Are lipids always light? Lipids in larval lampreys are enriched in 13C but depleted in 2H relative to muscle

doi:10.21203/rs.3.rs-1942444/v1

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Are lipids always light? Lipids in larval lampreys are enriched in 13C but depleted in 2H relative to muscle

https://doi.org/10.21203/rs.3.rs-1942444/v1

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Stable isotope ratios in organisms can be used to estimate source contributions to the organism. However, during lipid synthesis light isotopes of carbon (¹²C) and hydrogen (¹H) are preferentially incorporated into the lipids, potentially causing source contributions to be poorly estimated. Contrary to expectations and other published examples in animals, larval lampreys, which are basal vertebrates, have lipids which are enriched in heavy isotopes of carbon (¹³C), but still depleted in heavy hydrogen (deuterium; ²H). Four lamprey species were collected and their isotopes ratios of δ²H, δ¹³C, δ¹⁵N were measured in their muscle before and after lipid extraction. Larval lamprey of one species was collected every three months for a year from two streams in Maryland and the isotope ratios of muscle before and after lipid extraction, as well as the extracted lipid were measured. Muscle δ¹³C was positively related to C:N ratios in samples when lipids were not removed and δ²H was negatively associated with the percent hydrogen in a sample. As expected, the measured difference between muscle and lipid δ²H (Δ_MLδ²H) was the same for all months and was 111‰ (SE = ± 21, n = 35), but the Δ_MLδ¹³C was different between months (ANOVA, F_3,53 = 5.05, p < 0.005) and was always negative. Our work suggests that while lipids are often enriched in ¹²C relative to muscle, this is not a universal rule. The physiological mechanism(s) for generating heavy carbon-backbones in lipids remains unknown and requires exploration.

Stable isotopes are a powerful tool which have been used to deepen our understanding of numerous biological processes including physiology, nutrient cycling, food web structure, diet, and migration ¹. Stable isotopes ratios vary between organisms and their environment because lighter isotopes are favored during reactions^2,3. Lighter isotopes have a lower activation energy for reaction, and therefore proceed more rapidly than heavy isotopes^2,4. The difference between an organism and its starting materials (i.e., discrimination, although sometimes called fractionation³) is predictable and has been the subject of much study^5–7.

An organism’s stable isotope signature not only reflects materials incorporated from external sources, but also the manipulation of internal sources. For instance, while food sources can be used to build cellular components (e.g., proteins), they can also provide energy to synthesize lipids. Enzymatic synthesis of lipids favors lighter isotopes at a high rate and as a result, lipids are enriched (i.e., contain higher amounts than the starting materials) in light isotopes of carbon (¹²C)^6,8,9 and hydrogen (¹H)^10,11, which are the primary atoms in lipids. In contrast, nitrogen is much rarer in lipids⁸ and is not altered from dietary sources during lipid synthesis.

The enrichment of lipids with light isotopes of carbon⁶ and hydrogen¹² is well documented and is the result of conserved biochemical pathways^9,10. Lipids are ubiquitous in living organisms, and researchers using carbon and hydrogen stable isotope ratios need to account for lipids to make meaningful interpretations^6,13 either by extracting them from a sample or mathematically accounting for them prior to interpretation ^6,13,14. However, work in larval lampreys, which belong to the most basal extant vertebrate group¹⁵, suggests their lipids may be more enriched in ¹³C than the proteinaceous portions of the body^16,17.

Lampreys are jawless fishes with a complex life cycle including a larval period and a true metamorphosis¹⁸. The larval period usually lasts from 3–7 years and is used to collect sufficient lipid reserves to fuel metamorphosis¹⁹. Larval lampreys can accumulate large quantities of lipids in their bodies (~ 15% of their wet weight)^20,21 by feeding on detritus (e.g., decaying organic matter and bacteria)^22,23, often primarily allochthonous in origin (i.e., terrestrial)^24,25, and to varying extents algae in stream sediments¹⁹. After growing slowly, often reaching only a few grams in weight²⁶, larvae undergo a metamorphosis during which they do not feed but rely instead of lipid stores²¹.

Larval lampreys often have the most enriched δ¹³C (e.g., the ratio of ¹³C to ¹²C relative to a reference) in stream communities^27–29 despite being primary consumers which feed by collecting particulates²². Larval lamprey isotope ratios are often highly variable^17,30,31, although many studies have not accounted for lipids. However, if larval lamprey δ¹³C is mathematically corrected for lipids following published equations^6,8, larval lamprey δ¹³C become unexplainably high¹⁷, appearing to be more like those of marine predators instead of stream detritivores. Interestingly, although more limited work on larval lampreys using δ²H has been done^16,17,32, when lipids were removed δ²H values have risen²⁴, as would be expected based on theoretical expectations. If larval lamprey δ²H and δ¹³C are to be useful in predicting source dependence the effect of lipids needs to be understood. Additionally, the use of δ²H in stable isotope ecology is growing, and recommendations for interpreting their values are increasingly important to allow broader use¹².

Based on prior isotope ratio measurements in larval lampreys^16,17, we hypothesized that larval lamprey lipids would be enriched in¹³C relative to muscle, and therefore the δ¹³C would decline after lipids were extracted. In contrast, we hypothesized that larval lamprey lipids would be enriched in ¹H, as expected based on other organisms¹⁰, and the δ²H in muscle would increase after lipid extraction. Finally, because there is relatively little nitrogen in lipids, we hypothesized that muscle δ¹⁵N would be unchanged by lipid extraction.

Larval lamprey collection and sample preparation

Previously measured muscle samples of larval lampreys from four prior studies were included in our analysis^16,17,24,33. In addition, larval American brook lamprey (Lethenteron appendix) in August 2017 from Trout Brook (New York, USA), sea lamprey (Petromyzon marinus) were collected in Little Patuxent River (Maryland, USA) in May 2021, and least brook lamprey (Lampetra aepyptera) from Greenhill Run in September 2021 (Maryland, USA). Larvae were collected alive by backpack electrofisher (LR-24, Smith Root). To cause larvae to leave the sediment, a low-power setting (3 hz, 100 V, 2:2 pulse pattern) was used, and once larvae were observed a high-power setting (30 Hz, 120 V, continuous output) was activated, which immobilized them for collection. Pacific lamprey (Entosphenus tridentatus) larvae from Evans and Lampman (2019)³³ were tank reared their entire lives and in Washington, USA.

Captured larvae euthanized by overdose in MS-222 or clove oil and then dissected by filleting and skinning lateral muscle, being careful to avoid any organs or the notochord. Muscle samples were placed into a microcentrifuge tube and then dried at 60°C for 24 hours. After drying, a subsample was lipid extracted following a modified Folch method³⁴. Briefly, subsamples were washed with ~ 1 mL of 2:1, by volume, chloroform to methanol three times. Samples were spun in a centrifuge at 5000 rpm for five minutes between each wash before the supernatant was decanted and more chloroform methanol was added. All supernatants were collected and dried for 24 hours at 60°C. After three washes the muscle was considered lipid free, as the supernatant was clear, and the muscle had become brittle and lighter in color. The container with evaporated supernatant had a thick yellow to orange viscous liquid, assumed to be lipid.

In addition to these samples, larval least brook lamprey (Lampetra aepyptera) were sampled from two rivers (Henderson Run and Furnace Brook) near St. Mary’s City, Maryland in September and December of 2020, and then in March and May of 2021 (Fig. 1). Larvae were collected along a stream reach of ~ 100 m by backpack electrofisher, sampling habitat likely to contain larvae (soft sediments and depositional areas)¹⁹. Within one hour of collection, larvae were transported in stream water to the lab. Larvae were either immediately euthanized by overdose in MS-222 and then dissected, or if time was insufficient to process all larvae rapidly, were euthanized and then frozen at -20°C until they could be processed later. These muscle samples were treated identically as described above. The collection and handling of these lampreys was approved by St. Mary’s College of Maryland IACUC.

Stable isotope ratios are reported in δ (delta) notation, where δ^xY = [(R_sample/R_standard) – 1] * 1000. Y is the element, x is the heavy isotope measured, and R is the ratio of heavy to light isotope in the sample and universal standards. As a result, isotope ratios are reported in per mil (‰) and can be positive or negative. Positive values mean the sample is enriched in the heavy isotope, while negative values mean the sample is depleted in the heavy isotope relative to the standard.

Stable isotope samples for δ¹³C and δ¹⁵N were weighed and packed into tin capsules, while those for δ²H (sometimes denoted as δD) were packed in silver capsules. Lamprey samples collected in Maryland were sent to the Cornell University Stable Isotope Laboratory (COIL). At COIL, samples were analyzed for δ¹³C and δ¹⁵N on a Thermo Finnigan Delta Plus and δ²H samples were analyzed on a Delta V IRMS through a ConFlo III. Standard deviations for standards were 0.2‰ for δ¹³C, 0.3‰ for δ¹⁵N, or 2‰ for δ²H. Samples from prior studies were either analyzed by COIL on the same instruments or at the University of California (UC) Davis Stable Isotope Facility (SIF) using a PDZ Europa ANCA-GSL (EA) attached to a PDZ Europa 20‐20 isotope ratio mass spectrometer (IRMS).

Statistical Analysis

The effect of lipid extraction on the stable isotope ratios of muscle was calculated as follows: Δδ²H = δ²H_extracted – δ²H_untreated, Δδ¹³C = δ¹³C_extracted - δ¹³C_untreated, and Δδ¹⁵N = δ¹⁵N_extracted - δ¹⁵N_untreated. We fit the Δδ¹³C and Δδ¹⁵N against the C:N ratio (a common proxy for lipid content^6,8) with four models, all of which have been used to fit similar data in prior studies^6,8. Our simplest model was a linear model:

Δδ^xY = a * C:N + b, [eqn. 1]

where b is the y-intercept and therefore -b/a is the estimate of C:N in a lipid-free sample (x-intercept). We also ln-transformed the C:N data⁸:

Δδ^xY = a * ln(C:N) + b, [eqn. 2]

and as a result, e^(-a/b) would be the model estimate of C:N in a lipid-free sample.

We also fit the data with a non-linear model⁸:

Δδ^xY = (a * C:N + b) / (C:N + c), [eqn. 3]

where a is an asymptote (i.e., an estimate of the difference between lipid and muscle), -b/a is the estimate of C:N in a lipid-free sample (x-intercept). Lastly, we also fit a simplified version of Eq. 3:

Δδ^xY = (a * C:N + b) / (C:N), [eqn. 4]

where a remains the asymptote and -b/a the estimate of C:N in a lipid-free sample. For these models we pooled all samples. We examined model residuals to test for homoscedasticity and compared models using Akaike Information Criterion (AIC) values. If models had similar support based on AIC scores, the simpler model was selected as the top model.

For Δδ²H we used the equations 1, 2, and 4, but we used the percentage of hydrogen in the untreated sample instead of C:N. We chose to use percent hydrogen because this was measured on each δ²H sample (C:N was measured on a separate sample submitted for δ¹³C/δ¹⁵N). The percentage hydrogen in the body should increase with lipids³⁵ since lipids include numerous hydrogens, the other cellular components (e.g., proteins and carbohydrates) were not expected to increase. Model selection followed the same pattern as described for δ¹³C/δ¹⁵N.

To test the top non-linear models (which reach an asymptote), we calculated the difference between muscle and lipid in measured samples as follows: Δ_MLδ²H = δ²H_extracted – δ²H_lipid and Δ_MLδ¹³C = δ¹³C_extracted - δ¹³C_lipid and compared it to the model’s asymptote estimate. Lipid isotope ratios were only measured in lampreys collected in Maryland. Variation in Δ_MLδ^xY was larger in some months than others, so an analysis of variance (ANOVA) with Δ_MLδ^xY as the response and month as a predictor was used; a Tukey honest significant difference test was used to compare contrasts. Lastly, we assumed that after lipid extraction there was no lipid remaining in the samples so we used a t-test for each isotope ratio to compare the models estimate of C:N in lipid-free sample or the percentage hydrogen in a lipid-free sample with those measured in lipid extracted samples. All analyses were performed in R v4.2.1³⁶.

Isotope ratios and C:N or percent hydrogen

The range of C:N ratios in muscle was large, ranging from 3.4 to 32.5 (Fig. 1). Muscle δ¹³C was positively related to the C:N ratio, and the relationship often appeared non-linear (Fig. 1). Muscle δ¹⁵N sometimes declined with increasing the C:N ratio but the pattern was not universal, and variance was often high (Fig. 2). Muscle δ²H was often negatively related to C:N ratio in samples, although at times no relationship was obvious (Fig. 3). When muscle δ²H was regressed against the percentage of hydrogen in the measured sample, a negative linear relationship emerged in all sites (Fig. 4).

Regressing Δδ²H against the percent hydrogen resulted in failure of Eq. 3 to converge, and there was strong evidence that the relationship was linear (Fig. 5a; Table 1). The top model for Δδ²H (Eq. 1) was a linear model with a slope of 26 ± 3‰ (mean ± SE) and as a result there was no calculable maximum difference between muscle and lipid (Δ_MLδ²H). In contrast, fitting of Δδ¹³C provided strong support for non-linear models, which always had AIC values > 50 units different from the linear model (Table 1) and more homoscedastic residuals (data not shown). Eq. 4 had the lowest AIC, and all its terms were significant; the asymptote was − 6.15 ± 0.23‰ (mean ± SE), and the model estimated a lipid free sample would have a C:N of 3.59 (Fig. 5b; Table 1). Lastly, Δδ¹⁵N was best fit by a linear model (Table 1), and Eq. 3 failed to converge. The slope of Δδ¹⁵N in the linear model (Eq. 1) was different from zero (0.050 ± 0.006‰, mean ± SE), but the intercept was not (Fig. 5c; Table 1). Visually, as C:N approached seven, Δδ¹⁵N appeared to increase, suggesting for low C:N samples, lipids had only a small, or no, influence on Δδ¹⁵N (Fig. 5c). For samples with C:N < 10, mean Δδ¹⁵N after lipid extraction was 0.36‰ (SD ± 0.30, n = 98); inclusion of all data (n = 121) raised the mean and SD by ~ 0.1‰.

Table 1

The model, equation number used in the present study, their AIC values, number of samples (n), and parameter estimates (mean ± SE).
Isotope Ratio	Model	Eqn. #	AIC	n	a	b	c
δ²H	Δδ²H ~ a * %H + b	1	176	22	25.5 ± 2.9***	-154 ± 21***	NA
δ²H	Δδ²H ~ a * ln (%H) + b	2	174	22	-347 ± 47***	-191 ± 24***	NA
δ²H	Δδ²H ~ (a * %H + b) / (%H)	4	177	22	-6.15 ± 0.23***	-22.05 ± 1.31***	NA
δ¹³C	Δδ¹³C ~ a * C:N + b	1	385	121	-0.250 ± 0.022***	-0.607 ± 0.250***	NA
δ¹³C	Δδ¹³C ~ a * ln (C:N) + b	2	339	121	2.94 ± 0.36***	-2.87 ± 0.18***	NA
δ¹³C	Δδ¹³C ~ (a * C:N + b) / (C:N + c)	3	328	121	-6.79 ± 0.61***	-23.17 ± 1.76***	1.41 ± 1.30
δ¹³C	Δδ¹³C ~ (a * C:N + b) / (C:N)	4	328	121	-6.15 ± 0.23***	-22.05 ± 1.31***	NA
δ¹⁵N	Δδ¹⁵N ~ a * C:N + b	1	71	121	0.050 ± 0.0060***	0.064 ± 0.054	NA
δ¹⁵N	Δδ¹⁵N ~ a * ln (C:N) + b	2	87	121	-0.40 ± 0.13***	0.45 ± 0.065***	NA
δ¹⁵N	Δδ¹⁵N ~ (a * C:N + b) / (C:N)	4	103	121	0.90 ± 0.091***	-2.71 ± 0.52***	NA
Note: *** denotes p-values < 0.001

Seasonal difference between muscle and lipid

The measured difference between muscle and lipid δ²H (Δ_MLδ²H) was not different between months (ANOVA, F_{3, 31} = 1.56, p > 0.2) and was 111‰ (SE = ± 21, n = 35; Fig. 6a). However, Δ_MLδ¹³C was different between months (ANOVA, F_3,53 = 5.05, p < 0.005); March was different from all months, but no other month was different from another (Fig. 6b). The mean Δ_MLδ¹³C in March was − 3.52‰ (SE = ± 0.67, n = 15) and was lower than other months (mean = -5.58‰, SE = ± 0.20, n = 42; Fig. 6b). In March, the variance was also larger (6.8‰) than other months (May = 2.9‰, Sept = 0.87‰, Dec = 0.46‰) and March included samples where the muscle and lipid had identical δ¹³C values (Fig. 6b).

Δδ^xY model evaluation

The top model for Δδ²H (Eq. 1) estimated precent hydrogen in a lipid-free sample would be 6.02% (Table 1) and was not different from the measured value in the quarterly lipid-free samples (6.04%; T_20,117 = 0.06, p-value > 0.5). The top model for Δδ¹³C (Eq. 4) estimated Δ_MLδ¹³C to be -6.15 ± 0.23‰ (mean ± SE, n = 119; Table 1) which was lower than the measured value in March (-3.52 ± 0.67‰, n = 15) or all other months (-5.58 ± 0.20‰, n = 42). Although the model did not agree in the magnitude all values were negative and the model did not include month as a factor. The model for Δδ¹³C (Eq. 4) also overestimated the C:N ratio of a lipid-free sample (T_119,121 = 15.72, p-value < 0.0001); the model estimated C:N would be 3.59 ± 0.001 (mean ± SE) in lipid-free sample, but in measured samples it was 3.34 ± 0.02 (mean ± SE, n = 121).Visual inspection of the top model for Δδ¹³C and its residuals showed that it was not homoscedastic, fitting values farther from a C:N of 3.5 better than those close to it.

The carbon backbone of larval lamprey lipids is often enriched in ¹³C relative to muscle, but the attached hydrogens are depleted in the heavy isotope (²H; and therefore, enriched in the lighter isotope ¹H). It is unclear why lipid synthesis should prefer a lighter isotope in one case (hydrogen), but not another (carbon). Larval lampreys are known to have unusual lipid origins, relying on muscle instead of the liver cells for synthesis²⁰. Larval lampreys may retain lipid scaffolding from other organisms they feed on, such as bacteria and algae³⁷, and then saturate any double bonds that occur primarily with ¹H. Stream algae δ¹³C is variable and dependent on water velocity³⁸ and their lipids may be relatively enriched in δ¹³C compared to larval lamprey muscle.

Additionally, larval lampreys have low metabolisms, sometimes requiring a decade or more to grow only a few grams¹⁹. Larval lampreys consume only a small fraction of lignin or cellulose in their diet (< 10%)³⁹, ensuring much of the material they consume passes undigested through the gut; some ingested algae pass through larval lamprey guts and emerge alive^39,40. Presumably when feeding, larval lampreys quickly consume the few carbohydrates they encounter, and then rely upon energy from amino acids and lipids for most metabolic costs. Only after quickly utilizing the relatively fast reacting ¹²C and excreting it as ¹²CO₂ or incorporating it into their own amino acids, would larvae begin to synthesize lipids. Since ¹²C is expelled or sequestered the body fatty acids may be relatively enriched in ¹³C.

The seasonal difference in carbon between muscle and lipid (Δ_MLδ¹³C; Fig. 6b) suggests the enrichment in ¹³C occurs primarily after the spring algal bloom, and maybe related to a physiological shift in the larvae. Enriched ¹³C lipids appear to exist year-round in some larvae, but during spring months some individuals have lipids which can be produced with δ¹³C almost identical to muscle (Fig. 6b). As the year proceeds these lipids appear to be reworked and enriched with ¹³C (Fig. 6b). Larval lampreys deposit the greatest amount of lipids during periods of high diatom density⁴¹. Therefore, larval lipid isotopic signatures maybe the result of their low metabolism and the manipulation of lipids during prolonged periods of slow or negative growth.

We found lipid extraction increased δ¹⁵N (Fig. 5c), and our value (~ 0.4‰) was close to the value (0.3‰) reported in a metanalysis of lipid extraction impacts on δ¹⁵N⁴². Removal of lipids relies on solvents, and these solvents are likely to remove proteins that are lipophilic, which could alter δ¹⁵N⁴³. Our work reinforces findings from prior work that researchers should measure δ¹⁵N prior to lipid extraction⁴². The increase we observed in lamprey δ¹⁵N as presumed lipid levels increase (i.e., C:N ratios; Fig. 5c), suggest that either: 1) δ¹⁵N is also manipulated in the body or that the 2) larval lampreys with higher lipid stores were feeding on isotopically different food sources than those with lower lipid stores, possibly suggesting larvae seek out different food sources as they near metamorphosis. Even in captive rearing studies, larval lamprey isotopic values can be highly variable³³, suggesting that microhabitat and food sources may vary spatially.

The use of δ²H is an emerging field¹², and lipids are known to alter bulk isotope ratios^10,11, but the work in this study underscores the strong effects lipids can have on measures of δ²H (Figs. 3–4). The C:N ratio has often been used to correct for lipids in samples^6,8, but researchers measuring δ²H may not have access to C:N values. Even if C:N ratios are available, δ²H and C:N ratios are often measured in a different subsample, as in the present study, because fewer isotope facilities can measure δ²H/δ¹³C/δ¹⁵N simultaneously and the cost often increases for such an analysis. As a result, the C:N ratio of the δ²H sample is often unknown, albeit it is likely similar to the value report when measuring δ¹³C/δ¹⁵N because the samples were homogenized. However, it is challenging to ensure samples remain truly homogeneous given that melting point of lipids is near room temperature and lipids tend to separate from the protein component (T. M. Evans, personal observation). Additionally, δ²H samples are small (< 0.4 mg), making them vulnerable to small changes in the percentage of lipid within a sample. We utilized the percentage of hydrogen in a sample, which provided better fits than C:N values (Fig. 5a), and presumably better estimates of the effect of lipid on samples. As has been done for δ¹³C^6,8,14, to increase the efficacy of δ²H, a comprehensive examination across multiple groups correlating the percentage of hydrogen with the percentage of lipid and their effect on the isotope ratio is needed.

Our work suggests, that while lipids are often enriched in ¹²C relative to muscle⁶, this is not a universal rule. However, larval lampreys follow expectations for hydrogen^10,11, which is always enriched in the lighter isotope relative to the muscle. Scientists working with isotopes in larval lampreys should anticipate lipid effects. To ensure they interpret their stable isotope ratios meaningfully, larval lamprey samples should be run in duplicate before and after lipid extraction, as mathematical correction appears to depend on the season. These findings should not be assumed to be true of juvenile and adult lamprey lipids. The physiological mechanism(s) to explain larval lamprey lipids is unknown but is necessary to fully explain this phenomenon. Such work will provide deeper insights into how isotopes in animal bodies can be manipulated.

Acknowledgements

We thank St. Mary’s College of Maryland for providing support and sampling equipment and Dr. Jeffrey Lombardo for reading and making comments on an earlier draft.

Author contributions

T.M.E. Designed the study, collected, processed, and analyzed the samples, and wrote the manuscript. S.B. helped to collect and process samples in the lab and edited the manuscript.

Data availability

All data used in the present study are available in the supplementary file. In addition, the R scripts used to perform the analyses are available from the corresponding author (T. M. E.) with a reasonable request.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Are lipids always light? Lipids in larval lampreys are enriched in 13C but depleted in 2H relative to muscle

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Larval lamprey collection and sample preparation

Statistical Analysis

Results

Isotope ratios and C:N or percent hydrogen

Seasonal difference between muscle and lipid

Δδ^xY model evaluation

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Are lipids always light? Lipids in larval lampreys are enriched in 13C but depleted in 2H relative to muscle

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Larval lamprey collection and sample preparation

Statistical Analysis

Results

Isotope ratios and C:N or percent hydrogen

Seasonal difference between muscle and lipid

ΔδxY model evaluation

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Δδ^xY model evaluation