Unveiling the contribution of the reproductive system of individual Caenorhabditis elegans on oxygen consumption by single-point scanning electrochemical microscopy measurements

Metabolic analysis in animals is usually either evaluated as whole-body measurements or in isolated tissue samples. To reveal tissue specificities in vivo, this study uses scanning electrochemical microscopy (SECM) to provide localized oxygen consumption rates (OCRs) in different regions of single adult Caenorhabditis elegans individuals. This is achieved by measuring the oxygen reduction current at the SECM tip electrode and using a finite element method model of the experiment that defines oxygen concentration and flux at the surface of the organism. SECM mapping measurements uncover a marked heterogeneity of OCR along the worm, with high respiration rates at the reproductive system region. To enable sensitive and quantitative measurements, a self-referencing approach is adopted, whereby the oxygen reduction current at the SECM tip is measured at a selected point on the worm and in bulk solution (calibration). Using genetic and pharmacological approaches, our SECM measurements indicate that viable eggs in the reproductive system are the main contributors in the total oxygen consumption of adult Caenorhabditis elegans. The finding that large regional differences in OCR exist within the animal provides a new understanding of oxygen consumption and metabolic measurements, paving the way for tissue-specific metabolic analyses and toxicity evaluation within single organisms.

The radius of the glass sheet surrounding the disk electrode, known as Rg (radius of glass), [4] was measured by optical microscopy and found to be approx. 10 µm for all UMEs. The platinization processes was designed to increase the electrochemical response of the UME (increased signal-to-noise ratio) without drastically changing its geometry. It followed a procedure previously reported by us, [5] allowing for the active area to be approximated to a planar disk. This is important to facilitate the representation of the UME's geometry in finite element method (FEM) models without the need for electron microscopy techniques to characterize the true geometry of the UME surface. [6,7] As all studies were performed with normalized current values (self-referencing, see main manuscript), and the UME size is small (see below), the choice of the approximated UME geometry does not impact the final result.
Briefly, the platinization processes consisted in cycling the UME potential between 0.3 V and -0.5 V (vs Ag/AgCl/sat. KCl at a sweep rate of 0.100 V s -1 ) in a 1 mmol L -1 hexachloroplatinic acid hexahydrate (H2PtCl6·6H2O, CAS 18497-13-7, Sigma) in 0.5 mol L -1 H2SO4 solution. The potential sweep was repeated 30 times. A representative voltammogram for oxygen reduction at a platinized UME is shown in Figure S2c and suggests a 5-fold increase in effective radius (assuming a planar disk geometry). After platinization, UMEs presented a 3 to 5-fold increase in effective radius with resulting radii values between 1 and 2.5 µm. As discussed in the main text, the actual electrode geometry for these measurements does not have a large bearing on the analysis as long as the diffusion field at the UME is small compared to that at the nematode, so that the UME approximates to a point probe of concentration.  Figure S3 shows a representative hindered diffusion approach curve of the type obtained prior to SECM experiments presented in the manuscript, and the typical UME current dependence on the electrode/substrate separation can be observed. The approach curve was recorded using a platinized platinum UME at oxygen reduction reaction (ORR) conditions (-0.4 V vs Ag/AgCl/sat. KCl), where current is limited by oxygen diffusion. Currents were normalized by the value obtained in bulk solution. The UME is moved towards the insulating Petri dish surface at a constant speed while the current is recorded. At a given Z-distance (dependent on electrode size and geometry), [4] the normalized current starts to decrease due to hindrance of oxygen diffusion towards the UME caused by the small electrode/substrate gap. [8] By analyzing results shown in Figure S3, we conclude that the recorded current approximates to that recorded in bulk solution at the adopted largest working distance for the SECM map experiments (100 µm - Figure 2 in the main manuscript) .     Table S2).

Symbol Value Description 4
Number of electrons involved in the charge transfer reaction 20 and 40 m UME /Nematode separation 4 5 277 Oxygen concentration at bulk solution 4 5 2.2 × 10 DE + DI [10] Oxygen diffusion coefficient Simulations for the UME response near different sections of the nematode body were performed according to the experimental framework employed and described in the manuscript. The simulation domain employed is portrayed in Figure S4, where the UME body (B6), electroactive surface (B4) and the nematode body cross section (B5) can be seen.
Simulation domain size (> 500*Rb in both r and z dimensions) and mesh density (> 100.000 elements) were set as such that simulation results were independent of both. Boundary conditions for every boundary in the domain are specified in table S1. Rb was extracted from optical images from the animal's body ( Figure 1 of the manuscript and Figure S1), which were recorded with an inverted optical microscope attached to the SECM equipment. The value of d was set to 20 and 40 m. Ra was calculated using the ORR limiting currents recorded in bulk solution and assuming a planar disk geometry. Rg was calculated from optical images.
Simulations were performed in a 2D axis-symmetric geometry in COMSOL Multiphysics 5.4 using the transport of diluted species module using the stationary solver. Mass transport is described by diffusion only, assuming no migration due to the high electrolyte concentration in solution (M9 buffer solution). The flux (Ji) of each chemical species i is described by equation S1.

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At the UME surface (boundary B4) a concentration boundary condition of [O2] = 0 was in place to simulate the diffusion-limited ORR. The current at the UME was calculated by integrating the molecular flux of oxygen over the boundary B4.
Respiration by the animal was simulated by applying an inward oxygen flux at the animal surface (B5), as described in Table S1. Simulations were performed for varying values of kResp at the boundary, representing different possible respiration rates, and the impact of the O2 depletion layer created by boundary B5 on the UME response (B4) was investigated.
Experimental diffusion-limited currents for oxygen reduction at the UME near the worm were normalized (iNorm) by current values recorded in bulk and fitted to iNorm vs. kResp plots, as seen in Figure S5a.
Oxygen consumption rate (OCR) values were then calculated by integrating the O2 flux at B5. Figure S5b shows the relation between kResp and OCR for the head region of one animal simulated as a 20 m radius sphere (see Section SI-5 below). Calibration curves of iNorm vs OCR were constructed (Figure 3b, main manuscript) and used to find the local OCR. This was performed for every individual single-point measurement as it depends on region thickness.

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The kResp values used in the simulations were chosen empirically by simulating a large range (50 distinct values spanning from 1 × 10 DQ DI to 10 DI ) for the first sets of data (animals) examined. Further simulations where performed using 13 distinct kResp values (from 1 × 10 DQ DI to 1 × 10 DR DI ), covering all the respiratory rates encountered.

S-5 Comparison between 2D axis-symmetric and 3D models
The simulation geometry used (2D axis-symmetric) for the FEM model does not allow for the real animal geometry to be represented (irregular cylinder with varying radius). A full 3D model, without symmetry planes, is necessary to represent the true geometry  Although longer cylinder lengths impact more the current sampled at the center of the substrate (for both working distances), it is clearly that the UME response is dominated by the O2 consumption of regions of the substrate near the electrode, equivalent to smaller h/r values in Figure S6. As the cylinder gets longer, its length has a diminishing effect on the normalized current ratio, discerned by the lower slope of the curve at larger h/r ratios (longer cylindersca. 2.5). Even assuming a homogeneous O2 consumption along the substrate, the length (h) has a smaller bearing on the O2 concentration boundary layer than the radius (r). The intersection point between the two lines in Figure S6b (i/iSphere = 1) represents the case for a cylinder of h⁄r = 2, which equates in surface area to a sphere of radius r. At this point the 3D and 2D axissymmetric model are equivalent, despite the distinct geometry.
If the nematode geometry would be simplified to a constant radius cylinder it would have a h/r ratio between 15-20 (depending on the animal shape, the "average" radius can vary). Hence approximating the substrate to a sphere would represent at most a difference of <25% between S-12 the 2 geometries ( Figure S6b). This is the most these 2 models differ, as it assumes homogenous O2 consumption along the substrate, which is not the case for C. elegans (Figure 2, main manuscript). The concentration boundary around the middle body region will be dominated by the respiratory rate of the local reproductive system, minimizing the effect of the edges of the animal (head and tail), on the UME response. Similarly, in the head region measurements, the UME probes only the O2 concentration boundary layer at edge of the animal and is not impacted by respiration of the entire body. In reality, the UME probes the local O2 concentration over an area of the animal imminently close to it with regions further away impacting less its response. As OCR is dependent on the surface area and the 2D spherical model capture partially the nematode real geometry, the 2D axis-symmetric model is a reasonable approximation and adopted here for quantitative local OCR measurements.

S-6 UME as a local oxygen concentration probe
As the UME diffusion layer extends only to about 10 radii from the electrode, the O2 reduction current recorded at the electrode is proportional to the average O2 concentration at the edge of this concentration boundary layer, represented by the dashed line in Figure S7a. [13] As an approximation, the UME current can be seen as to be proportional to the O2 concentration value at a point at the center of the UME disk, but 10 radii away (green semi-circle, Figure   S7a). As the O2 concentration boundary layer around the worm extends several microns into the solution ( Figure S7b and Figure 3c of the main manuscript), the UME can be seen as a probe for the local oxygen concentration. Thus, the UME normalized current ratio can be calculated as the ratio between the O2 concentration at the electrode height (20 and 40 µm) in the substrate boundary layer in a simulation only considering the substrate geometry ( Figure   S7b) divided by bulk concentration values. For comparison, the local OCR measurement for a head region was simulated in the two cases: with and without considering the electrode geometry. The variation between the two approaches gave an error of < 15 %. This approximation can facilitate the simulation and analyses of turnover rates over substrates, as the only calculation needed is for the concentration distribution over the substrate diffusion layer, ignoring the electrode geometry and electrodic process, which can be complicated. Most substrates can be approximated to regular geometric forms, for most of which analytical or semi-analytical solutions for transport equations exist, [14,15] allowing these calculations to be performed in a simple manner, similarly to other approaches reported in the literature. [16][17][18] However, this simplified approach is only valid as long as the electrode geometry does not interfere with the concentration boundary layer created by the sample, which can happen if the electrode, for instance, has a large insulating glass sheet around the electroactive area that can hinder oxygen transport from solution to the animal's surface when in close proximity to the sample. To model this interaction between the UME and the substrate diffusion layer, a full model, considering

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the substrate and UME geometry (see S-4), is needed. This provides a complete insight in the electrode/substrate interaction, not possible in the simplified approach.