Feeding behavior increases strength and frequency of vibrational communication signals of Neoaliturus tenellus (Hemiptera: Cicadellidae)

Chapter 1

Feeding behavior increases strength and frequency of vibrational communication signals of Neoaliturus tenellus (Hemiptera: Cicadellidae).

Leafhoppers (Hemiptera: Cicadellidae) use substrate-borne vibrations to communicate, and characteristics of these vibrational signals help identify, locate, and assess suitability of potential mates. The strength and frequency composition of signals influence behavioral responses in conspecifics. Leafhoppers are known to produce these vibrational signals while probing, but it is unclear whether this dual activity degrades signal transmission, inducing a trade-off between nutrient acquisition and mate attraction. In contrast, we hypothesized that inserted mouthparts could provide an additional point of contact with the plant, improving signal transmission. Here, we combine simultaneous electropenetrography and accelerometer recordings to assess how beet leafhopper [Neoaliturus tenellus (Baker)] vibrational signals vary with probing behavior, including xylem ingestion, phloem salivation/ingestion, and probing of epidermis and mesophyll cells. Signals documented during six-hour recordings of male leafhoppers had significantly higher amplitude and dominant frequency when leafhoppers were probing than when they were not probing. However, the dominant frequency of signals was similar when leafhoppers were engaged in pathway phase, phloem ingestion, and xylem ingestion. Of the different probing behaviors, xylem ingestion had the strongest positive effect on signal amplitude, but phloem ingestion did not influence signal amplitude. Additional contact between the leafhopper and the plant surface, provided by leafhopper mouthparts, may improve vibration transmission, potentially increasing signal active space. In light of our finding that the act of probing plant tissues influences information conveyed in leafhopper vibrational signals, we suggest further research to evaluate the impacts these signal changes have on the behavior of other mates and natural enemies.

Introduction

Vibrational communication is prevalent among insects, with an estimated one hundred ninety-five thousand species using substrate-borne vibrations either exclusively or with other communication modality to convey information. Characteristics of these vibrations are highly species- and individual-specific, as they are under strong selection pressure from environmental conditions, natural enemy responses, and mate preference. Stronger signals have a greater effective transmission range and can be better perceived above background noise, thus increasing the chance of reaching the intended receiver and leading to successful communication. However, stronger signals are not always beneficial in the context of eavesdropping natural enemies.

For leafhoppers (Hemiptera: Cicadellidae), vibrational communication plays an essential role in mate location and courtship. Males usually produce a calling song to identify a potential mate, and a successful male call is then followed by a duet for mate localization, and eventually copulation. Many leafhoppers use a specialized organ, the tymbal, to produce vibrations that propagate through a plant and which can be perceived by conspecifics using mechanoreceptors located in the legs or abdomen. Vibrations are thought to travel from the leafhopper tymbal to the plant through any points of contact between the insect and the plant surface, after which they can be perceived up to several meters away on a continuous substrate.

To maximize signaling space on a plant, male leafhoppers often use a "call-fly" strategy, in which the male produces a series of vibrational signals on a leaf, waits for a female reply, and if no reply is perceived, he moves to another leaf on the plant and repeats this process until a duet and subsequent copulation occurs. This strategy is widespread within the Cicadellidae. Although, once sexually mature, male leafhoppers may be sexually active throughout the majority or entirety of their lifespan, suggesting that they must balance feeding and reproduction throughout their life. Indeed, males of multiple different leafhopper species have been observed to produce signals while probing plant tissues and this has been hypothesized to represent a way to minimize the trade-off between energy replacement/supplementation and mobility that leads to reproduction. However, it is not known if signal characteristics are affected by feeding. On one hand, the rostrum serves as an additional point of contact for the insect and could improve signal strength by increased connectivity with the plant. Furthermore, it is possible that higher frequencies (greater than one thousand Hertz) would propagate more effectively while probing due to increased connectivity, as herbaceous plants often function as low-pass filters in which higher frequencies attenuate more rapidly with distance than lower frequencies, although this relationship is difficult to predict. On the other hand, signaling is energetically demanding, and signals produced while probing could be lower in strength as a result of the additional energy expenditure. Therefore, we propose three mutually exclusive hypotheses: (i) a feeding-communication tradeoff, where probing reduces signal strength and frequency pattern, (ii) a feeding-communication facilitation where probing increases signal strength and frequency pattern, or (iii) a scenario where probing behavior has no influence on signal transmission.

Historically, leafhopper signaling while probing has only been visually observed, without clear determination of whether leafhoppers are actively feeding from the phloem or xylem, or if the stylets are located within other plant tissues from which they do not necessarily feed. Electropenetrography is a powerful tool for quantifying probing behavior of piercing-sucking insects, allowing precise quantification of feeding durations within different plant tissues over time. Electropenetrography involves passing an electrical current through an insect and a plant and analyzing patterns of voltage changes over time which correspond to known feeding activities within different plant tissues. While it is established that insect feeding activity is influenced by various types of vibrational stimuli, to our knowledge, Electropenetrography and substrate-borne vibrational recordings have not been applied simultaneously to assess interactions between feeding and communication in an individual insect. Pairing measurements of vibrational behavior with precise feeding observations would help identify impacts of feeding on vibrational communication.

The beet leafhopper, Neoaliturus tenellus, is an ideal model organism to assess impacts of feeding on communication. Due to its economic importance as a vector of beet curly top virus and potato purple top phytoplasma, considerable research effort has been dedicated to characterizing N. tenellus behaviors. Vibrational signals and mating behaviors of N. tenellus have previously been recorded, and the morphology of the vibration-producing tymbal organ has been characterized. Recently, male beet leafhopper signals from these recordings were analyzed in detail to help delineate species in the Neoaliturus species complex. While these original recordings were conducted using current technology at the time, recording the beet leafhopper using updated recording and spectral analysis techniques could provide a useful resource for future work regarding the beet leafhopper or leafhopper vibrational duetting more broadly. For leafhoppers, Electropenetrography waveforms are species-specific and need to be characterized and correlated with probing behaviors using histological methods for each different species that is recorded. Electropenetrography waveforms have been characterized and correlated with probing behaviors of N. tenellus, allowing identification of when N. tenellus is engaged in pathway phase, stylets moving through the epidermis and mesophyll, phloem ingestion and salivation, and xylem ingestion. Simultaneously recording N. tenellus probing behaviors and substrate-borne vibrations allows for an opportunity to identify if communication differs when leafhoppers are engaged in different probing behaviors.

In the current study, we combine Electropenetrography and recording of substrate-borne vibrations to evaluate the potential for feeding-induced changes in vibrational signal transmission of a leafhopper. Specifically, we assess how spectral and temporal characteristics of male N. tenellus vibrational signals differ when leafhoppers are engaged in different probing behaviors on beet, Beta vulgaris. We address the question of whether rostrum contact or insertion allows for improved signal transmission by quantifying metrics that capture the strength and frequency content of signals and comparing these values between non-probing and probing behaviors within individuals. We evaluate feeding in xylem and phloem separately, along with pathway probing behavior, and compare the number of signals while probing, and signal characteristics, to signal production when non-probing. In addition, to evaluate potential effects of Electropenetrography tethering on our observed behaviors, we record male vibrational signals in the absence of Electropenetrography to compare with signals made by isolated males. Lastly, we recorded the beet leafhopper male-female duet to illustrate the behavioral context in which male signals occur and to provide a base of comparison for future work on beet leafhopper vibrational communication.