Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode

  1. Xuan Tang
  2. Jiao Zhou
  3. Tuuli-Marjaana Koski
  4. Shiyao Liu
  5. Lilin Zhao  Is a corresponding author
  6. Jianghua Sun  Is a corresponding author
  1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. College of Life Science/Hebei Basic Science Center for Biotic Interactions, Institute of Life Science and Green Development, Hebei University, China

Abstract

Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees, is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes while a notable upregulated expression of a resilin-like mucin protein Muc91C was observed at the aECM layer of PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.

Editor's evaluation

This valuable work explores how pathogens can cause hypoxia in insect vectors and how responses to hypoxia can be exploited to promote vector competence. Using Pine Wood Nematode (PWN) infection of pine sawyer beetles the authors demonstrate that PWN loading activates hypoxia in the vector's tracheal system. The data were collected and analyzed using solid and validated methodology.

https://doi.org/10.7554/eLife.84621.sa0

eLife digest

Various parasites, bacteria and other disease-causing pathogens are transmitted by insects. A tiny worm called the pine wood nematode, for example, is spread by pine sawyer beetles which can carry up to 280,000 worms in their trachea, the network of tubes they use to breathe. This has resulted in millions of hectares of pine forests in Asia and Europe becoming infected with the deadly disease caused by the nematodes.

Pine wood nematodes, as well as other pathogens, can exploit the biological processes of the insects carrying them to make the insects transmit them more effectively. Precisely how nematodes and other disease-causing agents do this is unclear. One possibility is that they reduce the amount of oxygen being supplied to the trachea – a phenomenon known as hypoxia – which occurs naturally at specific stages in the life of an insect, and during infections.

To test this theory, Tang, Zhou, Koski et al. used genetics and imaging approaches to study how pine wood nematodes affect the trachea of pine sawyer beetles. The experiments found that when the nematodes infected the beetles, their trachea did indeed develop hypoxia. This, in turn, made the beetles’ airways more elastic and made the layer of structure lining the trachea, known as the apical extracellular matrix, thicker. These changes increased the amount of pinewood nematodes the trachea could hold, allowing the beetle to spread more worms from tree to tree.

Further experiments revealed that hypoxia in the trachea increased the levels of a protein called Muc91C in the apical extracellular matrix. When the levels of Muc91C were artificially decreased in the beetles, this made their airways less elastic and the apical extracellular matrix thinner.

This work suggests that pine wood nematodes exploit the beetles’ normal responses to loss of oxygen supply to make the beetles more effective at transmitting the nematodes between pine trees. Other pathogens carried by insects may also use this strategy to help increase their transmission. Further studies on the Muc91C protein may provide clues for potential drug targets to control pine wood nematodes and protect pine trees from disease.

Introduction

Insect vectors are obligatory for the dispersal of many pathogens, including several viruses and parasites deleterious to plant and animal health. Vector competence is pivotal for the successful pathogen arrival and infection of the target host, and is determined by a sophisticated set of direct interactions as pathogens enter, spread, replicate, and retain within vector cells and tissues (Bartholomay and Michel, 2018; Gray et al., 2019; Lataillade et al., 2020). Therefore, pathogens have evolved to modulate various defensive responses of vectors, such as autophagy or apoptosis, as has been shown in some leafhoppers, whitefly, and mosquitoes, to influence vector competence (Chen et al., 2017; O’Neill et al., 2015; Wang et al., 2020). Although understanding the responses that pathogens induce to manipulate vector competence is important for developing efficient management strategies, the underlying mechanism remains largely elusive.

A pathogen of pine trees, Bursaphelenchus xylophilus, commonly known as pine wood nematode (PWN), is transmitted by cerambycid beetles of the genus Monochamus and causes pine wilt disease in millions of hectares of pine forests across Asia and Europe (Akbulut and Stamps, 2012). PWN’s life cycle has two distinct developmental phases, namely the propagative stage (L1-L4) juveniles and reproductive adult that propagates inside pine trees and the dispersal stage (LIII and LIV juveniles) characterized by its close relationship with the vector beetle that transports the nematode to new host trees (Zhao et al., 2008; Zhao et al., 2014). The dispersal LIV enters the tracheal system of the adult beetle through the spiracles and inhabits the trachea for several days until the vector beetle has reached a new healthy host tree. The number of dispersal LIV carried by an adult beetle is a critical factor influencing the damage level of the pine wilt disease in the pine host tree (Togashi, 1985). Extraordinarily, each primary vector beetle, such as Monochamus alternatus in Asia, can harbor on average 15,000 and up to 280,000 nematodes in its tracheal system (Fielding and Evans, 1996). In contrast to circulative pathogens that move from gut lumen to salivary glands or ovary (Eigenbrode et al., 2018; Wei et al., 2017), and to other non-circulative pathogens that typically remain in the mouthparts (Uzest et al., 2007) and foregut of their vectors (Chen et al., 2011), PWNs merely reside and do not multiply in the tracheal systems. However, the effects of PWN on its unique resident niche (i.e., the vector tracheal system), and the mechanism behind the vector’s competence for loading of such large numbers of PWN are still poorly understood.

Hypoxia, which occurs when oxygen supply is inadequate, is common in mammal host tissues faced with infection by pathogens and induces several adaptive responses that improve host defense by maintaining the integrity of epithelial barriers and hindering the internalization process (Zeitouni et al., 2016). Recent studies suggest a modulator role of hypoxia in various biotic interactions. For example, the parasite Trypanasoma brucei has evolved a way to inhibit hypoxic responses to evade the host immune response in the bloodstream (McGettrick et al., 2016). In contrast, a mosquito gut bacterium benefits their hosts by activating hypoxic responses improving host gut growth (Coon et al., 2017; Valzania et al., 2018). As the trachea of the adult vector beetle is packed with nematodes, reduced gas transfer ability and its consequent hypoxia are conceivable.

The insect trachea, an interconnected network of air-filled epithelial tubes characterized with optimal diameter and length, exhibits intense cycles of compression and expansion for respiration, which resembles the deflation and inflation of vertebrate lungs (Westneat et al., 2003). The tracheal volumes changed by this behavior increase the internal pressure for improvement of air convection, thus facilitating gas exchange and diffusion (Hayashi and Kondo, 2018; Westneat et al., 2003). To increase oxygen supply, the tracheal system adapts to hypoxia by compensatory morphological and physiological changes (Harrison et al., 2018), such as extensively tracheal terminal branching (Centanin et al., 2010) and increased compression frequency of tracheal tubes (Greenlee et al., 2013). Mechanical properties of the tracheal tubes are therefore prerequisites for regulating the tracheal geometry and compression capacity and mainly depend on a viscoelastic material surrounding tracheal lumen, called apical extracellular matrix (aECM) (Dong et al., 2014; Hayashi and Kondo, 2018; Zuo et al., 2013). In Drosophila melanogaster, aECMs’ components, including serpentine, verm, and dumpy, affect aECM elasticity and have been reported to mediate the tubular diameter or length, possibly by restricting apical membrane growth (Luschnig et al., 2006; Wilkin et al., 2000). A study with the American cockroach (Periplaneta americana) suggested that chitin structures in aECM layer are responsible for the mechanical features that enable tracheal compression and volume change during respiration (Webster et al., 2011). Similarly, high inflation of lungs is related to the mechanical forces provided by aECM components (Berg et al., 1997; Wirtz and Dobbs, 2000). In addition, hypoxia has been shown to correlate with overall aECM composition and organization of blood vessels and respiratory networks (Jang et al., 2021; Kusuma et al., 2012), implying a possible link between hypoxia and the molecular components contributing to mechanical properties of the tracheal tubes. Taken together, we hypothesize that hypoxia may be involved in the retention of PWN in vector beetle’s tracheal system through the PWN exploiting mechanical properties of the vector beetle’s trachea to maintain a high vector capacity in a hypoxia-regulated manner.

Here, we revealed that the number of the nematodes in the trachea is negatively correlated with the oxygen level in the tracheal system of its vector beetle, leading to hypoxia. Both PWN loading and hypoxia enhanced the elasticity of tracheal tubes and increased the thickness of aECMs in tracheal epithelia. Based on transcriptome analysis of trachea with and without PWN, we identified aECM-related genes in the beetles and found a candidate aECM component, a resilin-like mucin protein Muc91C, the expression of which was substantially upregulated at the aECM layer after PWN loading and hypoxic treatment. Muc91C knockdown reduced elasticity of the tracheal tubes and aECMs thickness under hypoxic condition, indicating that Muc91C help regulate the aECM-related mechanical traits of the tracheal tubes. Importantly, RNAi of Muc91C in adult beetles significantly reduced PWN loading in trachea. These results demonstrate crucial roles of tracheal hypoxia and aECM-related tubular elasticity in improving vector competence for PWN transmission.

Results

PWN loading decreased oxygen levels in the tracheal system of vector beetle

To determine whether PWN loading reduces oxygen levels and caused hypoxia in trachea of vector beetles, we first measured the concentration of oxygen inside major thoracic tracheal tubes using an oxygen microsensor (Figure 1A). Radial oxygen profiles revealed a slight drop of oxygen pressure from 21 kPa to 18.9 kPa, respectively, when comparing pressure outside (i.e., in the ambient air) and inside the tracheal tubes of beetles without PWN. In contrast, there was a dramatic drop of oxygen pressure from 21 kPa outside of tubes to 3.8 kPa inside tubes when trachea was loaded with PWN (Figure 1B). Thus, our results showed that PWN loading induce oxygen shortage in trachea.

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

We further established the correlation between oxygen level and total number of nematodes in the trachea by measuring the oxygen concentration in the atrium cavity of the first and largest abdominal spiracles and calculating the number of nematodes in the beetle (Figure 1A). The partial pressure of oxygen was negatively correlated with the total number of nematodes in tracheal tissue per 0.3 g beetle (Figure 1C). The curve fit to this data revealed that when the total number of nematodes reached 10,000, partial pressure of oxygen in the cavity of tracheal spiracle dropped to 10 kPa (half of the corresponding value in the surrounding air usually 21 kPa), creating a mild hypoxia at approximately PO2 of 5–15 kPa. In addition, when the number of nematodes increased to 44,828, the oxygen pressure dropped dramatically to 4 kPa (Figure 1C), which is the threshold between mild and severe functional hypoxia in Drosophila (Harrison et al., 2018). Oxygen levels in the cavity of the atrium were further compared among three levels of PWN loading, differing in total number of nematodes in the trachea. The tracheal systems of beetles without PWN (null) or with light PWN loading (<10,000) had an oxygen pressure equal to that of the environment. By contrast, substantial decline to approximately 4 kPa of oxygen were detected in the tracheal spiracle with heavy PWN loading (>10,000). These results indicated PWN loading caused oxygen loss and light or heavy PWN loading induced hypoxia or anoxia in the trachea of vector beetles.

Nematode-induced hypoxia enhanced tracheal elasticity and thickened its apical extracellular matrix

Compared to those free of PWN, the trachea of vector beetles bearing PWN tended to be rubberlike (Figure 2—figure supplement 1A). The total number of the nematodes in the trachea correlated positively with the tracheal rubberization degree (Figure 2—figure supplement 1B). Trachea with the highest rubberization degree released five times more nematodes (reaching approximately 16,000 nematodes on average) compared to trachea with moderate or low rubberization. Preliminary observations further showed that the trachea tubes harboring PWN tended to be longer and narrower before breakage under external force (Figure 2—figure supplement 2, Figure 2—video 1, and Figure 2—video 2). This relationship indicated that PWN loading influenced the mechanical properties of the vector’s trachea.

The elasticity of trachea tubes was further quantified by comparing Young’s modulus values of tracheal tubes among the three levels of PWN loading (null, light, and heavy) mentioned above. Corresponding to their higher oxygen level, Young’s modulus values of trachea tubes without PWN and with light PWN loading were almost two times higher than the values in hypoxic trachea tubes with heavy PWN loading (Figure 2A). Given the negative correlation between elasticity and Young’s modulus values, this result suggested that heavy PWN loading enhanced tracheal elasticity. To confirm the influence of hypoxia on tracheal elasticity, we subjected beetles free of PWN to 1% O2 for 6 hr, 12 hr, and 24 hr. Compared with that of tracheal tubes exposed to normoxia and hypoxia conditions for 6 hr, the Young’s modulus values under hypoxic treatments for 12 hr and 24 hr decreased by nearly a half (Figure 2A). These results clearly demonstrated that heavy PWN loading and its induced hypoxia enhanced tracheal elasticity.

Figure 2 with 4 supplements see all
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

Due to the correlation between mechanical properties and aECM, we further investigated the tracheal structure with null and heavy PWN loading using transmission electron microscopy. Micrographs revealed that the secreted protein layer of tracheal aECM was significantly thickened by heavy PWN loading (Figure 2B and C). However, the heavy PWN loading had no effect on the morphology of the chitin layer of aECM. To examine the effect of hypoxia on tracheal aECM, we treated beetles free of PWN with 1% O2 for 6 hr, 12 hr, and 24 hr. Electron micrographs showed that the secreted protein layer of tracheal aECM under hypoxia for 12 hr and 24 hr was thicker than those under normoxia and hypoxia for 6 hr (Figure 2C). Therefore, our results indicated that PWN-induced hypoxia promoted the secretion of protein layers in aECM in trachea. Taken together, these results suggest that the increased secretion of protein layer of aECM was involved in enhanced tracheal elasticity by PWN-induced hypoxia.

PWN loading caused significant upregulation of Muc91C responsible for the thickened aECM layer in trachea

To confirm the role of proteins to the enhanced tracheal elasticity, we compared the tracheal transcriptomes between PWN-free and heavily PWN-loaded beetles and discovered 916 differentially expressed genes (DEGs). Out of these, 251 DEGs were identified as aECM-related genes (Figure 3A). Overall, the altered transcript profiles suggested dramatic epithelium reorganization in tracheal tubes of vector beetles after PWN loading. We then chose 45 genes related to mechanical properties to further scrutinize for their expression (Figure 3B). We examined chitinases, chitin synthases, and chitin deacetylases, which are involved in chitin catabolic processes, biosynthetic processes, and arrangement, respectively (Dong and Hayashi, 2015). The up- or downregulated pattern in the expression of these transcripts suggested a chitin metabolic equilibrium, which was consistent with the unchanged chitin layer among beetles with heavy PWN loading shown in TEMs (Figure 2C). Our gene list also included those genes functioning as aECM regulators. For example, metalloproteinases are proteinases that cleave proteins in the extracellular matrix (Glasheen et al., 2010). Cadherins, integrins, lachesin, and dumpy are transmembrane proteins participating in signal transduction between aECM and tracheal cells (Llimargas et al., 2004; Öztürk-Çolak et al., 2016; Wilkin et al., 2000). The changed expression of these transcripts after heavy PWN loading indicated altered regulatory cascades of aECM. We finally investigated the expression of the non-chitin structural components, including mucins, collagens, and laminin. Negatively regulated transcripts of collagens in heavy loading PWN samples and the location of laminin in basal lamina of tracheal cells (Dai et al., 2018) suggest they were not responsible for thickening the aECM layer in our observations (Figure 2C). Therefore, the genes encoding for the mucin family, a group of large glycosylated macromolecules, are possible candidate genes contributing to the elasticity of trachea in our study because their location and upregulation directly corresponded to the observed thickened non-chitin layer in aECMs visible in electron microscopy (Figure 2C). To investigate possible correlation between tracheal development at metamorphosis and after PWN loading, we further scrutinized the expression of the 45 aECM genes in tracheal tubes after 1 day, 3 days, 5 days, and 7 days post eclosion using RNA-seq data in a published study (Tang et al., 2022). All genes were downregulated between day 5 and day 7 post adult eclosion (Figure 3—figure supplement 1). Therefore, tracheal system of vector beetles responded to loading nematodes despite the completion of tracheal development. For Muc91C specifically, its expression was twofold upregulated 3 days after eclosion but was downregulated during the later stages post eclosion. However, PWN loading resulted in a sevenfold increase of Muc91C expression, a higher fold change than that during tracheal development.

Figure 3 with 1 supplement see all
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

Among the three mucin genes, Muc91C had the highest increase in expression after PWN loading, increasing more than sevenfolds (log2FC = 2.43), higher than the other two genes (Figure 3A). Next, we independently validated our RNA-seq results by repeating the experiment using new individuals and quantifying expression using reverse-transcription qPCR (RT-qPCR) for the genes that belong to the mucin family. In contrast to the other two mucin genes, the abundance of Muc91C transcripts positively correlated with the level of PWN loading in trachea (Figure 4A). Compared to that of trachea without PWN, the expression level of the Muc91C gene increased fivefold in trachea with heavy PWN loading. To test the effect of hypoxia on Muc91C, Muc5ACl, and Muc3A, gene expression levels were measured after exposure of beetles under 1% O2 for 6 hr, 12 hr, and 24 hr. Hypoxia treatment of 12 hr caused significant upregulation of Muc91C in trachea (Figure 4B). To exclude the influence of related tissues, we examined the tissue specificity of Muc91C in tracheal tubes, flight muscle, and midgut. Compared to fight muscle and midgut, Muc91C expression was mostly highly expressed in the trachea of adult beetles with or without PWN loading. Heavy PWN loading significantly increased Muc91C expression in all tested organs (Figure 4C), implying systematic hypoxia in beetles with heavy loading. We also further confirmed the protein levels of Muc91C in tracheal tubes with null, light, and heavy PWN loading by Western blot analysis. The PWN loading enhanced Muc91C in a nematode number-dependent manner (Figure 4D), similar with the tendency of oxygen loss in tubes with PWN (Figure 2C). These results indicated that heavy PWN loading in beetles’ trachea stimulates the expression of Muc91C via inducing hypoxia.

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

We further investigated the localization of Muc91C in tracheal tubes through immunohistochemistry analysis. Strong signals for Muc91C were detected beneath the layer of taenidial folds (Figure 5A) and above the apical membrane of the tracheal cell (Figure 5B) forming a continuously layer in tracheal tubes with heavy PWN, whereas slight fluorescence signal was observed in tracheal tubes without PWN. Thus, this result demonstrated that Muc91C is the component of the PWN-thickened aECM layer showed in TEM.

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.

Muc91C contributes to hypoxia-enhanced trachea elasticity and promote PWN loading

In order to determine whether increased Muc91C contribute to the tracheal elasticity, we investigate its gene structure and revealed that Muc91C encoded a resilin-like protein (Andersen, 2010), dominated by two long regions containing a series of short repeat motifs (Figure 6). Seventeen A repeats, with the consensus sequence PSSSYGAPA(S), were located in the N-terminal region. Additionally, 11 B repeats, with the consensus sequence GGYSSGGN, were located in the C-terminal region. As the only identified MaMuc91C homolog in insects, DmMuc91C was classified as a resilin-like protein (Andersen, 2010). In addition, a BLASTX search of MaMuc91C against Flybase revealed its similarity not only to DmMuc91C (CG7709-PB) but also to functional DmPro-resilin (CG15920-PA). Using DmPro-resilin and resilin-like DmMuc91C as query sequences in turn, we further characterized 17 resilin-related genes in the tracheal transcriptome of the PWN vector beetle and in the genomes of other insects. Phylogenetic analysis revealed that MaMuc91C and six proteins from other insects were assigned to clade I, which share repeat A motif with a consensus sequence of PS(Q)SSYGAP(A)S. Importantly, repeat A motifs in clade I shared two similar features with pro-resilin proteins. First, the glycine and proline residues included in the repeat A motifs in this clade potentially exhibit long-range elasticity by forming a stretchable beta-spiral structure (Ardell and Andersen, 2001; Tatham and Shewry, 2002). Secondly, tyrosine residues also included in repeat A motifs in clade I presumably facilitates the formation of dityrosine crosslinks with other repeats after secretion from the epidermal cells (Andersen, 2010; Qin et al., 2009). However, clade I lacked the chitin binding R&R Consensus sequence, a defining feature of true pro-resilin. Thus, these proteins were classified as resilin-like proteins. Based on the PTS domain and O-glycosylation sites (Zhao et al., 2020), we found that all the proteins in clade I (Figure 6) belonged to the mucin family and may be homologues of Muc91C, given their similarity with DmMuc91C. The other six resilin-related proteins were true pro-resilins and clustered into clade II, in which repeat A motifs were highly variable and all sequences contained the chitin binding R&R Consensus. No pro-resilin homologue was identified in tracheal transcriptome of M. alterantus. Regardless of the existence of R&R Consensus sequence, repeat A consensus in both pro-reslins and Muc91C likely confer long-range elasticity because previous studies have shown that synthetic peptide chains consisting of either 17 copies of A repeats (GGRPSDSYGAPGGGN) from DmPro-resilin or 16 copies of A repeats (AQTPSSQYGAP) from the AgaMuc91C (AGAP002367-PA), were able to form rubberlike elastic materials (Elvin et al., 2005; Lyons et al., 2007; Nairn et al., 2008). In addition, phylogenetic analysis based on M. alternatus and other insect species' mucin sequences showed that Muc91C proteins were clustered into a separate lineage (Figure 6—figure supplement 1). We therefore deduced that MaMuc91C encodes an insect-originated resilin-like protein and has a potential role in providing long-range elasticity.

Figure 6 with 1 supplement see all
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.

We further verified the key role of Muc91C in determining the thickness of aECM and its related elasticity under hypoxia condition. Firstly, we used TEM to observe the substructure of tracheal tubes treated with 1% O2 for 12 hr after performing gene knockdown of Muc91C in adult beetles. RNAi knockdown of Muc91C in adult beetles within 2 days after eclosion resulted in a 77.5% decrease in Muc91C mRNA levels and has no detrimental influence on adult survival (Figure 7—figure supplement 1A and B). Compared to dsGFP-injected beetles, the thickness of aECM layers in tracheal tubes were substantially reduced in dsMuc91C-injected beetles (Figure 7A). Next, we measured Young’s modulus values of trachea tubes treated with 1% O2 for 12 hr after dsRNA injection. Compared to the dsGFP control, the Young’s modulus values of tracheal tubes were significantly increased in the dsMuc91C samples, indicating their decreased elasticity after Muc91C knockdown (Figure 7B). Therefore, the enhanced tracheal elasticity induced by hypoxia relies on the expression of Muc91C.

Figure 7 with 1 supplement see all
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

To further confirm the role of Muc91C-induced elasticity in PWN loading, we carried out Muc91C knockdown in adult beetles and subsequently inoculated beetles with PWN. Injection of dsGFP and dsMuc91C resulted in similar proportions of death before dissection for PWN counting (Figure 7—figure supplement 1C). Compared to beetles injected with dsGFP, the number of nematodes in beetles injected with dsMuc91C was reduced by 50%, and the total number of nematodes in these beetles’ trachea reaching less than 10,000 (Figure 7C). Collectively, Muc91C was essential to promote heavy PWN loading by promoting tracheal elasticity during hypoxia.

Discussion

Vector competence is important for vector-borne pathogens’ transmission, but the underlying mechanism remains largely unexplored. Here, we demonstrate one of the strategies that PWN utilizes to maintain itself in its vector by modulating the vector beetle’s hypoxia responses. This strategy helps the nematode to reach high numbers even in a single vector beetle and may consequently promote the successful spread, establishment, and outbreak of pine wilt disease. Mechanically, we propose that PWN’s loading process in vector beetles follows a positive feedback loop (Figure 8). First, continuous entering of nematodes into trachea decreases oxygen levels, resulting in hypoxia, which in turn increases the elasticity of tracheal tubes by upregulating Muc91C. The more elastic tube structure, in turn, allows more nematodes to reside in the lumen until maximum elasticity is reached, resulting in decreased gas transfer due to physical blocking caused by nematodes. Finally, the resulting high level of CO2 drives nematodes away from the trachea (Wu et al., 2019). Thus, the nematodes strategically manage their loading and departure from the vector by manipulating oxygen supply in the vectors’ trachea.

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.

Hypoxia occurs universally across living organisms and plays multiple roles in hosts infected by pathogens. For instance, a protozoan parasite Leishmania donovani induces inflammatory hypoxia to limit the capacity of the host’s myeloid cells to kill the parasite (Hammami et al., 2017). Some commensal gut microbiota maintains epithelial hypoxia to limit the growth of enteric pathogens in the gut lumen of neonatal chicks (Litvak et al., 2019). However, hypoxia in pathogen–vector relationships has long been a neglected research topic. Our study shows that retention of nematodes caused hypoxia in tracheal tubes of vector beetles, providing empirical evidence for a previous hypothesis that the heavy PWN loading hinders the gas exchange of the beetle (Togashi and Sekizuka, 1982). Such hypoxia induced by PWN loading improved the vector’s tracheal capacity to harbor PWN. The correlation between hypoxia and vector competence can be extrapolated to the interaction between circulative pathogens and their vectors because their migration, replication, or propagation processes inside vectors may also increase oxygen demands and result in hypoxia.

Mechanical properties of tracheal tube are critical to luminal volume by regulating tubular size (Dong et al., 2014) and the dynamic volume changes during tracheal compression and expansion to increase the internal pressure for air convection and gases exchange, thus facilitating oxygen diffusion (Westneat et al., 2003). Our results show that after heavy PWN loading or hypoxic treatment, the thickness of ECM in the tracheal tubes of the vector beetle is remarkably enhanced. Regardless of an intricate relationship between ECM thickness and tubular diameter, the thickened ECM layer results in more elastic tubes that qualify a robust compression. Such improvement in compression capacity via tubular elasticity might be response to acute hypoxia or PWN loading, different from the chronic adaption that involve tracheal diametric expansion in Drosophila larvae incubated under constant hypoxic condition for generations (Henry and Harrison, 2004). In the PWN-beetle system, heavy PWN loading occupies luminal volume needed by tracheal ventilation for the beetle’s regular respiration or intensive respiration during dispersal flight in the search of suitable host pine trees. The enhanced tracheal elasticity adapts vector insects to hypoxic stress allowing more oxygen inhalation and provides adequate lumen volume to carry large numbers of nematodes. Therefore, altered mechanical properties of the beetles’ trachea after PWN loading likely maintain a balance between a limited fitness cost to beetles and high vector competence for effective transmission of PWN.

Along with enhanced tracheal elasticity in the vector beetle, our study also found remarkable global expression changes of aECM genes after PWN loading regardless of the termination of tracheal differentiation and maturation within the first 5 days after eclosion. For example, metalloproteinases degrading old cuticle and promoting apical membrane expansion (Glasheen et al., 2010), cadherins and integrins participating in signal transduction between aECM and tracheal cells (Hayashi and Kondo, 2018; Öztürk-Çolak et al., 2016) are significantly upregulated after heavy PWN loading. This result suggests that PWN loading causes dramatic epithelium reorganization in tracheal tubes of vector beetles. Therefore, our study provides an example that the tracheal system is able to immediately respond to the environment by epithelium reorganization through changed aECM components. Although aECM is crucial to determine mechanical properties of tubular organs including trachea, the role of its molecular components remains largely unknown. Using gene manipulation, we further identified Muc91C, which encodes a resilin-like protein in aECM, as the key component responsible for the thickening of non-chitin aECM layer and the improved tracheal elasticity. However, due to incomplete reduced elasticity under hypoxic condition in dsMuc91C-injected beetles, other tracheal cellular components such as proteins embedded in the membrane may also regulate tracheal elasticity. Structurally, Muc91C in M. alternatus contains two types of repeat motifs but lacks chitin-binding RR2, a consensus sequence in resilin monomers (pro-resilins) (Michels et al., 2016), suggesting that its elasticity trait resides in the repeat motifs. Consistent with the absence of pro-resilin in the tracheal tubes of vector beetles, Drosophila larval foregut is devoid of pro-resilin, but contains resilin-like Cpr56F (Lerch et al., 2020). Collectively, these examples suggest that resilin-like proteins are employed instead of resilin monomers in deformation of various cuticle-free organs. In addition to the repeated motifs, water retention caused by the glycosylation of mucin (Wagner et al., 2018) may also promote elasticity, given that water coating may plasticize Mucin91C and provide additional elasticity from the surface tension of the liquid, as proved in spider silk (Vollrath and Edmonds, 1989). Interestingly, remarkably upregulated Muc91C expression was also detected in flight muscles and midgut of beetles with heavy PWN loading. Because highly glycosylated macromolecular mucins are known to protect cells against microbial infection (Martens et al., 2018), higher systematic Muc91C level in various tissues may contribute to maintaining the integrity of mucosal barriers to alleviate the detrimental effects of nematodes invasion, such as increased ROS reported in previous studies (Zhou et al., 2018).

This study found that hypoxia promotes tracheal elasticity by upregulating the expression of Muc91C, suggesting that Muc91C is related to hypoxia adaptation. Similarly, significant upregulated expression of pro-resilin has been demonstrated in an intertidal crustacean, the copepod Tigriopus californicus, when exposed to extremely low oxygen levels (Graham and Barreto, 2019). Therefore, the sensitivity of gene expression of aECM components in epithelial tissues to hypoxia is likely a pervasive phenomenon in arthropods. Clustering into a separate lineage in the mucin phylogenetic tree (Figure 6—figure supplement 1), all insect Muc91C proteins investigated in this study had no human homologues and were only distantly related to other mucins, suggesting their independent evolutionary origin in insects. This result is consistent with a previous study reporting that Muc91C is the only mucin structurally similar to pro-resilins among 23 mucins and mucin-related proteins in D. melanogaster (Syed et al., 2008). In Asian longhorned beetles (Anoplophora glabripennis), for instance, AgMuc91C has a 84% sequence similarity with MaMuc91C (Figure 6). Therefore, further examination of Muc91C expression patterns and its responses to hypoxia in other pine sawyer beetles closely related to our study species, such as Monochamus saltuarius, can help to assess the role of Muc91C in facilitating heavy PWN loading and thus to pine wood disease. Overall, our study provides evidence of exploitation of hypoxia-enhanced tracheal elasticity by a plant parasitic nematode to improve vector competence in this unique pathogen–vector system and offers new insights for the development of novel targets to control the pine wilt disease.

Materials and methods

Collection and rearing of nematodes and beetles

Request a detailed protocol

Mature larvae of sawyer beetles (M. alternatus) were collected from dead pine trees in the Fuyang area, Hangzhou City, Zhejiang Province, and reared in an incubator (26°C, humidity 35%) until they pupated or for 7 days after eclosion. B.xylophilus were originally acquired from infected trees in Shannxi Province and maintained with Botrytis cinerea in potato dextrose agar (BD, 213400) plates at 25°C for generations until used in the experiments.

Nematode loading, tracheal dissections, and nematode quantification

Request a detailed protocol

50 ml Erlenmeyer flasks containing B. cinerea growing in autoclaved (121°C, 30 min) barley medium (10 g barley in 15 ml water) for 2 weeks at 25°C were inoculated with 1000 propagative nematodes. After the nematodes had fed for 5 days, autoclaved pine wood sawdust layer was spread on top of the barley layer of the rearing flasks and cultured in 4°C for another 2 weeks. When the color of the pupae’s eyes turned dark, they were put on the nematode-infected sawdust layer until 6 days after eclosion. Emerged adult beetles were dissected by ventral filleting, and the tracheal tubes located on both sides of the thorax were dissected and carefully deprived of surrounded muscle under a stereomicroscope (Olympus SZX16, Japan). The excised tracheal tubes and the left part of the body were washed in PBST (Solarbio, P1033) to irrigate the nematodes and presence of nematode was confirmed under a stereomicroscope (Olympus SZX16, Japan). The total number of isolated nematodes per beetle was counted in plates with an inverted microscope (Olympus CKX41, Japan).

Measurement of oxygen concentrations

Request a detailed protocol

The measurement of oxygen concentrations followed previous studies with modifications (Brune et al., 1995; Pettersen et al., 2005). The Oxygen Microsensors with tip diameters of 25 μm (Unisense OX-25-905248, Denmark) and 100 μm (Unisense OX-100-909667, Denmark), respectively, were used to penetrate into trachea tubes (which are visible after ventral filleting and gut removal) and poke into the atrium cavity of the largest spiracles located on both sides of the thorax. This protocol was used for beetles without or with nematodes to obtain high-resolution profiles of oxygen concentrations. Prior to use, the electrodes of the microsensors were polarized overnight in deionized and continuously aerated water. Calibration was carried out before and after each experiment and was done by measuring the current when the microelectrode was placed in water saturated with air (21 kPa O2) and measuring the background current in 1% Na2SO3 (0 kPa O2). The values were measured with an amperemeter (Unisense oximeter-5591, Denmark) connected to a computer with a software SensorTrace Logger (Unisense, Denmark). Oxygen microsensor was fixed on a micromanipulator. Once placed into tracheal tubes and poking into the atrium cavity under a stereomicroscope, the microsensor was paused for at least 20 s to ensure sufficient data collection. The microsensor was then pulled out and recording was continued for 15 s to record values of the ambient air. To take into account temperature-derived fluctuation in saturated oxygen concentration of different tests, we converted the concentrations to its corresponding partial pressure, and the average partial pressure of oxygen during the 20 s inside the trachea was calculated as the final data.

Hypoxic treatment

Request a detailed protocol

Hypoxic treatment was performed in a hypoxic chamber (FLYDWC-50; Fenglei Co., Ltd, China) placed in an artificial box to automatically control the ambient temperature, air flow, and pressure of oxygen (PO2). Adult beetles without nematodes were placed in the ventilated chamber, in which air flow was balanced with pure nitrogen to achieve the required 1% O2. The adult beetles were maintained in the chamber for 6 hr, 12 hr, or 24 hr at 25 ± 1°C in the dark.

Measurement of rubber-like degree

Request a detailed protocol

Adult beetles post 6 days after eclosion were dissected by ventral filleting, and the morphological trait of thoracic tracheal tubes without and with heavy PWN were observed under a stereomicroscope (Olympus SZX16, Japan). Tracheal tube, which lost its original orange metallic luster and became pink and leathery, is defined as a rubber-like tube. The number of rubber-like tubes and total number of thoracic tracheal tubes oxygenate to flight muscle were counted and the rubberization degrees were set into three degrees according to the ratio of the former two values (Ⅰ, none; Ⅱ, less than 50% rubber-like tubes; Ⅲ, 50–100% rubber-like tubes). Then, the number of PWN harbored in beetles was counted.

Measurement of mechanical properties with tensile testing

Request a detailed protocol

The two longest tracheal tubes linking the largest spiracle on both sides of the thorax and the spiracle basal to the head of the beetle were dissected as mentioned above and were maintained at room temperature (RT) in PBS (Solarbio, P1020). According to a previous study on muscle fibers (Hakim et al., 2018; Kaiser et al., 2019), tensile testing was performed with a micromechanical system (Aurora Scientific, ON, Canada) equipped with a length controller (322C), a 10 mN force transducer (405A), and dynamic control software suite DMC (ASI 600A). The measurements were performed inside a bath with PBS at room temperature. The dissected tracheal tubes were tied to the fixed hooks at the ends of length controller and force transducer using nylon fibers. Prior to testing, the unstretched lengths (L0) and radius (r) of the tubes were measured with caliper under an inverted microscope (Olympus CKX41, Japan). All samples were tested at a constant strain rate of 10% L0/3 s until failure or 2×L0 along the axial direction of tubes, as described in tensile testing on muscle fibers (Krysiak et al., 2018). The stiffness of the tubes was characterized by the Young’s modulus (E), which is a constant specifying the elastic properties of a material (Feynman et al., 1965). The Young’s modulus was determined from the linear region of the stress–strain curve, usually between 30% and 80% strain, using the equation of E = {(F/πr2)/(ΔL/L0)}.

Transmission electron microscopy of the tracheal tubes

Request a detailed protocol

The two longest tracheal tubes linking the largest spiracle on both sides of the thorax and the spiracle basal to head of the beetle were dissected and instantly fixed with 2.5% (vol/vol) glutaraldehyde (Coolaber, SL1790) and 4% (vol/vol) paraformaldehyde (Solarbio, P1112) with phosphate buffer (PB) (0.1 M, pH 7.4) (Macklin, H885798). The samples were then washed four times in PB and then tubes were postfixed with 1% (wt/vol) osmium tetraoxide in PB for 2 hr at 4°C, dehydrated through a graded ethanol series (30, 50, 70, 80, 90, 100%, 100%, 7 min each) into pure acetone (2×10 min). Samples were infiltrated in graded mixtures (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (16.2 g SPI-PON812, 10 g DDSA, and 8.9 g NMA), and then changed to pure resin. Finally, tubes were embedded in pure resin with 1.5% BDMA and polymerized for 12 hr at 45°C, 48 hr at 60°C. The ultrathin sections (70 nm thick) were sectioned with microtome (Leica EM UC6, Germany), double-stained by uranyl acetate and lead citrate, and examined by a transmission electron microscope (FEI Tecnai Spirit120kV, OR).

RNA-seq and transcriptome analysis

Request a detailed protocol

Each sample for RNA-seq was prepared by combining four individual beetles (two females and two males, 6 days after emergence) for one library construction. The two libraries included trachea of sawyer beetles with and without PWN. Tracheal tubes were extirpated, washed with PBS to remove the nematodes, and immediately flash frozen with TRIzol (Invitrogen, 15596018) and stored in –80°C before use. Total RNA was isolated from the frozen samples using TRIzol reagent according to the manufacturer’s protocol. mRNA was purified with a Dynabeads mRNA purification kit (Invitrogen, 61012). PolyA containing mRNAs was enriched with oligo (dT) magnetic beads (NEB, S1419S), fragmented with RNA fragmentation reagent, and subjected to the following procedure: first- and second-strand cDNA synthesis, purification, end repair, single-nucleotide addition, ligation of adapters, purification of ligated products, and PCR amplification for cDNA template enrichment. The RNA-seq library preparation kit for whole transcriptome discovery (compatible with Illumina, San Diego, CA) was used (Illumina Whole-Genome Gene Expression BeadChips). The 320–420 bp products were purified with the MiniElute gel extraction kit (QIAGEN, Hilden, Germany) and sequenced with the Illumina novaseq 6000 platform at Biomarker Technologies Co, LTD.

Identification and analysis of aECM-related genes from the transcriptome and phylogenetic analysis

Request a detailed protocol

Using the SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP), we predicted secreted proteins in DEGs based on the presence and extension of signal peptides. The retrieved proteins were manually confirmed by Nr, GO, KO annotations, and using pfam and a BlastX search against the amino acid sequences of known ECM-related genes from flybase (http://flybase.org/). Transmembrane domains were analyzed by TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The RR2 Consensus sequences were predicted using the cuticleDB website (http://bioinformatics2.biol.uoa.gr/cuticleDB/index.jsp). The O-glycosylation sites of mucin proteins were determined by NetOGlyc 4.0 (https://www.cbs.dtu.dk/services/NetOGlyc). To verify the classification of resilin-related proteins and other mucin proteins, phylogenetic analysis was performed using the full-length amino acid sequences of representative proteins from other species retrieved from the GenBank database. Multiple alignments of the amino acid sequences were performed using the ClustalW program in MEGA 10.0. The phylogenetic trees were constructed adopting neighbor-joining (NJ) method using 1000 bootstrap repetitions. All resilin-related and mucin sequences of other model species used in this study are provided as Figure 4—source data 1 and Supplementary file 1.

Real-time PCR

Request a detailed protocol

Total RNA was extracted from thoracic tracheal tubes of each beetle in different treatments using TRIzol reagent according to the manufacturer’s protocol and stored at –80°C until used in experiments. The total number of nematodes in corresponding tracheal samples were counted. RNA quality was assessed with an ND-1000 spectrophotometer (NanoDrop Technologies, DE). About 1 μg of RNA was used as template to produce cDNA with FastQuant RT kit with a genomic DNA eraser (Tiangen, KR106). RT-qPCR was performed using Talent qPCR PreMix (SYBR Green) (Tiangen, FP209) and on a 7300Plus Real-Time PCR system (Applied Biosystems, MA). At least five biological replicates were assayed for statistical analysis. The amount of mRNA was calculated by the change in cycle threshold (ΔΔCt) method and was normalized to control rp49 mRNA values. qPCR primers are listed in Supplementary file 2.

Western blot analysis

Request a detailed protocol

Total proteins were extracted from thoracic tracheal tubes of each beetle in different treatments using TRIzol reagent according to the manufacturer’s protocol and stored at –80°C until used in experiments. The protein extracts (100 mg) were electrophoresed on 4–12% SDS-PAGE precast gels (EASYBIO, China) and then transferred to 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore). The membrane was incubated with polyclonal antibody against target protein (anti-Muc91C, developed by BGI, China, 1:4000). Goat anti-rabbit IgG (EASYBIO, 1:5000) was used as the secondary antibody. Monoclonal antibody against Histone H3 (EASYBIO, 1:2000) was used as an internal control. Goat anti-mouse IgG (EASYBIO, 1:5000) was used as the secondary antibody. Protein bands were detected by SuperSignal West Atto Ultimate Sensitivity Substrate (Invitrogen, A38554).

Whole-mount immunohistochemistry

Request a detailed protocol

Thoracic tracheal tubes of each beetle in different treatments were dissected and instantly fixed with 4% paraformaldehyde overnight. After being washed with 1× PBS buffer, the samples were permeabilized with 0.5% TritonX-100/PBS at 37°C for 20 min, then blocked with 5% goat serum for 1 hr, and incubated with affinity-purified polyclonal rabbit antibody against Muc91C (produced by BGI, China, 1: 100) at 4°C for 24 hr. The samples were then washed three times for 5 min each with 0.5% TritonX-100/PBS, Alexa Fluor-488 goat anti-rabbit IgG (Invitrogen, A11008, 1: 500) was used as the secondary antibody. The cellular nucleus was stained with Hoechst 33342 (Invitrogen H3570, 1:1000) for 20 min. The cell membrane system was stained with CellTracker CM-DiI dye (Invitrogen, C7001, 1: 500) for 30 min. Finally, the samples were mounted with Fluoroshield mounting medium (Solarbio, S2100). Fluorescence was detected using an confocal laser-scanning microscope (SP8 Lightning, Leica). Taenidial folds are autofluorescence excitated by 488 nm or 561 nm lasers and have higher autofluorescence excitated by 561 nm laser. Images were processed using LAS X Navigator (Leica).

RNA interference

Request a detailed protocol

Double-stranded RNAs (dsRNAs) were synthesized with cDNA templates possessing T7 RNA polymerase promoter sequences on both ends (Supplementary file 2), according to the T7 RiboMax Express RNAi System Kit (Promega, P1700) protocol. Within 2 days after molting, approximately 60 adults beetle were injected at the joint of the second and the third abdominal segment with 30 μg of dsRNA (<5 μl) using a 7000-series modified microliter syringe (Hamilton, Bonaduz, Switzerland). Control beetles were injected with dsGFP as a negative control. The injected adult beetles were kept in standard conditions for 4 days, then treated with 1% O2 for 12 hr. The injected adult beetles used in the nematodes loading trial were kept in 50 ml Erlenmeyer flasks with barley medium containing dispersal L and LIV nematodes at 25°C for 5 days. When the adults were 7 days old, they were dissected to obtain trachea samples for the different assays, and disposed as described earlier.

Statistical analysis

Request a detailed protocol

The sample size is determined according to previous publications in the pine sawyer beetle species (Wu et al., 2019; Zhao et al., 2016; Zhou et al., 2018). For nematode loading, hypoxic treatments and RNAi treatments, individuals were randomly allocated into experimental group and control group, and no restricted randomization was applied. For nematode quantification, measurements of mechanical properties with tensile testing, researchers were blind to RNAi or hypoxic treatments before counting or measurements. All experiments were performed for at least three independent biological replicates.

A Pearson correlation coefficient was used to measure the correlation between PWN loading and partial pressure of oxygen. The semi-log fit using least square method was chosen for curve fitting. The fit formula was Y = 10.55 + (–9.629) × log(X) with an R2 value of 0.85. A Kolmogorov–Smirnov test was used to check the normality of data. When the assumption of normal distribution was met, Student’s t-test and one-way ANOVA with Dunn’s or Tukey’s multiple comparison were used for two-group and three-group comparisons, respectively. When the assumption of normal distribution was not met, Mann–Whitney U test and Kruskal–Wallis with Dunn’s multiple comparison were used for two-group and three-group comparisons, respectively. Statistical analysis was performed with GraphPad Prism 8.0 (GraphPad software, San Diego, CA).

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting file. Source data files have been provided for Figures 1, 2, 3, 4, 6 and 7. The RNA-seq data reported in this study was deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics, 2017) in National Genomics Data Center (Nucleic Acids Research, 2020), Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number CRA006464 and CRA008617 that are publicly accessible at https://ngdc.cncb.ac.cn/gsa/browse/CRA006464 and https://ngdc.cncb.ac.cn/gsa/browse/CRA008617.

The following data sets were generated
    1. Tang X
    (2023) Genome Sequence Archive
    ID CRA006464. RNA-seq of pine sawyer beetle with PWN.
The following previously published data sets were used
    1. Tang X
    (2022) Genome Sequence Archive
    ID CRA008617. RNA-Seq of tracheal tubes in adult pine sawyer beetle.

References

    1. Feynman RP
    2. Leighton RB
    3. Sands M
    (1965)
    The Feynman lectures on physics
    American Journal of Physics 33:750–752.
    1. Tatham AS
    2. Shewry PR
    (2002) Comparative structures and properties of elastic proteins
    Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences 357:229–234.
    https://doi.org/10.1098/rstb.2001.1031

Decision letter

  1. Sofia J Araujo
    Reviewing Editor; University of Barcelona, Spain
  2. Dominique Soldati-Favre
    Senior Editor; University of Geneva, Switzerland

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Sofia J Araujo as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife.

Specifically, the reviewers find that although the work is novel and of relevance it lacks strong experimental backup. The work would gain from deeper genetic and cell biological analysis and we hope you will find the reviewers' comments useful.

Reviewer #1 (Recommendations for the authors):

Tang et al. investigated if pathogen loading can increase hypoxia in the insect vector's tracheal system. To do so, the authors use pine wood nematodes (PWN) and their infection of Monochamus beetles via their tracheal system. They find that the number of the nematodes in the trachea is negatively correlated with the oxygen level in the tracheal system of its vector beetle, leading to hypoxia. They also observe that PWN loading and hypoxia enhanced the elasticity of tracheal tubes and increased the thickness of tracheal aECM. Analysing the transcriptome they identify changes in aECM related genes in infected beetles. They find that, upon infection, Muc91C has the highest increase in expression and that its downregulation in beetles reduces PWN loading and tracheal elasticity.

Strengths:

The analysis of the pathogen's strategy to maintain itself in its vector by modulating the vector's hypoxic responses. The observation that nematode entry decreases oxygen levels, inducing hypoxia, which in turn increases tracheal tube elasticity by Muc91C upregulation.

The demonstration that heavy PWN loading, or hypoxic treatment, enhances the elasticity of the tracheal tubes of the vector beetle.

The molecular analysis of the tracheal response to infection and aECM gene expression changes.

Weaknesses:

The molecular analysis of Muc91C in the tracheal system of the beetles would gain with a more detailed cellular observations.

This work clearly demonstrates the effect of PWN loading on the elasticity of tracheal tubes. However, its major weakness lies on the lack of a detailed analysis of Muc91C expression and localization.

Enhanced tracheal elasticity induced by hypoxia upon infection relies on the expression of Muc91C. Where does Muc91C localize in relation to the chitinous aECM? Are the effects observed due to protein levels?

I understand that these experiments may be difficult to perform in Monochamus beetles, but the authors could try to find answers to these questions using Drosophila melanogaster.

Reviewer #2 (Recommendations for the authors):

The manuscript by Xuan Tang and colleagues contains the experimental analyses of the relationship between the pine sawyer beetle and the pinewood pathogen nematode (pwn). The authors find that the load of pwn in the beetle correlates with a hypoxia response, in turn inducing the expression of the mucin Muc91C, a presumptive cuticular protein, in the tracheal system. They claim that the underlying mechanism involves hypoxia-induced Muc91C-dependent changes in tracheal cuticle elasticity. Other mucins do not seem to be implicated in this process. The circuitry nematode-load, hypoxia, Muc91C-expression and increased elasticity ends with the release of the nematodes on pines, where they act as a pest.

The addressed biological, ecological problem is really exciting. The model of the sequence of events is intriguing and, partly, backed up with solid data, also from previous works (Wu et al., 2019 cites in the manuscript).

Besides some minor points that I will mention below, there is a major one that puzzles me. It is about the relationship between tracheal development/differentiation, gene expression (among others of Muc91C) and the analyses of beetles in different experiments (hypoxia, transcriptome etc). For instance, nematodes loading on pupae is described on lines 450ff: when the "colour of.. eyes turned dark, nematodes were added": which tracheal stage is this? Changes in Muc91C expression in hypoxia assays and transcriptomics were then performed on adult beetles, when tracheal development/differentiation is supposed to be largely terminated, at least in other insects. Hence, how can Muc91C alter tracheal physics after the overall architecture of the tracheae is established? One possibility is that I am wrong and the tracheal development/differentiation continues also in adult beetles even seven days post eclosion (as stated in the Materials and methods section). Another possibility is that newly forming tracheal branches/tips respond to hypoxia and nematode loads, but not portions of the tracheae established already during metamorphosis.

A related problem is the equation of elasticity of the tracheal cuticle and oxygen concentration: is there any logical argument that allows us to suppose that a more elastic extracellular matrix might facilitate oxygen diffusion? According to the authors, the tracheal cuticle becomes even thicker as a response to nematode loading and hypoxia. If I remember well, hypoxia-incubated Drosophila larvae tend to produce tracheal tubes with a larger diameter (after moulting, not as an immediate response) for better aeration. A subtle problem arises also, when considering Muc91C incorporation into the cuticle and cuticle property changes after tracheal development/differentiation: the tracheal cuticle formed at metamorphosis would have a stiffer "more normal" consistency than the "newly" added cuticle: the old cuticle would restrict the elasticity of the new one like a concrete wall the elasticity of a balloon. Again, as mentioned above: we need a more detailed description of the course of tracheal development/differentiation in relation to gene expression (Muc91C).

Another point concerns the description of putative elastic proteins in insects: Muc91C contains repeats that may be responsible for elasticity. However, this has not been shown (while it has been shown for Resilin). The authors should be careful in this regard. Muc91C as a mucin is decorated by sugars that may bind water, this swelling may confer "elasticity" to the cuticle rather than its repeats. Increased water content in the cuticle may also be responsible for "thicker" cuticles in nematode loaded or hypoxic tracheae: increased thickness may be an artefact of preparation. NB: the two TEM images in Figure 5 C do not have comparable quality. Preparation, the thickness of the sections or contrasting differ too much.

A problem might also be the function of Muc91C: first, can the authors exclude that it is not expressed in tissues associated with the tracheal system when transcriptomics were carried out? In Drosophila, the Muc91C expression is detected in the nervous system and the fat body. Second: RNAi against Muc91C seems to produce viable beetles. I would expect a reduction in overall fitness, though. May this, if the case, affect the interaction with pwn?

The cloudy situation of the relationship tracheal development/differentiation, gene expression and organ response should be clarified: we need a thorough description of tracheal development/differentiation; the course of tracheal development/differentiation should be next correlated with gene expression (for instance Muc91C). This is a crucial experiment as this would be the first example that the main tracheal system may respond to the environment (here pwn and hypoxia) even after differentiation. Moreover, it would strengthen or serve to refute the argument of Muc91C being expressed and functional beyond terminal differentiation of the tracheal system. Overall, I consider this issue as very crucial as the current version reports only on a phenomenon rather than on a biological process.

The robustness of their conclusion on the relevance of Muc91C function would increase if additional genes (only mucs are not sufficient) expression were knocked-down (such as the chitin synthase coding gene on the list in Figure 3) and the resulting phenotypes analysed.

We need also clarification regarding the rubberization issue (lines 163ff): it is unclear how this was measured. The description in the material and methods section and in the figure legend are insufficient.

Reviewer #3 (Recommendations for the authors):

Tang and colleagues demonstrated the importance of hypoxia-mediated enhancement of tracheal elasticity in vector beetle M. alternatus to transport more nematodes to new host pine trees. Tracheal infection by PWN provoked hypoxia inside the trachea and this made the host tracheal aECM thicker than the non-infected host, increasing tracheal elasticity. The authors then revealed the significance of Muc91C gene, which encodes a resilin-like mucin protein and is highly up-regulated upon nematode infection, by RNAi experiment. Indeed, tracheal aECM was not developed (not thickened) in the Muc91C-RNAi insect and it's elasticity was significantly lower than control insect. Interestingly, when the development of tracheal aECM was disrupted by Muc91C-RNAi, the total amount of trachea-residing PWN was significantly decreased, indicating that PWN-mediated tracheal hypoxia is critical to vector competence to carry more nematodes.

Although the authors have come up with fairly clear results, there are some shortcomings. A key point of this paper is the elasticity and thickness of aECM of tracheal tissue, but they just showed TEM images and mathematical constant (Young's modulus) to demonstrate them. This is ambiguous and very insufficient to interpret the importance of tracheal elasticity. The author should present more rigid and scientific results to explain this key context (e.g., at least need to do immunostaining of resilin-like mucin protein between PWN-infected and non-infected insects or other measuring methods).

Nevertheless, they found a novel functional gene (Muc91C), which is partially important to tracheal development such as thickness and elasticity.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode" for further consideration by eLife. Your revised article has been evaluated by Dominique Soldati-Favre (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) The authors show that Muc91C localizes underneath the chitinous aECM in tracheal tubes upon infection. This is a very good result, however, the images are not clear and the manuscript would gain from higher magnification images focusing on a small section of the membrane and showing the tracheal cells in more detail.

2) Reference to the elastic property of repeat A: the way the authors state that Muc91 repeat A has been shown to "regulate elasticity" is, to my understanding, wrong. First, repeat A from pro-resiling has been shown to confer elasticity, not of Muc91. Second, elasticity does not come from the repeats, but from their ability to form di-tyrosine bridges, and this has not been shown.

3) Young's modulus: did I understand correctly that the forces were measured along the main axis of the tracheal tubes? To me, they should be measured perpendicular to the main axis. Please describe the method more in detail. Forces may be different in different directions.

Reviewer #1 (Recommendations for the authors):

The authors show that Muc91C localizes underneath the chitinous aECM in tracheal tubes upon infection. This is a very good result, however, the images are not clear and the manuscript would gain from higher magnification images focusing on a small section of the membrane and showing the tracheal cells in more detail.

Reviewer #2 (Recommendations for the authors):

– Reference to the elastic property of repeat A: the way the authors state that Muc91 repeat A has been shown to "regulate elasticity" is, to my understanding, wrong. First, repeat A from pro-resiling has been shown to confer elasticity, not of Muc91. Second, elasticity does not come from the repeats, but from their ability to form di-tyrosine bridges, and this has not been shown.

– My argument that Muc91 may be involved in retaining water in the cuticle and thereby contributing to elasticity was refuted. Fine with me, if this was done with a valid argument. Muc91 as a mucin may bind water and thereby contribute to elasticity, and not (only) via its repeats.

– Young's modulus: did I understand correctly that the forces were measured along the main axis of the tracheal tubes? To me, they should be measured perpendicular to the main axis. Please describe the method more in detail. Forces may be different in different directions.

https://doi.org/10.7554/eLife.84621.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1 (Recommendations for the authors):

Tang et al. investigated if pathogen loading can increase hypoxia in the insect vector's tracheal system. To do so, the authors use pine wood nematodes (PWN) and their infection of Monochamus beetles via their tracheal system. They find that the number of the nematodes in the trachea is negatively correlated with the oxygen level in the tracheal system of its vector beetle, leading to hypoxia. They also observe that PWN loading and hypoxia enhanced the elasticity of tracheal tubes and increased the thickness of tracheal aECM. Analysing the transcriptome they identify changes in aECM related genes in infected beetles. They find that, upon infection, Muc91C has the highest increase in expression and that its downregulation in beetles reduces PWN loading and tracheal elasticity.

Strengths:

The analysis of the pathogen's strategy to maintain itself in its vector by modulating the vector's hypoxic responses. The observation that nematode entry decreases oxygen levels, inducing hypoxia, which in turn increases tracheal tube elasticity by Muc91C upregulation.

The demonstration that heavy PWN loading, or hypoxic treatment, enhances the elasticity of the tracheal tubes of the vector beetle.

The molecular analysis of the tracheal response to infection and aECM gene expression changes.

Thanks for your positive comments.

Weaknesses:

The molecular analysis of Muc91C in the tracheal system of the beetles would gain with a more detailed cellular observations.

This comment is really helpful. In this revised version, we conducted immunostaining of Muc91C in the tracheal system of the beetles. We found heavy nematode loading resulting in substantial upregulation of Muc91C beneath the layer of taenidial folds and above the apical membrane of the tracheal cell, as shown in line 290-295 and Figure 4E & F. This result is consistent with the nematode-thickened aECM layer showed in TEM, thus strongly demonstrating that Muc91C is the component of the PWN-thickened aECM layer showed in TEM.

This work clearly demonstrates the effect of PWN loading on the elasticity of tracheal tubes. However, its major weakness lies on the lack of a detailed analysis of Muc91C expression and localization.

Enhanced tracheal elasticity induced by hypoxia upon infection relies on the expression of Muc91C. Where does Muc91C localize in relation to the chitinous aECM? Are the effects observed due to protein levels?

I understand that these experiments may be difficult to perform in Monochamus beetles, but the authors could try to find answers to these questions using Drosophila melanogaster.

This comment is essentially the same with Comment #1 above.

To examine Muc91C expression at the protein level, we carried out Western blot and found nematode loading significantly enhance Muc91C in the trachea of beetles, as shown in line 285-290 and Figure 4D. By performing immunostaining using Muc91C antibody, we found Muc91C were expressed beneath the layer of chitinous taenidial folds and above the apical membrane of the tracheal cell, as shown in line 290-295 and Figure 4E & 4F. This is consistent with the thickened aECM layer in TEM analysis. Therefore, we confirm that the observed effects are due to protein levels thus revealing a strong association between elasticity and the expression of Muc91C.

Reviewer #2 (Recommendations for the authors):

The manuscript by Xuan Tang and colleagues contains the experimental analyses of the relationship between the pine sawyer beetle and the pinewood pathogen nematode (pwn). The authors find that the load of pwn in the beetle correlates with a hypoxia response, in turn inducing the expression of the mucin Muc91C, a presumptive cuticular protein, in the tracheal system. They claim that the underlying mechanism involves hypoxia-induced Muc91C-dependent changes in tracheal cuticle elasticity. Other mucins do not seem to be implicated in this process. The circuitry nematode-load, hypoxia, Muc91C-expression and increased elasticity ends with the release of the nematodes on pines, where they act as a pest.

The addressed biological, ecological problem is really exciting. The model of the sequence of events is intriguing and, partly, backed up with solid data, also from previous works (Wu et al., 2019 cites in the manuscript).

Thanks for your positive comments.

It is about the relationship between tracheal development/differentiation, gene expression (among others of Muc91C) and the analyses of beetles in different experiments (hypoxia, transcriptome etc). For instance, nematodes loading on pupae is described on lines 450ff: when the "colour of.. eyes turned dark, nematodes were added": which tracheal stage is this? Changes in Muc91C expression in hypoxia assays and transcriptomics were then performed on adult beetles, when tracheal development/differentiation is supposed to be largely terminated, at least in other insects. Hence, how can Muc91C alter tracheal physics after the overall architecture of the tracheae is established? One possibility is that I am wrong and the tracheal development/differentiation continues also in adult beetles even seven days post eclosion (as stated in the Materials and methods section). Another possibility is that newly forming tracheal branches/tips respond to hypoxia and nematode loads, but not portions of the tracheae established already during metamorphosis.

We totally agree with this comment that analysis of the relationship between gene expression and tracheal development would provide basic knowledge underlying this study. Actually, there is a fascinating developmental synchronization between PWN and the vector beetle in previous studies (Zhao et al., 2014) and the life cycles of the two species are shown in Figure 7. The formation of dispersal LIV exclusively induced by late pupa beetle with black eyes and newly molted adult beetle (Zhao et al., 2013). Thus, the PWN loading to tracheal system of the beetle occurs after beetle eclosion. In parallel with this study, we have actually investigated the morphological and transcriptomic changes of tracheal development during metamorphosis of the vector beetle morphological observation has shown that, the pattern of tracheal network has been already formed at the late pupal stage (the time when propagative PWNs were introduced in the present study). Tracheal development continues within the first five days post eclsion and is almost terminated after five days post eclosion, given the dramatic changes of tracheal gene expression occurring within five days after eclosion. These results have been submitted to BioRxiv (Tang et al., 2022). Therefore, developmental pattern observed in vector beetles support the second possibility in this comment that newly forming tracheal branches/ tips respond but not portions of the trachea established already during metamorphosis. In the current study, we added analysis of the temporal changes for the gene expression of the 45 aECM-related genes (including Muc91C) at different time points post eclosion, using the released RNA-seq data as shown in line 251-261 and Figure 3—figure supplement 1. We found that all genes were expressed at low levels after five days post-elcosion, suggesting a termination of tracheal development before the transcriptome and hypoxia treatment were carried out.

A related problem is the equation of elasticity of the tracheal cuticle and oxygen concentration: is there any logical argument that allows us to suppose that a more elastic extracellular matrix might facilitate oxygen diffusion? According to the authors, the tracheal cuticle becomes even thicker as a response to nematode loading and hypoxia. If I remember well, hypoxia-incubated Drosophila larvae tend to produce tracheal tubes with a larger diameter (after moulting, not as an immediate response) for better aeration.

The facilitation of a more elastic ECM on oxygen diffusion is likely due to its role on tracheal ability to compress. In the revised text, we interpreted the association between aECM and oxgen diffusion in Introduction (line 75-96). In insects, including beetles, the tracheal system exhibits rapid cycles of compression and expansion, analogous to the inflation and deflation of vertebrate lungs. The tracheal volumes changed by this behavior increase the internal pressure for improvement of air convection and gases exchange, thus facilitating oxygen diffusion (Westneat et al., 2003). In addition, hypoxia increases the frequency of this behavior to promote gas exchange (Greenlee et al., 2013). There is evidence showing that the compression capacity is largely determined by tracheal mechanical properties. In the American cockroaches, the chitin structures in ECM layer are responsible for mechanical features that enable volume changes during respiration (Webster et al., 2011). Similarly, high inflation of lungs is related to the mechanical forces provided by ECM components (Berg et al., 1997; Wirtz and Dobbs, 2000).

In the revised text, we discussed the thickened tracheal cuticle in response to nematode loading and hypoxia by adding “Regardless of an intricate relationship between ECM thickness and tubular diameter, the thickened ECM layer results in more elastic tubes that qualify a robust compression. Such improvement of compression capacity via tubular elasticity might be response to acute hypoxia or PWN loading, different from the chronic adaption that involve tracheal diametric expansion in Drosophila larvae incubated under constant hypoxic condition for generations (Henry and Harrison, 2004).” in Discussion (line 449-455).

A subtle problem arises also, when considering Muc91C incorporation into the cuticle and cuticle property changes after tracheal development/differentiation: the tracheal cuticle formed at metamorphosis would have a stiffer "more normal" consistency than the "newly" added cuticle: the old cuticle would restrict the elasticity of the new one like a concrete wall the elasticity of a balloon. Again, as mentioned above: we need a more detailed description of the course of tracheal development/differentiation in relation to gene expression (Muc91C).

To address the concern about tracheal plasticity, we explained

PWN-induced epithelium reorganization in the Discussion (line 463-473). As mentioned above, tracheal development is terminated within the first five days after eclosion. However, PWN loading causes substantial expressional changes of 251 ECM related genes, suggesting dramatic epithelium reorganization in tracheal tubes of vector beetles. For example, in parallel with the increased expression of Muc91C, metalloproteinases degrading old cuticle and promoting apical membrane expansion (Glasheen et al., 2010), cadherins and integrins participating in signal transduction between aECM and tracheal cells (Hayashi and Kondo, 2018; Öztürk-Çolak et al., 2016) are significantly upregulated as well. Therefore, aECMs are already well-developed during metamorphosis and undergo reconstruction after PWN loading, resulting in a more elastic tracheal cuticle structure for supporting respiration.

Another point concerns the description of putative elastic proteins in insects: Muc91C contains repeats that may be responsible for elasticity. However, this has not been shown (while it has been shown for Resilin). The authors should be careful in this regard. Muc91C as a mucin is decorated by sugars that may bind water, this swelling may confer "elasticity" to the cuticle rather than its repeats. Increased water content in the cuticle may also be responsible for "thicker" cuticles in nematode loaded or hypoxic tracheae: increased thickness may be an artefact of preparation.

We have clarified the causal links between Muc91C and elasticity in line 350-356. Muc91C of the beetle contains two series of short repeated motifs located in the N-terminal (A repeats) and C-terminal (B repeats) region and the A repeats are shared by Muc91C homologues of other insects, as shown in Figure 5 of the current version of manuscript. The direct contribution of repeat A in elastic properties has been confirmed in several studies (Elvin et al., 2005; Lyons et al., 2007; Nairn et al., 2008), demonstrating that synthetic peptide chains containing either the A-repeat-containing region from D. melanogaster pro-resilin or a series of the consensus sequence, AQTPSSQYGAP, from the A. gambiae Muc91C (AGAP002367-PA) form rubberlike elastic materials. Actually, in a seminal paper about resilin-like gene products in insects (Andersen, 2010), DmMuc91C and AgaMuc91C are both classified as a resilin-like protein due to their abundance in short repeats containing proline and glycine, a common feature of proteins with long-range elasticity. In the revised version, we also carried out immunostaining of Muc91C as shown in line 290-295 and Figure 4E & 4F. We detected strong signals of Muc91C beneath the layer of taenidial folds and above the apical membrane of the tracheal cell, forming a continuous layer in PWN-loaded trachea. Thus, this result indicates that the enhanced expression of Muc91C instead of increased water content accounts for the thickened ECM observed by TEM.

NB: the two TEM images in Figure 5 C do not have comparable quality. Preparation, the thickness of the sections or contrasting differ too much.

The samples for were treated and prepared according to the same protocol. All the ultrathin sections share the same thickness (70nm), as shown in line 603. We have adjusted the contrasting of the TEM images in Figure 6A as suggested in this revised version.

A problem might also be the function of Muc91C: first, can the authors exclude that it is not expressed in tissues associated with the tracheal system when transcriptomics were carried out? In Drosophila, the Muc91C expression is detected in the nervous system and the fat body.

This concern makes sense. To exclude the influence of related tissues, we carefully removed the surrounded muscle under microscope when sampling the trachea for transcriptomes. These details of operation have been added in the Materials and methods in line 531. To elucidate the tissue specificity of Muc91C, we conducted qPCR for Muc91C expression in tracheal tubes, flight muscle and midgut. Compared to the other two tested tissues, Muc91C expression was mostly highly expressed in the trachea of both infested or non-infested adult beetles. These results have been added in line 280-285 and Figure 4C. In addition, the immunostaining of Muc91C has shown its primary localization in tracheal tubes but not in other associated tissues, as demonstrated by Figure 4E & 4F.

Second: RNAi against Muc91C seems to produce viable beetles. I would expect a reduction in overall fitness, though. May this, if the case, affect the interaction with pwn?

The viability of beetles was not affected by RNAi against Muc91C and we add this results in this revised vision. In RNAi efficiency assessment, TEM imaging and tensile testing, dsGFP or dsMuc91C were injected into beetle adults within two days after molting and treated with 1% O2 for 12h after dsRNA injection. All treated beetles survived until tracheal dissection, as shown in line 377 and Figure 6—figure supplement 1B. In the RNAi experiments for PWN loading, dsGFP and dsMuc91C resulted in similar proportions of death before dissection for counting, as shown in line 387-388 and Figure 6—figure supplement 1C.

Collectively, RNAi against Muc91C did not reduce the fitness of beetles.

The cloudy situation of the relationship tracheal development/differentiation, gene expression and organ response should be clarified: we need a thorough description of tracheal development/differentiation; the course of tracheal development/differentiation should be next correlated with gene expression (for instance Muc91C). This is a crucial experiment as this would be the first example that the main tracheal system may respond to the environment (here pwn and hypoxia) even after differentiation. Moreover, it would strengthen or serve to refute the argument of Muc91C being expressed and functional beyond terminal differentiation of the tracheal system. Overall, I consider this issue as very crucial as the current version reports only on a phenomenon rather than on a biological process.

This comment is very constructive and essentially the same with the comment #1. By thoroughly assessing the time course of metamorphosis, our work (Tang et al., 2022) has confirmed that the tracheal development/differentiation is almost terminated within the first five days after eclosion. As the comment suggested, we analyzed the temporal changes for the gene expression of the 45 ECM-related genes (including Muc91C) at different time points post eclosion, using the released RNA-seq data. In the revised text, we add this analysis in line 251-261 and Figure 3—figure supplement 1. We found that all genes were expressed at low levels after five days post-elcosion. For Muc91C specifically, its expression was 2-fold upregulated three days after eclosion but was downregulated during the later stages post eclosion. These results are consistent with the dynamics of other tracheal genes exhibiting upregulation mainly during the first five days post eclosion. Therefore, the current study provides an example that the tracheal system is able to respond to the environment. Moreover, PWN loading results in a 7-fold increase of Muc91C expression, a higher fold-change than that during tracheal development. This result thus supports the argument of Muc91C being expressed and functional beyond terminal differentiation of the tracheal system.

The robustness of their conclusion on the relevance of Muc91C function would increase if additional genes (only mucs are not sufficient) expression were knocked-down (such as the chitin synthase coding gene on the list in Figure 3) and the resulting phenotypes analysed.

Further studies should be carried out to systematically assess the function of diverse ECM components in nematode-altered tracheal elasticity as suggested. In this study, we focused on Muc91C, the foremost component that directly linked with the hypoxia- or PWN enhanced thickness and elasticity of the non-chitinous ECM layer on the basis of TEM observation, immunostaining and gene structure analysis. The incomplete reduced elasticity under hypoxic condition in dsMuc91Cinjected beetles suggested complementary roles of other components. In the revised text, we discussed the coordination among Muc91C and other ECM in determining tracheal elasticity in the Discussion (line 463-473).

We need also clarification regarding the rubberization issue (lines 163ff): it is unclear how this was measured. The description in the material and methods section and in the figure legend are insufficient.

Clarification regarding the rubberization issue has been added in the Materials and methods section in line 566-575, as suggested.

Reviewer #3 (Recommendations for the authors):

Tang and colleagues demonstrated the importance of hypoxia-mediated enhancement of tracheal elasticity in vector beetle M. alternatus to transport more nematodes to new host pine trees. Tracheal infection by PWN provoked hypoxia inside the trachea and this made the host tracheal aECM thicker than the non-infected host, increasing tracheal elasticity. The authors then revealed the significance of Muc91C gene, which encodes a resilin-like mucin protein and is highly up-regulated upon nematode infection, by RNAi experiment. Indeed, tracheal aECM was not developed (not thickened) in the Muc91C-RNAi insect and it's elasticity was significantly lower than control insect. Interestingly, when the development of tracheal aECM was disrupted by Muc91C-RNAi, the total amount of trachea-residing PWN was significantly decreased, indicating that PWN-mediated tracheal hypoxia is critical to vector competence to carry more nematodes.

Thanks for your positive comments.

Although the authors have come up with fairly clear results, there are some shortcomings. A key point of this paper is the elasticity and thickness of aECM of tracheal tissue, but they just showed TEM images and mathematical constant (Young's modulus) to demonstrate them. This is ambiguous and very insufficient to interpret the importance of tracheal elasticity. The author should present more rigid and scientific results to explain this key context (e.g., at least need to do immunostaining of resilin-like mucin protein between PWN-infected and non-infected insects or other measuring methods).

Nevertheless, they found a novel functional gene (Muc91C), which is partially important to tracheal development such as thickness and elasticity.

As the Reviewer suggested, we carried out immunostaining assay of Muc91C protein in PWN-infected and non-infected beetles and found PWN loading significantly enhance the expression of Muc91C protein, which formed a continuous layer beneath the layer of taenidial folds and above the apical membrane of the tracheal cell, as shown in line

290-295 and Figure 4E & F. In addition, by conducting Western blot for Muc91C in tracheal tubes with null, light and heavy PWN, we found Muc91C expression were positively correlated with PWN loading number, as shown in line 285-290 and Figure 4D. Therefore, we have provided solid evidence that the expression of Muc91C is responsible for thickened aECM and enhanced tracheal elasticity in beetles with heavy PWN loading.

References

Andersen SO. Studies on resilin-like gene products in insects. Insect Biochemistry and Molecular Biology, 2010, 40: 541-551. DOI: 10.1016/j.ibmb.2010.05.002

Berg JT, Fu Z, Breen EC, Tran H-C, Mathieu-Costello O, West JB. High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma. 1997, 83: 120-128. DOI:10.1152/jappl.1997.83.1.120

Elvin CM, Carr AG, Huson MG, Maxwell JM, Pearson RD, Vuocolo T, Liyou NE, Wong DCC, Merritt DJ, Dixon NE. Synthesis and properties of crosslinked recombinant pro-resilin. Nature, 2005, 437: 999-1002. DOI:10.1038/nature04085

Glasheen BM, Robbins RM, Piette C, Beitel GJ, Page-McCaw A. A matrix metalloproteinase mediates airway remodeling in Drosophila. Developmental Biology, 2010, 344: 772-783. DOI: 10.1016/j.ydbio.2010.05.504

Greenlee KJ, Socha JJ, Eubanks HB, Pedersen P, Lee WK, Kirkton SD. Hypoxia-induced compression in the tracheal system of the tobacco hornworm caterpillar, Manduca sexta. Journal of Experimental Biology, 2013, 216: 2293-2301. DOI:10.1242/jeb.082479

Hayashi S, Kondo T. Development and function of the Drosophila tracheal system. Genetics, 2018, 209: 367-380. DOI:10.1534/genetics.117.300167

Henry JR, Harrison JF. Plastic and evolved responses of larval tracheae and mass to varying atmospheric oxygen content in Drosophila melanogaster. The Journal of Experimental Biology, 2004, 207: 3559-3567. DOI:10.1242/jeb.01189

Lyons RE, Lesieur E, Kim M, Wong DCC, Huson MG, Nairn KM, Brownlee AG, Pearson RD, Elvin CM. Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method. Protein Engineering, Design and Selection, 2007, 20: 25-32. DOI:10.1093/protein/gzl050

Nairn KM, Lyons RE, Mulder RJ, Mudie ST, Cookson DJ, Lesieur E, Kim M, Lau D, Scholes FH, Elvin CM. A synthetic resilin Is largely unstructured. Biophysical Journal, 2008, 95: 3358-3365. DOI:10.1529/biophysj.107.119107

Öztürk-Çolak A, Moussian B, Araújo SJ. Drosophila chitinous aECM and its cellular interactions during tracheal development. Developmental Dynamics, 2016, 245: 259-267. DOI:10.1002/dvdy

Tang X, Koski T-M, Sun J. The entry of pinewood nematode is linked to programmed tracheal development of vector beetles. bioRxiv, 2022: 2022.2010.2029.514345. DOI:10.1101/2022.10.29.514345

Webster MR, De Vita R, Twigg JN, Socha JJ. Mechanical properties of tracheal tubes in the American cockroach (Periplaneta americana). Smart Materials and Structures, 2011, 20: 094017. DOI:10.1088/0964-1726/20/9/094017

Westneat MW, Betz O, Blob RW, Fezzaa K, Cooper WJ, Lee WK. Tracheal respiration in insects visualized with synchrotron x-ray imaging. Science, 2003, 299: 558-560. DOI: doi:10.1126/science.1078008

Wirtz HR, Dobbs LG. The effects of mechanical forces on lung functions. Respiration Physiology, 2000, 119: 1-17. DOI:10.1016/S0034-5687(99)00092-4

Zhao L, Zhang S, Wei W, Hao H, Zhang B, Butcher RA, Sun J. Chemical signals synchronize the life cycles of a plant-parasitic nematode and its vector beetle. Current Biology, 2013, 23: 2038-2043. DOI: 10.1016/j.cub.2013.08.041

Zhao L, Mota M, Vieira P, Butcher RA, Sun J. Interspecific communication between pinewood nematode, its insect vector, and associated microbes. Trends in Parasitology, 2014, 30: 299-308. DOI:10.1016/j.pt.2014.04.007

[Editors’ note: what follows is the authors’ response to the second round of review.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

1) The authors show that Muc91C localizes underneath the chitinous aECM in tracheal tubes upon infection. This is a very good result, however, the images are not clear and the manuscript would gain from higher magnification images focusing on a small section of the membrane and showing the tracheal cells in more detail.

This comment is really helpful. In this revised version, we added higher magnification images of Muc91C in tracheal system with null or heavy nematode loading, by scanning through 63x objective and setting confocal zoom factor to 4x, thus focusing on a small section of the membrane, as shown in line 319-327 and Figure 5A &B, at the bottom right panels. The images provide robust evidence that heavy nematode loading caused substantial upregulation of Muc91C beneath the layer of chitinous taenidial folds and above the apical membrane of the tracheal cell.

Reviewer #2 (Recommendations for the authors):

– Reference to the elastic property of repeat A: the way the authors state that Muc91 repeat A has been shown to "regulate elasticity" is, to my understanding, wrong. First, repeat A from pro-resiling has been shown to confer elasticity, not of Muc91. Second, elasticity does not come from the repeats, but from their ability to form di-tyrosine bridges, and this has not been shown.

Repeat A in elastic pro-resilins has been proved to confer elasticity. In the revised version, we described in details the correlation between repeat A of Muc91C and elasticity in line 344-350 by adding related literature, and made revision to the presentation of causality in line 359-364. Repeat A in Muc91C of M. alternatus (PSSSYGAPS) contains glycine and proline residues, which can form a stretchable β-spiral structure (Ardell and Andersen, 2001; Tatham and Shewry, 2002). Besides, the tyrosines residues in repeat A of Muc91C facilitate the formation of di-tyrosine crosslinks with other repeats (Andersen, 2010; Qin et al., 2009). Thus, repeat A of Muc91C is likely to confer long-range elasticity. This has been further evidenced by an empirical study showing that peptide chains synthesized from 16 copies of similarly repeat A (AQTPSSQYGAP) in Muc91C of A. gambiae can from rubber-like elastic materials (Lyons et al., 2007; Nairn et al., 2008).

– My argument that Muc91 may be involved in retaining water in the cuticle and thereby contributing to elasticity was refuted. Fine with me, if this was done with a valid argument. Muc91 as a mucin may bind water and thereby contribute to elasticity, and not (only) via its repeats.

We highly appreciate this comment that contributes to a more comprehensive understanding of our results, and agree with you that mucins may bind water because of the decoration by sugars (Wagner et al., 2018) and the Muc91C which may plasticized by water coating may provide additional elasticity from the surface tension of the liquid, as proved in spider silk (Vollrath and Edmonds, 1989), in addition to repeated sequences. This possible contribution of water in elasticity of Muc91C was added in Discussion line 496-500.

– Young's modulus: did I understand correctly that the forces were measured along the main axis of the tracheal tubes? To me, they should be measured perpendicular to the main axis. Please describe the method more in detail. Forces may be different in different directions.

We added more details in the methods describing measurement of mechanical property of tracheal tubes in line 600-602 and provided more explanation of Young’s modulus in 603-604. The forces were measured along the main axis (the axial direction), according to the procedures of tensile testing on muscle fibres (Krysiak et al., 2018). We used Young’s module to characterize the elasticity of tracheal tubes, because this constant can specify the stiffness of a material irrespective of directionality of the pulling force used (Feynman et al., 1965).

References

Andersen SO. Studies on resilin-like gene products in insects. Insect Biochemistry and Molecular Biology, 2010, 40: 541-551. DOI:10.1016/j.ibmb.2010.05.002

Ardell DH, Andersen SO. Tentative identification of a resilin gene in Drosophila melanogaster. Insect Biochemistry and Molecular Biology, 2001, 31: 965-970. DOI:10.1016/S0965-1748(01)00044-3

Feynman RP, Leighton RB, Sands M. The feynman lectures on physics; vol. ii. American Journal of Physics, 1965, 33: 750-752.

Krysiak J, Unger A, Beckendorf L, Hamdani N, von Frieling-Salewsky M, Redfield MM, Dos Remedios CG, Sheikh F, Gergs U, Boknik P, Linke WA. Protein phosphatase 5 regulates titin phosphorylation and function at a sarcomere-associated mechanosensor complex in cardiomyocytes. Nature Communications, 2018, 9: 262. DOI:10.1038/s41467-017-02483-3

Lyons RE, Lesieur E, Kim M, Wong DC, Huson MG, Nairn KM, Brownlee AG, Pearson RD, Elvin CM. Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method. Protein Engineering, Design and Selection, 2007, 20: 25-32. DOI:10.1093/protein/gzl050

Nairn KM, Lyons RE, Mulder RJ, Mudie ST, Cookson DJ, Lesieur E, Kim M, Lau D, Scholes FH, Elvin CM. A synthetic resilin is largely unstructured. Biophysical Journal, 2008, 95: 3358-3365. DOI:10.1529/biophysj.107.119107

Qin G, Lapidot S, Numata K, Hu X, Meirovitch S, Dekel M, Podoler I, Shoseyov O, Kaplan DL. Expression, cross-Linking, and characterization of recombinant chitin binding resilin. Biomacromolecules, 2009, 10: 3227-3234. DOI:10.1021/bm900735g

Tatham AS, Shewry PR. Comparative structures and properties of elastic proteins. Philosophical Transactions of the Royal Society B: Biological Sciences, 2002, 357: 229-234. DOI:10.1098/rstb.2001.1031

Vollrath F, Edmonds DT. Modulation of the mechanical properties of spider silk by coating with water. Nature, 1989, 340: 305-307. DOI:10.1038/340305a0

Wagner CE, Wheeler KM, Ribbeck K. Mucins and their role in shaping the functions of mucus barriers. Annual Review of Cell and Developmental Biology, 2018, 34: 189-215. DOI:10.1146/annurev-cellbio-100617-062818

https://doi.org/10.7554/eLife.84621.sa2

Article and author information

Author details

  1. Xuan Tang

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Contributed equally with
    Jiao Zhou and Tuuli-Marjaana Koski
    Competing interests
    No competing interests declared
  2. Jiao Zhou

    State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Writing – review and editing
    Contributed equally with
    Xuan Tang and Tuuli-Marjaana Koski
    Competing interests
    No competing interests declared
  3. Tuuli-Marjaana Koski

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. College of Life Science/Hebei Basic Science Center for Biotic Interactions, Institute of Life Science and Green Development, Hebei University, Baoding, China
    Contribution
    Validation, Writing – review and editing
    Contributed equally with
    Xuan Tang and Jiao Zhou
    Competing interests
    No competing interests declared
  4. Shiyao Liu

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Lilin Zhao

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. University of Chinese Academy of Sciences, Beijing, China
    Contribution
    Conceptualization, Funding acquisition, Writing – review and editing
    For correspondence
    zhaoll@ioz.ac.cn
    Competing interests
    No competing interests declared
  6. Jianghua Sun

    1. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
    2. College of Life Science/Hebei Basic Science Center for Biotic Interactions, Institute of Life Science and Green Development, Hebei University, Baoding, China
    Contribution
    Conceptualization, Supervision, Funding acquisition, Investigation, Writing – review and editing
    For correspondence
    sunjh@ioz.ac.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9465-3672

Funding

National Natural Science Foundation of China (32088102)

  • Jianghua Sun

National Natural Science Foundation of China (32061123002)

  • Jianghua Sun

National Key Research and Development Program of China (2021YFC2600100)

  • Jianghua Sun

National Natural Science Foundation of China (32230066)

  • Lilin Zhao

National Natural Science Foundation of China (31970466)

  • Jiao Zhou

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was funded by the National Natural Science Foundation of China (32088102, 32061123002, 32230066 and 31970466) and National Key Research and Development Program of China (2021YFC2600100). We are grateful to Yujia Xiang and Jin Ge for the assistance in bioinformatic analysis. We thank Kathryn E Bushley for reviewing the manuscript.

Senior Editor

  1. Dominique Soldati-Favre, University of Geneva, Switzerland

Reviewing Editor

  1. Sofia J Araujo, University of Barcelona, Spain

Version history

  1. Preprint posted: March 31, 2022 (view preprint)
  2. Received: November 1, 2022
  3. Accepted: February 21, 2023
  4. Version of Record published: March 30, 2023 (version 1)

Copyright

© 2023, Tang, Zhou, Koski et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 559
    Page views
  • 121
    Downloads
  • 0
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Xuan Tang
  2. Jiao Zhou
  3. Tuuli-Marjaana Koski
  4. Shiyao Liu
  5. Lilin Zhao
  6. Jianghua Sun
(2023)
Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode
eLife 12:e84621.
https://doi.org/10.7554/eLife.84621

Further reading

    1. Cell Biology
    2. Developmental Biology
    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

    1. Developmental Biology
    2. Stem Cells and Regenerative Medicine
    Irina AD Mancini, Riccardo Levato ... Jos Malda
    Research Article

    During evolution, animals have returned from land to water, adapting with morphological modifications to life in an aquatic environment. We compared the osteochondral units of the humeral head of marine and terrestrial mammals across species spanning a wide range of body weights, focusing on microstructural organization and biomechanical performance. Aquatic mammals feature cartilage with essentially random collagen fiber configuration, lacking the depth-dependent, arcade-like organization characteristic of terrestrial mammalian species. They have a less stiff articular cartilage at equilibrium with a significantly lower peak modulus, and at the osteochondral interface do not have a calcified cartilage layer, displaying only a thin, highly porous subchondral bone plate. This totally different constitution of the osteochondral unit in aquatic mammals reflects that accommodation of loading is the primordial function of the osteochondral unit. Recognizing the crucial importance of the microarchitecture-function relationship is pivotal for understanding articular biology and, hence, for the development of durable functional regenerative approaches for treatment of joint damage, which are thus far lacking.