Anatomy of an agricultural antagonist: Feeding complex structure and function of three xylem sap‐feeding insects illuminated with synchrotron‐based 3D imaging

Many insects feed on xylem or phloem sap of vascular plants. Although physical damage to the plant is minimal, the process of insect feeding can transmit lethal viruses and bacterial pathogens. Disparities between insect‐mediated pathogen transmission efficiency have been identified among xylem sap‐feeding insects; however, the mechanistic drivers of these trends are unclear. Identifying and understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases. Here, we applied synchrotron‐based X‐ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap‐feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue‐green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy‐winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae). The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex. The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions. Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities. These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases.Here, we applied synchrotron-based X-ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap-feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue-green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy-winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae).The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex.
The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions.Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities.
These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

| INTRODUCTION
Insects within the suborder Auchennoryncha such as sharpshooter leafhoppers, spittlebugs, and cicadas feed on the xylem sap of plants.Several distinctive challenges are associated with this lifestyle.While phloem is rich in sugar and amino acids, xylem sap is nutritionally poor; as such, these insects must extract a large amount of fluid from the plants to meet their metabolic needs (Raven, 1983).It has been estimated that daily rates of ingestion may reach 1000 times the mass of the insect (Mittler, 1967) and that the rate of fluid flow through their mouthparts likely surpasses 1 m/s (Ranieri et al., 2020).Furthermore, xylem sap is energy intensive to extract: while phloem is under positive pressure and can be extracted passively, xylem sap is under negative pressure (i.e., tension), and extraction requires the application of large amounts of force to overcome the pressure differential between the plant and insect (Bergman et al., 2021;Raven, 1983).Many questions remain unanswered regarding this unusual feeding strategy, such as how xylem sap-feeding insects generate the forces necessary to overcome the extreme negative pressures of the xylem vessels and how they are able to sustain such a large daily fluid intake.Understanding xylem sap ingestion in these insects is of applied relevance because they can transmit the xylem-limited bacterial plant pathogen Xylella fastidiosa over the course of feeding (Sicard et al., 2018).The threat of X. fastidiosa to agriculture continues to increase globally, and without curative methods available to limit disease impacts, further investigation to better understand the biology of this system is necessary.
Xylem sap-feeding insects are nested within the monophyletic suborder Auchenorryncha (Cryan & Urban, 2012) within the order Hemiptera; the suborder includes members of the Aphrophoridae (superfamily Cercopoidea, spittlebugs and froghoppers) and Cicadidae (cicadas) as well as members of the Cicadellidae (leafhoppers; Cryan & Urban, 2012).The most notable vector of X. fastidiosa in the United States has historically been the leafhopper Graphocephala atropunctata (Cicadellidae: Cicadellinae) (Tumber et al., 2014); however, the spread of diseases caused by X. fastidiosa in California have increased following the introduction of an invasive leafhopper, Homalodisca vitripennis (Cicadellidae: Cicadellinae) (Redak et al., 2004;Tumber et al., 2014).The meadow spittlebug Philaenus spumarius (Cercopoidea: Aphrophoridae) is an important insect vector of X. fastidiosa in Europe (Cornara et al., 2019) and is also present in California, along with G. atropunctata and H. vitripennis.Collectively, they represent a diverse set of insects that have all evolved to feed on xylem sap, which presents an opportunity for exploring the functional similarities across xylem sap-feeding taxa.
Differences in transmission efficiency among these X. fastidiosa vectors have been known for some time.For example, in grapevines, G. atropunctata is more efficient than H. vitripennis in transmitting X. fastidiosa (Almeida & Purcell, 2003;Daugherty & Almeida, 2009;Purcell, 1979;Purcell & Saunders, 1999).Disparate anatomy and relative sizes of components of the feeding complex between taxa may have a role in these differential transmission rates.The structure of the functional foreguts of xylem sap-feeding insects have been examined through dissection with traditional microscopy, scanning electron microscopy (SEM), and microcomputed tomographic (micro-CT) imaging (Backus et al., 2012, Backus & McLean, 1982;Bergman et al., 2021;Killiny & Brodersen, 2022;Leopold et al., 2003;Ranieri et al., 2020;Ruschioni et al., 2019;White et al., 2021).However, a high-resolution comparative examination of the 3D structure between species remains to be conducted and represents a necessary step toward understanding the physiology of ingestion and bacterial transmission.Here, through the application of synchrotron-based Xray micro-CT imaging, we generated 3D images and digital reconstructions of key internal features of the feeding complex of xylem sap-feeding insects G. atropunctata, H. vitripennis, and P. spumarius, including the cibarial dilator musculature, the food canal, the precibarial valve, and salivary organs.The application of this technique revealed new anatomical details critical to illuminate aspects of the process of xylem sap ingestion and highlighted important differences between taxa with functional implications.
1.1 | General anatomy of the feeding complex Xylem sap-feeding insects have a specialized feeding complex to aid in sap extraction and ingestion.These three insect species have a homologous suite of components within the head integrated to form the feeding complex (Figure 1): the stylets and food canal, the precibarium and the precibarial canal, the cibarium, the salivary organs, and their associated musculature.The distalmost part of the feeding apparatus is made of interlocking maxillary stylets forming the food and salivary canals, which are surrounded by two mandibular stylets (stylet fascicle; Backus, 1985; Figure 2, Supporting Information: Figures S1, S2).The interlocking portion is positioned within the crease of the three segmented labium.The labrum is a segment adjacent to the labium and stylet fascicle, approaching a cone-like shape.To initiate feeding, the insect inserts these modified mouthparts into the plant.Next, movement of the stylets within the plant allows the insect to probe for a suitable xylem vessel.
The hypopharynx and the epipharynx are fused to form the precibarium, which houses the precibarial valve and precibarial canal (Backus & McLean, 1982;Ruschioni et al., 2019).Near the proximal interlocking juncture of the stylets, a hypopharyngeal extension (Almeida & Purcell, 2006) connects the precibarium and the food canal so that the fluid within the food canal flows into the precibarial canal, with a ~120°angle formed at this juncture.The cibarium (Figure 2) is a chamber connecting the precibarium and the esophagus (Nault & Ammar, 1989).Contraction of the cibarial dilator muscles creates negative pressure, which pulls fluid into the cibarium; the collapse of the cibarial diaphragm following muscular relaxation pushes the fluid in the cibarium through the esophageal valve toward the esophagus and into the midgut.Here, we report several new anatomical features revealed through this 3D digital visualization of the feeding complex.

| Functional anatomy of the precibarium and precibarial valve
Although the rate of fluid flow is dictated mainly by the rate and degree of contraction of the cibarial pump, the direction of fluid flow is controlled in the insect by a few physical mechanisms: the precibarial valve and the esophageal valve.These valves are located on either side of the cibarial pump: the esophageal valve is located proximally and the precibarial valve on the distal end.The precibarial valve bisects the length of the precibarial canal and controls the flow of fluid from the food canal within the stylets into the cibarium (Backus & McLean, 1982).The esophageal valve is a passive one-way valve that permits flow of fluid from the cibarium to the esophagus/ midgut and prevents reversal of flow (Ruschioni et al., 2019).
However, the precibarial valve is actively controlled by the contraction of precibarial valve musculature.Therefore, the closing mechanism of the valve within the precibarium has critical implications for the flow of fluid in this system.Furthermore, colonization of bacteria within the precibarium has been found to correlate with successful transmission of X. fastidiosa during feeding (Almeida & Purcell, 2006).
As such, understanding the mechanistic role of the precibarium and the precibarial valve in fluid flow is therefore critical to illuminating the transmission dynamics of this pathogen.Furthermore, the mechanism of closure of the precibarial valve has been debated.The precibarial valve of leafhoppers was first described in Backus and McLean (1982) as a unit of cuticle located half-way along the precibarium.The base of this unit (subsequently described elsewhere as the "flap") was attached to the proximal edge of a "pit," and the pit was described as being connected to the apodeme of the precibarial valve muscle (see Figure 2 of Backus & McLean, 1982).The mechanism for valve closure suggested was that when the muscle contracts, the flap hinges downward (i.e., the far end of the valve swings toward the hypopharynx), effectively closing the food canal (Backus & McLean, 1982).Ruschioni et al. (2019) provided a disparate interpretation of the anatomy of the precibarial valve with consequences for its underlying function.In contrast to Backus and McLean (1982), a larger portion of the precibarium was considered to work as the valve in addition to the cuticular flap.The cuticular flap, as in Backus and McLean (1982), is described as articulating with the edge of the pit; however, here the rim of the pit is referred to as the "ring" and the pit itself is called the "bell-like invagination."The precibarial valve muscle is called the "basin-like muscle," and the muscle was interpreted to attach to a "basin-like structure" that sits proximally to the flap along the epipharynx (see Figure 3 in Ruschioni et al., 2019).This is notably in contrast to the Backus and McLean (1982) interpretation, where this muscle was attached to the bell-like invagination (i.e., the pit).In

| X-ray micro-CT imaging and data analyses
The specimens were scanned at beamline 8.3.2 of the Advanced Light Source at the Lawrence Berkeley National Laboratory.X-ray images were generated over the course of two to five scans for each specimen using a 4x objective lens.Four specimens of G. atropunctata, four specimens of H. vitripennis, and two specimens of P. spumarius were scanned, reconstructed (Gürsoy et al., 2014), and measured.Specimens were prepared for scanning using the protocol outlined in Wood and Parkinson (2019).They were stored in 70% ethanol, stained overnight before scanning in Lugol's solution, and washed in 70% EtOH for 30 min.Specimens were then placed within a pipette tip containing 70% EtOH for scanning.The pipette tip was affixed to a wooden rod and sealed on either end with epoxy.~1750 unique images were generated for each specimen respectively and used to form reconstructed 3D images with a voxel size of 1.605 μm.
The digital volume was visualized using Avizo Lite v. 9.2.0 (Thermo Fisher Scientific) and Dragonfly version 2021.3 (Object Research Systems), and a semiautomated volume thresholding tool was used to demarcate different anatomical features.Two dimensional measurements were performed in Dragonfly using the measurement tool.

Volume was calculated using a region of interest demarcation tool in
Dragonfly in which voxels were selected based on a threshold of greyscale values (which is an indication of relative density).The cibarial dilator muscles and the large, continuous cavity within the stylet fascicle (i.e., the food canal) and precibarium (i.e., the precibarial canal) were selected using this tool to measure the volume of these regions.As each voxel is a cube with a known edge length (in this case, 1.605 µm), the volume value given by Dragonfly represents the sum of the area of the voxels selected.The volumes of interest were each converted to a mesh and exported separately as a stereolithography 3D object file (STL).Meshlab (Cignoni et al., 2008) was used to remove external voxels, simplify larger meshes, and convert the files to wavefront (OBJ) (Clark et al., 2023).The meshes were then visualized and imaged using Maya (Autodesk).

| The precibarium and precibarial valve
In all specimens examined, the cuticular precibarial valve flap is down, completely blocking the canal within the valve so that the fluid in the distal and proximal portions of the precibarium are separated (Figure 3 and Supporting Information: Figures S3, S4).The precibarial valve muscle is attached to the proximal portion of a pit near the base of the cuticular flap (Figure 3 and Supporting Information: Figures S3, S4).

| The cibarium and the cibarial dilator muscles
In each insect examined, we found that these units consist of approximately 30 separate fan-shaped muscles arranged in a

| The salivary complex
Xylem sap-feeding insects rely on an internal salivary complex to produce and deliver saliva through the stylets.The stylet fascicle contains a salivary canal at the dorsal boundary between the interlocking maxillary stylets.At the juncture where the stylets diverge, the anterior salivary duct (ASD) connects the salivary canal within the stylet fascicle to the salivarium (Figure 6).The salivarium has two structures connected to the proximal end: a reservoir ventrally and a common salivary duct (CSD) dorsally.The reservoir is attached to a v-shaped bipinnate muscle unit (Supporting Information: Figure S5).The internal tubing (lumen) of the CSD is surrounded by wall cells (Supporting Information: Figure S6).The CSD diverges in two away from the base into the efferent salivary duct (ESD) and wraps sinusoidally before diverging laterally from the base.Terminology to describe the morphology of the salivary complex was adapted from Wang et al. (2021).

| Interspecific disparities in scaling
The dimensions of the food canal and the precibarial canal vary among species due to the notable size differences between these insects (Table 1).For instance, the food canal in G. atropunctata, on average, was around half the size and volume of that in H. vitripennis.Furthermore, we found striking differences in the size of the muscles between the specimens of the different species examined here.The length of the cibarial dilator musculature of H. vitripennis was approximately 150% that of G. atropunctata, and the muscle volume of H. vitripennis was almost an order of magnitude larger than that of G. atropunctata (with an average of 1.54 mm 3 in H. vitripennis, 0.42 mm 3 in P. spumarius, and 0.16 mm 3 in G. atropunctata, see Table 1 and Supporting Information: Table S1).

| The precibarium and the precibarial valve
The 3D images generated here allowed us to observe that the flap within the precibarial valve completely closes in each taxon, effectively sealing the distal precibarial canal off from the rest of the system (Figures 2 and 4).This would prevent fluid backflow and air transmission within the canal, which would improve feeding efficiency (Figure 7).The data also indicate that the precibarial valve is closed when the precibarial valve muscle is relaxed.We used this high-resolution visualization of the 3D morphology to evaluate hypotheses related to the mechanism of valve closure that have been proposed.Our 3D imaging data in all three insect taxa examined show that the muscle is not attached to the basin-like structure (Figure 3 and Supporting Information: Figures S3, S4 The anterior salivary duct is connected to the proximal end of the salivarium (purple).The salivarium is connected at the distal end to the reservoir (gray) and the common salivary duct (light blue).Scale bar is 0.25 mm.Terminology used to describe the morphology of the salivary complex was adapted from Wang et al. (2021).
to the position of the muscle attachment and our observation of full valve closure, we can infer that movement of liquid into the bell-like invagination may not be necessary to fully close the food canal as proposed by Ruschioni et al. (2019).However, we hypothesize that the bell-like invagination and basin-like structure may still have a role in feeding.These areas, for instance, may enable recirculation of fluid moving toward the valve when it is closed.Our 3D imaging data reveals the morphology of the closed valve; as such, the specific angles of the flap in relation to the hypopharynx during feeding in vivo remain to be determined.High resolution imaging data from different sap ingestion stages will be necessary to address some of these questions.

| The cibarium and the cibarial dilator muscles
The cibarial dilator muscles consist of two bipinnate units symmetric across the central apodeme (Chapman, 1998;Snodgrass, 1935).The T A B L E 1 Average measurements of the feeding complex across specimens examined in this study.F I G U R E 7 Simplified schematic of the anatomy of the feeding complex (left) and a diagram indicating how the different parts of the feeding complex function to facilitate ingestion (right).The 3D imaging results show that the precibarial valve is capable of full closure, which would seal the distal precibarial canal and food canal off from the rest of the system.This would facilitate feeding efficiency and may have implications for the dynamics of bacterial transmission.The precibarial valve muscle is attached at the base of the flap, so that contraction of this muscle would pull the valve open.
muscles on the proximal membrane of the cibarium have been previously described as a two-unit muscle system (Chapman, 1998;Snodgrass, 1935).We found that these units are each comprised of a series of ~15 separate fan-shaped muscles (Figures 4 and 5).As the insects would be able to control the contraction of these muscles independently, instead of as a single unit, this would theoretically provide an exponentially higher number of possible coordination patterns to achieve different degrees of cibarial inflation, both in terms of total volume and the spatial distribution of the fluid.This would permit increased control over the rate and volume of fluid flow through the feeding complex.It is also possible that this may provide a level of redundancy in case of damage.This multi-muscle control scheme suggests that the nature of cibarial inflation is more dynamic than under a two-muscle system, and further in vivo study of cibarial contraction rates, fluid flow, muscle physiology, and cibarial pumping mechanics are necessary to understand the implications of the morphological disparities identified here.

| The salivary complex
The salivary organs remain poorly studied for this group of insects.
We identified a number of organs in the distal salivary production complex, including the ASD, the salivarium, the CSD, reservoir, ESD, lumen, and wall cells (Figure 6 and Supporting Information: Figures S5, S6).The overall structure of the organs and their arrangement are similar to that in Nilaparvata lugens, a phloem sap-feeding planthopper in which these structures have also been digitally reconstructed in 3D (Wang et al., 2021).Saliva is produced in glands upstream and is transported through two salivary ducts.These merge to form a CSD, which is connected to a cup-shaped vessel known as the salivarium.A reservoir is connected to the proximal end of the salivarium.Dilator muscles (i.e., dilator of the salivary syringe) surround the reservoir.In N. lugens, the reservoir expands upon dilator contraction, pumping saliva from the CSD through the salivarium and into the reservoir, which is likely the same mechanism for inflation of the salivarium employed in the insects examined here (Supporting Information: -Figure S5).The salivarium is connected distally to the ASD, which in turn is connected to the stylet fascicle.Relaxation of the dilator muscles would move saliva from the reservoir toward the ASD and the stylet fascicles.

| Interspecific disparities in scaling
Observed interspecific scale disparities in components of the feeding complex such as the cibarial dilator muscles, the food canal, and the precibarial canal likely translate to variation in fluid transport rates.
The observed disparities of X. fastidiosa transmission efficiency between these vectors (Almeida, 2007;Almeida & Purcell, 2003;Cornara et al., 2016;Daugherty & Almeida, 2009) may be due to consequences of differences in size and structure of the feeding complex between these insect vectors; further study integrating muscle force application capabilities and fluid flow dynamics with these 3D morphological reconstructions will investigate this.
Dimensional measurements presented here were similar to other studies that have used SEM data (e.g., Almeida & Purcell, 2006;Rapicavoli et al., 2015) and 3D imaging data (Killiny & Brodersen, 2022;Ranieri et al., 2020) to visualize aspects of the sharpshooter feeding complex.The digital approach for measuring volume used here represents a promising tool in terms of speed and accuracy for calculating volume of critical areas of fluid flow and for calculating force application capabilities of the musculature.The method of preparation, although well-suited for visualizing insect soft tissue in 3D (Wood & Parkinson, 2019) can cause dehydration which may reduce the fidelity of the volume measurements of the samples (Metscher, 2009); alternative staining approaches may provide more accurate volume measurements (e.g., Swart et al., 2016).Nevertheless, the approach applied here represents the most highresolution and accurate technique for measuring 3D volume of these anatomical features to date.

| CONCLUSIONS
Here, we applied synchrotron-based micro-CT imaging to visualize the feeding complex of three important xylem sap-feeding insect vectors of plant pathogens in 3D.We found that the volume of the cibarial dilator musculature in H. vitripennis was much larger compared to the other insects examined (e.g., over an order of magnitude larger than the cibarial dilator musculature of G. atropunctata), which indicates a dramatic difference in force application capabilities for fluid pumping between these insects.We found that the precibarial valve's flap closes completely, revealing an additional level of control over fluid flow in the feeding complex and a factor that may affect pathogen transmission.The attachment site of the precibarial valve muscle was found to differ from previous interpretations, which resulted in an updated description of the mechanism for how the precibarial valve functions.We were also able to identify organs involved in salivary production and transport.
These findings illuminate important functional aspects of the feeding complex, demonstrating the power of 3D imaging, modeling, and volumetric measurement techniques for improving our understanding of these and other important insect vectors of plant pathogens.
Future work can integrate the 3D anatomical models of the food canal and precibarial canal presented here into computational fluid dynamics simulations to analyze the influence of the disparate morphological features between insects identified here as well as illuminate how the full closure of the precibarial valve would impact the dynamics of sap flow and bacterial transmission.
The dynamics of fluid flow about the precibarial valve remain understudied, and a full high-resolution 3D visualization of the anatomy is necessary to understand this process.It has been unclear if the precibarial valve fully closes.This has critical implications for the dynamics of fluid flow within the feeding canal, as the precibarial valve is proximal to the point at which the canal interfaces with the atmosphere and the xylem vessel.If the flap within the precibarial valve cannot fully seal the end of the canal from the rest of the system, fluid could continuously flow from the proximal areas of the canal, such as the cibarium, into the food canal of the stylet fascicle.This could, for example, cause fluid to leak from the tip of the stylets and allow air to enter the cibarium after extraction from the xylem vessel.However, if the precibarial valve flap completely seals off these two areas, it would limit backflow from the proximal precibarial canal and keep the liquid inside the canal proximal to the precibarial valve under tension when the stylets are extracted from the xylem vessel.We used our 3D imaging data to determine whether or not the precibarial valve fully closes.F I G U R E 1 3D structure of the head of (a) Homalodisca vitripennis, (b) Graphocephala atropunctata, and (c) Philaenus spumarius generated through synchrotron-based micro-CT imaging in frontal (top) and lateral (bottom) views.Includes the internal structure of the feeding complex (rightmost column in a, b, and c).Scale bars are 0.25 mm.

F
I G U R E 4 Front view of Homalodisca vitripennis (a), Philaenus spumarius (b), and Graphocephala atropunctata (c) revealing the structure of the cibarial dilator muscles in pink.External anatomy of the insect is at 90% transparency.Data represents a 3D reconstruction generated from micro-CT data.Scale bar is 0.25 mm.micro-CT, microcomputed tomographic.F I G U R E 5 The cibarial dilator muscle unit in front (left), lateral (middle), and posterior (right) view, with and without the cibarium (above and below, respectively).Central apodeme bisects the two muscle units.Color gradient used to show 30+ individual muscle units with independent apodeme attachment sites.Scale bar is 0.25 mm.bipinnate structure (Figures 4 and 5) that have an apodeme connected along the long axis of the cibarium representing the direction of force application.The opposing end of each muscle is attached to the inside of the head exoskeleton.
).It is attached to the cuticle of the epipharynx at the proximal end of the ring by the base of the flap.As such, contraction of the "basin-like muscle" would not pull the basin-like structure to open the valve as suggested by Ruschioni et al. (2019).Instead, contraction of the muscle would pull the epipharynx directly at the point of attachment (at the base of the flap, i.e., the ring) to open the valve.Furthermore, given the revision F I G U R E 6 Front view (left), closeup front (middle), and lateral view (right) of the organs of the salivary system of Homalodisca vitripennis (a), Graphocephala atropunctata (b), and Philaenus spumarius (c) along with the stylet fascicle (pink), labium, and labrum (green).
Specimens measured in Dragonfly version 2021.3 (Object Research Systems).