1. Preface 2. Primary circuit 3. Experimental setup 4. signal sources
II Theoretical Backgrounds
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
The EPG systems used now and in the past can divided in DC and AC systems, or a combination of these two. One may proceed reading directly to section III for a review. However, to fully understand these systems reading the first two sections is wise.
In the EPG technique, an insect or any other 'organism' with piercing mouthparts (such as a mite or a thick) and a plant or other ‘feeding substrate’ (animal, prey or artificial substrate behind a membrane, e.g.) are made part of an electrical circuit by two electrodes, one to the plant and one to the insect. These electrodes accomplish the connection to the electronic measuring circuit, called primary circuit. The measuring principles and their suitability in experimental research are discussed here. Moreover, this article aims to discuss a number of confusing papers with incorrect statements about Ohm's law relations in the primary circuit and the supposed unsuitable design of the EPG system on this website in particular. The impact of the primary circuit components with different values on the EPG signals is of crucial importance in this discussion. Also, some basic properties of the primary circuit need performing experimental procedures of EPG recording as described in the Manual‑Giga‑8dd 20.pdf (page Downloads/Manuals).
2. Primary circuit
All EPG systems have a primary circuit (Fig. 1), the properties of which are very similar. Nevertheless they have different details that determine what is recorded and what is not, or less completely. The role of the components and their values in understanding the signals. All systems have a user controlled voltage supply (Vs) that is either an AC or a DC source, or combines the two sources. One lead of the source is connected to the electrical ground and the other lead to the plant electrode. The plant electrode is mostly inserted into the potting soil, or otherwise electrically connected to the feeding substrate of the insect. The insect electrode is connected to the input resistor (Ri), which is connected to electrical ground thus completing the primary circuit. The insect-Ri junction is the measuring point (M) that is connected to the input terminal of head stage amplifier (Amp, mostly fixed at 50x gain). The amplifier and all subsequent device circuitry are no part of the primary circuit, and do not affect the input voltage Vi across Ri, i.e. the EPG signal. As soon as the insect stylets penetrate the plant, the circuit is closed and the fluctuating voltage at the measuring point is amplified, which is the EPG signal that is recorded on a computer hard disk (HD), and visualized on the computer screen. The signal voltage fluctuations Vi is a fraction of the circuit voltage V, which is the sum of the adjustable voltage supply Vs and voltages originated between the electrodes Vobe (see section II, chapter 7) and this Vi/V fraction is depends on Ri and the sum of resistances between the electrodes Rbe (Fig. 1). The Vi voltage fluctuations appear in a number of distinct patterns with respect to amplitude, frequency, and voltage level, which are referred to as the EPG waveforms. For a number of insects these waveforms have been correlated experimentally with the insect's stylet penetration (probing) activities and stylet tip positions in the plant tissues and cells. Between the head stage amplifier and the computer circuits are processing signal, which may be a simple magnification or more complex processing depending on the EPG system used. Fig. 1. The primary circuit (simplified, more detailed in Fig. 3)
3. Experimental setup
The EPG device has a main control box and a separate EPG probe unit (Fig. 2). This probe contains the most noise sensitive parts of the primary circuit; the the head stage amplifier and its input socket at the measuring point (M), the head stage amplifier and the ground connected input resistor Ri.
The EPG probe is metal film covered outside, thus shielding the internal components against noise mainly from 110 or 220VAC power cables in the lab at 60 or 50Hz. Also the plant and insect are shielded by placing them with the EPG probe inside a Faraday. The EPG probe is cable (75cm) connected to the control box, which can remain outside the Faraday cage since the head stage output signal is not noise sensitive. The control box contains the adjustable voltage supply (Vs) an additional gain control, and an analog to digital (AD) converter. The AD converter now integrated in the control box circuitry. The digital output makes the signal suitable for computer storage and display. The control box to enable control button handling as it stays outside the cage but should be grounded to the Faraday cage (alligator clip). A (micro) USB socket is used for data transfer to the computer and also, to power the EPG device when connected to the USB socket of the computer.
Fig. 2. Experimental EPG configuration. Only one of the 8 probes shown
4. Signal sources
The fluctuating signal voltages in the plant-insect combination are due to: 1) generated by voltage sources, called electromotive force (emf) components, and 2) electrical resistance (R) changes, called R-components. The emf-components originate mainly from membrane potentials of plant cells when punctured by the stylet tips and from streaming potentials caused by the fluid movements in the two capillary stylet canals. Muscle and neural potentials in the insect do not contribute to the EPG signal. The R-components are mainly caused by valve movements in the food and salivary ducts. Valve opening causes a reduced resistance, i.e. a signal amplitude increase. Presumably resistance fluctuations may be due of salivary composition changes. In fact resistance fluctuations (R-components) reduce the emf signal components to a greater or lesser extent on the one hand and modulate the stable level of V on the other hand. If the stable level of V is positive its modulated signal will be positive but if this level is negative the modulated signal will be negative too. If this level is 0Volt there will be no R-components in the signal due to this modulation. Consequences of this effect will be discussed in XX
II Theoretical Backgrounds
5. Physical-electrical properties
The relevant properties in primary circuit (Fig. 1) of the DC EPG system are discussed here in more detail (Fig. 3) and these are playing a role in AC and mixed EPG systems as well. A number of voltage sources (Fig. 3, blue) can be distinguished: the voltage supply (Vs), the plant and insect electrode potentials (Pe1 and Pe2), the membrane potentials of punctured plant cells (Pm), and the streaming potentials in capillary stylet canals (Ps).
Next to these distinguished separate voltage sources it is useful to indicate groups of sources for Ohm’s law relationships considerations. The latter two separate sources Pm and Ps will then be indicated as the emf-component Vemf source, which also makes sense biologically. With the electrode potentials together they can be indicated as the voltage originated between electrodes (Vobe) source. The Vobe together with the voltage supply Vs are then indicated as the circuit voltage V, the sum of all voltage sources in the primary circuit. Finally, for voltage in the Ohm’s law relations it is useful to define Vc, the (more or less) constant part of V relative to Vemf, the fluctuating part of V. These voltages are listed in Table 1 and depicted in figure 3.
Similarly, the resistances (Fig 8, red) can be distinguished in electrode resistances (Re1 and Re2), the stylet tip resistance (Rst), and the stylet canal resistance (Rsc) including the valve movement resistance fluctuations. The sum of all these resistances (i.e. between Vs and the measuring point (M) is indicated as the resistance between the electrodes (Rbe). The Rbe value can be calculated from experimental measurements. The insect resistance as such cannot but will close to Rbe in general. It is important to realize the emf and R component sources (Vemf and Rbe) are fluctuating and that the fluctuation ranges should be considered.
In addition to the main voltage sources and resistances above, a number of minor sources and resistances could be mentioned such as bi-metal and root potentials and . For resistances there are soil and plant resistance. The values of these additional voltage sources and resistances will be neglected here since they are small, relatively stable, and therefore have little impact on EPG signal amplitudes. Insect nerve and muscle potentials come from organs that are hemolymph embedded, which short circuits their voltage contributions.
Fig. 3. Detailed version of the primary electrical circuit in Fig.1 with relevant voltage sources and electrical resistances, some of which are stable or constant, and other sources fluctuate. Vi is not a source but the measured (recorded) part of the circuit voltage V , and V = Vobe + Vs (see Fig 8).
Since the electrode potentials form an important voltage source they should be considered seriously (Neher, 1974). They are caused by the two rather different metal-electrolyte interfaces of the electrodes: copper-soil and silver-hemolymph, respectively (Fig. 3, Pe1 and Pe2), which are representing a galvanic element (battery-like item). Presumably their contribution in each measurement is relatively stable but once compensated for by adjustment of Vs a slow drift or polarization may occur over time due to chemical changes at the electrode interfaces (electrolysis). Their initial value is very unpredictable and may be negative or positive. Recent measurements (unpublished) have confirmed this and values appeared within a range of ±150 mV. Anyhow, the role of electrode potentials has seriously been underestimated and Backus et al. (2020) that even stated that their role 'has been firmly disproven', without giving any data or references. The main role of the voltage supply Vs is to compensate for unwanted electrode potential values. Therefore its range is made in proportion to the experienced electrode potential values ± 0.5V DC (Giga-8, later models). Neglecting compensation by Vs adjustments will result in undesirable variability in R component contributions and interferences with emf components.
6. Ohm´s law relations
In order to discuss how the measured/recorded voltage Vi (Fig. 1) depends on the relevant voltage sources and resistances as sufficiently presented in the simplified diagram of the primary circuit here (Fig. 4). The Vobe emf components and the Rbe elements are focused in relation to Ri. The role and use of voltage supply Vs and the calibration pulse source Vcal (in gray) will be discussed later.
Ohm's law: V = I R or I = V / R [ 1 ]
current I is the same in each serial part: I = Vobe / Rbe = Vi / Ri [ 2 ]
the emf component sensitivity is: Vi/V = Ri / ( Ri + Rbe) = Viemf/Vemf [ 3 ] ___________________________________________________________________________________
7. Sensitivity to emf- and R-components
The emf-component sensitivity is the recorded Viemf fraction (%) of Vemf, which identical to Vi/V (equation [ 3 ]). The Vi/V values in primary circuits with Ri in a range of 105-1011Ω and two supposed constant Rbe values of 107Ω and 109Ω are calculated and plotted. No matter the actual value of V is not constant but in fact fluctuates, the Vi/V ratio will remain constant. The emf-component sensitivity Vi/V values for a constant Rbe of 107Ω and 109Ω are plotted (Fig. 5, Y-axis) versus input resistances (Ri) from 105-1011Ω (Fig. 5, X-axis) are shown as a sigmoid curve for each Rbe (Fig. 5, bold yellow and blue s-curve, respectively). This sigmoid emf sensitivity has been referred to earlier (Tjallingii 1988, Backus 2019, 2020).
The R component sensitivity is not as straight forward as emf component sensitivity. The R component amplitude is caused by Rbe fluctuation, and equals the modulated amplitude of the circuit voltage V. In terms of Vi/V at 108Ω Ri, an ΔRbe fluctuation between 109 and 107Ω, causes a fluctuation between a ViMN/V (on blue s-curve) and ViMX/V (on yellow s-curve), i.e. ΔVi/V (double black arrow). The ΔVi/V values for all Ri values are represented by the blue dotted Gaussian curve, which should be considered as the R component sensitivity plotted vs. Ri. Thus, with increasing Ri the R component sensitivity increases first to a certain maximum and then decreases. The maximum sensitivity occurs when the Ri value is halfway the (log) Rbe fluctuation range. Here the fluctuation range is 107-109Ω, and at an Ri of 108Ω is the maximum R component sensitivity and the maximum amplitude of the R component is about 80% on the Vi/V scale. The fluctuation range (ΔRbe) in this example is presumably much higher than in real insects waveforms. A smaller – more realistic – Rbe range of 107‑107.2Ω (Fig. 6, additional thin blue s-curve for Rbe 107.2Ω) shows that the maximum amplitude decreases considerably (Fig. 6, Gaussian dashed blue curve for ΔRbe 107‑107.2). Though lower, maximum Vi/V amplitude is also shown at an Ri value halfway (log) 107 and 107.2Ω fluctuation range of Rbe, similar to the maximum amplitude of the larger ΔRbe 107‑109Ω fluctuation (bold dashed blue curve).
The ΔVi/V R‑component relations are exactly the same for DC and AC based primary circuits. The only difference is that in the DC system the circuit voltage V is variable (Fig 4) due the superimposed emf-components and in the AC system the voltage supply V is constant.
So far the value indicated by Vobe across Rbe (Fig. 4; Fig. 5, blue double arrow Vbe/V) is not discussed. For a supposed constant 109Ω Rbe its value is 91%. The Vbe voltage as such is not measured at the measuring point and therefore, it plays no role in recorded EPG signals. Backus (2019, 2020) incorrectly suggests that Vbe represents the R‑component sensitivity. The true R‑component sensitivity is ΔVi/V, which does depend on a single Rbe value but on the Rbe fluctuation range (ΔRbe). . The assumption that Vbe represents the R‑component sensitivity should be rejected, therefore. Possibly this wrong concept might be explained by the other aspect added to Fig. 6, which is the ‘relative responsiveness factor f, used in the earlier discussion (Tjallingii, 1988) about the part of the maximum signal amplitude caused by a Rbe fluctuation. In Fig. 5 the maximum amplitude with an Rbe of 107Ω at Ri 108Ω is represented by the yellow ViMX arrow and when Rbe increases to 109Ω the amplitude reduction is represented by the black ΔVi/V arrow. The relative responsiveness factor f was defined as (%):
The R-component is part of the maximum amplitude caused by the ΔRbe fluctuation. Factor f was plotted in the same graph (Tjallingii, 1988) as the emf sensitivity each using an own Y-axis; ΔVi/ViMX for f and Vi/V for emf-components sensitivity, which this was clearly explained in text.. Whether or not this might have caused the confusion and incorrect assumption that f or Vbe/V (Fig. 9) would represent the R-components sensitivity remains guessing.
The f value is represented by the distance between ViMN and ViMX as part of ViMX/V (yellow s-curve) for all Ri values (X-axis). For the wide 107‑109Ω Rbe fluctuation range f the becomes maximally 99% (at Ri=105Ω) creating an R‑component dominance for low Ri values. For the small Rbe fluctuation range the maximal f the becomes only 37% (at Ri=105Ω). Thus the emf-component will slightly dominate. The 50:50 emf-/R-component sensitivity discussed by Backus (2020) will not be reached when Rbe fluctuates less than a 10-fold (RbeMX ≤ 10 RbeMN; f ≤ 50%). Lowering Ri will increase the R component proportionally (as shown by the f value) but the fluctuation range is crucial in determining how much. We have no data so far about fluctuation ranges and how they differ between insects of different size and (likely) between specific EPG waveforms within insects. Empirically, we are observing differences between DC primary circuit differing in Ri but these can be modified very much by Vs adjustments (unpublished results). More experiments will be needed understand the role of Ri in real EPG recordings. The question what Ri will be optimal for certain Rbe fluctuation ranges cannot be answered presently.
8. emf- and R-component interactions
8.1 Entangling and interference
Most R- and emf-components in EPG signals do not contribute separately. Their fluctuations are often very similar and coinciding since they have an underlying biological activity in common. For example, when after puncturing of a phloem sieve element the cibarial valve in the stylet 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 (emf-component) voltage as well. Thus both components coincide and simultaneously contribute to an increased signal voltage, at least when both components are positive. Then the emf- and R-component are reinforcing each other. However, when the emf- and R-component have an opposite sign, R-component with positive increase but emf-component with an increased negative amplitude, they will interact and may eliminate each other. The characteristic negative E2 pulses are very clear when Vc (and so the circuit voltage V) is adjusted negative (Fig. 7, bottom trace). When Vc is adjusted to about 0Volt, only the small emf-component E2 pulses are shown (middle trace). No or only very small R-component effects are shown at 0Volt Vc level since a modulation of 0Volt results in a 0Volt response. When Vc is adjusted positive the negative emf-pulses are eliminated by the positive R-component pulses (Fig. 5, top trace). During opening of the valve in the salivary duct (morphology not very clear) the saliva moves in the plant direction, which appears to cause a negative streaming potential.
In fact Rbe fluctuations mean a reduction to greater or lesser extent of the constant Vc voltage level on the one the one hand and of the Vemf on the other (very small R-component at 0Volt Vc level as mentioned above). An Rbe decrease will always cause a more negative response when Vc or Vemf are negative and a more positive effect when Vc or Vemf is positive. In contrast, the sign and amplitude of Vemf is completely Vc independent (see next section).
8.2 Simulated interactions
In a spread-sheet model the emf-/R-component responses in the signal Vi are simulated, both separate and interacting. It is assumed that in case of insect resistance only (Fig. 8A; Vemf=0), emf only (Fig. 8B; ΔRbe=0), or R and emf both (Fig. 8C) are fluctuating in a sine wave like mode. The Vemf fluctuation presented here is around three constant Vc levels of ‑0,15V, 0V, and 0.15V (V=Vc±Vemf). The Rbe fluctuation is around the average Rbe of 107Ω (log scale, Rbe=10^(7±ΔRbe)). Moreover, the simulated signal responses are calculated for three Ri values. (Fig. 8; 106, 107, and 108Ω; see Settings).
When no emf-components occur (Fig. 8A; Vemf=0Volt) the response signals of the simulated Rbe fluctuation show a positive fluctuation for the positive Vc level, a flat line for the 0Volt Vc level, and a negative fluctuation for the negative Vc level, respectively (Fig.8A). The Vc level effect is that for the positive and negative Vc level the sine wave phases are in opposite direction (inverted). When Vc is positive an Rbe increase during the first sinus phase causes a Vi signal decrease according to Ohm’s law (blue curves for all 3 Ri values). When Vc is negative an inverted version of the response signal is shown: Vi increases first, i.e. becomes less negative when the Rbe increases (yellow curves), thus the Vi sine wave derived phases resemble the Rbe fluctuation. At the 0Volt Vc level the response signals (purple curves) are represented by a flat lines; no Vc voltage to be modulated. These Vc/Rbe relations are crucial in EPG recording. With increasing Ri (Fig. 8A graphs to the right) the response curve amplitude (peak to peak [p-p] Vi voltage) first increases at Ri 107Ω, and then decreases at Ri 108Ω, thus confirming the maximum ΔVi/V at Ri halfway the Rbe fluctuation (Fig. 5 and 6, dashed Gaussian curves). Also, the sinusoid response shape is Ri dependant: the best sinus fit at Ri 107Ω and a poorer sine wave fit for the lower (106Ω) and higher (108Ω) Ri. For the 0Volt Vc adjustment the flat line responses remains the same.
When no R-components occur (Fig. 8B; ΔRbe=0Ω) the simulated Vemf fluctuations the Vi signal responses show that for all Vc levels and Ri values there is perfect sinus fit which is Vc level independent. The Ri value increases the Vi level as well as the p-p amplitude. No sine wave inversions or deformations.
When both emf- and R-components occur (Fig. 8C) the response signals show sign, shape, and amplitude differences between Vc levels, and Ri values. At Ri 106 and 107Ω the positive and negative Vc levels show more or less the same response curve signals as without emf-components (Fig.8A), although at the Vc 0Volt level there is some signal, not a flat line. At Ri 108Ω the positive Vc response signal (blue curve) differs due to since emf-/R-component counteracting effects, during the second sine wave phase, especially. The negative Vc response the (Ri 108Ω, yellow curve) shows no such interaction but some emf-/R-component reinforcement. When the higher 109Ω Ri is used the interaction effects are reduced since emf-components will become more dominant then (Fig, 8D, cf left panel). But when the Rbe fluctuation increases, e.g. to 10(7±1)Ω, the R-component impact will increase too (Fig, 8D, cf middle panel). When the sign of either the Vemf or the ΔRbe fluctuation is different, the response curves are inverted; compare Fig. 8D middle and right panel. At the 0Volt Vc level (Fig. 8, purple curves) these impact differences are also present but much smaller (not visible in the Fig. 8 signals at the scale used).
There are serious interactions between emf- and R-components which depends on the Vc voltage level and Vemf fluctuation range, the average Rbe value and Rbe fluctuation range, and the ratio of Rbe and Ri. The simulation data shown here are from only one low average 107Ω Rbe and Ri values of 106, 107, 108, and 109Ω but for 10 or 100 times higher Rbe and Ri values results are very similar. The serious interactions between emf- and R-components depend - in addition to the Vc voltage level and Vemf fluctuation range - on the average Rbe value and Rbe fluctuation range, and the ratio of Rbe and Ri. With incresing Ri values there is a trend of increasing amplitude R-component amplitude to the point where Ri=Rbe then decreasing for higher Ri. Also the sign of Vemf and Rbe fluctuations very much determines the interaction impact on the signal (Fig. 8D middel and right panel). The simulation data shown here are from only one low average 107Ω Rbe and Ri values of 106, 107, 108, and 109Ω but for 10 or 100 times higher Rbe and Ri values results are very similar. The sine wave example fluctuation is a simplification but it demonstrates that real interactions may be even more complicated.
9. Electrical current
So far only voltages and resistances have been considered in the primary circuit: the electrical current was neglected so far. Clearly, lower insect and input resistances (Rbe and Ri) in the primary circuit means a higher electrical current (equation [ 2 ]). When a lower input resistance Ri is used in an attempt to optimize 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 prevented as much as possible (Neher, 1974). Organisms are affected more by electrical current than by voltage as such (think of birds on high voltage wires). Insects with resistances of 106‑109Ω facilitate an increasing current with decreasing Ri values (Fig. 13); the smaller the insect resistance (Rbe) 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. The impact of such a theoretical current increase is difficult to predict. Gustatory sense-nerve cell 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 be safe.
Fig. 15. 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 system
The AC EPG system was the first one, called the´feeding monitor´. This system introduced by McLean and Kinsey (1964) used an alternating current (60Hz AC) voltage supply (Fig.16A; Vs). In fact the AC voltage supply was a ‘carrier wave’, the amplitude of which was modulated (AM) by resistance fluctuations in the plant-insect, similar to AM radio in which audible frequencies modulate the carrier wave amplitude. Also similar to AM technology, the signal was processed into the demodulated output signal reflecting the resistance fluctuations of the plant-insect. The emf-components do not change the carrier wave amplitude and are blocked by a capacitor (Fig.16A Ci) between the measuring point and the amplifier input. Therefore the AC system is sensitive to resistance fluctuations only and can be considered as an 'R-EPG system'. Later, several other systems were made with different Ri and carrier wave frequencies (Brown & Holbrook,1976; Kawabe et al.,1981; Backus & Bennett, 1992)
Fig. 16. The primary EPG circuit of two EPG systems. A) AC system. The amplitude modulated carrier wave of the AC voltage supply amplitude is processed to the output signal (out, reflectung the original resistance changes. B) DC system. The DC voltage supply plus all other voltage sources are (partly) reflected in the output signal in addition to the and the R modulated voltage source fluctuations (out). Thus the signal contains both R- and emf-components, switch (s) closed (Ri=1GΩ) When switch s is open (Ri=10TΩ) the system then functions as an emf-EPG-system (see 10.2.2.). Only newer Giga-8dd models have this remote controlled switch. In older models (Giga-8d) hand operated switches have been used but in which switching mostly may cause disturbance of insect behavior.
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). The DC voltage in the primary circuit is also amplitude modulated and therefore, also reflects the R components. However, it appeared (Tjallingii, 1985) that the EPG signal additionally contains plant-insect generated contributions, i.e. emf components. Because of sensitivity to both, R and emf fluctuations, the DC system can be considered as an ‘R+emf-EPG system’.
10.2.2 emf system. In standard DC R+emf-EPG systems a remote controlled switch has been mounted (Fig.16B; s) to change this DC into an ‘emf-EPG system’. In emf mode this system is not sensitive to plant-insect resistance (Rbe) fluctuations because Ri in the primary circuit is then switched to a 1000x higher Ri value (Fig. 17B, switch s open); i.e. to 10TΩ (=1013Ω) instead of the standard 1GΩ Ri. The amplifier’s own internal input resistance then determines the Ri value. When switch s is closed the internal 10TΩ Ri does not affect the external shunt resistance of 1GΩ Ri; in terms of Ohm´s law, only lowest Ri value then counts. In fact the DC modulations by ΔRbe are still present in the signal but they are negligibly small.
10.2.3 differential system The head stage amplifier in DC and AC systems is has mostly been used in a ´single ended´ configuration (Fig.1 and 3), in which one of the two input terminals is used. In a differential configuration (now in model Giga-8dd) both input terminals of the head stage amplifier are used (Fig. 17) without altering the Ohm’s law relationships the primary circuit. In both configurations the signal voltage Vi (Fig.1 and 3) is measured across Ri but in single ended mode Ri is connected to ground whereas in differential mode it is connected to Fig. 17. Differential circuit configuration . the second (negative) input terminal junction with the voltage supply (Vs) and the + terminal of Vs is connected to ground, thus resulting in exactly the same position for the plant-insect in the circuit.
The advantage of this differential EPG system is that the plant electrode is connected to ground and the voltage supply is incorporated in each EPG probe (Fig. 2). This allows to record from more than one insect on a single plant while keeping Vs adjustments of individual insects. Also, the plant and Faraday cage are now Fig. 17. Differential circuit configuration excluding short circuit risk by plant-cage contacts. Moreover, this configuration allows using the EPG system in the field.
10.3 AC-DC systems
10.3.1 Dual AC-DC-EPG system This system was proposed (Tjallingii, 2000) and constructed (Kindt et al., 2005; Tjallingii et al., 2009). The aim was to separate simultaneously recorded R and emf components in and analyze their feature differences. In the primary circuit an AC voltage (carrier wave/oscillator) and a DC voltage (each adjustable) are supplied superimposed to the soil/plant (Fig. 18). The system has two device output branches, one with the normal DC R+emf-EPG system signal, and one with the AC R-EPG system signal. The resulting signals by this dual system (Kindt et al., 2005; Tjallingii et al., 2009) were interesting as such, but did not show any clear advantage in routine experiments as compared to results obtained by the standard DC EPG system. Although in specialized studies on fine details of certain events and waveforms future use may show some value, this device seems to have no additional value in normal EPG studies.
A system with one output for emf-component signals and one output for concurrent R-component signals of the same insect is not possible. The emf-component signals require a very high Ri value and R-component signals require a much lower Ri value related to the plant-insect resistance range. A primary circuit cannot have two different Ri values at the same time. The dual AC/DC system design comes as close as possible to these properties. A dual system with a 1G/10TΩ switch might be another option but then with Ri switched to emf mode (Ri 10TΩ) the AC system properties will be disabled (Fig. 18, lower branch). Thus allowing no concurrent, only subsequent signal comparison.
Fig. 18. Dual AC/DC EPG system. The voltage supply is a superimposed AC and DC voltage. After the amplifier the signal is split. In the upper branch a low pass filter removes the modulated AC carrier wave and the remaining signal is identical to the signal in Fig. 4B (although there was no switch to emf mode in the experimental prototype device used then). The output signal still contains the DC R components. In the lower branch only the carrier wave and its modulations will pass the high pass filter and the output signal only contains R components.
10.3.2 AC-DC correlation monitor This (Backus and Bennett, 2009) is another device using a mixed voltage supply (Vs). The article’s description seems to concern a test version for a later commercialized device that seems nearly similar, except for some details in the signal processing circuitry. These details are not given by purpose because the authors declared these ‘patent protected’ (Backus et al. 2019). The 2009 block diagram of the circuit (Fig. 19) shows 3 signal outputs, one is the DC R+emf output similar to the upper branch of the dual AC-DC system (Fig. 18) and the other two are identical AC outputs that can be adjusted differently by the user. Similar to the dual AC-DC-EPG system the circuit is split after the (extra, but not essential) buffer amplifier. The upper branch provides the DC, R+emf-EPG signals after removing the carrier wave frequency and its modulated amplitude by a low-pass filter. The middle and lower branch signals contain both the DC (R+emf) and the AC (R only) signals. After passing an AC amplifier (of which the difference between ‘gain’ and ‘range’ adjustments remains unclear) are passing into a circuit with coupling capacitor. When this capacitor is enabled (switch open) only the AC R-components of the signal are passing whereas the DC R- and emf-components are blocked. This signal then passes the rectifier circuit and the signal is identical to the AC output signal in dual AC-DC EPG system above. When disabled (switch closed) the AC and DC R-component and the DC emf-component signals will pass mixed through the rectifier circuit and to the AC output of that branch. Since this signal often contains a substantial DC voltage level, a DC offset source is added to prevent ‘fold-over’ effects by the rectifier, that would distort the signals. In order adjust the DC offset properly an extra offset monitoring output with the pre-rectification signal has presumably been added (Fig. 19, the a red dashed - - - ? - - -).
Fig. 19. Modified block diagram after Backus & Bennett (2009) with additionally 1) Extended Ri choices of the head stage amplifier (as derived from the original text) and 2) a DC offset monitoring output (- - ? - - -) as described in later desig versions (Backus, 2019; Lucini and Panizzi, 2019).
Presumably the 2019 version has only two branches, a DC and one AC branch with the (supposed) additional offset monitoring output. Another 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.
Further discussion of device sentivities to emf- and R-component in section II and Appendix
- 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
- 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
- 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
- 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
- For routine EPG recording no comparison or separate emf- and R-components recording is needed
- 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
- 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).