DOI: 10.5061/dryad.xksn02vpp
title: Data from: Symbiont infection and psyllid haplotype influence phenotypic plasticity during host switching events

description: Many herbivorous insect species exhibit phenotypic plasticity when using
 multiple hosts, which facilitates survival in heterogeneous host
 environments. Physiological host acclimation is an important part of it,
 yet the effects of host acclimation on insect feeding behavior are not
 well studied, particularly for insect vectors of plant
 pathogens. We studied the combined effects of host acclimation
 and infection with a plant pathogenic symbiont on feeding behavior
 of Bactericera cockerelli, an oligophagous psyllid widespread in
 both crop and natural habitats that feeds primarily on Solanaceae and
 transmits an economically important plant pathogen, Candidatus
 Liberibacter solanacearum (CLso). We used a factorial design and
 the electrical penetration graphing technique to disentangle the effects
 of host acclimation, CLso infection, and psyllid haplotype on the
 within-plant feeding behavior of B. cockerelli during conspecific and
 heterospecific host switches. This approach allows to connect phenotypic
 plasticity with the role of B. cockerelli as a vector by quantifying the
 frequency and duration of behaviors involved in CLso
 transmission. We found significant reductions in multiple metrics
 of B. cockerelli feeding efficiency, exacerbated by infection
 with CLso, which could lead to reduced transmission of this pathogen.
 Psyllid genotype was also important; the Central haplotype exhibited less
 dramatic changes in feeding efficiency than the Western haplotype during
 heterospecific host switches. Our study shows that host
 acclimation and heterospecific host switching directly alter feeding
 behaviors underlying pathogen transmission, and that the magnitude of
 feeding efficiency reductions depends on both host genotype and infection
 status.
description: Plants were taken from the greenhouse and placed in two Faraday
 cages hosted on opposite benches in the same room, one with a Giga-8
 DC-EPG amplifier and the other one with a Giga-4 DC-EPG amplifier (EPG
 Systems, Wageningen, The Netherlands). The plastic saucers beneath the
 pots were filled with water to ensure soil electrical conductivity during
 recordings. We randomly selected a leaf and gently turned it upside down
 to expose the abaxial surface, then secured it to a small piece of
 Styrofoam using a glass slide laid across the surface and affixed by a
 rubber band (if necessary, thin wooden sticks were also used to position
 this arrangement for easy leaf access). We collected fourth instar nymphs
 from select colonies in the morning before setting up EPG recordings. To
 simulate stress associated with foraging for a new host, nymphs were
 allowed to sit without plant access for 4 hours in a 10x1.5 cm petri dish
 (Fisher Scientific, Pittsburgh, PA, USA) sealed with parafilm. After this
 period, each nymph was taken with a paint brush and placed on a 10-µl
 pipette tip connected to a vacuum pump (NestEcho, Chaozhou, China) tube to
 prevent the insects from moving. To create electrical
 circuits that included a plant and psyllid, we tethered each nymph by
 attaching a 12.5 μm thick, 2.5 cm long gold wire (Sigmund Cohn Corp., Mt.
 Vernon, New York, USA) to the pronotum using a small drop of conductive
 water-based silver glue (EPG Systems) under a Bausch & Lomb
 StereoZoom 4 Microscope configured on a boom stand (Cambridge Instruments,
 now Leica, Deerfield, IL, USA) and illuminated by LED-6W dual gooseneck
 lights (AmScope, Irvine, CA, USA). The opposite end of the wire was glued
 with solvent-based silver glue (Dag 503 62% silver coating, Ladd Research,
 Essex Junction, VT, USA) to a copper wire electrode welded to a brass
 nail. Tethered nymphs were connected by the brass nail to the EPG probes
 wired to the DC-EPG amplifiers and placed on the previously prepared
 leaves, hanging a few millimeters away from the leaf, and a second
 electrode was inserted into the soil of each potted plant, close to the
 main stem to ensure contact with the roots, to close the electrical
 circuit.   Once all nymphs were in place, the Stylet+ d
 Windows software (EPG systems) was started, and nymphs immediately lowered
 to touch the leaves. During the initial probes, the output voltage of each
 channel was set to about 2V on the DC-EPG amplifier. We recorded the
 feeding behavior of psyllids from 12 channels simultaneously using the two
 DC-EPG amplifiers over a 14-hour period, during which LED lights on top of
 the Faraday cages were kept on by a timer to maintain a 16:8 L:D
 photoperiod in the room, with the goal of getting at least 20 recordings
 per psyllid/plant combination for each experiment. Each week three to four
 sets of recordings were carried out on a new set of plants, which were
 assigned randomly to EPG channels, and each day a different leaf was
 selected on each plant, and new nymphs were used.
description: # Data from: Symbiont infection and psyllid haplotype influence phenotypic
 plasticity during host switching events
 [https://doi.org/10.5061/dryad.xksn02vpp](https://doi.org/10.5061/dryad.xksn02vpp) This dataset includes raw electropenetrography (EPG) recordings used to disentangle the effects of host acclimation, Candidatus Liberibacter solanacearum (CLso) infection, and psyllid haplotype on the within-plant feeding behavior of Bactericera cockerelli during conspecific and heterospecific host switches. This approach allows to connect phenotypic plasticity with the role of B. cockerelli as a vector by quantifying the frequency and duration of behaviors involved in CLso transmission. ## Description of the data and file structure The EPG recordings have the following naming scheme: Folder "Central_Lso+_mt_yp" refers to EPG recordings of psyllids of the Central haplotype_infected by CLso_reared on MicroTom tomato_trasferred to Yellow Pear tomato. The same naming scheme applies to individual recordings (subfolders), which are uniquely identified by a recording number at the end of the file name. Inside each subfolder there are 14 hourly recordings labelled as ".D01" to ".D14" The EPG recordings are structured as follows: ├── Psyllid haplotype_CLso infection status_rearing host_transfer host (Main folder) ├── Psyllid haplotype_CLso infection status_rearing host_transfer host_recording number (Subfolder) ├── Psyllid haplotype_CLso infection status_rearing host_transfer host_recording number_hour number (hourly recordings) For example: ├── Central_Lso+_mt_yp ├── Central_Lso+_mt_yp_01 ├── Central_Lso+_mt_yp_01.D01 The EPG recordings have been used for three experimental comparisons: | **Experimental comparison** | **Psyllid haplotype** | **Infection status** | **Rearing host** | **Novel host** | **Novel host** | | :-------------------------- | :-------------------- | :------------------- | :--------------- | :------------- | :------------- | | 1 | Western | uninfected | Bell Pepper | Yellow Pear | MicroTom | | | Western | uninfected | MicroTom | Yellow Pear | Bell Pepper | | 2 | Western | CLso+ | MicroTom | Yellow Pear | Bell Pepper | | | Western | uninfected | MicroTom | Yellow Pear | Bell Pepper | | 3 | Western | CLso+ | MicroTom | Yellow Pear | Bell Pepper | | | Central | CLso+ | MicroTom | Yellow Pear | Bell Pepper | ## Code/Software The EPG recordings (hourly files) can be opened by using the "Stylet+ a" Windows software (EPG systems) to mark the beginning of each of the following waveforms: NP: non-probing behaviour; C: stylet pathways in mesophyll and parenchyma; D: initial contact with phloem cells, marked as ‘11’; E1: phloem salivation; E2: passive phloem sap ingestion; G: active xylem ingestion. Files with waveform sequences were then uploaded to the Microsoft Excel Workbook for automatic parameter calculation of EPG data originally developed for aphid waveforms by Sarria et al. (2009) to obtain the following variables: number, duration per insect and duration per event for NP, C, D (as pd-L in the worksheet), E1, E2 and G waveforms; number of single E1 (i.e., E1 followed by C instead of E2); number of sustained E1 (sE1) and E2 (sE2), that is, E1 and E2 events longer than 10 min, respectively; percentage of probing time spent in C, G, E1 and E2 phases and percentage of sE2 over total E2. A separate workbook was used for each insect/plant treatment, and the variables thus cal- culated by individual insects were averaged by treatment using the Summarize with PivotTable function of Excel.