α helices, β strands, and loop regions of each domain are marked in red, yellow, and green, respectively. In the structural comparison, each domain is marked in pink, while the template domains are marked in white. Structural features of the GGDEF domain, using WspR (PDB id: 3I5C) from <span class="html-italic">P. aeruginosa</span> as the template. (<b>B</b>) The structural comparison between the GGDEF domains of LQ-3 and WspR. The SSSD and GGDEF motifs are marked in blue, corresponding to the colored boxes marking the conserved amino acid residues in the GGDEF domain. (<b>C</b>) The structural features of the EAL domain, using RmcA (PDB id: 5M3C) from <span class="html-italic">P. aeruginosa</span> as the template. (<b>D</b>) The structural comparison between the EAL domains of LQ-3 and RmcA. (<b>E</b>) Structural features of the PilZ domain, using MotI (PDB id: 5VX6) from <span class="html-italic">B. subtilis</span> as the template. (<b>F</b>) Structural comparison between the PilZ domains of LQ-3 and MotI. The RxxxR and (D/N)x(S/A)xxG motifs are marked in orange, corresponding to the colored boxes marking the conserved amino acid residues in the PilZ domain.</p>
Full article ">Figure 7
<p>Assay of biocontrol-related phenotypes of c-di-GMP-metabolism-related proteins in LQ-3. (<b>A</b>,<b>B</b>) The swarming motility test of LQ-3 (pUBXC) and its mutant strains. (<b>C</b>) The pellicle biofilm detection of LQ-3 and corresponding mutant strains. (<b>D</b>) The colony biofilm detection of LQ-3 and corresponding mutant strains. (<b>E</b>) Colonization ability detection of LQ-3 and its mutant strains in wheat roots. The statistical analysis was performed using GraphPad Prism 8 software by one-way ANOVA with the Turkey test (<span class="html-italic">p</span> &lt; 0.05). LQ-3(pUBXC): wild-type strains containing the pUBXC plasmid; <span class="html-italic">dgcK</span>, <span class="html-italic">dgcP</span>: c-di-GMP diguanylate cyclases (DGCs); <span class="html-italic">pdeH</span>: c-di-GMP phosphodiesterases (PDEs); <span class="html-italic">ydaK</span>, <span class="html-italic">ykuI</span>: c-di-GMP receptor.</p>
Full article ">
24 pages, 43777 KiB  
Article
Socket Array Irregularities and Wing Membrane Distortions at the Eyespot Foci of Butterfly Wings Suggest Mechanical Signals for Color Pattern Determination
by Yugo Nakazato and Joji M. Otaki
Insects 2024, 15(7), 535; https://doi.org/10.3390/insects15070535 - 16 Jul 2024
Viewed by 742
Abstract
Eyespot foci on butterfly wings function as organizers of eyespot color patterns during development. Despite their importance, focal structures have not been examined in detail. Here, we microscopically examined scales, sockets, and the wing membrane in the butterfly eyespot foci of both expanded [...] Read more.
Eyespot foci on butterfly wings function as organizers of eyespot color patterns during development. Despite their importance, focal structures have not been examined in detail. Here, we microscopically examined scales, sockets, and the wing membrane in the butterfly eyespot foci of both expanded and unexpanded wings using the Blue Pansy butterfly Junonia orithya. Images from a high-resolution light microscope revealed that, although not always, eyespot foci had scales with disordered planar polarity. Scanning electron microscopy (SEM) images after scale removal revealed that the sockets were irregularly positioned and that the wing membrane was physically distorted as if the focal site were mechanically squeezed from the surroundings. Focal areas without eyespots also had socket array irregularities, but less frequently and less severely. Physical damage in the background area induced ectopic patterns with socket array irregularities and wing membrane distortions, similar to natural eyespot foci. These results suggest that either the process of determining an eyespot focus or the function of an eyespot organizer may be associated with wing-wide mechanics that physically disrupt socket cells, scale cells, and the wing membrane, supporting the physical distortion hypothesis of the induction model for color pattern determination in butterfly wings. Full article
(This article belongs to the Section Insect Physiology, Reproduction and Development)
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<p>Wing color patterns of the Blue Pansy butterfly <span class="html-italic">J. orithya</span>. (<b>a</b>) Dorsal wings. Males have a single blue form (right), and females have blue and brown forms (middle and left, respectively). In each wing, there are two eyespots, the anterior and posterior ones. Potential eyespot foci are named the first, second, third, fourth, fifth, and sixth focal areas from the anterior to posterior sides. An asterisk indicates the background area where physical damage was made to induce ectopic color patterns. (<b>b</b>) Dorsal cover scale image. Dorsal cover scales were removed at once from the wings with transparent adhesive tape for the purpose of illustrating wing veins and compartments. Scales along the wing veins are not present in this image, highlighting the wing veins. The anterior and posterior eyespot foci are located in the compartments M<sub>1</sub> and CuA<sub>1</sub>, respectively.</p>
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<p>Forewing eyespots. (<b>a</b>) The anterior eyespot (an arrow). (<b>b</b>) Magnification of the anterior eyespot shown in (<b>a</b>). (<b>c</b>) Focal area of (<b>b</b>). (<b>d</b>) Focal area without scales. An arrow indicates the location of the focal area, nearly at the end of the midline. This is a contralateral wing from the wing shown in (<b>a</b>–<b>c</b>). The following e and f are also contralateral wings. Thus, the wing margin is to the right. (<b>e</b>) Magnification of (<b>d</b>). (<b>f</b>) Magnification of (<b>e</b>). An arrow indicates a relatively large socket interval in the focal area. (<b>g</b>) The posterior eyespot (an arrow). (<b>h</b>) Magnification of the posterior eyespot shown in (<b>g</b>). (<b>i</b>) Focal area of (<b>h</b>). Scale directions appeared to be somewhat irregular. (<b>j</b>) Focal area without scales. An arrow indicates the location of the focal area, nearly at the end of the midline. This is a contralateral wing from the wing shown in (<b>g</b>–<b>i</b>). The following (<b>k</b>,<b>l</b>) are also contralateral wings. Thus, the wing margin is to the right. (<b>k</b>) Magnification of (<b>j</b>). (<b>l</b>) Magnification of (<b>k</b>). An arrow indicates a relatively large socket interval in the focal area.</p>
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<p>Anterior eyespot in the hindwing (female). (<b>a</b>) The anterior eyespot of an individual. (<b>b</b>) Magnification of the focal area of the posterior eyespot shown in (<b>a</b>). White focal scales are positioned without disorganization. (<b>c</b>) The eyespot area without scales. An arrow indicates the focal area at the end of the midline. This eyespot is identical to the one shown in (<b>a</b>,<b>b</b>). (<b>d</b>) Magnification of (<b>c</b>). (<b>e</b>) Magnification of (<b>d</b>). An arrow indicates an area of socket array irregularities. (<b>f</b>) Magnification of (<b>e</b>). An arrow indicates an area of socket array irregularities. (<b>g</b>) The anterior eyespot of another individual. (<b>h</b>) Magnification of the focal area of the posterior eyespot shown in (<b>g</b>). Blue and white focal scales are positioned without disorganization. (<b>i</b>) The eyespot without scales. An arrow indicates the focal area at the end of the midline. This eyespot is identical to the one shown in (<b>g</b>,<b>h</b>). (<b>j</b>) Magnification of (<b>i</b>). Arrows indicate distortions of the wing membrane. (<b>k</b>) Magnification of (<b>j</b>). Arrows indicate distortions of the wing membrane. (<b>l</b>) Magnification of (<b>k</b>).</p>
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<p>Posterior eyespot in the hindwing (male). (<b>a</b>) The posterior eyespot. (<b>b</b>) Magnification of the focal area of the posterior eyespot shown in (<b>a</b>). White focal scales are disorganized in direction. (<b>c</b>) The eyespot area without scales. The arrow indicates the focal area at the end of the midline. This eyespot is identical to the one shown in (<b>a</b>,<b>b</b>). (<b>d</b>) Magnification of (<b>c</b>). An arrow indicates an area of disorder. (<b>e</b>) Magnification of (<b>d</b>). Arrows indicate striking distortions of the wing membrane, one of which is associated with the unusual arrangement of socket arrays. (<b>f</b>) Magnification of (<b>e</b>). Arrows indicate striking distortions of the basal membrane. (<b>g</b>) Magnification of (<b>f</b>). (<b>h</b>) Another magnification of (<b>f</b>).</p>
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<p>Anterior and posterior eyespots in the unexpanded forewing (female). (<b>a</b>) The whole unexpanded forewing. (<b>b</b>) Magnification of the anterior eyespot shown in (<b>a</b>). (<b>c</b>) Magnification of the posterior eyespot shown in (<b>a</b>). (<b>d</b>) The anterior eyespot without scales. This is a contralateral wing from the wing shown in (<b>a</b>–<b>c</b>). Panels (<b>e</b>–<b>i</b>) also show images of the contralateral wing. Thus, the wing margin is to the left. (<b>e</b>) Magnification of (<b>d</b>). An arrow indicates the focal area, which is nearly at the end of the midline. (<b>f</b>) Magnification of (<b>e</b>). An arrow indicates a socket interval disorder. (<b>g</b>) Posterior eyespot without scales. (<b>h</b>) Magnification of the focal area of the posterior eyespot shown in (<b>g</b>). An arrow indicates the focal area, which is nearly at the end of the midline. (<b>i</b>) Magnification of (<b>h</b>). Arrows indicate irregularities in socket intervals.</p>
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<p>Anterior and posterior eyespots in the unexpanded hindwing (female). (<b>a</b>) The whole unexpanded hindwing. (<b>b</b>) Magnification of the anterior eyespot shown in (<b>a</b>). (<b>c</b>) Magnification of the posterior eyespot shown in (<b>a</b>). (<b>d</b>) The anterior eyespot area without scales. This is a contralateral wing from the wing shown in (<b>a</b>–<b>c</b>). (<b>e</b>) Magnification of (<b>d</b>). An arrow indicates the focal area. (<b>f</b>) Magnification of (<b>e</b>). An arrow indicates a socket interval disorder. (<b>g</b>) The posterior eyespot area without scales. This wing is obtained from an individual different from that of (<b>a</b>–<b>f</b>). (<b>h</b>) Magnification of the focal area of the posterior eyespot shown in (<b>g</b>). An arrow indicates the focal area, which is nearly at the end of the midline. (<b>i</b>) Magnification of (<b>h</b>). An arrow indicates disorders of socket intervals. (<b>j</b>) The posterior (fifth) eyespot area without scales from yet another individual. (<b>k</b>) Magnification of the focal area of the posterior eyespot shown in (<b>j</b>). An arrow indicates the focal area. (<b>l</b>) Magnification of (<b>k</b>). An arrow indicates irregularities in socket intervals.</p>
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<p>Potential focal areas in the unexpanded forewing. (<b>a</b>) The first focal area. (<b>b</b>) Magnification of the first focal area shown in (<b>a</b>). (<b>c</b>) Magnification of (<b>b</b>). (<b>d</b>) The third focal area. (<b>e</b>) Magnification of (<b>d</b>). (<b>f</b>) Magnification of (<b>e</b>). (<b>g</b>) The fourth focal area. (<b>h</b>) Magnification of the focal area shown in (<b>g</b>). (<b>i</b>) Magnification of (<b>h</b>).</p>
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<p>Potential focal areas in the unexpanded hindwing. (<b>a</b>) The first focal area. (<b>b</b>) Magnification of the first focal area shown in (<b>a</b>). (<b>c</b>) Magnification of (<b>b</b>). (<b>d</b>) The third focal area. (<b>e</b>) Magnification of (<b>d</b>). (<b>f</b>) Magnification of (<b>e</b>). (<b>g</b>) The fourth focal area. (<b>h</b>) Magnification of the focal area shown in (<b>g</b>). (<b>i</b>) Magnification of (<b>h</b>).</p>
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<p>Damage-induced hindwing color patterns. (<b>a</b>) A whole male wing with a damage-induced black spot. An arrow indicates the induced spot. (<b>b</b>) Magnification of the damage-induced black spot shown in (<b>a</b>). An arrow indicates the damaged point. (<b>c</b>) Magnification of (<b>b</b>). Scales are disordered in direction and density. (<b>d</b>) Damaged area without scales. The damaged area is indicated by an arrow. (<b>e</b>) Magnification of (<b>d</b>). Arrows indicate socket irregularities in the direction and substantial distortions of the wing membrane. (<b>f</b>) Magnification of (<b>e</b>). (<b>g</b>) A wing with a damage-induced orange area (an arrow). (<b>h</b>) Magnification of (<b>g</b>). There are black scales at the center (an arrow). (<b>i</b>) Magnification of (<b>h</b>). (<b>j</b>) A damaged area without scales. The arrow indicates the area of socket array irregularities. (<b>k</b>) Magnification of (<b>j</b>). An arrow indicates the area of socket array irregularity. (<b>l</b>) Magnification of (<b>k</b>). An arrow indicates an isolated socket with large intervals.</p>
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<p>Damage-induced color patterns in the unexpanded hindwing. (<b>a</b>) A whole male wing with a damage-induced black spot. An arrow indicates the induced spot. (<b>b</b>) Magnification of the damage-induced black spot shown in (<b>a</b>). (<b>c</b>) Magnification of (<b>b</b>). (<b>d</b>) Damaged area without scales. The damaged area is indicated by an arrow. (<b>e</b>) Magnification of (<b>d</b>). An arrow indicates the damaged area of the wing membrane. (<b>f</b>) Magnification of (<b>e</b>). There are many socket array irregularities (an arrow). (<b>g</b>) Wing with a damage-induced orange area (an arrow). (<b>h</b>) Magnification of (<b>g</b>). An arrow indicates the damaged area. (<b>i</b>) Magnification of (<b>h</b>). An arrow indicates the damaged area. (<b>j</b>) A damaged area without scales. An arrow indicates the area of socket array irregularities. (<b>k</b>) Magnification of (<b>j</b>). An arrow indicates the area of socket array irregularities. (<b>l</b>) Magnification of (<b>k</b>). An arrow indicates the irregular socket intervals.</p>
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<p>Female anterior eyespot focal areas of the nonrandom sampling group via light microscopy and SEM. Four representative samples are shown. Each line of panels contains images of a single individual. (<b>a</b>) Eyespot focal area of individual No. 30. (<b>b</b>,<b>c</b>) Wing membrane distortions at a branching point of socket arrays (arrows). (<b>d</b>) Magnification of (<b>b</b>). (<b>e</b>) Magnification of (<b>c</b>). (<b>f</b>) Eyespot focal area of individual No. 36. (<b>g</b>) Socket arrays with many branching points. Arrows indicate ridges or furrows parallel to socket arrays. (<b>h</b>) Socket arrays with irregularities and wing membrane distortion (an arrow). (<b>i</b>) Socket arrays with displaced sockets (arrows). (<b>j</b>) Magnification of (<b>h</b>). (<b>k</b>) Eyespot focal area of individual No. 39. (<b>l</b>) Socket arrays with many branches. (<b>m</b>) A displaced socket with wing membrane distortion (an arrow). (<b>n</b>) Magnifiction of (<b>m</b>). (<b>o</b>) A socket with the wing membrane distortion at a branching point of socket arrays. (<b>p</b>) Eyespot focal area of individual No. 42. (<b>q</b>) Socket arrays with an irregular socket at a branching point (an arrowhead). Ridges perpendicular to socket arrays are indicated (arrows). (<b>r</b>) Socket arrays with perpendicular ridges (arrows) and a parallel furrow (an arrowhead). (<b>s</b>,<b>t</b>) Magnification of q.</p>
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<p>Female anterior eyespot focal areas of the random sampling group via light microscopy and SEM. Four representative samples are shown. Each line of panels contains images of a single individual. (<b>a</b>) Eyespot focal area of individual No. 12. (<b>b</b>) Socket arrays with irregularities. Long (an arrow) and short (an arrowhead) socket intervals are indicated. (<b>c</b>) Socket arrays with an extra socket (an arrow) and wing membrane distortion (an arrowhead). (<b>d</b>,<b>e</b>) Magnification of (<b>c</b>). (<b>f</b>) Eyespot focal area of individual No. 17. (<b>g</b>) Socket arrays with many branching points. (<b>h</b>) Socket arrays with a long socket interval (an arrow) and a displaced socket at a branching point (an arrowhead). (<b>i</b>) Magnification of (<b>h</b>) (left side). (<b>j</b>) Magnification of (<b>h</b>). (<b>k</b>) Eyespot focal area of individual No. 23. (<b>l</b>) Socket arrays with a relatively small number of branching points. (<b>m</b>) Magnification of (<b>l</b>). (<b>n</b>) Socket array with an irregular socket (an arrow). (<b>o</b>) Magnification of (<b>n</b>). (<b>p</b>) Eyespot focal area of individual No. 43. (<b>q</b>) Socket arrays with branching points. (<b>r</b>) Magnification of (<b>q</b>). (<b>s</b>) Magnification of (<b>q</b>). A displaced socket at a branching point is indicated by an arrow. (<b>t</b>) Magnification of (<b>s</b>). An isolated large socket is shown.</p>
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<p>Physical distortions and mechanical signals. (<b>a</b>) Illustration of the physical distortions of the wing membrane detected in this study. The distortion is located at the tip of the midline. (<b>b</b>) Possible mechanical forces from the wing veins, the marginal band organizer, and the discal organizer (left). The prospective eyespot organizer then pushes back to balance forces (right). (<b>c</b>) Feedback and reinforcement of the mechanical signals from the prospective eyespot organizer. Reinforcement indicates further secretion of the cuticle to bind to the pupal cuticle focal spot more tightly and the polyploidization of organizing cells.</p>
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13 pages, 2386 KiB  
Article
Pattern Matters in the Aposematic Colouration of Papilio polytes Butterflies
by Huile Lim, Ian Z. W. Chan and Antónia Monteiro
Insects 2024, 15(7), 465; https://doi.org/10.3390/insects15070465 - 22 Jun 2024
Viewed by 840
Abstract
Many toxic animals display bright colour patterns to warn predators about their toxicity. This sometimes leads other sympatric palatable organisms to evolve mimetic colour patterns to also evade predation. These mimics, however, are often imperfect, and it is unclear how much their colour [...] Read more.
Many toxic animals display bright colour patterns to warn predators about their toxicity. This sometimes leads other sympatric palatable organisms to evolve mimetic colour patterns to also evade predation. These mimics, however, are often imperfect, and it is unclear how much their colour patterns can vary away from the model before they become ineffective. In this study, we investigated how predation risk of the palatable Common Mormon butterfly (Papilio polytes) is affected by two alterations of its wing pattern that make it progressively more distinct from its model, the Common Rose (Pachliopta aristolochiae). We deployed butterfly paper models in the field, where all models displayed the same colours but had different patterns. In the first modification from the Wildtype pattern, we exchanged the position of the red and white colour patches but kept the overall pattern constant. In the second modification, we created an eyespot-like shape from the pre-existing pattern elements by moving their positions in the wing, altering the overall wing pattern. Both modifications increased attack risk from predators relative to Wildtype patterns, with the eyespot-like modification having the highest predation risk. Our results show that avian predators can distinguish between all three patterns tested, and that pattern is important in aposematic signals. Predators learn to avoid aposematic colours, not in isolation, but as part of specific patterns. Full article
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<p>The three butterfly patterns deployed in this study. (<b>a</b>) <span class="html-italic">Papilio polytes</span> Wildtype pattern, (<b>b</b>) Flipped pattern, and (<b>c</b>) Eyespot pattern.</p>
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<p>Plots of mean smoothed reflectance spectra of the colours on the dorsal side of real and artificial (paper) models of <span class="html-italic">P. polytes</span>. The Eyespot model is represented but similar reflectance spectra were obtained for the Flipped models (<a href="#app1-insects-15-00465" class="html-app">Supplementary Figures S1 and S2</a>). The lines represent the mean, and the shaded areas represent the standard deviation of the spectral data (six measurements per colour were taken). (<b>a</b>) Black reflectance spectra, (<b>b</b>) red reflectance spectra, and (<b>c</b>) white reflectance spectra.</p>
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<p>An example of our model setup. The butterfly paper models are suspended on a wire above a stick so that the paper models flit in the wind. (<b>a</b>) Top view. (<b>b</b>) Side view.</p>
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<p>Individual points represent where each of the paper butterflies were deployed at each site in Singapore. (<b>a</b>) MacRitchie Reservoir Park (1.3488° N, 103.8224° E). (<b>b</b>) Clementi Forest (1.3294° N, 103.7802° E). (<b>c</b>) Kent Ridge Park (1.2939° N, 103.7913° E).</p>
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<p>Bite marks or obvious tugging on the models (with or without mealworm present) were regarded as predation. (<b>a</b>,<b>b</b>) Red rectangles show distinct “V” shaped marks resembling those made by bird beaks. (<b>c</b>) The green wire has been pulled (top rectangle) and a long shred of Blu-Tack on the ground (bottom rectangle).</p>
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<p>Survival curves showing the number of days that the models of each type survived in the field before being attacked. On average, the Eyespot types survived for the shortest number of days, followed by Flipped types, and finally, the Wildtype. Black crosses represent censored datapoints. **: <span class="html-italic">p</span>-value &lt; 0.005, ***: <span class="html-italic">p</span>-value &lt; 0.001.</p>
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17 pages, 3327 KiB  
Article
Automated Knowledge Extraction in the Field of Wheat Sharp Eyespot Control
by Keyi Liu and Yunpeng Cui
Information 2024, 15(7), 367; https://doi.org/10.3390/info15070367 - 21 Jun 2024
Viewed by 741
Abstract
Wheat sharp eyespot is a soil-borne fungal disease commonly found in wheat areas in China, which can occur throughout the entire reproductive period of wheat and has a great impact on the yield and quality of wheat in China. By constructing a domain [...] Read more.
Wheat sharp eyespot is a soil-borne fungal disease commonly found in wheat areas in China, which can occur throughout the entire reproductive period of wheat and has a great impact on the yield and quality of wheat in China. By constructing a domain ontology for wheat sharp eyespot control and modeling the domain knowledge, we aim to integrate and share the knowledge in the field of wheat sharp eyespot control, which can provide important support and guidance for agricultural decision-making and disease control. In this study, the literature in the field of wheat sharp eyespot control was used as a data source, the KeyBERT keyword extraction algorithm was used to mine the core concepts of the ontology, and the hierarchical relationships among the ontology concepts were extracted through clustering. Based on the constructed ontology of wheat sharp eyespot control, the schema of knowledge extraction was formed, and the knowledge extraction model was trained using the ERNIE 3.0 knowledge enhancement pretraining model. This study proposes a model and algorithm to realize knowledge extraction based on domain ontology, describes the construction method and process framework of wheat sharp eyespot control domain ontology, and details the training and reasoning effect of the knowledge extraction model. The knowledge extraction model constructed in this study for wheat sharp eyespot control contains a more complete conceptual system of wheat sharp eyespot. The F1 value of the model reaches 91.26%, which is a 17.86% improvement compared with the baseline model, and it can satisfy the knowledge extraction needs in the field of wheat sharp eyespot control. This study can provide a reference for domain knowledge extraction and provide strong support for knowledge discovery and downstream applications such as intelligent Q&A and intelligent recommendation in the field of wheat sharp eyespot control. Full article
(This article belongs to the Special Issue Text Mining: Challenges, Algorithms, Tools and Applications)
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<p>Ontology construction process.</p>
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<p>Ontology concept.</p>
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<p>Object properties and data properties.</p>
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<p>Ontology of wheat sharp eyespot control.</p>
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<p>ERNIE 3.0 model framework.</p>
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<p>Model training process.</p>
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<p>Inference workflow.</p>
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19 pages, 3634 KiB  
Article
Microbiological and Mechanism Analysis of Novel Wheat Seed Coating Agents-Induced Growth Promotion of Wheat Seedlings
by Chao Chen, Wei Wang, Shuying Li, Shun He, Shufeng Zheng and Daoqing Xu
Agronomy 2024, 14(6), 1209; https://doi.org/10.3390/agronomy14061209 - 3 Jun 2024
Viewed by 647
Abstract
TFC (10% thifluzamide–fludioxonil–clothianidin) is a novel wheat seed-coating agent. In the field, we confirmed that 10% TFC plays a positive role in preventing soil-borne diseases and promoting wheat seedling growth. However, its effects on rhizosphere microecology and the underlying molecular mechanism are not [...] Read more.
TFC (10% thifluzamide–fludioxonil–clothianidin) is a novel wheat seed-coating agent. In the field, we confirmed that 10% TFC plays a positive role in preventing soil-borne diseases and promoting wheat seedling growth. However, its effects on rhizosphere microecology and the underlying molecular mechanism are not fully understood. Field trials revealed a positive effect on the biomass, plant height, and root length of wheat sharp eyespots in a Yingshang field, with 95.3% control efficiency. The effects of 10% TFC on the rhizosphere soil microbiome of young wheat plants were evaluated using high throughput sequencing technology. The results demonstrated that seed-coating agents significantly changed bacterial and fungal communities, and reduced the number of bacteria but increased the number of fungi. Sequence analysis revealed that the abundance of Proteobacteria, Actinobacteria, and Patescibacteria in bacteria and Ascomycota, Mortierellomycota, and Basidiomycota in fungi were significantly enriched, which have been reported as being beneficial for plant growth and pathogen resistance. In contrast, the abundance of Mucoromycota in fungi was reduced, and most of the related genera identified were pathogenic to plants. In this study, 15-day-old wheat plant tissues treated with 10% TFC were subjected to global transcriptome analysis by RNA sequencing to provide insights into the effects of 10% TFC on seedling growth. The comparative analysis of Triticum aestivum L. libraries identified 8286 differentially expressed genes (DEGs), of which 2290 and 5996 genes were up- and downregulated in seedling growth in the presence of 10% TFC, respectively. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses were performed for up- and downregulated DEGs separately, showing that these DEGs were enriched for terms related to the phenylpropanoid biosynthesis pathway, the protein products of which promote cell differentiation and seedling growth. This research provides comprehensive insights into its effects on wheat seedling growth and the rhizosphere microecology of seed coatings and provides important insights into their regulation and into understanding the potential benefits of seed coatings in disease management and plant growth promotion. Full article
(This article belongs to the Section Agroecology Innovation: Achieving System Resilience)
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<p>Effects of seed-coating treatments on plant growth ((<b>a</b>,<b>e</b>) Fengtai; (<b>b</b>) Fengyang; (<b>c</b>) Feixi; and (<b>d</b>) Yingshang) and soil-borne diseases ((<b>f</b>) Fengtai; (<b>g</b>) Fengyang; (<b>h</b>) Feixi; and (<b>i</b>) Yingshang). CK: uncoated seeds; TFC: 10% TFC-coated seeds; DFT: 27% DFT-coated seeds.</p>
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<p>Effects of seed-coating treatment on fungal Chao1, Shannon, Simpson, and Pielou’s diversity indices (<b>a</b>), PCoA of fungal community structure (the points with different colors indicate different groups) (<b>b</b>), phylum level fungal community composition (<b>c</b>), and heatmap of species clustering at the genus level of fungi in the different soil samples (<b>d</b>). CK: uncoated seeds; T: 10% TFC-coated seeds. Asterisks indicate significant differences (<span class="html-italic">* p</span> &lt; 0.05).</p>
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<p>Effects of seed-coating treatment on bacterial Chao1, Shannon, and Simpson diversity indices (<b>a</b>) and phylum-level bacterial community composition (<b>b</b>). CK: uncoated seeds; T: 10% TFC-coated seeds.</p>
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<p>Effects of seed-coating treatment on archaeal Chao1, Shannon, Simpson, and Faith’s PD diversity indices (<b>a</b>), PCoA of archaeal community structure (the points with different colors indicate different groups) (<b>b</b>), and phylum-level archaeal community composition (<b>c</b>). CK: uncoated seeds; T: 10% TFC-coated seeds. Asterisks indicate significant differences (<span class="html-italic">* p</span> &lt; 0.05).</p>
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<p>The linear discriminant analysis effect size (LEfSe) results for CK and 10% TFC treatments in the soils. (<b>a</b>) Cladogram of fungal taxa in the soil; (<b>b</b>) cladogram of bacterial taxa in the soil; (<b>c</b>) cladogram of archaeal taxa in the soil; (<b>d</b>) Venn diagram of fungal biomarkers in each treatment in the soils; (<b>e</b>) Venn diagram of bacterial biomarkers in each treatment in the soils; (<b>f</b>) Venn diagram of archaeal biomarkers in each treatment in the soils; (<b>g</b>) Venn diagram of fungal taxonomic distributions for treatments among different combinations; (<b>h</b>) Venn diagram of bacterial taxonomic distributions for treatments among different combinations; (<b>i</b>) Venn diagram of archaeal taxonomic distributions for treatments among different combinations. CK: uncoated seeds; T: 10% TFC-coated seeds. Only taxa that had met the linear discriminant analysis significance threshold of &gt;2.0 are shown. Every circle indicates a treatment; the numbers of OTUs shared between different treatments is interpreted using the number in the overlapping circles, while the number in the non-overlapping area represents the number of unique OTUs of the specific treatment.</p>
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<p>The linear discriminant analysis effect size (LEfSe) results for CK and 10% TFC treatments in the soils. (<b>a</b>) Cladogram of fungal taxa in the soil; (<b>b</b>) cladogram of bacterial taxa in the soil; (<b>c</b>) cladogram of archaeal taxa in the soil; (<b>d</b>) Venn diagram of fungal biomarkers in each treatment in the soils; (<b>e</b>) Venn diagram of bacterial biomarkers in each treatment in the soils; (<b>f</b>) Venn diagram of archaeal biomarkers in each treatment in the soils; (<b>g</b>) Venn diagram of fungal taxonomic distributions for treatments among different combinations; (<b>h</b>) Venn diagram of bacterial taxonomic distributions for treatments among different combinations; (<b>i</b>) Venn diagram of archaeal taxonomic distributions for treatments among different combinations. CK: uncoated seeds; T: 10% TFC-coated seeds. Only taxa that had met the linear discriminant analysis significance threshold of &gt;2.0 are shown. Every circle indicates a treatment; the numbers of OTUs shared between different treatments is interpreted using the number in the overlapping circles, while the number in the non-overlapping area represents the number of unique OTUs of the specific treatment.</p>
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<p>Differential gene expression analysis of wheat seedlings in response to 10% TFC. (<b>a</b>) Correlation analysis of the samples used for sequencing. The sample numbers are indicated, and the values in the squares are the Pearson correlation coefficients, calculated using R Studio. Dark colors indicate high expression, while lighter colors indicate lower expression. (<b>b</b>) Volcano plot of DEGs. <span class="html-italic">X</span>-axis: log<sub>2</sub>-fold change (10% TFC/control). <span class="html-italic">Y</span>-axis: the negative log<sub>10</sub>- adjusted <span class="html-italic">p</span>-value (FDR). Red data points indicate upregulated transcripts and blue data points indicate downregulated transcripts. (<b>c</b>) Number of up- and downregulated DEGs. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.</p>
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<p>Major pathways differentially regulated by 10% TFC in wheat seedlings, as indicated by (<b>a</b>) GO and (<b>b</b>) KEGG enrichment analysis.</p>
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12 pages, 8522 KiB  
Article
Trehalase Inhibitor Validamycin May Have Additional Mechanisms of Toxicology against Rhizoctonia cerealis
by Xiaoyue Yang, Yan Shu, Shulin Cao, Haiyan Sun, Xin Zhang, Aixiang Zhang, Yan Li, Dongfang Ma, Huaigu Chen and Wei Li
J. Fungi 2023, 9(8), 846; https://doi.org/10.3390/jof9080846 - 14 Aug 2023
Cited by 4 | Viewed by 1575
Abstract
Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative [...] Read more.
Sharp eyespot is a crucial disease affecting cereal plants, such as bread wheat (Triticum aestivum) and barley (Hordeum vulgare), and is primarily caused by the pathogenic fungus Rhizoctonia cerealis. As disease severity has increased, it has become imperative to find an effective and reasonable control strategy. One such strategy is the use of the trehalose analog, validamycin, which has been shown to have a potent inhibitory effect on several trehalases found in both insects and fungi, and is widely used as a fungicide in agriculture. In this study, we demonstrated that 0.5 μg/mL validamycin on PDA plates had an inhibitory effect on R. cerealis strain R0301, but had no significant impact on Fusarium graminearum strain PH-1. Except for its inhibiting the trehalase activity of pathogenic fungi, little is known about its mechanism of action. Six trehalase genes were identified in the genome of R. cerealis, including one neutral trehalase and five acidic trehalase genes. Enzyme activity assays indicated that treatment with 5 μg/mL validamycin significantly reduces trehalase activity, providing evidence that validamycin treatment does indeed affect trehalase, even though the expression levels of most trehalase genes, except Rc17406, were not obviously affected. Transcriptome analysis revealed that treatment with validamycin downregulated genes involved in metabolic processes, ribosome biogenesis, and pathogenicity in the R. cerealis. KEGG pathway analysis further showed that validamycin affected genes related to the MAPK signaling pathway, with a significant decrease in ribosome synthesis and assembly. In conclusion, our results indicated that validamycin not only inhibits trehalose activity, but also affects the ribosome synthesis and MAPK pathways of R. cerealis, leading to the suppression of fungal growth and pesticidal effects. This study provides novel insights into the mechanism of action of validamycin. Full article
(This article belongs to the Special Issue Fusarium, Alternaria and Rhizoctonia: A Spotlight on Fungal Pathogens)
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<p>Sensitivity of the strain R0301 to validamycin. (<b>a</b>) Validamycin inhibited trehalase activity in the mycelium of <span class="html-italic">R. cerealis</span>. (<b>b</b>) Validamycin inhibited the hyphal growth of <span class="html-italic">R. cerealis</span> on PDA and PTA plates (25 °C, 3–8 days). The concentration of validamycin was set to 0, 0.5, 1, 2, 4, and 8 μg/mL. (<b>c</b>) <span class="html-italic">F. graminearum</span> was treated at 0, 5, 20, 40, 80, 160, and 320 μg/mL under the same conditions.</p>
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<p>Phylogenetic tree analysis of trehalase proteins. (<b>a</b>) The phylogenetic tree based on the amino acid sequence was constructed by the MEGAX program using the maximum likelihood method. The amino acid sequences from different species were aligned by MAFFT. (<b>b</b>) Expression differences and (<b>c</b>) expression levels of transcriptome data from <span class="html-italic">R. cerealis</span>.</p>
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<p>Transcriptome data analysis of trehalase. (<b>a</b>) Volcano plot showing the distribution of DEGs at |log2(fold-change)| ≥ 2 and FDR (false discovery rate) <span class="html-italic">p</span> &lt; 0.05 before and after validamycin treatment. The red dots in the plot represent the statistically significant upregulated DEGs, the green dots represent the statistically significant downregulated DEGs, and gray dots indicate the DEGs without statistical significance. (<b>b</b>) KOG classification of DEGs. (<b>c</b>) Pie charts showing the top 20 upregulated and (<b>d</b>) downregulated DEG enriched GO terms.</p>
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<p>Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations of DEGs in R0301 vs. R0301 treated with validamycin. (<b>a</b>) Accumulated bar chart indicates the number of genes enriched by each KEGG pathway. Blue represents genes that are upregulated and orange represents genes that are downregulated. (<b>b</b>) Bubble charts showing the top 20 DEGs that are upregulated or (<b>c</b>) downregulated before and after validamycin treatment that are enriched in the KEGG pathway. The color of the bubble represents the corrected <span class="html-italic">q</span> value. The size of the bubble indicates the number of DEGs. The KEGG pathways are listed according to their molecular interactions and reaction networks within the ‘metabolism’, ‘genetic information processing’, ‘environmental information processing’, ‘cellular processes’, ‘human diseases’ and ‘organismal systems’ categories with different types of shapes.</p>
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<p>Expression values of related pathway genes before and after treatment with validamycin. (<b>a</b>) Heatmap of the expression values of genes related to starch and sucrose metabolism. (<b>b</b>) Heatmap of expression values of genes related to ribosome biogenesis in eukaryotes. (<b>c</b>) Heatmap of the expression values of genes related to ribosomes. Red indicates that the expression was relatively high, while blue indicates that the expression was relatively low. White indicates that the gene expression between the conditions analyzed was unchanged.</p>
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17 pages, 4344 KiB  
Article
Efficacy of Different Fungicide Spraying Techniques on the Infestation with Kabatiella zeae and Formation of Fusarium Mycotoxins in Forage Maize
by Tim Birr, Andreas Tillessen, Joseph-Alexander Verreet, Mario Hasler and Holger Klink
Agriculture 2023, 13(6), 1269; https://doi.org/10.3390/agriculture13061269 - 19 Jun 2023
Cited by 2 | Viewed by 1837
Abstract
The application of fungicides in maize by the commonly used overhead spraying technique is more challenging than in small-grain cereals. Especially in later development stages, when the plant has reached a considerable height, lower plant organs (e.g., ears) may be insufficiently protected, since [...] Read more.
The application of fungicides in maize by the commonly used overhead spraying technique is more challenging than in small-grain cereals. Especially in later development stages, when the plant has reached a considerable height, lower plant organs (e.g., ears) may be insufficiently protected, since a large part of the applied fungicide is deposited on the upper leaves. In contrast, lower plant organs can be reached by the dropleg spraying technique, which allows sub-canopy applications. This study investigated the efficacy of fungicide applications during flowering in forage maize using the overhead and dropleg spraying techniques as well as a combination for the control of Kabatiella zeae and mycotoxin-producing Fusarium species, which can affect leaves and ears, respectively. The efficacy was evaluated in field trials under natural K. zeae and artificial Fusarium inoculum conditions in Northern Germany by measuring disease severities of K. zeae on maize leaves, dry matter yields, and concentrations of the Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) at harvest. Fusarium inoculations of main ears at full flowering resulted in significantly higher DON and ZEN concentrations compared to natural Fusarium infections, whereas the dry matter yield was not affected. The strongest disease progressions of K. zeae were determined after flowering on the upper leaves and leaves around the main ear. Disease severities were significantly reduced on the upper leaves by the overhead application and the combination of the overhead and dropleg spraying technique, whereby the three spraying techniques were equally able to decrease the infestation on the yield-essential leaves around the main ear. No differences in dry matter yield were found between the application techniques, but they were significantly higher than in the untreated control. The contamination with DON and ZEN was most effectively reduced by sub-canopy applications using the dropleg technique, whether as a solo application or in combination with the overhead technique. The main ears were reached better with the applied fungicide, reducing Fusarium infections, and therefore, contamination with mycotoxins. The dropleg technique offers an opportunity for improved protection of lower plant organs, especially in tall growing crops. In combination with the overhead spraying technique, the protection of various plant organs along the entire plant with the applied fungicide could be advantageous, especially when different parts of the plant are affected by different fungal diseases. Full article
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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<p>Symptoms of eyespot disease on maize leaves. (<b>A</b>) Typical eyespots caused by <span class="html-italic">Kabatiella zeae</span>. (<b>B</b>) Eyespots spread over the entire leaf during disease progression. (<b>C</b>) Eyespots join together into large necrotic areas causing premature drying and maturity of leaves.</p>
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<p>Symptoms of <span class="html-italic">Fusarium</span> rot diseases in maize. (<b>A</b>) Ear rot—White-colored mold layers on main ear. (<b>B</b>) Rotted rudimentary ear. (<b>C</b>) Stalk rot—Disintegrated and reddish discolored pith of the stalk.</p>
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<p>Schematic comparison between the (<b>A</b>) overhead and (<b>B</b>) dropleg spraying technique used for the fungicide applications in forage maize at BBCH 65. (<b>A<sub>I,</sub>A<sub>II</sub></b>) In the conventional spraying treatment (T3) fungicides were applied from above the crop in a basically vertical direction down into the crop with a standard nozzle spacing of 0.5 m on a horizontal spray boom and a distance 0.5 m between spray boom and the top of the crop. (<b>B<sub>I,</sub>B<sub>II</sub></b>) In the dropleg spraying treatment (T4) two nozzles in a twin-spray-cap at the lower end of the dropleg were guided in the crop centrally between two maize rows with a distance of 1 m underneath the spray boom spraying towards the ground and sideways.</p>
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<p>Boxplots and means (yellow rhombus) of (<b>A</b>) dry matter yield (dt/ha), (<b>B</b>) DON and (<b>C</b>) ZEN concentrations (µg/kg) in forage maize samples at silage maturity of the cultivar “SY Werena” in the two fungicide-untreated controls without (T1) and with (T2) silk channel inoculation of main ears with macroconidia of <span class="html-italic">Fusarium culmorum</span> seven days after silk emergence at BBCH 65 summarized for the three trial locations. Five statistics are represented in each boxplot from bottom to top: The smallest observation, lower quartile, median, upper quartile, and largest observation, respectively. Different letters describe significant differences in dry matter yield, DON and ZEN concentrations between the non-inoculated (T1) and inoculated (T2) fungicide-untreated control. Statistical significance was evaluated at <span class="html-italic">p</span> ≤ 0.05. DON = deoxynivalenol; ZEN = zearalenone.</p>
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<p>Boxplots and means (yellow rhombus) of disease severities of <span class="html-italic">Kabatiella zeae</span> (percentage of leaf area affected by symptoms) on the upper, middle and lower leaf segment of forage maize at BBCH 65, BBCH 75 and BBCH 85 of the cultivar “SY Werena” in the fungicide-untreated control (T2) summarized for the three trial locations. Five statistics are represented in each boxplot from bottom to top: The smallest observation, lower quartile, median, upper quartile, and largest observation, respectively.</p>
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<p>Boxplots and means (yellow rhombus) of disease severities of <span class="html-italic">Kabatiella zeae</span> (percentage of leaf area affected by symptoms) on the (<b>A</b>) upper, (<b>B</b>) middle (L-2 to L+2) and (<b>C</b>) lower leaf segment of forage maize at BBCH 85 of the cultivar “SY Werena” depending on different fungicide spraying techniques (treatments T2–T5) summarized for the three trial locations. Treatments: T2—Fungicide-untreated control, T3—Overhead spraying technique, T4—Dropleg spraying technique, T5—Combination of T3 and T4. The fungicide Prosaro<sup>®</sup> was applied (T3–T5) with 1.0 L/ha and a spray volume of 400 L/ha at BBCH 65. Five statistics are represented in each boxplot from bottom to top: The smallest observation, lower quartile, median, upper quartile, and largest observation, respectively. Different letters describe significant differences in disease severities between treatments T2–T5. Statistical significance was evaluated at <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Boxplots and means (yellow rhombus) of dry matter yield (dt/ha) of forage maize of the cultivar “SY Werena” depending on different fungicide spraying techniques (treatments T2–T5) summarized for the three trial locations. Treatments: T2—Fungicide-untreated control, T3—Overhead spraying technique, T4—Dropleg spraying technique, T5—Combination of T3 and T4. The fungicide Prosaro<sup>®</sup> was applied (T3–T5) with 1.0 L/ha and a spray volume of 400 L/ha at BBCH 65. Five statistics are represented in each boxplot from bottom to top: The smallest observation, lower quartile, median, upper quartile, and largest observation, respectively. Different letters describe significant differences in dry matter yield between treatments T2–T5. Statistical significance was evaluated at <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Boxplots and means (yellow rhombus) of (<b>A</b>) DON and (<b>B</b>) ZEN concentrations (µg/kg) in forage maize samples at silage maturity of the cultivar “SY Werena” depending on different fungicide spraying techniques (treatments T2–T5) summarized for the three trial locations. Treatments: T2—Fungicide-untreated control, T3—Overhead spraying technique, T4—Dropleg spraying technique, T5—Combination of T3 and T4. The fungicide Prosaro<sup>®</sup> was applied (T3–T5) with 1.0 L/ha and a spray volume of 400 L/ha two days after silk channel inoculation of main ears with macroconidia of <span class="html-italic">Fusarium culmorum</span> at BBCH 65. Five statistics are represented in each boxplot from bottom to top: The smallest observation, lower quartile, median, upper quartile, and largest observation, respectively. Different letters describe significant differences in DON and ZEN concentrations between treatments T2–T5. Statistical significance was evaluated at <span class="html-italic">p</span> ≤ 0.05. DON = deoxynivalenol; ZEN = zearalenone.</p>
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15 pages, 3164 KiB  
Article
A Novel Wall-Associated Kinase TaWAK-5D600 Positively Participates in Defense against Sharp Eyespot and Fusarium Crown Rot in Wheat
by Haijun Qi, Xiuliang Zhu, Wenbiao Shen and Zengyan Zhang
Int. J. Mol. Sci. 2023, 24(5), 5060; https://doi.org/10.3390/ijms24055060 - 6 Mar 2023
Cited by 1 | Viewed by 2223
Abstract
Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. [...] Read more.
Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat. Full article
(This article belongs to the Special Issue Molecular Insights into Plant-Biotic Interactions and Crop Yield)
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<p>Chromosomal distribution of the typical <span class="html-italic">TaWAK</span> genes in wheat. The 140 <span class="html-italic">TaWAK</span> genes were unevenly distributed on 20 wheat chromosomes. The bar indicates the length of chromosome in megabases (MB).</p>
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<p>The transcript profiles of 140 <span class="html-italic">TaWAKs</span> in the wheat RNA-sequencing (RNA-seq) data. (<b>A</b>) The transcript levels of 140 <span class="html-italic">TaWAKs</span> upon <span class="html-italic">R. cerealis</span> infection in the resistant recombinant inbred lines (RILs) derived from the cross ‘Shanhongmai’ × ‘Wenmai 6′. (<b>B</b>) The transcript profiles of 140 <span class="html-italic">TaWAKs</span> upon <span class="html-italic">F. pseudograminearum</span> infection. The RNA-seq data upon <span class="html-italic">F. pseudograminearum</span> infection were checked from the online RNA-seq data (<a href="http://www.wheat-expression.com/" target="_blank">http://www.wheat-expression.com/</a> accessed on 25 August 2022) [<a href="#B32-ijms-24-05060" class="html-bibr">32</a>].</p>
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<p><span class="html-italic">TaWAK-5D600</span> is involved in wheat responses to both sharp eyespot and <span class="html-italic">Fusarium</span> crown rot. (<b>A</b>) The transcript levels and fold change of the significantly upregulated 27 <span class="html-italic">TaWAK</span> genes in the RILs-R response to <span class="html-italic">R. cerealis</span> infection. (<b>B</b>) The transcript levels and fold change of the 27 <span class="html-italic">R. cerealis</span> induced <span class="html-italic">TaWAK</span> genes upon <span class="html-italic">F. pseudograminearum</span> infection in the online RNA-seq data (<a href="http://www.wheat-expression.com/" target="_blank">http://www.wheat-expression.com/</a> accessed on 25 August 2022) [<a href="#B32-ijms-24-05060" class="html-bibr">32</a>]. (<b>C</b>) Transcript levels of <span class="html-italic">TaWAK-5D600</span> in sharp eyespot-resistant wheat line CI12633 at non-treatment and 4, 7, and 10 dpi with <span class="html-italic">R. cerealis</span> Rc207. (<b>D</b>) The transcript patterns of <span class="html-italic">TaWAK-5D600</span> in FCR-mildly-resistant wheat line CI12633 at non-treatment and 1 and 2 dpi with <span class="html-italic">F. pseudograminearum</span> WHF220. <span class="html-italic">TaWAK-5D600</span> transcript level at non-treatment is set to 1. <span class="html-italic">TaActin</span> gene was used as the internal control (<span class="html-italic">t</span>-test: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01).</p>
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<p>Phylogenetic tree, conserved-domain, and gene-structure analyses of <span class="html-italic">TaWAK-5D600</span>. (<b>A</b>) A phylogenetic tree of TaWAK-5D600 and other 18 WAK proteins from different plants. The position of TaWAK-5D600 was indicated by a red blot. (<b>B</b>) Gene structure of <span class="html-italic">TaWAK-5D600</span>; black boxes represent exons and black lines indicate introns. (<b>C</b>) Schematic diagram of the TaWAK-5D600 protein. The conserved protein domains of TaWAK-5D600 were represented by different colored boxes. (<b>D</b>) Subcellular localization of TaWAK-5D600 in wheat protoplasts cells (bars = 20 μm).</p>
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<p>Silencing of <span class="html-italic">TaWAK-5D600</span>-compromised wheat resistance both to sharp eyespot and <span class="html-italic">Fusarium</span> crown rot. (<b>A</b>) Typical BSMV symptoms on wheat leaves at 15 dpi infected with BSMV and transcripts of BSMV coat protein (<span class="html-italic">CP</span>) gene detecting by RT-PCR assays. (<b>B</b>) The silencing efficiency of <span class="html-italic">TaWAK-5D600</span> detecting by RT-qPCR assay. The transcript level of <span class="html-italic">TaWAK-5D600</span> in BSMV:GFP (control) wheat seedlings was set to 1. (<b>C</b>) Sharp eyespot symptoms on <span class="html-italic">TaWAK-5D600</span>-silenced and BSMV:GFP (control) wheat plants at 30 dpi with <span class="html-italic">R. cerealis</span>. (<b>D</b>) Disease indexes (DIs) of <span class="html-italic">TaWAK-5D600</span>-silenced and BSMV:GFP (control) wheat plants at 30 dpi with <span class="html-italic">R. cerealis</span> in two independent batches (<span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> &lt; 0.01). (<b>E</b>) <span class="html-italic">Fusarium</span> crown rot symptoms on <span class="html-italic">TaWAK-5D600</span>-silenced and control wheat plants at 30 dpi with <span class="html-italic">F. pseudograminearum.</span> (<b>F</b>) Disease index (DI) of <span class="html-italic">TaWAK-5D600</span>-silenced and control wheat plants at 30 dpi with <span class="html-italic">F. pseudograminearum</span> WHF220 in two independent batches (<span class="html-italic">t</span>-test: ** <span class="html-italic">p</span> &lt; 0.01). Bars indicate SEs of the mean.</p>
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<p>Transcript profiles of <span class="html-italic">TaWAK-5D600</span> and defense-related genes in BSMV:GFP (control) and BSMV:TaWAK-5D600-infected wheat seedlings. Relative transcript abundances of <span class="html-italic">TaWAK-5D600</span> and the tested genes <span class="html-italic">TaSERK1</span>, <span class="html-italic">TaMPK3</span>, <span class="html-italic">TaPR1</span>, <span class="html-italic">TaChitinase3</span>, and <span class="html-italic">TaChitinase4</span> in BSMV: TaWAK5D600-infected CI12633 seedlings were quantified relative to those in BSMV:GFP (control) seedlings. Statistically significant differences were calculated based on three replications via a Student’s <span class="html-italic">t</span>-test (** <span class="html-italic">p</span> &lt; 0.01). <span class="html-italic">TaActin</span> was used as an internal control.</p>
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9 pages, 1596 KiB  
Perspective
Algal Ocelloids and Plant Ocelli
by Felipe Yamashita and František Baluška
Plants 2023, 12(1), 61; https://doi.org/10.3390/plants12010061 - 22 Dec 2022
Cited by 4 | Viewed by 5229
Abstract
Vision is essential for most organisms, and it is highly variable across kingdoms and domains of life. The most known and understood form is animal and human vision based on eyes. Besides the wide diversity of animal eyes, some animals such as cuttlefish [...] Read more.
Vision is essential for most organisms, and it is highly variable across kingdoms and domains of life. The most known and understood form is animal and human vision based on eyes. Besides the wide diversity of animal eyes, some animals such as cuttlefish and cephalopods enjoy so-called dermal or skin vision. The most simple and ancient organ of vision is the cell itself and this rudimentary vision evolved in cyanobacteria. More complex are so-called ocelloids of dinoflagellates which are composed of endocellular organelles, acting as lens- and cornea/retina-like components. Although plants have almost never been included into the recent discussions on organismal vision, their plant-specific ocelli had already been proposed by Gottlieb Haberlandt already in 1905. Here, we discuss plant ocelli and their roles in plant-specific vision, both in the shoots and roots of plants. In contrast to leaf epidermis ocelli, which are distributed throughout leaf surface, the root apex ocelli are located at the root apex transition zone and serve the light-guided root navigation. We propose that the plant ocelli evolved from the algal ocelloids, are part of complex plant sensory systems and guide cognition-based plant behavior. Full article
(This article belongs to the Special Issue Plant Signaling, Behavior and Communication)
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<p><b>Algal Eyespot of Chlamydomonas.</b> Chlamydomonas alga with two flagella associated with the basal bodies which intracellularly organize intracellular bundles of microtubules (known as rootlets) of which the D4 bundle anchors the eyespot. This eyespot is constructed from chloroplast thylakoid membranes and carotenoid globules, aligned under the plasma membrane which is enriched with photoreceptor channelrhodopsin. Besides the bundles of microtubules, the basal body also organizes the centrin-based contractile nucleo-basal body connector anchoring the nucleus. M4, M2 and D2 rootlets are not shown in this simplified scheme.</p>
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<p><b>Algal Ocelloid of Dinoflagellates.</b> Camera-like ocelloid of warnowiid dinoflagellates is composed of cornea-like mitochondrion enclosing hyaloplasm acting as lens and chloroplast-based retinal body. Similarly, as in the algal eyespot, the chloroplast plays the central role in the microbial vision. Adapted according [<a href="#B43-plants-12-00061" class="html-bibr">43</a>].</p>
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<p><b>Bacterial Vision: Cyanobacterium Synechocystis.</b> The whole cyanobacterial cell acts as a lens, focusing light beams on a small patch of the plasma membrane which controls the type-IV pili-based motility apparatus anchored in the plasma membrane via T4P complexes. Under the plasma membrane are thylakoid membranes. This model was adapted according to [<a href="#B49-plants-12-00061" class="html-bibr">49</a>].</p>
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<p><b>Root Apex Ocelli.</b> Arabidopsis root apex expresses phot1 blue-light photoreceptor in cortex cells of the transition zone. The phot1 photoreceptors are arranged in the U-shape arrangements under the root epidermis cells which are devoid of phot 1 and are proposed to act as a lens cells, focusing the light on the underlying cortex cells. The root apex ocelli are proposed to allow root skototropism when roots grown within the illuminated portion of Petri dish can recognize the dark area and navigate the root growth towards it.</p>
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<p><b>Microtubules-MTOC in Rudimentary Cell Vision of Eukaryotic Cells.</b> Albrecht-Beuhler’s rudimentary cellular vision is accomplished via microtubules conveying infrared wavelengths along microtubules towards the perinuclear centrosome of animal cells. In the plant cells, the centrosome is not corpuscular but is distributed diffusely along the whole nuclear surface.</p>
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12 pages, 2458 KiB  
Article
Screening Winter Wheat Genotypes for Resistance Traits against Rhizoctonia cerealis and Rhizoctonia solani Infection
by Karol Lisiecki, Grzegorz Lemańczyk, Dariusz Piesik and Chris A. Mayhew
Agriculture 2022, 12(12), 1981; https://doi.org/10.3390/agriculture12121981 - 23 Nov 2022
Cited by 4 | Viewed by 1724
Abstract
Rhizoctonia cerealis and Rhizoctonia solani are considered to be among the most harmful soil-borne pathogens for crop plants globally. The lack of effective protection and the requirement to minimize the use of chemical pesticides necessitate the need to develop alternative protective methods. One [...] Read more.
Rhizoctonia cerealis and Rhizoctonia solani are considered to be among the most harmful soil-borne pathogens for crop plants globally. The lack of effective protection and the requirement to minimize the use of chemical pesticides necessitate the need to develop alternative protective methods. One such method is resistance breeding against biotic and abiotic stresses. Here, we present studies on the presence of resistance traits in winter wheat genotypes that evaluate the plants’ resistance to the above two pathogens, in both field and laboratory environments. In the field environment, the incidence and severity of sharp eyespot were studied using 132 winter wheat cultivars, where random samples at the BBCH 75–77 were collected for analysis. The degree of the intensity of sharp eyespot was determined, applying the 0–4° scale. The susceptibility of the 132 cultivars of winter wheat to R. cerealis (AG-D subgroup I) and R. solani (AG-5) was also studied under laboratory conditions. In the laboratory, test pieces of potato dextrose agar colonized by the test isolates were placed onto filter paper soaked with distilled water and then placed into Petri dish. Infection on the roots, coleoptiles and leaves was then assessed after 15 days for R. cerealis and after 10 days for R. solani. None of the tested winter wheat genotypes were found to be asymptomatic to the pathogens. A moderate susceptibility was observed for such genotypes as Anthus, Baryton, Bellenus, Borderland Benatka, Blonde, Cubus, Estero, and Flairway. However, the classification of those associated with moderate susceptibility in laboratory tests resulted in severe symptoms in field tests. Hence, field experiments provide the most reliable measurements to determine the effects of pathogens on the plants. Full article
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)
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<p>Correlation matrix between the studied variables depending on the species of pathogen used (R. c.—red color—<span class="html-italic">R. cerealis</span>; R. s.—blue color—<span class="html-italic">R. solani</span>; x, y axes—percentage of disease severity caused by Rhizoctonia species, Roots—roots disease index (%), Stem base—stem base disease index (%), Leaves—leaves disease index (%), Disease index—the disease index of plants in field trials (%), Percentage—the percentage of infected population showing symptoms (%), Damping-off (%), *—significance level 0.05, ***—significance level 0.001).</p>
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<p>The heatmap of the infestation distribution of the tested plants in individual experimental variants, i.e., the type of pathogen and the type of experiment (laboratory, field). The colors successively correspond to the clusters to which the plants were assigned using the hierarchical K-means method: green—potentially resistant, yellow—moderate, red—susceptible/non-resistant. Rc.L—<span class="html-italic">R. cerealis</span> in the laboratory conditions, Rc.F—<span class="html-italic">R. cerealis</span> in the field conditions, Rs.L—<span class="html-italic">R. solani</span> in the laboratory conditions, Rs.F—<span class="html-italic">R. solani</span> in the field conditions.</p>
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<p>Distribution of the studied genotypes of plants using the method of hierarchical K−means in laboratory conditions in the inoculation of <span class="html-italic">R. cerealis</span> (clustering in the form of a phylogenetic tree). The colors correspond to the assigned clusters according to the resistance type; red−susceptible, yellow−less susceptible, green−least susceptible.</p>
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<p>Distribution of the studied genotypes of plants using the method of hierarchical K−means in field conditions with the inoculation of <span class="html-italic">R. cerealis</span> (clustering in the form of a phylogenetic tree). The colors correspond to the assigned clusters according to the resistance type; red−susceptible, yellow−less susceptible, green−least susceptible.</p>
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<p>Distribution of the studied genotypes of plants using the method of hierarchical K−means in laboratory conditions with the inoculation of <span class="html-italic">R. solani</span> (clustering in the form of a phylogenetic tree). The colors correspond to the assigned clusters according to the resistance type; red−susceptible, yellow−less susceptible, green−least susceptible.</p>
Full article ">Figure 6
<p>Distribution of the studied genotypes of plants using the method of hierarchical K−means in field conditions with the inoculation of <span class="html-italic">R. solani</span> (clustering in the form of a phylogenetic tree). The colors correspond to the assigned clusters according to the resistance type; red−susceptible, yellow−less susceptible, green−least susceptible.</p>
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24 pages, 3335 KiB  
Article
Rediscovering Monogenoids (Platyhelminthes) Parasitizing Pomacentrid and Chaetodontid Fishes from Cayo Arcas Reef, Gulf of Mexico
by Edgar F. Mendoza-Franco, Nuno Simões, Víctor M. Vidal-Martínez and M. Leopoldina Aguirre-Macedo
Diversity 2022, 14(11), 985; https://doi.org/10.3390/d14110985 - 16 Nov 2022
Cited by 2 | Viewed by 1884
Abstract
During a research of gill ectoparasites on damselfishes (Pomacentridae) and butterflyfishes (Chaetodontidae) from the Cayo Arcas reef in the Campeche Bank (Gulf of Mexico), the following monogenoids (Platyhelminthes) were found: Paraeuryhaliotrema pomacentris n. gen., n. sp. (Dactylogyridae) on beaugregory Stegastes xanthurus (Poey, 1860) [...] Read more.
During a research of gill ectoparasites on damselfishes (Pomacentridae) and butterflyfishes (Chaetodontidae) from the Cayo Arcas reef in the Campeche Bank (Gulf of Mexico), the following monogenoids (Platyhelminthes) were found: Paraeuryhaliotrema pomacentris n. gen., n. sp. (Dactylogyridae) on beaugregory Stegastes xanthurus (Poey, 1860) (Pomacentridae) that is characterized, in part, by possessing a haptor armed with a dorsal, ventral anchor-bar complexes, seven pairs of similar hooks; two pairs of eyespots; overlapping gonads; a copulatory complex composed of a male copulatory organ (MCO) and an accessory piece; MCO tubular with a bulbous base from which arises a coiled shaft in the clockwise direction; and a dextral vaginal pore; Neohaliotrema variabilis n. sp. on bicolor damselfish Stegastes partitus (Poey, 1868) (type host), beaugregory Stegastes xanthurus (Poey, 1860) and beaugregory Stegastes leucostictus (Müller & Troschel, 1848); Neohaliotrema manubrium n. sp., Neohaliotrema aliamanubrium n. sp. and Neohaliotrema bifidum n. sp., Neohaliotrema bychowskii Zhukov, 1976 and Neohaliotrema macracanthum Zhukov, 1976 on Sergeant-major Abudefduf saxatilis (Linnaeus, 1758) (Pomacentridae); Neohaliotrema similium n. sp. on S. xanthurus; Haliotrema brevicirrus Zhukov 1976 on spotfin butterflyfish Chaetodon ocellatus Bloch 1787 (Chaetodontidae); Microcotyle multilineatus n. sp. (Microcotylidae) on brown chromis Chromis multilineata (Guichenot 1853) (Pomacentridae). The new species are described and illustrated; new illustrations and measurements of the haptoral structures, and new redescription and illustrations are provided for N. bychowskii, N. macracanthum and H. brevicirrus, respectively. The present study represents the first knowledge about ectoparasitic monogenoids of fishes in the Cayo Arcas reef from the Gulf of Mexico. Full article
(This article belongs to the Special Issue Diversity of Macroparasites in Marine Fishes)
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Figure 1

Figure 1
<p><span class="html-italic">Paraeuryhaliotrema pomacentris</span> n. gen., n. sp. from the beaugregory <span class="html-italic">Stegastes xanthurus</span>. (<b>A</b>) Whole mount (composite, dorsal view) (<b>B</b>) Copulatory complex (dorsal view) (<b>C</b>) Ventral anchor (<b>D</b>) Dorsal anchor (<b>E</b>) Ventral bar (<b>F</b>) Dorsal bar (<b>G</b>) Hook (<b>H</b>) Haptor.</p>
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<p>Whole mount of <span class="html-italic">Neohaliotrema variabilis</span> n. sp. (composite, ventral view) from the beaugregory <span class="html-italic">Stegastes partitus</span> showing position of hook pairs 1–7.</p>
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<p>Sclerotized structures of <span class="html-italic">Neohaliotrema variabilis</span> n. sp. from the beaugregory <span class="html-italic">Stegastes partitus</span>. (<b>A</b>,<b>B</b>) Ventral anchors (<b>C</b>,<b>D</b>) Dorsal anchors (<b>E</b>) Hook pair 1 (<b>F</b>) Hook pairs 4 (<b>G</b>–<b>I</b>) Ventral bars (<b>J</b>) Dorsal bar. (<b>K</b>,<b>L</b>) Hook pairs 3. (<b>M</b>) Copulatory complex. (<b>N</b>,<b>O</b>) Ventral bars (<b>P</b>) Hook pairs 2 (<b>Q</b>,<b>R</b>) Hook pairs 7.</p>
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<p><span class="html-italic">Neohaliotrema manubrium</span> n. sp. from the Sergeant-major <span class="html-italic">Abudefduf saxatilis</span> (<b>A</b>) Whole mount (composite, ventral view) (<b>B</b>) Copulatory complex (<b>C</b>) Hook pair 1 (<b>D</b>) Hook pair 2 (<b>E</b>) Hook pair 3 (<b>F</b>) Ventral bar (<b>G</b>) Dorsal bar (<b>H</b>) Egg (<b>I</b>) Ventral anchor (<b>J</b>) Dorsal anchor (<b>K</b>) Hook.</p>
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<p>Haptoral structures of <span class="html-italic">Neohaliotrema aliamanubrium</span> n. sp. from Sergeant-major <span class="html-italic">Abudefduf saxatilis</span> (<b>A</b>) Ventral anchor (<b>B</b>) Dorsal anchor (<b>C</b>) Hook pair 7 (<b>D</b>) Ventral bar (<b>E</b>) Dorsal bar (<b>F</b>) Hook pair 1 (<b>G</b>) Hook pair 2 (<b>H</b>) Hook pair 4 (<b>I</b>) Hook pair 3.</p>
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<p>Haptoral structures of <span class="html-italic">Neohaliotrema bifidum</span> n. sp. from the Sergeant-major <span class="html-italic">Abudefduf saxatilis</span> (<b>A</b>) Haptor (ventral view) showing positions of hook pairs (1–7) (<b>B</b>) Ventral anchor (<b>C</b>,<b>D</b>) Ventral anchors (<b>E</b>) Dorsal anchor (<b>F</b>) Dorsal anchor (<b>G</b>–<b>M</b>) Hook pairs 1–7, respectively.</p>
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<p>Haptoral structures of <span class="html-italic">Neohaliotrema similium</span> n. sp. from the beaugregory <span class="html-italic">Stegastes xanthurus</span> (<b>A</b>) Dorsal anchor (<b>B</b>) Ventral anchor (<b>C</b>) Dorsal bar (<b>D</b>) Ventral bar (<b>E</b>) Hook pair 3 (<b>F</b>) Hook pair 2 (<b>G</b>) Copulatory complex (<b>H</b>) Hook pair 1 (<b>I</b>) Hook pair 4 (<b>J</b>) Hook pair 7.</p>
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<p>Haptoral structures of <span class="html-italic">Neohaliotrema bychowskii</span> Zhukov, 1976 from Sergeant-major <span class="html-italic">Abudefduf saxatilis</span> (<b>A</b>) Hook pair 1 (<b>B</b>) Hook pair 2 (<b>C</b>) Ventral bar (<b>D</b>) Dorsal bar (<b>E</b>) Hook pair 4 (<b>F</b>) Hook pair 3 (<b>G</b>) Ventral anchor (<b>H</b>) Dorsal anchor.</p>
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<p><span class="html-italic">Haliotrema brevicirrus</span> Zhukov, 1980 from the Spotfin butterflyfish <span class="html-italic">Chaetodon ocellatus</span> (<b>A</b>) Whole mount (composite, ventral view) (<b>B</b>,<b>C</b>) Copulatory complexes (<b>D</b>) Ventral anchor (<b>E</b>) Ventral bar (<b>F</b>) Dorsal bar (<b>G</b>) Dorsal anchor (<b>H</b>) Dorsal anchor with base slightly bent toward the midline of haptor (<b>I</b>) Hook.</p>
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<p><span class="html-italic">Microcotyle multilineatus</span> n. sp. from the Brown chromis, <span class="html-italic">Chromis multilineata</span>. (<b>A</b>) Whole mount (composite, ventral view) (<b>B</b>) Genital atrium (<b>C</b>) Spines (<b>D</b>) Deeper focus of the genital atrium (<b>E</b>) Clamp.</p>
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21 pages, 4325 KiB  
Article
Butterfly Wing Color Pattern Modification Inducers May Act on Chitin in the Apical Extracellular Site: Implications in Morphogenic Signals for Color Pattern Determination
by Joji M. Otaki and Yugo Nakazato
Biology 2022, 11(11), 1620; https://doi.org/10.3390/biology11111620 - 6 Nov 2022
Cited by 5 | Viewed by 4515
Abstract
Butterfly wing color patterns are modified by various treatments, such as temperature shock, injection of chemical inducers, and covering materials on pupal wing tissue. Their mechanisms of action have been enigmatic. Here, we investigated the mechanisms of color pattern modifications using the blue [...] Read more.
Butterfly wing color patterns are modified by various treatments, such as temperature shock, injection of chemical inducers, and covering materials on pupal wing tissue. Their mechanisms of action have been enigmatic. Here, we investigated the mechanisms of color pattern modifications using the blue pansy butterfly Junonia orithya. We hypothesized that these modification-inducing treatments act on the pupal cuticle or extracellular matrix (ECM). Mechanical load tests revealed that pupae treated with cold shock or chemical inducers were significantly less rigid, suggesting that these treatments made cuticle formation less efficient. A known chitin inhibitor, FB28 (fluorescent brightener 28), was discovered to efficiently induce modifications. Taking advantage of its fluorescent character, fluorescent signals from FB28 were observed in live pupae in vivo from the apical extracellular side and were concentrated at the pupal cuticle focal spots immediately above the eyespot organizing centers. It was shown that chemical modification inducers and covering materials worked additively. Taken together, various modification-inducing treatments likely act extracellularly on chitin or other polysaccharides to inhibit pupal cuticle formation or ECM function, which probably causes retardation of morphogenic signals. It is likely that an interactive ECM is required for morphogenic signals for color pattern determination to travel long distances. Full article
(This article belongs to the Section Developmental Biology)
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Figure 1

Figure 1
<p>Wing color patterns and experimental procedures. (<b>A</b>) Dorsal wings of three females (left) and one male. The posterior eyespot (ES), distal parafocal element (PFE), and submarginal band (SMB) are indicated. (<b>B</b>) Ventral hindwings of individuals shown in A. ES, PFE, and SMB are indicated. (<b>C</b>) Side view of a pupa. This pupa is immediately before eclosion. Forewing color patterns are seen through the pupal cuticle. (<b>D</b>) Ventral view of a pupa. The position of a gauge probe is shown by a red circle. (<b>E</b>) A pupa with a gauge probe for the mechanical load test. (<b>F</b>) A pupa with a forewing lift configuration with an injection needle. Lifted wing surfaces are covered with a piece of transparent plastic film.</p>
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<p>Results of mechanical load tests for pupae treated with various modification-inducing treatments. (<b>A</b>) Pupal hardness at 6 h and 24 h post-pupation under modification-inducing treatments. The hardness is expressed in gram-force. The number of treated pupae is shown for each treatment. Sodium chloride treatment was compared with other treatment modes (including no treatment). Asterisks indicate statistical significance: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. (<b>B</b>) Fold change values from 6 h to 24 h post-pupation. At the bottom, <span class="html-italic">p</span>-values between 6-h and 12-h hardness values are shown.</p>
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<p>Color pattern modifications induced by FB28 and other modification inducers. (<b>A</b>) FB28. (<b>B</b>) Previously known modification inducers, tungstate and heparin. These treatments cause similar modifications of PFEs toward the proximal positions (closer to eyespots). (<b>C</b>) Dextran sulfate. This treatment acts on the thickening and dislocation of PFEs (farther from eyespots). (<b>D</b>) Comparison between a hindwing treated with FB28 and a hindwing without treatment. The dorsal (<b>left</b>) and ventral (<b>right</b>) sides of the hindwing are shown. PFEs are indicated by arrows.</p>
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<p>Results of mechanical load tests for pupae treated with FB28. (<b>A</b>) Pupal hardness 6 h and 24 h post-pupation. The hardness is expressed in gram-force. The number of treated pupae is shown for each treatment. Sodium chloride treatment was compared with FB28 treatment. Asterisks indicate statistical significance: * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01. (<b>B</b>) Fold change values from 6 h to 24 h post-pupation. At the bottom, <span class="html-italic">p</span>-values between 6-h and 12-h hardness values are shown.</p>
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<p>FB28 fluorescent signals under ultraviolet light from forewing-lifted pupae after injection. Post-injection hours are indicated. (<b>A</b>–<b>D</b>) A pupa. HD, AN, FW, and HW indicate the head, antennae, forewing and hindwing, respectively. The white arrow in B indicates a pupal cuticle focal spot. (<b>E</b>–<b>H</b>) Another pupa. E and H are under white light. The red arrows in F and G indicate fluorescent signals at the peripheral area, which may correspond to PFE in H. (<b>I</b>–<b>L</b>) Yet another pupa. Arrows in I indicate pupal cuticle focal spots, which correspond to the foci of eyespots shown in (<b>K</b>,<b>L</b>).</p>
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<p>Confocal optical sections and three-dimensional reconstructions of the pupal hindwings stained with MitoRed for mitochondria (red) and BODIPY FL C<sub>5</sub>-ceramide for membranous structures (green) together with FB28 (blue). White arrows indicate the pupal cuticle focal spot. Blue arrows indicate FB28-positive cells below or between the epithelial cells. (<b>A</b>) An optical horizontal section at the surface area of the hindwing. The light blue-green area on the right is a pupal cuticle focal spot embedded within the procuticle layer. A black cross at the center of the spot indicates positions of cross-sectioning lines for B and C. Red signals on the left are mitochondria below the cuticle due to a tilt of the sample. A white cross at the center of this panel indicates the positions of the cross-sectioning lines for D and E. At the position of the white cross, another spot is located (out of focus). (<b>B</b>,<b>C</b>) Cross sections of A that transverse the spot. (<b>D,E</b>) Cross sections of A that transverse another spot (not visible in A). (<b>F</b>) A three-dimensional reconstruction image of A.</p>
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<p>FB28 fluorescent signals from the post-pupation pupal and adult cuticle structures. (<b>A</b>) A pupal case of sagittal cut under white light. (<b>B</b>) The same specimen of A under ultraviolet light. Blue fluorescent signals are detected from many parts, but arrows indicate representative ones. (<b>C</b>). A pupal case with the forewing-lift operation. An arrow indicates the FB28 fluorescent signal from the thin cuticle covering the dorsal surface of the hindwing. (<b>D</b>) A dissected pupal case. The inner side of the cuticle covering the dorsal forewing is shown at the bottom left side of this panel, showing blue fluorescence. (<b>E</b>) Leg joint showing blue fluorescence. (<b>F</b>) Thorax muscle after removal of the exoskeleton, showing strong blue fluorescence. (<b>G</b>) Dorsal side of an adult hindwing. Cover scales at the anterior part were removed. Arrows indicate wing joint and wing base showing blue signals. (<b>H</b>) Ventral side of an adult hindwing. Arrows indicate fluorescent signals from the wing basal membrane due to scale removal. (<b>I</b>) Ventral side of an adult hindwing. Fluorescent signals were detected from the wing basal membrane. Cover and ground scales at the anterior part were removed.</p>
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<p>Color pattern modifications induced by covering materials. Only the right dorsal hindwing was treated with a covering material (<span class="html-italic">t</span>), and the left hindwing was not treated (<span class="html-italic">nt</span>). The top panels show the entire dorsal hindwings, and the bottom panels show magnification of the posterior eyespots on the dorsal hindwings. Eyespot (ES), parafocal element (PFE), and submarginal band (SMB) are indicated by red arrows. (<b>A</b>,<b>B</b>) Individuals treated with plastic film. (<b>C</b>,<b>D</b>) Individuals treated with glass plate. (<b>E</b>–<b>H</b>) Individuals treated with silicone glassine paper.</p>
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<p>Double treatments with a covering material and the injection of FB28. Only the right dorsal hindwing was treated with a covering material. The top panels show the dorsal view of the treated individuals (the ventral view in C), and the bottom panels show magnification of the posterior eyespots on the hindwings. Eyespot (ES), parafocal element (PFE), and submarginal band (SMB) are indicated by red arrows. (<b>A</b>–<b>C</b>) Individuals treated with plastic film and FB28. (<b>D</b>) Individuals treated with glass plate and FB28. (<b>E,F</b>) Individuals treated with silicone glassine paper and FB28.</p>
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<p>Double treatments with a covering material and dextran sulfate. Only the right dorsal hindwing was treated with a covering material. The top panels show the dorsal view of treated individuals, and the bottom panels show magnification of the posterior eyespots on the dorsal hindwings. Eyespot (ES), parafocal element (PFE), and submarginal band (SMB) are indicated by red arrows. (<b>A</b>,<b>B</b>) Individuals treated with plastic film and dextran sulfate. (<b>C</b>,<b>D</b>) Individuals treated with glass plate and dextran sulfate. (<b>E</b>,<b>F</b>) Individuals treated with silicone glassine paper and dextran sulfate.</p>
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15 pages, 2121 KiB  
Article
Developmental Plasticity in Butterfly Eyespot Mutants: Variation in Thermal Reaction Norms across Genotypes and Pigmentation Traits
by Ana Rita Amaro Mateus and Patrícia Beldade
Insects 2022, 13(11), 1000; https://doi.org/10.3390/insects13111000 - 31 Oct 2022
Cited by 3 | Viewed by 2259
Abstract
Developmental plasticity refers to the property by which a genotype corresponds to distinct phenotypes depending on the environmental conditions experienced during development. This dependence of phenotype expression on environment is graphically represented by reaction norms, which can differ between traits and between genotypes. [...] Read more.
Developmental plasticity refers to the property by which a genotype corresponds to distinct phenotypes depending on the environmental conditions experienced during development. This dependence of phenotype expression on environment is graphically represented by reaction norms, which can differ between traits and between genotypes. Even though genetic variation for reaction norms provides the basis for the evolution of plasticity, we know little about the genes that contribute to that variation. This includes understanding to what extent those are the same genes that contribute to inter-individual variation in a fixed environment. Here, we quantified thermal plasticity in butterfly lines that differ in pigmentation phenotype to test the hypothesis that alleles affecting pigmentation also affect plasticity therein. We characterized thermal reaction norms for eyespot color rings of distinct Bicyclus anynana genetic backgrounds, corresponding to allelic variants affecting eyespot size and color composition. Our results reveal genetic variation for the slope and curvature of reaction norms, with differences between eyespots and between eyespot color rings, as well as between sexes. Our report of prevalent temperature-dependent and compartment-specific allelic effects underscores the complexity of genotype-by-environment interactions and their consequence for the evolution of developmental plasticity. Full article
(This article belongs to the Special Issue Contributions of Women in Insect Science)
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Figure 1
<p>Wing traits measured in adult butterflies from four genotypes. (<b>a</b>) The photos represent the typical phenotype of ventral wing surfaces of <span class="html-italic">Bicyclus anynana</span> females from four genetic stocks (WT, Fr, BE, and Choc) reared at 19 °C (top panel) or 27 °C (bottom panel). For each individual, we obtained measurements of the areas of the black and golden rings of two eyespots on the forewing (eA and eP, for the anterior and posterior eyespots, respectively) and two on the hindwing (e2 and e5, for the second and fifth eyespots, respectively), as well as forewing and hindwing areas. (<b>b</b>) The diagram on top displays the symbols used to represent the different traits we measured. For each of the two eyespots measured on each wing, the more anterior is represented by a circle on the top of the wing, and the more posterior by a circle on the bottom of the wing. The color of the circles at the center of each icon corresponds to either the black or golden rings.</p>
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<p>Variation in eyespot rings in relation to developmental temperature and genotype. Areas of the black and golden rings (different colors) of each of the four target eyespots (symbols to the left; see <a href="#insects-13-01000-f001" class="html-fig">Figure 1</a>) in male (left) and female (right) butterflies from each of the four target genotypes (names to the right) developed at different temperatures (19, 23, or 27 °C). Values correspond to the mean and standard deviation of eyespot area relative to the area of the corresponding wing. We tested for the effect of temperature (T) and genotype (G) on ring area, using wing area as a covariate (see <a href="#sec2-insects-13-01000" class="html-sec">Section 2</a>). Statistical significance of effects of G, T, and GxT for each ring area (different colors) is represented with <sup>ns</sup> for non-significant (<span class="html-italic">p</span> &gt; 0.05), * for <span class="html-italic">p</span> &lt; 0.05, ** for <span class="html-italic">p</span> &lt; 0.01, and *** for <span class="html-italic">p</span> &lt; 0.001. Raw data in <a href="#app1-insects-13-01000" class="html-app">supplementary File S1</a> and details of the statistical analysis in <a href="#app1-insects-13-01000" class="html-app">supplementary File S2</a>.</p>
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<p>Principal component analysis (PCA) of eyespot ring area for different temperatures and genotypes. (<b>a</b>) PCA loadings for each of the eight pigmentation traits (two color rings for each of four target eyespots; symbols on the left), reflecting their contribution to defining the first four principal components identified (termed Dim 1–4; with eigenvalues and % variation explained). The analysis was performed separately for the female and male datasets. (<b>b</b>) Mean values along Dim 1 and Dim 2 (with bars representing standard deviation) as a function of developmental temperature (19, 23, or 27°C) for each of the four genotypes (WT, Fr, BE, and Choc in different line styles; legend in bottom right corner) in females (red) and males (blue). We tested for the effect of temperature (T) and genotype (G) on each Dim (see <a href="#sec2-insects-13-01000" class="html-sec">Section 2</a>). Statistical significance of effects of G, T, and GxT for each Dim is represented with <sup>ns</sup> for non-significant (<span class="html-italic">p</span> &gt; 0.05), * for <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. Further details in <a href="#app1-insects-13-01000" class="html-app">supplementary File S3</a>.</p>
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<p>Thermal reaction norms for eyespot ring area for butterflies differing at specific pigmentation loci. Means and standard deviations for eyespot ring areas (symbols to the right) relative to the corresponding wing areas as a function of developmental temperatures in females from different genetic lines (<b>a</b>), and for allelic variants for the BE (<b>b</b>) and Fr (<b>c</b>) lines. For BE and Fr, we compare sibling “mutant” (heterozygous at the respective locus) and “wild type” (homozygous for the wild-type allele, represented by WT and solid line in the figures). We tested for the effect of temperature (T) and genotype (G) on eyespot ring area using wing area as a covariate (see <a href="#sec2-insects-13-01000" class="html-sec">Section 2</a>). Statistical significance for GxT interactions displayed as: <sup>ns</sup> (non-significant) <span class="html-italic">p</span> &gt; 0.05, * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001. Reaction norms for males in <a href="#app1-insects-13-01000" class="html-app">File S4</a>, details of statistical analysis in <a href="#app1-insects-13-01000" class="html-app">Files S2 (for (<b>a</b>)) and S4 (for (<b>b</b>,<b>c</b>)), raw data in File S1</a>.</p>
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