Born suckers: the cibarial pump of Philaenus spumarius scales across ontogeny to ensure functional equivalence Free

The dilute sap within a plant's xylem vessels is under negative pressure (tension), as it consists primarily of water that is being pulled from the soil and through the plant before evaporating into the atmosphere. These negative pressures can be immense, routinely exceeding −1 MPa. Despite xylem sap being both nutrient poor and hard to extract, some insects within the sub-order Auchenorrhyncha (order Hemiptera) feed exclusively on this liquid. These insects likely all evolved from a single xylem-feeding ancestor and as a group they represent the sole origin of this mode of feeding in the animal kingdom (Bell-Roberts et al., 2019). To extract xylem sap, they possess an enlarged muscular cibarial pump that can produce negative pressures exceeding those within a xylem vessel. The cibarial pump consists of a bipennate cibarial dilator muscle (CDM) which attaches to an apodeme extending from the midline of a flexible diaphragm covering the top half of a rigid cibarial chamber (Fig. 1). Contraction of the CDM lifts the apodeme and attached diaphragm away from the floor of the chamber, drawing liquid inward. The negative pressure generated within the cibarial chamber can be calculated by dividing the component of the CDM's force acting in the direction of the cibarial diaphragm's displacement by the area of the diaphragm (Fig. 1B–D). As the biomechanical and energetic challenges involved in this mode of feeding are clearly enormous, there may be geometric or energetic constraints on cibarial pump function associated with changes in the insect's size.

A previous allometric analysis of cibarial pump function by Novotny and Wilson (1997) examined a wide range of adult xylem-feeding hemipteran species and concluded that feeding on xylem sap may become more energetically favourable with increasing body size, potentially promoting the evolution of larger-bodied xylem-feeding insects. Based on analysis of external cibarial and mouthpart morphology, this earlier study concluded that ‘cibarial pump load’ L (volumetric flow rate of xylem sap Q divided by CDM volume) scaled with body volume (equivalent to body mass M_b) as M_b^−0.17, while the pressure difference (Δp) required to move xylem at flow rate Q through the insects’ tube-like stylets scaled as M_b^−0.21. Thus, both L and Δp were predicted to diminish as M_b increases, enhancing the efficiency of xylem extraction in larger insects. Crucially, however, this study's focus on adult xylem-feeding bugs did not consider the substantial change in size that occurs in all these species as they develop from a first instar nymph into an adult: all xylem-feeding bugs begin their lives small! We re-examined these conclusions from an intraspecific analysis of cibarial pump function across ontogeny.

One of the most studied xylem-feeding insects is Philaenus spumarius, a globally distributed spittlebug species that is an important vector of the plant disease Xylella fastidiosa (Cornara et al., 2017). Adult P. spumarius have been found to produce pressures of −1.3 MPa within their cibarium (Bergman et al., 2021), allowing them to extract the xylem sap from over 300 herbaceous and woody plant species (Bodino et al., 2020; Hamilton, 1982; Weaver and King, 1954). While the first instar hatches out with a body mass of just ∼0.2 mg, it eventually grows into an adult weighing between 8 and 9.5 mg, equivalent to a 40–45 times increase in body mass (Horsfield, 1978). Yet even the newly hatched first instars apparently feed by inserting their mouthparts into xylem vessels (Horsfield, 1978) and so must encounter the same hydraulic challenges as the adults. Here, we used micro-computed tomography (micro-CT) scans of P. spumarius heads (third instar to adult) to investigate whether the morphology of their cibarial pump displays isometric scaling across ontogeny (where variables scale with body mass M_b according to the square-cube law: length M_b^0.33, area M_b^0.67, volume M_b^1.0), and whether changes in body size are related to changes in the pump's function.

Nymphs and adults of Philaenus spumarius (Linnaeus 1758) (n=1 third instar, n=6 fourth instars, n=8 fifth instars and n=5 adults, as determined from eye width comparisons with a developmental data series in Weaver and King, 1954) were collected from around the UBC Point Grey campus, Vancouver, BC, Canada, between May 2019 and June 2023. Nymphs were first placed on a piece of tissue paper to remove any adhering spittle mass, then all specimens were weighed individually to 0.01 mg on an electronic balance (XPE205 DeltaRange, Mettler Toledo, Greifensee, Switzerland). They were then narcotized in pure CO₂ and photographed on a stereomicroscope to determine their maximum head width (measured from a dorsal image as the maximum distance across the eyes) before their heads were removed and prepared for micro-CT scanning.

Data were analysed and stats were performed in R v.3.5.1 (http://www.R-project.org/) running in RStudio v.1.1.463 (https://posit.co/products/open-source/rstudio/). Each variable was tested for normality using quantile–quantile plots and the Shapiro–Wilk normality test. Body mass and all the variables of interest were log transformed before generating linear regression models with 95% confidence intervals for each variable using the stats (v.3.5.1) package.

The dimensions of the cibarial chamber scale isometrically across development, with neither the volume of the chamber nor the cross-sectional area of the cibarial diaphragm scaling with exponents distinguishable from M_b^1.0 or M_b^0.67, respectively (Fig. 2A,B, Table 1). However, the dimensions of the CDM do differ from isometry in two aspects: CDM volume scales less than predicted with an exponent of M_b^0.85, and this may be attributed to the lower exponent for CDM muscle fibre length (M_b^0.23) (Fig. 2C,E, Table 1). CDM physiological cross-sectional area scales as M_b^0.64 (Fig. 2D), but this exponent is not significantly different from isometry, indicating that relatively shorter muscle fibres explain the reduced CDM volume rather than a reduction in their cross-sectional area and force production capacity. Likewise, the scaling of pennation angle is independent of body mass (M_b^−0.07), with an average empty-cibarium corrected angle of 28.39±1.2 deg (Fig. 2F). Finally, the scaling of maximum suction pressure calculated from these variables is also independent of body mass, scaling with M_b^0.05, which is not significantly different from M_b⁰ (Fig. 2G).

The previous intraspecific analysis of adult xylem-feeding insects focused on two metrics to evaluate the impact of changes in scale on cibarial pump energetics: Pump load L [volumetric flow of xylem sap (Q)/CDM volume] and pressure drop across the mouthparts for a given Q (Δp) (Novotny and Wilson, 1997). Pump load L was predicted to scale as M_b^−0.17 (Novotny and Wilson, 1997), indicating that the energetic requirements of xylem sap extraction decreased with increasing size. However, this result assumes that Q is proportional to the insect's metabolic rate, assumed to be M_b^0.75, while CDM volume was estimated from measurements of the exterior of the frontoclypeus, giving a scaling exponent of M_b^0.94 for cercopids and cicadas. The P. spumarius data presented here, however, show that their CDM volume scales across development as M_b^0.85, significantly lower than predicted from isometry. Using this exponent, and assuming pumping frequency is mass independent, Q should scale with cibarial chamber volume (M_b^0.90), giving an L of M_b^0.05. Xylem excretion rate (also equal to Q) has also been measured empirically across all P. spumarius life stages by Horsfield (1978) and plotting these data shows Q scales with mass as M_b^0.89. Using this value of Q then gives an L of M_b^0.04. These exponents are both close to zero, suggesting it is no more difficult for a newly hatched first instar spittlebug to extract xylem sap than it is for the adult. The close agreement between the scaling exponents for measured values of Q and cibarial chamber volume (0.89 and 0.90, respectively) further supports the assumption that pumping frequency is mass independent.

For P. spumarius, the length of their food canal (the distance from the tip of the stylets to where it joins with the cibarial chamber) l_FC scales as M_b^0.30 [the average of M_b^0.29 from this study (Fig. 2H) and M_b^0.31 for external stylet length measured by Hoffman across all instars; Hoffman, 1983] while its internal radius r is assumed to follow geometric similarity: M_b^0.33 (Novotny and Wilson, 1997). If it is assumed cibarial pumping frequency is mass independent, then Q should scale with the same exponent as cibarial chamber volume (M_b^0.90), resulting in Δp scaling as M_b^−0.13. Or, if Q scales the same as the rates of xylem excretion measured across ontogeny by Horsfeld (M_b^0.89; Horsfield, 1978), Δp then scales as M_b^−0.14. Thus, P. spumarius likely experience a more moderate decrease in Δp across their development than predicted from looking across adult xylem-feeding species (Novotny and Wilson, 1997). However, the importance of this parameter Δp on the energetic requirements of xylem feeding needs additional context: the pressure gradient needed to drive xylem sap through the insects’ stylets is very small relative to negative pressures encountered within the xylem vessel (Δp<0.025 MPa or less than 2% of the maximum calculated cibarial pressure generated by an adult P. spumarius; Ranieri et al., 2020). Thus, it may be concluded that a reduction in the proboscis’ already relatively minor resistance to xylem sap flow will not substantially reduce the cost of xylem sap extraction as body mass increases.

This study reveals that all P. spumarius nymphs generate the same maximum cibarial pressures as the adult bug, and that the energetic requirements of xylem sap extraction are essentially independent of body mass. Thus, contrary to a previous intraspecific analysis of adult xylem-feeding insects (Novotny and Wilson, 1997), across development there is no size-dependent change in the costs associated with this feeding strategy. This conclusion is consistent with the observation that P. spumarius nymphs and adults both insert their mouthparts into xylem vessels (Horsfield, 1978) and largely feed on the same plant species (Weaver and King, 1954). While some changes in host plant preference have been observed to occur across development (Cornara et al., 2017), this can be attributed to changes in the length of the mouthparts: larger individuals possess a longer proboscis housing longer stylets, giving them access to xylem vessels located more deeply within a plant's stem or beneath a thicker layer of trichomes (Hoffman and McEvoy, 1985). Repeating this study on those xylem-feeding species that undergo a switch in feeding location (e.g. from subterranean roots to aerial stems) or plant host species (e.g. from herbaceous plants to trees) when they transition from nymph to adult (e.g. cicadas) could be an important test to determine whether this isometric pattern of cibarial development is common to all xylem-feeding bugs, or whether there are cases where cibarial development does not result in functional equivalence to accommodate changes in the xylem tensions encountered at different stages of development.

Our thanks to the two anonymous reviewers for their constructive comments and suggestions.