Fluid dynamics in the functional foregut of xylem-sap feeding insects: A comparative study of two Xylella fastidiosa vectors

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Highlights

Abstract

Xylem sap sucking insects are adapted to ingest fluids under tension. Although much has been learned about such feeding strategy, this adaptation still poses several unresolved questions, including how these insects ingest against strong xylem sap tension. Xylem sap-feeding insects are vectors of the plant pathogenic xylem-limited bacterium Xylella fastidiosa. This bacterium colonizes the cuticular lining of the foregut of vectors in a persistent manner. We used micro-computed tomography and scanning electron microscopy to investigate the foregut morphometry of two X. fastidiosa vector species: Philaenus spumarius and Graphocephala atropunctata (Hemiptera: Aphrophoridae and Cicadellidae, respectively). On the basis of morphometric data, we built a hydrodynamic model of the foregut of these two insect species, focusing on the precibarium, a region previously shown to be colonized by X. fastidiosa and correlated with pathogen acquisition from and inoculation to plants. Our data show that space in the P. spumarius functional foregut could potentially harbor twice as many cells as similar space in G. atropunctata, although the opposite trend has been observed with biological samples. Average flow velocity of ingested fluid depended on the percentage of the cibarium volume exploited for suction: if the entire volume were used, velocities were in the range of meters per second. In contrast, velocities on the order of those found in the literature (about 10 cm/s) were attained if only 5% of the cibarium volume were exploited. Simulated bacterial colonization of the foregut was analyzed in relation to hydrodynamics and pressure needed for insects to ingest. Our model is designed to represent the diameter reduction of the food canal in both insect species when infected with X. fastidiosa. Results indicated that full bacterial colonization significantly increased the mean sap-sucking flow velocity. In particular, the colonization increased the maximum section-averaged velocity in the G. atropunctata more than two times and the net pressure needed to mantain the flow in the precibarium when colonized is relevant (about 0.151 MPa) if compared to a standard xylem sap tension (1 MPa). Bacterial colonization also influenced the sucking process of the G. atropunctata, by hindering the formation of a recirculation zone (or eddy), that characterizd the flow in the distal part of the precibarium when bacteria were absent. On the other hand, considering the pressure the insect must generate to feed, X. fastidiosa colonization probably influences fitness of the G. atropunctata more than that of P. spumarius.

Introduction

Xylem sap-feeders are insects adapted to obtain nourishment from an energetically costly and nutritionally dilute substrate (Raven, 1983). These insects have an efficient muscular pump, the cibarium, to suck plant sap under tension. The cibarium is located between the stylets and the esophagus, after which the anatomical foregut or alimentary canal proper starts. The food canal in the stylets are connected to the cibarium through a narrow channel: the precibarium, which is lined with chemosensory papillae separated into two groups by the precibarial valve (Backus and McLean, 1983). Because of the low nutrient content in xylem sap, these insects ingest a large amount of sap. They also generally excrete large volumes of liquid that may reach, in some species, up to 1,000 times their body mass in a 24 h period (Mittler, 1967, Horsfield, 1978). In other words, xylem sap ingestion is energetically expensive, but the mechanics and energy requirements to feed on such diet are yet to be understood.

Electrophysiology and electromyography studies have revealed that xylem sap-feeders have a complex feeding physiology. The rate of the cibarial muscle activity varies, with an average of 1.22 ± 0.05 Hz for the leafhopper G. atropunctata (Hemiptera: Cicadellidae) (Almeida and Backus, 2004) and 0.7 Hz for the spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) (Cornara et al., 2018). Cibarial muscle contraction period of 0.175–0.350 s was recorded for another leafhopper, Homalodisca vitripennis (Dugravot et al., 2008). During each cibarial muscle contraction event, sap fluid passes through the stylets and precibarium, entering the cibarial chamber. Muscle relaxation increases pressure in the chamber, resulting in sap being pushed into the midgut. Additionally, sap could move back to the stylets, through the precibarium; however, that movement does not occur due to the presence of a pressure sensitive check valve (precibarial valve), which blocks flow backwards (Ruschioni et al., 2019). The speed of sap flow into the mouthparts has been estimated to be ~7.8–8 cm/s in G. atropunctata (Purcell et al., 1979) to 30–50 cm/s in H. vitripennis (Andersen et al., 1992), estimates obtained by considering volumes excreted, dimensions of the food canal of these leafhopper species, and sap fluid behaving like water. In other words, available estimates assume that sap flow through the food canal occurs constantly over a period of time. However, from a simplified perspective, sap ingestion occurs at distinct, rhythmically repeating stages, namely fluid sucking from plants into the cibarial chamber, followed by pushing of that fluid into the midgut. Focusing on the actual sap flow in the precibarium, here we analyzed only the first of these two stages of ingestion.

Although the functional morphology of the foreguts of different xylem sap feeders has been studied (Raven, 1983, Backus and McLean, 1983), this particular feeding adaptation still poses a number of questions. First, in piercing the plant tissue with the stylets to feed on xylem, insects must avoid embolization/cavitation of the vessels so that ingestion can occur; the role of salivary sheaths to prevent cavitation during stylet penetration of vessels has been hypothesized (Backus and Lee, 2011, Crews et al., 1998), but remains enigmatic. Moreover, xylem sap is typically under considerable tension; the negative pressure may vary depending on plant site, time of day, and plant condition, and it is often measured down to −3 MPa (Pockman et al., 1995, Kim, 2013). Ingestion in this condition requires the generation of strong pressures, but how these insects generate such pressures is not yet understood. These species have large cibarial muscles and a structurally reinforced precibarium (Backus, 1985, Malone et al., 1999); such morphological adaptations would be compatible with requirements to suck against such tensions (Malone et al., 1999, Novotny and Wilson, 1997). Nevertheless, the maximum tension that muscles can generate has been proposed to be on the order of 0.1 MPa (Raven, 1983, Kim, 2013, Young and Schmidt-Nielsen, 1985). Yet feeding ratios (function of xylem sap nutritional components and tension) of these insects support a capability to pump against −1.8 MPa (Andersen et al., 1992).

The discrepancy among these observations is intriguing. Numerical techniques, based on physical models and boundary conditions derived or deduced from measurement, represent a more precise way to use the measured data, and could help to better understand the observations. Detailed knowledge on the feeding mechanism of xylem sap-sucking insects is also of applied importance because all these species are vectors of the xylem-limited plant pathogenic bacterium Xylella fastidiosa (Sicard et al., 2018), and the development of hydrodynamic models of vector foreguts could be critical in future studies on vector-pathogen interactions. This bacterium has a unique feature among pathogens spread by arthropods. It multiplies in the insect foregut without being circulative in the hemolymph (Almeida et al., 2005). The retention sites in vectors are localized in the precibarium and the cibarium (Almeida and Purcell, 2006, Purcell et al., 1979, Brlansky et al., 1983), but the impacts of bacterial colonization on insect feeding, fitness, and how bacterial inoculation of plants occurs remain to be determined.

The spittlebug P. spumarius and the leafhopper G. atropunctata are important vectors of X. fastidiosa in Europe and California, USA, respectively. The biology of X. fastidiosa transmission by these insects is similar, despite the fact that they belong to different families (Cornara et al., 2016). There are few estimates of X. fastidiosa populations on the cuticular surface of the cibarium and precibarium of insect vectors, but recent studies with P. spumarius indicate that cell populations are reasonably small, with 102-103cells per insect (Cornara et al., 2016, Saponari et al., 2014). On the other hand, populations in G. atropunctata may be small during early stages of colonization, but normally reach ~104cells per insect (Retchless et al., 2014, Labroussaa et al., 2017). The role of different fluid dynamics in the foregut has been hypothesized as a possible explanation (Cornara et al., 2016). Another relevant factor is the role of bacterial colonization on vector fitness. Scanning electron microscopy (SEM) observations of colonized individuals of both insect species reveal the presence of large biofilms on the precibarium (Almeida and Purcell, 2006, Brlansky et al., 1983, Alves et al., 2008), compatible with the assumption that sap-sucking would be negatively impacted by reductions in lumen diameter in that canal. Interestingly, X. fastidiosa cells form a colony of polarly attached cells on the surface of insect vectors (e.g. Almeida and Purcell, 2006, Brlansky et al., 1983, Newman et al., 2004). Whether acquisition of bacteria by insects leads to fitness reduction also remains to be determined.

We propose that sap fluid dynamics in the foregut of insect vectors may explain some of these biological observations, help understand how X. fastidiosa colonizes vectors, and the potential impacts of these interactions on vector feeding and acquisition/inoculation of X. fastidiosa. To test our hypothesis we compared the morphometry and geometry of the precibarium profiles of P. spumarius and G. atropunctata. Photographs of the two insects are reported in Fig. 1.

On the basis of the micro-computed tomography (μCT) reconstructions of the precibarium profiles of these vector species, we developed two hydrodynamic models per insect: i) one not colonized by X. fastidiosa (NC); and another ii) with full X. fastidiosa cell colonization (C), represented by a bacterial biofilm covering the length of the precibarium. We focused on the fluid dynamics associated with sap intake through the precibarium, as that region has been correlated with X. fastidiosa inoculation to plants (Almeida and Purcell, 2006). While limited in scope, the analyses of these models provide novel insights on these interactions; future experimental and quantitative work will be needed to incorporate other components of the system such as sap tension in the plant host as well as insect operation of valves and fluid movement into the midgut.

Section snippets

Insects

Philaenus spumarius and G. atropunctata adults used in the experiments were obtained from the University of California’s greenhouses in Berkeley, from rearing colonies established from individuals collected from field populations in Alameda and Sonoma counties, Northern California. General methods and protocols as for maintaining insects were as previously described (e.g. Cornara et al., 2016, Zeilinger et al., 2018). Morphometric data was generated using measurements from μCT and SEM samples.

Foregut profile

The precibarium (Pr) is a narrow canal, starting from the hypopharyngeal extension, which inserts into the food canal formed by the stylets (St), and ends in the cibarial chamber (Ci) (Fig. 2A, B). μCT and SEM observation reveal that the precibial profile of both species is generally narrow in the distal part (also termed the D-sensillum field; Backus and Morgan, 2011) while it enlarges quickly in the proximal half (also termed the epipharyngeal basin and precibarial trough; Backus and Morgan,

Discussion

The detailed morphometry and the numerical simulations provided a description of the fluid dynamics that occurs in the precibarial canal of the functional foregut of the studied insects. Our analysis of the fluid dynamics provide new details on the flow of ingested sap, assuming that no cavitation occurs, that sap behaves like water, and that the precibarium is evenly tubular. The assumptions on the sap tension at the inflow and on the length of the stylets prevented the model from generating

Data availability

The μCT Images mentioned in section Materials and Methods, limited to the head of the two insects discussed in this paper, are available as images in jpg format at the linkhttps://figshare.com/s/f023261da7c354fdd990,https://doi.org/10.6084/m9.figshare.7322165.

CRediT authorship contribution statement

Emanuele Ranieri: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Gianluca Zitti: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing. Paola Riolo: Writing - review & editing, Supervision. Nunzio Isidoro: Writing - review & editing, Supervision. Sara Ruschioni: Writing - review & editing. Maurizio Brocchini:

Acknowledgments

We acknowledge Brandon Walters from Micro Photonics for his support and help in μCT software analysis. We also acknowledge Guangwei Min and Reena Zalpuri (University of California Berkeley Electron Microscopy Facility) for assistance with microscopy. The research was funded by the California Department of Food and Agriculture PD/GWSS Research Program. We thank E.A. Backus (USDA Agricultural Research Service, Parlier, CA, USA), C. Loudon (University of California, Irvine, CA, USA), and one

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