Stearoyl-CoA desaturases sustain cholinergic excitation and copulatory robustness in metabolically aging C. elegans males

Abstract

Regulated metabolism is required for behaviors as adults age. To understand how lipid usage affects motor coordination, we studied male Caenorhabditis elegans copulation as a model of energy-intensive behavior. Copulation performance drops after 48 h of adulthood. We found that 12-24 h before behavioral decline, males prioritize exploring and copulation behavior over feeding, suggesting that catabolizing stored metabolites, such as lipids, occurs during this period. Because fat-6/7-encoded stearoyl-CoA desaturases are essential for converting the ingested fatty acids to lipid storage, we examined the copulation behavior and neural calcium transients of fat-6(lf); fat-7(lf) mutants. In wild-type males, intestinal and epithelial fat-6/7 expression increases during the first 48 h of adulthood. The fat-6(lf); fat-7(lf) behavioral and metabolic defects indicate that in aging wild-type males, the increased expression of stearoyl-CoA desaturases in the epidermis may indirectly modulate the levels of EAG-family K⁺ channels in the reproductive cholinergic neurons and muscles.

Keywords: Behavioral endocrinology; Behavioral neuroscience; Molecular mechanism of behavior.

Graphical abstract

Figure 1

FAT-6:YFP expression in Aging Males (A) RT-qPCR results of stearoyl CoA desaturases fat-5, fat-6 and fat-7 in day 1 (black columns) and day 2 (gray columns) wild-type adult males (3 males/replicate). Each individual gene was normalized to the day 1 equivalent. Numbers of independent biological replicates assayed are listed below the data. Error bars represent SD. p values were determined using the unpaired t-test. (B and B′) Cartoon of CRISPR/Cas9 FAT-6:YFP knock-in design and confocal images of FAT-6:YFP expression in adult hermaphrodites; panel shows whole worm intestinal and head epidermal expression. (C) FAT-6:YFP fluorescence in day 1 and day 2 males was quantified by a series of rectangular ROIs drawn over the epidermis and intestine. (D) Intestinal FAT-6:YFP fluorescence in aging wild-type males. (E) FAT-6:YFP fluorescence in panel (D), was quantified by a series of rectangular ROIs drawn over the intestine; the series of ROIs corresponding to the first half of the intestine were designated anterior. The posterior was composed of the second half of the intestine extending to the end of the male body. p values were determined using one-way ANOVA with Bonferroni’s multiple comparisons after test. (F) Fixative Nile Red staining of aging wild-type males. (G) Fixative Nile red staining in (F) was quantified by a series of rectangular ROIs drawn over the epidermis and intestine. p values were determined using one-way ANOVA with Bonferroni’s multiple comparisons posttest. For (C), (E) and (G), bars and whiskers represent mean and SD. Numbers of animals assayed are listed at the bottom. A.U. (Arbitrary Units).

Figure 2

Metabolic Characterization of fat-6(lf); fat-7(lf) males (A) Fixative Nile Red fluorescence quantification of day 1-3 males that were either starved or grown with and without oleic acid (O.A)-fed bacteria. O.A supplemented males were grown from eggs to day 1 adult males on agar plates that contain OP50 bacteria fed with 0.3 mM oleic acid. To wean the males from oleic acid, males were moved to agar plates that contained OP50 bacteria that were not fed with oleic acid; day 2 males were weaned off oleic acid for 24 h, and day 3 males were weaned off oleic acid for 48 h. For day 1 starved males, well-fed L4 males were washed of bacteria and then placed on 100 μg/mL ampicillin agar plates that lack bacterial food and assayed as adults 24 h later. For day 2 and day 3 starved males, day 1 well-fed males were washed of bacteria and then placed on 100 μg/mL ampicillin agar plates for 24 and 48 h that lack bacterial food. p values for daily comparisons, and separately between days, were determined using one-way ANOVA with Bonferroni’s multiple comparison posttest. (B) Oxygen consumption of S-basal buffer and 50 males in S-basal buffer per independent sample, as measured by change in voltage over time (10 min). N= 3 independent paired samples of wildtype and fat-6(lf); fat-7(lf) day 1 males. Solid lines are measured data; fitted dashed lines were determined by linear regression. The slopes of fitted lines of fat-6(lf); fat-7(lf) oxygen consumption is steeper than wildtype in all three trials. p values (determined in Prism) represent the chance of the slopes from the fitted lines being identical. (C) RT-qPCR results, one male per biological replicate, of the metabolic genes ctl-1, T20H4.5, cco-1, ech-2, and fat-5. Error bars represent SD. p values were determined using the Kruskal-Walis test. Number of biological replicates are listed below the bars. (D and E) Intestinal FAT-6:YFP expression. Starved animals were grown on NGM plates lacking OP50; a glycerol ring around the edge of the agar was used to contain animals on the NGM plate. p values were determined using the unpaired t-test. Bars and whiskers represent mean and SD. Number of animals assayed are listed below data. A.U. (Arbitrary Units).

Figure 3

Age-dependent behavioral choice in male C. elegans (A) The bacterial consumption rate of aging wild-type males quantified by fluorescence OP50 within the intestinal lumen. Bars and whiskers represent mean and SD. p values were determined using the unpaired t-test. (B) Raw data stacks of male choice behaviors colorized in yellow, blue, and red for exploring, feeding, and mating behaviors, respectively. Stacks consist of 32 bior tricolored horizontal lines each depicting the duration of exploring, feeding, and mating behaviors of a single male over the time course of 1 h. (C) The average proportion of time spent on exploring (E), feeding (F), and mating (M) behavior is plotted as a ternary graph, and each data point is an individual male. As a data point reaches a vertex, the proportion approaches 1. A data point localized to a vertex represents 1 h of performing a single behavior. (D) Markov-simulated data is depicted in red and co-plotted with the raw data in black. (E) A Markov model state diagram of aging wild-type males shows an age-specific proportion, the probability of transitioning to a future state based on its present and past state, colorized in yellow, blue, and red for exploring, feeding, and mating behaviors. The green upward arrows adjacent to day 2 proportions represent increases in day 2 proportions when compared to day 1. The red downward arrow adjacent to day 2 proportions represents decreases in day 2 proportions when compared to day 1. (F)fat-6(lf); fat-7(lf) Markov-simulated data in red is depicted and co-plotted with the raw data in black. (G) A Markov model state diagram, of aging fat-6(lf); fat-7(lf) males, shows an age-specific proportion, the probability of transitioning to a future state based on its present and past state, colorized in yellow, blue, and red for exploring, feeding, and mating behaviors. The green upward arrows adjacent to day 2 proportions represent increases in day 2 proportions when compared to day 1. The red downward arrow adjacent to day 2 proportions represents decreases in day 2 proportions when compared to day 1. (H) Markov-simulated data for wildtype in black and fat-6(lf); fat-7(lf) in red on day 1 of adulthood. (I) A Markov model state diagram comparing aging wild-type and fat-6(lf); fat-7(lf) males from (E) and (G). (J) The number of behavioral transitions leading to exploring, as a future state, in day 1 adults. The behavioral transitions across age were quantified from observed data in (B).P-values determined using the Mann Whitney non-parametric test. (K) The number of behavioral transitions leading to feeding, as a future state, in day 2 adults. The behavioral transitions across age were quantified from observed data in (B).P-values determined using the Mann Whitney non-parametric test. A.U. (Arbitrary Units).

Figure 4

Mating performance and fitness of fat-6(lf); fat-7(lf)males (A) The mating potency of wild-type and fat-6(lf); fat-7(lf) males. The percentages of successful matings are listed at the top of the bars. Numbers of animals assayed are listed at the bottom. p values were determined using Chi-square and Fisher exact test. (B) The mating fitness of competing wild-type and fat-6(lf); fat-7(lf) males. Wild-type males carrying a fluorescent protein (pck-2:YFP) were competed against wild-type males lacking a fluorescent protein. Because both strains were found to be equivalent, wild-type males carrying a fluorescent protein were used in competition assays against fat-6(lf); fat-7(lf) males. The percentage of females mated is listed at the top of the bars. Numbers of animals assayed are listed at the bottom. p values were determined using Chi-square and Fisher exact test. (C and D)The serial mating assay results of wild-type and fat-6(lf); fat-7(lf) males. The number of females impregnated is seen from 12 h, bottom black rectangle, to 72 h in rectangular subdivisions. Each column of rectangles represents a single male across 72 h. Males are organized, from left to right, by the total sum of females impregnated. (D′ and D″) The percent of mated males and number of females impregnated, in the serial mating assays (C and D), across 72 h, was quantified and re-visualized in 12-h increments. Bars and whiskers represent mean and SD. p values for (D′) were determined using Chi-square and Fisher exact test. p values for (D″) were determined using unpaired t-test. (E) Quantification of 72 h of serial copulation of intestinal and/or epidermal fat-6 rescue in fat-6(lf); fat-7(lf) males. Bars and whiskers represent mean and SD. p values were determined using one-way ANOVA with Bonferroni’s multiple comparison posttest. (F and F′)Analysis of mating video recordings of fat-6(lf); fat-7(lf) males. We quantified the number of spicule insertion attempts and the time on the vulva. Numbers of animals assayed are listed at the bottom. Bars and whiskers represent mean and SD. p values were determined using unpaired t-test.

Figure 5

Characterization of cholinergic neurons in fat-6(lf); fat-7(lf)males (A) Day 1 wild-type and fat-6(lf); fat-7(lf) males show similar response to the AChR agonist arecoline (ARE). (B) Wild-type and fat-6(lf); fat-7(lf) day 1 males were placed on NGM pads infused with 15 mM aldicarb. We quantified the time until protraction of the spicule, which is a proxy for muscle contraction. Bars and whiskers represent mean and SD. p values were determined using unpaired t-test. (C) G-CaMP6 calcium sensor fluorescence was digitally recorded in wild-type and fat-6(lf); fat-7(lf) day 1 males mounted on 10% noble agar containing Polybead polystyrene 0.1 μm microspheres. Ellipse-shaped ROIs were drawn for 12 cholinergic neurons. (D) Example of cholinergic Ca²⁺-induced fluorescence changes in a wild-type and a fat-6(lf); fat-7(lf) male. Fluorescence waves, color-coded to their respected neurons, were plotted across time. The colors of the lines match the colored neuron names in (C). (E) Amplitude of neuronal calcium waves, measured as the average difference between the local min and max of waves. (F) Signal duration of neuronal calcium waves, measured as the average duration of fluorescence above the threshold, which is set by taking the mean fluorescence of the neuron. For (E) and (F), each dot represent one neuron recording of one male, bars and whiskers represent mean and SD. p values were determined using unpaired t-test.

Figure 6

Dysregulated unc-103 in fat-6(lf); fat-7(lf) males (A) RT-qPCR results of ERG-like/UNC-103, EAG/EGL-2, and BK/SLO-1 K⁺ channels in wild-type and fat-6(lf); fat-7(lf) males (3 males/replicate). The result of the unc-15 reference gene is also shown. Bars and whiskers represent mean and SD. p values were determined using the Mann Whitney non-parametric test. (B) The percent of males displaying protracted spicules. L4 males were transferred to new plates and kept in groups of 15. Spicule protraction was quantified 12–15 h after transfer. p value was determined using a Chi-square and Fisher exact test. Values at the bottom of the bars denote number of males assayed. Values above the data denotes % spicule protracted. (C) Single-worm RT-qPCR results of unc-103 isoforms in wild-type and fat-6(lf); fat-7(lf) males. Attempts were made to measure all isoforms of ERG-like/UNC-103 K⁺ channels, but we could not reach comparable levels of all isoforms except isoform A/D in mutants. Bars and whiskers represent mean and SD. (C′)Data in C displayed as fold change normalized to wild-type unc-103 isoform A and D. (D) Single-worm RT-qPCR results of the EAG K⁺ channel gene egl-2. unc-103 mutant males were separated based on spicule protraction phenotypes (Prc/NonPrc).Data was normalized to the median of the wild-type dataset. Bars and whiskers represent mean and SD. p values were determined using an unpaired t-test. (D′) Partitioning by fold change, based on (D), shows the major contribution to the mean. Triple mutantegl-2 levels are biased to larger fold changes (>5-fold) versus Non-Prc unc-103(lf) (1.0-3.0). No p value was provided as this panel serves only as a different visual representation of data seen in (D). For (A)-(D′) Values below the data denote the number of biological replicates.

Figure 7

FAT-6/7 maintains lipid droplets as day 2 adult male lipid oxidation occurs (A) Tissues required for mating behavior are colorized with a yellow epidermis, orange muscles, pink neurons used for vulva sensing and spicule insertion, and blue ventral cord neurons used for the control of locomotion and male tail stability. The ventral cord neurons are connected either through gap junctions or synapses to neurons involved in spicule insertion. For example, VB11, DB7 and VA11 are connected to the PDB, PDC, or PVV neurons controlling copulatory locomotion and posture after sensing the vulva through HOA or HOB. PVV neurons in turn also connect either through synapses or gap junctions to PCB controlling appropriate sensing of the vulva and spicule insertion. Male specific CA9 is indirectly connected either by gap junctions or synapses to the spicule insertion cholinergic neurons SPC, PCB, and PCC (Brittin et al., 2021; Cook et al., 2019; Jarrell et al., 2012; Sulston, 1976; Sulston et al., 1980, 1983; Sulston and Horvitz, 1977). (B) A summary of expected lipid metabolism in day 1 adult males. Dotted arrows represent pathways not expected or unlikely to occur. Thicker arrows indicate prioritized lipid metabolism. The golden and gray box represents the conditions in epidermal and neuromuscular tissues, respectively. Day 1 males consume dietary lipids and prioritize the synthesis of lipid storage in the epidermis. Consumed lipids from the intestine are not oxidized under ad libitum conditions. Lipolysis and β-oxidation persist to maintain homeostasis. Metabolic byproducts can be used in fat synthesis, de novo, to form lipid storage. (C) A summary of expected lipid metabolism in day 2 adult males. Dotted arrows represent pathways not expected or unlikely to occur. Thicker arrows indicate prioritized lipid metabolism. The golden and gray box represents the conditions in epidermal and neuromuscular tissues, respectively. Despite ad libitum conditions, day 2 adult males decrease feeding behavior. As dietary fat ingestion decreases, lipolysis of lipid stores is prioritized. Under these conditions, fatty acid desaturases play a role in maintaining lipid droplet stores as the oxidation of lipid droplets becomes a source of metabolic fuel. (D) A summary of expected lipid metabolism in day 1 adult fat6(lf); fat-7(lf) mutant males. Dotted arrows represent pathways not expected or unlikely to occur. Thicker arrows indicate prioritized lipid metabolism. The golden and gray box represents the conditions in epidermal and neuromuscular tissues, respectively. Under ad libitum conditions, the inability to create lipid storage results in dietary fat ingestion and immediate oxidation. To compensate for increased fat-6(lf); fat-7(lf) lipid oxidation, muscle ERG-like/UNC-103 and EAG/EGL-2 channels are upregulated, resulting in the modulation of muscle excitation involved in posture and spicule movement. In the circumstance of fat-6; fat-7 deficiency, calcium activity of motor neurons increase with dysfunctional lipid catabolism on day 1.