Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode
Figures
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig1-v1.tif/full/617,/0/default.jpg)
Negative correlation between oxygen level and pine wood nematode (PWN) loading in tracheal system of M. alternatus.
(A) Schematic representation of experimental setup used for microelectrode (top right) measurements inside a major trachea tube of the thorax (bottom left) and the atrium cavity of the largest spiracle (bottom right). (B) Partial oxygen pressure in the tracheal tubes of vector beetles with and without PWN (N = 38 and 76 tracheal tubes in five adults, respectively). ***p<0.0001 (Mann–Whitney U test). The internal lines are medians, edges of the boxes are interquartile ranges, and whiskers are minimum and maximum ranges. (C) The relationship between oxygen partial pressure in atrium of vector beetle (N = 5, 17 and 14 adults for null, light, and heavy PWN loading) and the number of nematodes. Left panel: fitted curve is drawn in red line. The gray line, which shows 10,000 PWN, divides beetle samples into light or heavy PWN loading. The blue line shows the estimated loading number of nematodes when partial pressure drops to 4 kPa. Right panel: partial pressure of oxygen in atrium of beetles in three different degrees of PWN loading. ***p=0.0001, ****p<0.0001 (Kruskal–Wallis nonparametric test, Dunn’s multiple comparison test). The internal lines are medians, edges of the boxes are interquartile ranges, and whiskers are minimum and maximum ranges.
-
Figure 1—source data 1
Raw data for partial pressure of oxygen and corresponding PWN number.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig1-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig2-v1.tif/full/617,/0/default.jpg)
Effects of pine wood nematode (PWN) loading and hypoxia on mechanical properties and apical extracellular matrix (aECM) of the trachea tubes of M. alternatus.
(A) Calculated Young’s modulus in different experimental groups according to values in tensile test in the longitudinal direction on the longest tubes linking two homolateral spiracles. Groups represent tubes from beetles without PWN (Null), light (<10,000 nematodes), heavy (>10,000 nematodes) PWN loading, and treated with 1% O2 for 6 hr, 12 hr, and 24 hr, respectively (N = 22–41 tracheal tubes for each treatment). **p<0.01, ***p<0.001, and ****p<0.0001 (Kruskal–Wallis nonparametric test, Dunn’s multiple comparison test). Internal lines are medians, edges of the boxes are interquartile ranges, and whiskers are minimum and maximum ranges. (B, C) Transmission electron micrographs (TEMs) for axial views of the longest tracheal tubes. (B) Representative images were shown for global view of tracheal tubes without (null) and with heavy PWN loading. Bars, 20 μm. Experiments were performed at least three times. (C) Electron microscopy images showed the thickened tracheal aECM (red arrow) between the chitin layer (taenidial ridges) and the body of tracheal cell (blue arrow) after different treatments (without PWN, i.e., null, heavy PWN, and 1% O2 for 6 hr, 12 hr, and 24 hr, respectively). Bars, 1 μm. The right bottom panel shows the statistical data of the thickness of aECM corresponding to TEMs. Data were measured in six tracheal tubes of three adults for each treatment. ***p<0.001 and ****p<0.0001 (Kruskal–Wallis nonparametric test, Dunn’s multiple comparison test). Data are represented as mean ± SEM.
-
Figure 2—source data 1
Raw data for Young’s modulus and aECM thinkness in different experimental groups.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig2-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig2-figsupp1-v1.tif/full/617,/0/default.jpg)
Morphological changes of trachea of M. alternatus after pine wood nematode (PWN) loading.
(A) Morphology of thoracic tracheal tubes without and with heavy PWN under stereoscopic microscope. Images in the upper (scale bar = 500 μm) or lower (scale bar = 50 μm) panel show trachea in vivo and in PBS solution, respectively. White arrow pinpoints PWN inside trachea tubes. Experiments were performed at least three times. (B) The relationship between total number of PWN per M. alternatus and degree of rubberization. According to the proportion of rubber-like tubes in all thoracic tracheal tubes, we sorted the trachea of beetles with PWN into three rubberization degrees (Ⅰ, none; Ⅱ, less than 50% rubber-like tubes; Ⅲ, 50–100% rubber-like tubes), and counted the number of PWN released from trachea. 39 adults, NⅠ = 14, NⅡ = 8, NⅢ = 17. Labels with different letters are significantly different at p<0.0001 (Kruskal–Wallis test, followed by Dunn’s multiple comparison test).
-
Figure 2—figure supplement 1—source data 1
Raw data for the relationship between PWN number and rubberization degree.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig2-figsupp1-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig2-figsupp2-v1.tif/full/617,/0/default.jpg)
Pulling test of trachea of M. alternatus after pine wood nematode (PWN) loading.
Snapshots from videos imaged under stereotype microscope showed the pulling result of thoracic tracheal tubes with or without PWN. Red circles show one representative tubes in two groups before and after pulling.
Pulling test of trachea of M. alternatus without pine wood nematode (PWN).
Pulling test of trachea of M. alternatus with pine wood nematode (PWN).
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig3-v1.tif/full/617,/0/default.jpg)
Transcriptome analysis showed changed expression pattern of apical extracellular matrix (aECMs)-related genes in trachea treat with pine wood nematode (PWN).
(A) Volcano plot of RNA-seq data from trachea of beetles with and without PWN. Gray dots indicate all differentially expressed genes (DEGs), and blue dots indicate aECM-related DEGs. The red dots indicate the three mucins. (B) Heat map of differential expressed genes related to mechanical property of tracheal tubes of beetles with and without PWN. The red font indicates the three mucins.
-
Figure 3—source data 1
Raw data for FPKM values of 45 selected aECM genes in trachea with and without PWN.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig3-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig3-figsupp1-v1.tif/full/617,/0/default.jpg)
Heat map of differential expressed genes related to mechanical property of tracheal tubes of beetles after 1 day, 3 days, 5 days, and 7 days post eclosion (PAE 1, PAE 3, PAE 5 and PAE 7, respectively).
-
Figure 3—figure supplement 1—source data 1
Raw data for FPKM values of 45 selected aECM genes during tracheal maturation post adult eclosion.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig3-figsupp1-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig4-v1.tif/full/617,/0/default.jpg)
Heavy pine wood nematode (PWN) loading and hypoxia enhance Muc91C expression at the mRNA and protein level.
(A) Relative RNA abundance of three genes belonging to mucin family in trachea of beetles with null, light, (<10,000 nematodes) or heavy (>10,000 nematodes) PWN loading. Data are represented as mean ± SEM. Columns labeled with different letters indicate statistically significant differences in mean relative abundance (N = 7–8 for each gene, one-way ANOVA with Tukey’s multiple comparisons, p=0.0003, 0.1992, and 0.1353 for Muc91C, Muc5ACl, and Muc3A, respectively). (B) Relative RNA abundance of three genes belonging to mucin family in trachea of beetles under normoxia or 1% O2 for 6 hr, 12 hr, or 24 hr. Data are represented as mean ± SEM. Columns labeled with different letters indicate statistically significant differences in mean relative abundance (N = 5 for each gene, one-way ANOVA with Tukey’s multiple comparisons, p=0.0009, 0.9389, and 0.3662 for Muc91C, Muc5ACl, and Muc3A, respectively). (C) Relative RNA abundance of Muc91C in tracheal tubes, flight muscle, and midgut of beetles with null or with heavy PWN loading. Data are represented as mean ± SEM. *p<0.05, ***p<0.001 (N = 4, Mann–Whitney nonparametric test). Columns labeled with different letters indicate statistically significant differences in mean relative abundance (N = 4, one-way ANOVA with Tukey’s multiple comparisons, p<0.0001 for beetles with or without PWN). (D) The protein level of Muc91C in tracheal tube with null, light, and heavy PWN. Histone H3 is used as internal control. Three biological replicates of each treatment are shown.
-
Figure 4—source data 1
Raw data for mRNA level of Muc91C in different experimental groups and tissues.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig4-data1-v1.xlsx
-
Figure 4—source data 2
Raw data for protein level of Muc91C in tracheal tubes with PWN.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig4-data2-v1.zip
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig5-v1.tif/full/617,/0/default.jpg)
The localization of Muc91C at the apical extracellular matrix (aECM) layer in tracheal tubes.
Immunostaining images of tracheal tube with null (left panels) and heavy pine wood nematode (PWN) (right panels) loading. Boxed regions (pink) in the top-left fluorescence images (bars, 50 μm) are shown at high magnification at below panels (bars, 20 μm), illustrating the localization of Muc91C (green), taenidial folds (red in A), tracheal cell body (red in B), and nuclei (blue). Bright-field images and merged bright-field and fluorescent images are shown at the right of each panel. The red arrowheads in bright-field image show residual PWN in the tracheal tube. Experiments were performed at least three times.
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig6-v1.tif/full/617,/0/default.jpg)
Phylogenetic relationship among resilin-related proteins in insects.
The phylogenetic analysis was performed using the full-length amino acid sequences of representative resilin-related proteins. Am, Apis melifera; Mp, Myzus persicae; Nv, Nasonia vitripennis; Aga, Anopheles gambiae; Dm, Drosophila melanogaster; Ma, Monochamus alternatus; Agl, Anoplophora glabripennis; TC, Tribolium castaneum. The blue and yellow branches represent Muc91C (Clade I) and Pro-resilin (Clade II), respectively. The domain architectures are listed at the middle panel. Brown boxes indicate signal peptide. Blue boxes indicate repeats A. Yellow boxes indicate repeats B. Orange boxes indicate RR2. The consensus sequences of repeats A are listed at the right panel. MaMuc91C is highlighted in pink.
-
Figure 6—source data 1
Raw data for sequences for MaMucins.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig6-data1-v1.docx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig6-figsupp1-v1.tif/full/617,/0/default.jpg)
A phylogenetic tree constructed using M. alternatus mucin sequences (orange dots) and other species' mucin sequences.
Muc91C proteins were highlighted in red font. The full-length amino acid sequences of representative proteins were retrieved from the GenBank database. The phylogenetic tree was constructed with the MEGA 10.0 software based on a multiple alignment of the amino acid sequences performed by ClustalW, and adopting the NJ method. A bootstrap analysis of 1000 replications were used, and 50% cut-off bootstrap values were applied to condense the tree. The sequence numbers of mucin or mucin-like proteins are listed in Supplementary file 1.
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig7-v1.tif/full/617,/0/default.jpg)
The role of Muc91C in tracheal elasticity and pine wood nematode (PWN) loading.
(A) Electron microscopy images showed the thickened tracheal apical extracellular matrix (aECM) (red arrow) under 1% O2 for 12 hr after dsGFP or dsMuc91C injection. Bars, 1 μm. The right panel shows the statistical data of the thickness of aECM corresponding to TEMs. Data were measured in six tracheal tubes of three adults for each treatment. ***p<0.001 (Mann–Whitney nonparametric test). Data are represented as mean ± SEM. (B) Young’s modulus of trachea treated 1% O2 for 12 hr after dsGFP or dsMuc91C injection (N = 10 tracheal tubes for each treatment). *p<0.05 (Mann–Whitney nonparametric test). The internal lines are medians, edges of the boxes are interquartile ranges, and whiskers are ranges. (C) Number of residual PWNs released from every 0.3 g beetle incubated with PWN (N = 6 and 7 adults, respectively). dsGFP or dsMuc91c is injected before incubation. **p<0.01 (Student’s t-test, unpaired and two-tailed). Data are represented as mean ± SEM.
-
Figure 7—source data 1
Raw data for aECM thickness, Young’s Module values and PWN numbers of dsMuc91C-treated beetles.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig7-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig7-figsupp1-v1.tif/full/617,/0/default.jpg)
RNAi efficiency of the Muc91C gene and survival rates in dsRNA-treated adult beetles.
(A). Relative abundance of Muc91C in GFPi and Muc91Ci beetles. Data are presented as mean ± SEM. N = 5. p-Value is determined by Mann–Whitney U test. (B). Accumulated survival rate of adults in RNAi efficiency assessment, TEM imaging, and tensile testing. Adults were injected with dsRNA and treated with 1% O2 for 12 hr. (C). Survival rate of adults in pine wood nematode (PWN) loading assay. Adults were injected with dsRNA and reared in flasks with barley medium containing PWN. N = 10. Difference of proportions was analyzed by chi-square test.
-
Figure 7—figure supplement 1—source data 1
Raw data for mRNA level of Muc91C and survival rate of dsMuc91C-injected beetle.
- https://cdn.elifesciences.org/articles/84621/elife-84621-fig7-figsupp1-data1-v1.xlsx
![](https://iiif.elifesciences.org/lax:84621%2Felife-84621-fig8-v1.tif/full/617,/0/default.jpg)
Schematic diagram of the feedback regulation of pine wood nematode (PWN) loading through O2 and CO2 level in the tracheal system of M. alternatus.
When LIV dispersal nematodes continuously enter the vector beetle through spiracles, the consequent hypoxia enhances the elasticity of tracheal tubes by upregulating Muc91C. The more elastic tube structure, in turn, allows more nematodes to reside in the lumen. When tracheal tubes reach to their maximum elasticity, nematodes influence gas exchange of beetles and the resultant high level of CO2 drives nematodes away from the trachea.
Additional files
-
Supplementary file 1
Species and GenBank accession no. of pro-resilins and mucins.
- https://cdn.elifesciences.org/articles/84621/elife-84621-supp1-v1.docx
-
Supplementary file 2
Primers for real-time qPCR and RNAi experiments.
- https://cdn.elifesciences.org/articles/84621/elife-84621-supp2-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/84621/elife-84621-mdarchecklist1-v1.docx