Measuring Principles

(ongoing changes)

I   Introduction

1. Preface   2. Primary circuit   3. Experimental setup

II  Theoretical Backgrounds

4. signal sources     5. Physical-electrical properties     6.  Ohm´s law relations     7. Sensitivity to emf- and R-components     8. emf- and R-components entangling                 9. Electrical current

III  Review of EPG measuring systems

1 AC EPG systems   2.  DC EPG systems  3. AC-DC systems  4. Conclusions

Appendix: Other objections to Giga-8 systems

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## I   Introduction

1. Preface

EPG systems used now and in the past can divided into DC, AC, and combined systems. One may go directly to Section III for a review but to understand that section better it is wise reading first Sections I and II.

In the EPG technique, an insect with piercing mouthparts and a plant are made part of an electrical circuit by connecting each to an electrode, one to the insect and one to the plant. Also, other 'arthopodes' with piercing mouthe part (such as a mites or thicks) and other ‘feeding substrates’ (such as a prey animal or an artificial substrate behind a membranecan) be used. The electrodes are connected to the electronic measuring circuit, called 'primary circuit' .

In addition to the measuring principles this article aims to discuss a number of confusing papers (e.g. Backus et al. 2019; Backus & Patterson 2023) containing incorrect interpretations of the Ohm's law relations in the primary circuit. The authors claim that the present and past DC-EPG system design would have unsuitable properties. Though some properties of the primary circuit may still need further experiments, most properties and their signal consequencies are fairly well known at present.

2. Primary circuit

All EPG systems have a similar primary circuit (Fig. 1), but some properties are different and determine what aspects are recorded in a greater or lesser extent. The user controlled voltage supply (Vs) source provides an AC, DC, or mixed AC and DC voltage to the circuit. One lead of the Vs source is connected to ground and the other lead to the plant, mostly the soil in which the plant is growing. The insect on the plant is attached to a thin flexible gold wire electrode that leads to the signal measuring point (M) of the circuit, which is the input of the head stage amplifier (Amp; for DC systems 50x gain).

The fluctuating voltage between the measuring point and ground - Vi across the input resistor Ri (typically 109Ohm) - represents the EPG signal recorded and visualized on the computer. In fact, the amplifier internal resistance (parallel to Ri; Fig.1 dotted line) is much higher (about 1012Ohm) than Ri, and negligible, therefore. The properties of the amplifier is not part of the primary circuit and have no effect on the EPG signal (this also applies to any subsequent circuit for signal processing). Though signal processing in DC systems is restricted to simple amplification, it is more complex in  AC systems (see chapter 10). The voltage fluctuations in the signals shows certain patterns that have been distinguished and described as EPG waveforms characterized by amplitude, frequency, and voltage level. For a number of insects the waveforms have been correlated experimentally with the insect's stylet penetration (probing) activities and the stylet tip positions in the plant tissues and cells.

3. The experimental setup

The EPG device (see page Products) has a main control box and a (mostly 8) separate EPG (measuring) probe units (Fig. 2). The head stage amplifier or EPG probe input at (M) is the most noise sensitive part of the primary circuit. This measuring EPG probe should be mounted inside a Faraday cage to protect the head stage amplifier against noise from 220VAC 50Hz (or 110VAC 60Hz) power cables in the lab. The cage and the device are grounded to each other (alligator clip) or to the water supply or central heating system. Recent DC devices are powerer by a USB cable, used for data transfer to the computer as well.

Plants, insects, and measuring probes should be shielded by placing them inside the Faraday. The measuring probes (coax-)cables (75cm) are are shielded and and allow the control box to be put outside the Faraday cage. This provides noise free operation of its pushbuttons for individual channel (insect) settings: the adjustable voltage supply (Vs, between ±125mV) pushbutton, the -50mV calibration pulse, the gain control (50 to 100x), and the Ri switch button between 1GOhm and 10TOhm (internal amplifier input resistor), All current settings are displayed on the smal device screen. The output signals to the computer are digitized (internal AD converter) and HD stored and real time displayed on the PC monitor using data acquisition software..

II   Signal sources

4. Biological voltage sources

The direct biological sources of the signal voltages are:  1) sources generated by the plant-insect combination; called electromotive force (emf) components, and  2) due to changes of the electrical plant-insect resistance; called R-components. In fact the R-components are not a voltage source as such, but they modulate the amplitude of the internal plant-insect emf-components as well as external plant-insect voltages in the primary circuit.

The emf-components mainly originate from fluid movements in the two capillary stylet canals, these are called streaming potentials. Also, the membrane potentials of plant cells when punctured by the stylets play a role but these provide the less fluctuating voltage levels of the signal: extracellular versus intracellular stylet tip positions. Other internal insect and plant voltage sources such as muscle and nerve potentials in the insect and fluctuating plant cell potentials (e.g. sieve element depolarizations) do not contribute to the signal since they are short-circuited by their surrounding hemolymph and plant tissue conductance respectively.

The R-components, mainly caused by movements of the valves associated with the food and salivary stylet canals. The effect of valve opening is a reduction of the insect resistance - thus increasing the measured fraction of the internal emf-component voltages - and valve closing causing the opposite effect. Some (presumably small) resistance fluctuations may be caused by resistance changes due to a fluctuating composition of saliva and plant sap. The other important voltages sources that are modulated are the external (not biologically related) voltages from the user operated voltage source Vs and the (most of the time rather stable) electrode potentials caused by the insect and plant electrode interfaces acting as a galvanic element due to their metal-electrolyte differences.  More source details and interactions between voltage sources and resistances are discussed in more detail in the next two sections.

5.  Voltage sources in the circuit

The voltage sources playing a role in EPG signals are depicted in Figure 3. The relevant (blue) voltage sources are:

• the electrode potentials Pe1 and Pe2 due to the insect and soil electrode interfaces*, the silver glue/epidemal fluids and copper/soil fluids, respectively. Together these are acting as a galvanic element that is sometimes substantial (between ±200mV*) but they are rather unpredictable and may differ between chanels (insect-plant replicates) and though rather stable later they mostly seem to decrease initially.
• the streaming potentials (Ps) in capillary stylet canals due to fluctuating fluid movements in the capillary stylet canals
• the membrane potentials of punctured plant cells (Pm; -50 to -120mV) of plant cells when punctured by the insect’s stylets. Pm is rather constant but may differ between cell clusters.

Pm and Ps together are representing the emf-components (Vemf) in the EPG signal.

A few other voltage sources shown in Fig.3 are not or less relevant, such as: the negligibly small bi-metal potential(s) by metal-metal wire connections at the insect and the soil electrode side; the bio-potentials (Pb) from insect nerves and muscles that are short circuited internally by the surrounding hemolymph; and the small and rather constant root(-soil) potential (Pr). All preceding voltage sources are summed and indicated here as the voltage sources between the electrodes (Vsbe), the ‘voltage between the electrodes’.

Finally, there is the external supplied voltage source Vs (range ±500mV), which is user controlled.

The voltage supply Vs has two functions:

1. it is used to compensate for the unpredictable electrode potentials (Pe1 and Pe2) adjusting the signal amplitude and voltage level
2. it can be used to distinguish R-components (see next section) in waveforms when used during recording, thus enabling some signal feature details to assist in identifying some waveforms.

In the Ohm’s law considerations of next text sections it is easy distinguish the sum of Vemf and the electrode potentials Pe1&2 as the 'voltage sources between the electrodes' Vsbe, as well as the sum of Vsbe and Vs as V, called 'circuit voltage'. Thus, V is the sum of all major voltage sources in the primary circuit which play a role in the Ohm's law  considerations in the following sections.

* Note: Electrode potentials should be taken seriously as a voltage source (Neher, 1974; Wkipedia). In Backus et al. (2009, 2018, 2020, and 2023) the electrode potentials are neglected or denied, which resulted in a neglect of their compensation by the user controlled DC voltage supply (Vs) and poor signals.

6. Resistances in the circuit

Similar to voltage sources, the resistances in the primary circuit (Fig. 4) mainly contribute to the signal when they fluctuate. Their fluctutions modulate the also fluctuating Vemf amplitude and moreover, they modulate the more stable voltages of the circuit voltage V. Stable resistances as such will cause a stable level of the signal voltage related to the proportions of resistances in the primary circuit in accordance to Ohm's law (next section). The relevant (red) resistances in the primary circuit are:

• the two electrode resistances, the silver glue-insect cuticle contact (Re1) and the copper-soil/plant contact (Re2) arew relatively low and mostly stable. These hardly play a role in the signal, although the insect electrode resistance may sometimes be high or changes to a higher level due to a poor silver glue attachment, affecting the signal properties.
• the internal insect body resistance (Rin) is small and stable.
• the main stable insect resistance is formed by the insect's stylet canals (Rsc) and their associated valves
• the main fluctuating insect resistance is caused by the valves associated with the food canal (Rfv) which causes a main R-component in EPG signals. This is the morphologcal and functionional well known cibarial valve. However, a valve associated with the salivary duct/canal (Rsv) seems to cause another main R-component but morphologcal and functionional its existance is not clear, in spite of the supposed valve activity indications from EPG signals (phloem waveforms E1 and E2 in aphids).
• the stylet tip resistance (Rst) may strongly increase (Rst) for a short period (<1s) when membrane of the plant cell is sealing the stylet tip opening just before the stylets break through the membrane. The increase may reach values of up to 5GΩ and seems similar to the "giga seal" known from voltage clamp experiments in plant physiology.
• Finally, there remain three resistances to be mentioned: the plant root-soil resistance (Rrs), the soil electrode resistance (Re2), and the internal resistance of the voltage supply (Rvs) source. These resistances are small and stable and don't seem to play any role in the signal.

The sum of all resistances above (except Rvs) can indicated as the "resistance between the electrodes (Rbe)", nearly identical to the total circuit resistance (R). The Rbe fluctuation and the amplifier input resistance Ri the primary circuit are determining the ratio between R- and emf-components in the primary circuit; see discussion in next sections.

6. Ohm´s law relations

 To understand how the measured/recorded EPG signal voltage Vi (Fig. 1) depends on the voltage sources and resistances in the primary circuit (Fig. 3 and 4) in accordance of Ohm's law, a simplified diagram of the electrical relevant elements is shown in Fig. 5. _____________________________________________________________________________ Ohm's law:                                V  = I R     or        I = V / R                      [ 1 ]   current I in each serial part:           I  = Vbe / Rbe  =  Vi / Ri                        [ 2 ]             emf -component sensitivity*:      Vi/V = Ri / (Ri + Rbe)                                                                                               = Viemf/Vemf  = Vical/Vcal                [ 3 ]   circuit voltage:                             V  = Vsbe + Vs  =  Vbe + Vi                   [ 4 ] _____________________________________________________________________________

7. Sensitivity to emf- and R-components

The emf-component sensitivity is the response voltage Viemf as the fraction (%) of the original Vemf amplitude, i.e. Viemf/Vemf, which is equal to the Vi/V (equation [ 3 ]). Regardless the fluctuating voltage V, the fraction Vi/V will remain the same at a certain (constant) Rbe value. The Vi/V values (Y-axis) of two assumed Rbe values (107Ω and 109Ω) are calculated and plotted against Ri values in a range of 105-1011Ω (X-axis). The emf-component sensitivity Vi/V are represented by two sigmoid curves, (Fig. 6, yellow 107Ω and blue 109Ω). These emf sensitivity curves have been referred to earlier (Tjallingii 1988, Backus 2019, 2020). Note that the assumed constant Rbe values are used here to explain the emf- and R-sensitivities, in reality Rbe fluctuates, see next section. For example, at Ri=107.6Ω (arbitrary value to show) the emf-sensitivity for the insect resistance Rbe of 107Ω is shown by the black double Vi/V arrow (dashed) between the 0% and the yellow 107Ω Rbe s-curve. The signal voltage Vi in Vi/V is measured voltage across the 107.6Ω Ri. The complimentary Vbe/V value - between the yellow s-curve and the 100% represents the voltage across Rbe is not measured across Ri and is not part of the EPG signal. Nevertheless it was incorrectly claimed by Backus (2019; see further next section) that Vbe/V would represent the R-component of the signal.

The R-component sensitivity is not as straight forward as the emf-component sensitivity. The R-component fluctuation amplitude is caused by Rbe fluctuation. An insect resistance (Rbe) fluctuation range of 107-109Ω will cause a signal fluctuation (amplitude) between the yellow and the blue s-curve (in Vi/V%) at each Ri in the X-axis range (Fig. 6). At an input resistance Ri of 108Ω the Vi/V fluctuation is represented by the ΔVi/V (double black arrow). The absolute value of ΔVi/V fluctuation for all Ri values in the 105-1011Ω range is represented by the black dashed Gaussian curve. The Gaussian curve should be considered as the R-component sensitivity at each Ri. With increasing Ri the R-component sensitivity increases first to a maximum at Ri 108Ω and then decreases. Thus, the maximum R-component sensitivity ΔVi/V (Rbe modulation of V) appears occurs at an Ri value halfway the (log) Rbe 107-109Ω fluctuation, i.e. when Ri=108Ω, about 80% on the Vi/V scale.

What is the role of Vbe? This is the voltage across Rbe (Fig. 5) and represented as a black double arrow (Fig.6, Vbe/V; just arbitrarily chosen here for demonstration at 107.6Ω Ri). Vbe has an inverse sigmoid relationship to Ri on Vi/V scale (not shown here); Rbe vs. Ri. However, Vbe is not part the measured signal and the assumption that Vbe would represents the R‑component sensitivity (Backus, 2019, 2020) is incorrect should be rejected.

The large 107-109Ω ΔRbe fluctuation range in the example here is very unlikely to occur in waveforms during real insect recordings. Though no data of real Rbe fluctuations have been published, a 107-107.25Ω (about 8MΩ) ΔRbe fluctuation range during a spittle bug waveform has recently been measured experimentally (Cornara, unpublished). The spittle bug R-component amplitude maximum at 107.125Ω Ri (value halfway ΔRbe) is only about 12% on the Vi/V scale (Fig.7, maximum of dashed black Gaussian curve and black ΔVi/V double arrow between the yellow and dashed blue s-curve). For aphid E2 pulses (see Fig. 8) a ΔRbe of 109.02-109.04Ω (about 50M) fluctuation range has been measured (Tjallingii, unpublished); maximal 2% Vi/V scale.

The wrong interpretation of Vbe might have been come from misinterpreting the earlier discussed ‘relative responsiveness f ' to R-components (Tjallingii, 1988). The responsiveness f expresses the R-component fraction ΔVi/V of the Vi/V belonging to the lowest Rbe in its fluctuation range, here 107Ω (yellow s-curve):
. Plotting f versus Ri (range 105-1011Ω) results in an inverse s-curve (Fig. 7, two red curves). For a ΔRbe fluctuation between 107-109f increases with decreasing Ri; to nearly 100% at 105Ω Ri and decreases to 0% at 1011Ω Ri. Though the R-component sensitivity (Fig. 7, black dashed Gaussian curve) has an optimum of about 9% at 107.125Ω, f is highest for the lowest Ri. The ΔVi/V R-component decreases too as the R-component sensitivity shows (black ΔVi/V double arrow). Similarly, the f factor depends on the ΔRbe fluctuation, and for the more realistic 107-107.25Ω Rbe fluctuation f will increase to only about 37% ΔVi/V scale at 105Ω Ri (Fig.7, right Y-axis), which is only 0.55-0.99% Vi/V scale (left Y-axis). This very small signal therefore, needs a very high gain to be visusalized. A gain of up to 12000x had to be applied when using such low Ri values in the Backus 2019).  As in Fig.7, the original graph (Tjallingii, 1988) f was also in the same graph and using a separate Y-axis, ΔVi/V and Vi/V respectively. Though possibly confusing, this difference was clearly explained in the 1988 text. The Vi signal decreases when Ri becomes smaller and depending on the fluctuation range may need a higher gain. The common Ri in DC EPG devices is 109Ω and the usual gain of 50-100x is adequate.

The R‑component sensitivity ΔVi/V is the same for DC and AC based EPG devices (primary circuits). The only difference is that the circuit voltge level V in DC systems is a mixture of the fluctuating Vsbe (due to Vemf) and the supplied voltage Vs, whereas V in AC systems does not depend on other voltages in the primary circuit than the user adjusted constant AC voltage (amplitude). Stable voltages such as Pe1&2 was filtered out in AC systems in the past (section 10.1). In DC systems the R-component can be modified (increased or decreased) by Vs adjustments without affecting the emf-components. A higher Vs adjustment will result in a higher voltage V level (Fig. 3) and its ΔRbe modulation amplitude. The question at what Ri a 50:50 emf-:R-component recording will occur is irrelevant therefore: the R-component can be modified manually during recording by Vs adjustment, which can be very helpful in waveform identification (next section).

8. Interactions between emf- and R-components

8.1 Entangling and interference

Most R- and emf-components in EPG signals are not separately contributing to the signal. Their fluctuations are often similar and coinciding since they have an underlying biological activity in common. For example, when the cibarial valve in the food canal opens the electrical insect resistance (Rbe) will decrease and causes a higher R‑component voltage. Meanwhile the phloem sap flux in the stylet canal increases and causes a higher streaming potential (Vemf voltage) as well. Thus both components coincide and contribute entangled to the signal. When the emf- and R-component have the same sign they will reinforce each other but if they have an opposite sign they will counteract.

For example, the characteristic E2 spikes are clear when the V level is made negative by Vs adjustment (Fig. 8, bottom V‾ trace). When the V level is adjusted by Vs to a 0Volt level (V0 trace) only the small negative emf-components of these E2 spikes are shown (V0 trace). Further positive V level increase shows no spikes (V+ trace). Then the negative emf-component of the E2 spikes are compensated by the positive going R-component of these spikes. And when the V level is increased further, the E2 spikes appear again, now positive (trace V++). The sign switch and the amplitude changes point to a clear R-component of the E2 spikes.

Measured calibration pulse responses applied during a negative (V‾) and positive voltage Vs adjustment (V++) allowed calculation of Rbe. A 1.9GΩ Rbe was derived from the -50mV calibration pulse responses during the E2 level and 1.8GΩ Rbe was calculated from E2 spike responses before and during the calibration pulses. Thus during the E2 spike Rbe appeared to decrease 100MΩ as compared to Rbe during the E2 level between the spikes. These measurements were shown by E2 spikes in Myzus persicae on Raphanus sativus (Tjallingii unpublished).

8.2  Simulated interactions

In order to demonstrate the interactions between emf- and R-component a spread-sheet model was used. The calculated Vi responses (Fig. 9) as caused by a sinewave fluctuating Vemf source voltage (ΔVemf 0±5 mV) is recorded at 5 voltage V levels (-20, -10, 0, 10, and 20 mV; as set by Vs adjustment) using a constant or sinewave fluctuating Rbe. The calculated responses for a constant Rbe (Fig. 9A) show that the Vemf responses Vi will be equal and only the voltage level differs. When only the insect resistance fluctuates (sinewave ΔRbe=10(8.8±0.05)) without a emf-component (Vemf= 0V), the Vi response amplitude and sign will be determined completely by the voltage level V (Fig. 8B). When V is positive an Rbe increase will cause a Vi decrease thus resulting in a sine wave phase shift between Vi and ΔVemf (cf. Fig. 9B for positive Vi response and all Fig. 9A responses). However, when the V level is 0V there is no Vi signal (no voltage to modulate), and when the V level is negative the lowering of Rbe causes a higher Vi amplitude and the phase shift with the Vemf disappears.

When emf- and R-components both occur in phase (Fig. 9C) the Vi response show sign, shape, and amplitude differences between V levels due to interaction emf and Rbe primarary circuit properties. The calulated responses show similar interactions as the observed in E2 aphid signals (Fig. 8). At the 0Volt level only the emf-components play a role, and at negative V levels, the in phase emf and Rbe fluctuation responses are similar and reinforcing each other; the more negative the stronger. At positive V levels the two components are counter acting, which at the +10mV level leads to a nearly an almost flat line indicating a nearly complete compensation of the components amplitudes. The reinforcing-counteracting shift level depends on the fluctuation ranges ΔVemf and ΔRbe as well, of course. Anyhow, the V level enables to visualise the emf- and R-components of EPG signals.

Figure 9. Simulated signal responses Vi as caused by emf-Rbe interactions in accordance to Ohm’s law relationships

8.3 Conclusions

There are serious interactions between emf- and R-components which depends on the voltage level and sign of the circuit voltage V, the ΔVemf voltage fluctuation range, and the ΔRbe resistance fluctuation range, as well as on the device input resistance Ri (Chapter 7). The level of the circuit voltage V depends on DC contributions by Pe1&2, and the adjustable voltage supply Vs. The latter is a useful tool to control emf- and R-components interactions, thus visualizing waveform indentification (Fig. 8). The sine wave simulation demonstrates these simplified interaction principles.

9. Electrical current

So far only voltages and resistances have been considered in the primary circuit. However, the electrical current through plant and insect plays an important role as well. A lower input resistance Ri - the only resistance in the primary circuit that can be controlled by the user - means a higher electrical current through plant and insect (equation [ 2 ]). When a lower input resistance Ri is choosen in an attempt to increase the R‑component sensitivity (ΔVi/V) in large insects with low Rbe, for example, the current will increase. In insect- and plant-physiology measurement methods the electrical current should dercreased as much as possible (Neher, 1974). Organisms are affected more by electrical current than by voltage as such (think of birds that have no problem landing on high-voltage wires). Insects with resistances of 106‑109Ω facilitate a current increase when selecting a decreased Ri value (Fig. 13). The smaller the insect and input resistance (Rbe and Ri) the steeper the curves. Using an Ri of 106Ω (1MΩ) and an insect with the same 1MΩ resistance (Rbe) the current will be 500 times larger than when an Ri of 1GΩ is used. Though the real impact of such a theoretical current increase is difficult to predict. Gustatory sense-nerve cells in the pharyngeal cavities will potentially be vulnerable when exposed to this current. Especially, when the cibarial valve is closed the taste cells in the sensilla pores will conduct the major part of the electrical current, possibly affecting taste perception. Intracellular plant cell punctures may affect ion fluxes (ion channels) through the plasmalemma and electrical signal transmission in plants. Although the current values are still in the nano-ampere (nA) range and possibly the impact may be limited, avoiding increase in electrical current by using a higher Ri device will always be safe.

Fig. 10. Electrical current through insect and circuit voltage V= 50mV for six Ri and Rbe values

.

III  Review of EPG measuring systems

10.  EPG systems

10.1  AC EPG systems

The first EPG system was an AC device, called the ´feeding monitor´. This system introduced by McLean and Kinsey (1964) used an alternating current (60Hz AC) voltage supply (Fig.11A; Vs). This supply voltage serves as a ‘carrier wave’, the (peak-peak) voltage was amplitude modulated (AM) by the plant-insect resistance (Rbe) fluctuations, similar to AM radio in which audible frequencies modulate the high frequency carrier wave amplitude. Also similar to AM technology, the signal was processed into a demodulated output signal reflecting the resistance fluctuations of the plant-insect. The emf-components as such do not change the carrier wave amplitude. Only the amplitude modulated high frequency carrier wave signals will pass the capacitor (Fig.11A, Ci) between and reach the head stage amplifier. Consequently, the AC system is only sensitive to AM resistance fluctuations of the carrier wave and not to low frequency Vemf and ΔRbe modulated V level signal components. The AC system therefore, can be considered as a pure 'R EPG system'. Subsequent signal processing (discussed in 10.3) does not change this property. Several other AC systems have been constructed later with different Ri values and carrier wave frequencies (Brown & Holbrook,1976; Kawabe et al.,1981), more recently in combination with a DC EPG system design (Backus & Bennett, 1992; 2009; see 10.3).

Fig. 11. The primary EPG circuit of two systems. A) The AC system with an adjustable AC (originally 60 Hz) voltage supply (Vs). This system is sensitive to R-components only. B) The DC system with a adjustable DC voltage supply (Vs). This system is sensitive to emf- and R-components (Chapter 7).

10.2  DC EPG systems

10.2.1  Standard DC EPG system

DC EPG systems were developed later (Schaefer, 1966; Smith & Friend, 1970; Tjallingii, 1978, 1985, 1988) and they are using a DC voltage supply (Vs). In the standard DC EPG system (Fig. 11A) circuit voltage level V is also "amplitude modulated" by Rbe fluctuations. The voltage level V is reduced more or less by Rbe (Ohm's law, equation [ 3 ]) and therefore, with the sensitivity expressed by ΔVi/V (chapter 7) but additionally, it appeared that plant-insect generated voltage sources are contributing to the EPG signal (Tjallingii, 1985), the emf components. Because of sensitivity to both, Rbe and emf fluctuations, the DC system can be considered as an ‘R+emf EPG system’.

10.2.2  Pure emf DC EPG system

The standard DC EPG system (R+emf) has been modified (Giga-8d model, 2018; EPG Systems, Netherlands) by adding a remote controlled switch (Fig.11B; s) to change this DC into a 'pure emf EPG system’. In emf mode the switch is open and no longer sensitive to (Rbe) fluctuations because the Ri in the primary circuit is then switched to a 1000x higher value (Fig. 11B); due to the internal Amp (OpAmp, operational amplifier chip) resistance of 10TΩ (=1013Ω) that now determines Ri between the measuring point (M) and ground instead of the standard 1GΩ Ri. When the switch is closed the amp internal 10TΩ Ri is parallel to the amp external 1GΩ Ri  and the resulting shunt resistance will very closely approach the 1GΩ Ri. With the 10TΩ Ri the modulations of V by ΔRbe are still present in the signal but then are negligibly small. Thus, with the switch open (emf mode) the DC EPG system is a 'pure emf EPG system'.

10.2.3 Differential DC EPG system

The head stage amplifier (Amp) in DC and AC systems has mostly been used in a ´single ended´ configuration  in which only one of the two amplifier (OpAmp) terminals is used (Fig.1, and 11). When using both amplifier terminals Ri can be connected between these and the head stage amplifier will measure the voltage (difference) across Ri and this is called a differential configuration (Fig. 12; now standard in recent model Giga-8dd). There is altering in the Ohm’s law relationships of the primary circuit. In all both configurations, single and differential, the signal voltage Vi (Fig.11 and 12) is measured across Ri but in single ended mode Ri is connected to ground whereas in differential mode the plant is connected to ground (Fig. 12). This configuration has the advantage of that the voltage supply Vs is now via Ri connected to the insect and the adjustment is now on the insect side (Fig.12) instead of on the plant side (Fig.11). Using a device with 8 channels (for recording up to 8 insects concurrently)  all plant and the Faraday cage are connected to ground Vs adjustment of each channel is done per insect, not per plant. Electrical isolation between plants     Fig. 12. Differential circuit configuration            and between plants and Faraday cage (thus short circuiting signal of individual insects) is no longer a problem. Also, this allows recording from more than one insect on a the same (single) plant, and EPG recording in the field.

10.3  AC-DC EPG systems

Several combinations of AC and DC systems have been developed and used in order to study how emf- and R-components in waveforms are related to each other.

10.3.1  Dual AC-DC-EPG system

This system was proposed (Tjallingii, 2000) and it was used in a study on thrips (Kindt et al., 2005) and aphids (Tjallingii et al., 2009). The aim was to separate the simultaneously recorded R and emf components in and analyze their feature differences and synchrony. In the primary circuit a high frequency AC voltage (carrier wave; cf. Fig.11A) and a DC voltage (cf. Fig 11B) - each separately adjustable - are superimposed supplied to the soil/plant in the Vs voltage (Fig.13). After the head stage amplifier (Amp.; 50x gain) the signal is split in two, each connected to an output branch.

In the upper branch the modulated AC carrier wave signal components are blocked by low pass (LP) filter leaving the normal R+emf EPG system signal reaching the DC output, after an extra amplifier (up to 2x, thus up 100x total gain). In the lower branch the voltage level V and the low frequency Vemf and Rbe modulation components in the signal are blocked by a high pass (HP) filter. Subsequently, after an extra amplifier (similar to the upper branch) the signal processing

was identical to the AC EPG system (see 10.1). Though the resulting signals by this dual system were interesting as such, they did not show any clear advantage in routine experiments as compared to results obtained by the standard DC EPG system. In specialized studies comparing the fine details of certain events and waveforms this EPG system may be valuable (Tjallingii et al., 2009) but in general, this device seems to give little added value in applied EPG studies.

One EPG system device with a pure emf-component output as well as a second output for a the concurrent pure R-component of the same insect is not possible. Recording pure emf-component signals requires a very high Ri (see 10.2.2) value and recording R-component signals requires a low Ri near the ΔRbe fluctuation range. A primary circuit cannot have two different Ri values at the same time. The dual AC/DC system design has an Ri suitable for both components and by adjusting Vs a V level can be selected with a low R-component contribution (see Fig.8, middle V0 trace). A dual system with a 1G/10TΩ switch might be another option but when Ri switched to emf mode (10TΩ Ri) the AC output will not show an R-component signal (Fig. 18, lower branch). By switching the device will alternate between pure emf-components upper branch DC output (10TΩ) Ri and pure R-component normal mode at the lower branch AC output, thus allowing no concurrent comparison but only subsequent pure emf- and pure R-component signals.

Fig. 13. Dual AC/DC EPG system. Voltage supply Vs is superimposed AC and DC voltage. After the head stage amplifier the signal is split in two branches. Upper branch low pass (LP) filter removes the ΔRbe modulated high frequency carrier wave, leaving the Fig.11B DC system signal (no emf mode switch). This standard DC R+emf signal after optional amplification (Amp) is connected to the DC output. No switch to emf mode. The lower branch signal after a high pass (HP) filter (and optional extra gain (Amp) only is fed into the final signal processing (see 10.3.2) by a rectifier and smoothening filter that goes to the AC output. This signal is identical to the Fig.11A signal with R-components only.

10.3.2  AC-DC correlation monitor

More recently this device was built that also uses a mixed voltage supply (Vs) and it was claimed to record better 'balanced' emf- and R- signal components (Backus and Bennett, 2009, 2019) than the DC and other AC-DC systems. In the published block diagram (2009) may suggests that this concerns a test version for a later commercialized device version. HIER Except for some details in the signal processing circuitry it appears similar although some details were not given because the authors declared these ‘patent protected’ (Backus et al. 2019). The 2009 block diagram of the circuit (Fig.14) shows that the device applies a mixed (superimposed) AC (carrier wave oscillator) and DC voltage to the plant, both voltage adjustable. The input resistor (Ri) value can be selected in a range of 106-1013Ω. The signal after the head stage amplifier goes to a buffer amplifier, which seems redundant since the head stage amplifier will act as such already. Then signal is split into 3 branches, each to an additional amplifier, the upper one indicated as 'DC' and the other two as 'AC' amplifiers. Though their gain function is obvious, the 'DC' and 'AC' indications are unclear. These seem to refer to the respective DC and AC branches, rather than to DC and AC amplifier properties. In general shuch indications refer to the frequency range amplifiers are handing: 'DC' from 0Hz to a high frequency, and 'AC' to a specified frequency from higher than 0Hz to a much higher frequency, comparable to a LP and a HP filter respectively. Also the '& Range' indication of the amplifiers is unclear as the output range of an amplifier is limited and a high gain automatically reduces the output range (Volt). Thus 'Range' seems a redundant indication here.

Fig. 14. Modified block diagram after Backus & Bennett (2009) with more Ri choices at the head stage amplifier and a DC offset monitoring output (- - ? - - -), as described in later desig versions (Backus, 2019; Lucini and Panizzi, 2019).

The upper branch is identical to the Fig.13 upper branch of the dual AC-DC system providing an R-/emf-component signal to the DC output after passing the low pass filter. The other two branches are identical (the middle is focussed here first) but can be adjusted differently: i.e. with or without using the coupling capacitor and both branches are connected to a separate AC output. With coupling capacitor the DC signal is blocked and only the modulated carrier wave is allowed to pass (c.f. HP filter in Fig.13) and the signal enters the rectifier (smoothening filter assumed) and the upper envelop of the signal going to the AC output reflects the R-components only. Without the coupling capacitor (switch closed) both, the DC, R+emf EPG signal and the AC, R EPG signal will not be separated and both will be processed (rectified and smoothened). Since this signal often contains a substantial DC voltage level, a DC offset source is added to lift the signal to a completely positive level which will prevent signal distortion by ‘fold-over’ effects of the rectifier. In order adjust the DC offset properly an extra offset monitoring output with the pre-rectification signal has presumably been added (2019 version; Fig. 19, the a red dashed - - - ? - - - ). The additional Log amplifier will transform the amplification to a log scale, reducing the higher voltage output signals due to the DC offset lift, if needed. Presumably the 2019 version has only two branches, the top DC and the bottom AC branch.

The complication that occurs when the the DC and AC signals are mixed in the bottom branch (without coupling capacitor) is that this signal contains two R-components, one times from the DC signal (as at the DC output) and again in the modulated carrier wave AC signal (as at the Fig.13, AC output). Therefore, in addition to the R-/emf-component interactions (chapter 8) the R-components in the DC signal will interfere (reinforcing or counteracting) with R-components in the AC signal. Especially when the DC ofsett is needed to lift the signal before rectification this will be a problem. Lifting the voltage level of the signal can be avoided completely by using the DC voltage supply Vs (called 'Power supply' in Fig 14). Any unwanted circuit voltage V level is mostly caused by the electrode potentials ((Fig.3; Pe1 and Pe2) in the primary circuit. However, these electrode potentials are incorrectly denied and confused with the very small bimetal potentials (Pb1 and Pb2, Fig.3; Backus et al. 2018, 2020, and 2023)

HIER

Another monitor feature – not visualized in the 2009 block diagram – is a choice switch (head stage amplifier in modified Fig.19, block diagram) to select an Ri value, in the range of 1MΩ to 10TΩ. The Ri selection plays an important role in both articles. The intention is that the user can/should select an Ri value matching the supposed ‘inherent resistance' of the used insect in order to achieve a balanced R-emf component sensitivity of the monitor. However, there is no inherent insect resistance as such; the R-component amplitudes depend on the resistance fluctuation range during each specific waveform. This fluctuation range is not known (see section II, chapter 2). Moreover, a lower Ri value than 1GΩ will cause lower emf-component amplitudes and will increase the electrical current through the plant/insect, which is undesirable. In the coupling capacitor enabled mode (recommended?; Backus and Bennett, 2009; Backus et al. 2019) the mixed AC-DC signal contains two R-component contributions, one is the modulated AC carrier waves and the other is the modulated DC circuit voltage. These two R component contributions may interact - either reinforce or counteract each other - depending on the magnitude and sign of the DC circuit voltage. Such interactions do not occur in the two separated signals in dual AC/DC EPG system. No such interactions will occur when this AC-DC monitor is used in the coupling capacitor disabled mode. Therefore, this recommended mixed AC-DC mode should not be used and the AC-DC correlation monitor design has certainly no advantage over the dual AC/DC EPG system.

9.4 Conclusions

1. EPG systems differ in R- and emf-component sensitivity
• The AC EPG-system is exclusively sensitive to R‑components
• The regular DC system (with Ri=1GΩ) is sensitive to both, R- and emf-components
• The DC emf-EPG system (with Ri ≥10TΩ) is exclusively sensitive to emf-components
2. The AC-DC dual system separates concurrently recorded AC R-component signals and DC R+emf-component signals, which makes these signals real time comparable
3. Combining a pure R-component (AC) and pure emf-component (DC) 2 branch system is not possible.
4. The mixed AC-DC correlation monitor, similar to the previous device but the intention to mix the AC and DC system signals by enabling the coupling capacitor will cause undesirable AC and DC R‑components interferences in the signal. The developers of monitor claim its superior properties but the opposite seems the case, unfortunately
5. The 1GΩ Ri value of the regular DC system provides a compromise between minimizing the electrical current and allowing recording of emf-components a R-components from a wide range of insects. Although the R-component sensitivity at 1GΩ is somewhat reduced for insects with low resistance fluctuation ranges Vs adjustments will allow enough visualization
6. For routine EPG recording no comparison or separate emf- and R-components recording is needed
7. The importance of R-components is often not clear. Although suggestions and assumptions about biological phenomena related to specific R-components have been made, only little convincing experimental evidence has been provided so far. In a whitefly study (Jiang and Walker, 2001) some waveform fluctuations during pathway were suggested to be caused by insect resistance fluctuation; stylet penetration depth and partial withdrawal. Also, the aphid waveform E2 spikes – assumed to represent salivary valve opening movements  (Tjallingii 1978, 1985; then called waveform D and D+E, respectively) – showed an important R-component. These E2 R‑components can perfectly be shown by adjusting Vs more negative; no Ri lowering or matching is needed
8. The R- and emf-components in most waveforms are very much entangled and therefore and very similar thus some emf-component dominance will mostly not affect the overall waveform features

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Appendix: Other claimed DC system property failures

Backus and Bennett (2009) and Backus et al. (2019) objections regarding the regular DC system (Giga models) and refutation (RE; Tjallingii)

1)  The regular DC system does not supply an accurate DC voltage (Pearson et al., 2014): when the voltage supply (Vs) on a Giga-8dd or -8d device was set to 0Volt, an offset voltage of a different level was observed. In older DC systems (Giga-4 and -8): when the Vs knob was put on the 0Volt position the Vs output was not 0Volt.

RE: This criticism is correct, but irrelevant: In the primary circuit, the insect and plant (soil) electrodes are providing electrode potentials. These are always unpredictable and need compensation by Vs adjustments. Whatever the initial Vs voltage is, adjustments should be done after recording has started and only during a probe (stylet penetration) when the primary circuit is completed. Any initial Vs value (0Volt according to Pearson et al.) is arbitrary and will be overruled by the need of a Vs compensation adjustment.

2)  For EPG recording of large insects an AC EPG system should be used and for small insects a DC EPG system (Backus et al., 2018) because experimental evidence showed that AC and DC supplied voltages interfered size specific with feeding behavior of these insects .

RE: The experimental evidence shown by is not convincing because: a) insects with the AC treatment were still subjected to the DC voltage of the electrode potentials. This voltage source superimposes a DC voltage on top of the AC voltage thus biasing the AC treatment conditions. The DC treatment group (without AC voltage supply) could have been subjected to an extra DC voltage, which could have been unexpectedly high. Feeding behavior of both insect sizes may have been affected thus making the results unreliable. was set at different voltage levels before recording. Therefore, the experimental procedure was incorrect. b) The numbers of replicates were very low in these experiments. Thus experimentally and statically these results are unreliable.

Note: Regarding the underlying insect size related ´inherent electrical resistance', it seems likely that large insect have a lower average electrical resistance in general. But what counts for R-components, is not their average electrical resistance, but the fluctuation range within each waveform, which is merely unknown. An experimental approach to investigate these fluctuation ranges may be valuable in order to understand the biological backgrounds.

3)  The operational amplifiers (OpAmp) used in the DC EPG system are inferior since these would be 'notorious' for drift (Backus 2020).

RE: The OpAmps (head stage amplifier chip) in all DC EPG systems are all high quality and similar to those used in the Backus' device primary circuit. Moreover, if a small drift might occur it will be completely negligible in relation to the electrode potentials. and their possible polarization (which is a still not well studied aspect).

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