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Biosynthetic constraints on amino acid synthesis at the base of the food chain may determine their use in higher-order consumer genomes [1]

['Javier Gómez Ortega', 'School Of Biological Sciences', 'Monash University', 'Clayton', 'Victoria', 'David Raubenheimer', 'The University Of Sydney', 'Charles Perkins Centre', 'School Of Life', 'Environmental Sciences']

Date: 2023-02

Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited, and for Drosophila melanogaster this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumer’s exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. Here, we update the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, we found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, we also found that exome matched AA profiles are generally conserved across taxa and that the composition of these profiles might be explained by energetic and elemental limitations on microbial AA synthesis. Thus, it appears that ecological constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that this constrains biomass composition.

Here, we attempt to improve dietary amino acid proportions (protein quality) for male and female reproduction in the fruitfly. We do this by tailoring the fly’s diet to contain amino acids in the proportions found in all the expressed proteins of either male or female Drosophila. In doing so, we discover that, despite functional differences between the sexes, their pattern of genome-encoded amino acid utilisation is remarkably conserved. In fact, this amino acid profile is also conserved in other species’ genomes from bacteria to humans. We hypothesise that this conservation represents an evolutionary strategy for organisms to make the most of limited amounts of dietary protein.

The amount and type of food that organisms consume shapes their fitness. Many species, including the fruitfly Drosophila melanogaster, suffer protein-limitation, which means they must evolve strategies to make the most of the protein they consume. We previously discovered that this protein limitations manifests at the level of individual amino acids for egg production in fruitflies.

Funding: This work was funded in part by the ARC (FT150100237), the NHMRC (1182330) to M.D.W.P., as well as the ARC (FT170100259) to C.K.M. (ARC is the Australian Research Council - www.arc.gov.au ; NHMRC is the National Health and Medical Research Council - www.nhmrc.gov.au ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Although we showed exome matching to be biologically effective, its current implementation does not incorporate weightings for the substantial differences we know to exist in genes’ sizes and their degree of expression [ 23 ]. Many studies have documented considerable differences in gene expression profiles when comparing transcriptomes between sexes, across life-history stages, or in response to biotic and abiotic stimuli [ 24 – 28 ]. For instance, in Drosophila, more than 8,000 genes, representing at least 50% of the genome, have been reported to be differentially expressed when comparing adult males with fertilised females–an observation that is unsurprising given the much heavier anabolic burden of reproduction for females than for males [ 25 ]. Thus, we predicted that we could improve the precision of exome matching by incorporating weightings for gene expression changes and in doing so, would establish a new way of tailoring diets to match an organism’s individual AA demands for life-stage and health status. Here, we set out to test this prediction and, in doing so, uncover that there is a surprisingly small variation in the way transcriptome weightings modify predicted AA usage. These data may reflect fundamental energetic and nutrient constraints on body composition across taxa.

To predict limiting AAs, our exome matching protocol involves two steps. First, we calculate each AA’s relative abundance in every protein of an organism’s in silico translated exome. Second, we find the average proportional representation for each AA across all proteins encoded by the exome. This genome-wide averaged AA proportion can then be compared to the AA proportion in the food to identify the essential AA that is most underrepresented in the diet and thus predicted to be limiting. We demonstrated that supplementing the diets of flies and mice with the limiting AA that was identified in this way can improve growth and reproduction and modify feeding behaviour [ 14 , 21 , 22 ]. Thus, for every organism whose genome has been sequenced, exome matching can theoretically be used as a tool to guide precision nutrition for better health.

Among the main components in the diet, protein is the limiting nutrient for the growth and reproduction of many organisms. It is, therefore, a principal constraint on evolutionary fitness [ 1 , 4 – 6 ]. For example, the abundance of protein-rich food has been shown to increase population size or stimulate body growth of birds such as the galah (Eolophus roseicapilus) or the goldfinch (Carduelis carduelis) and mammals like the house mouse (Mus musculus) or several species of squirrels (Sciurus and Tamiasciurus spp.) [ 7 – 13 ]. In the fruit fly Drosophila melanogaster, we have found that female reproduction is reduced by decreasing overall dietary protein concentration [ 14 – 16 ]. We also found that this protein limitation is determined by the concentration of the single most limiting essential Amino Acid (AA) in the diet, which can be identified by comparing the proportion of AAs that is available in food against the proportion of AAs encoded by the fly’s exome–a procedure we called exome matching [ 14 – 16 ]. Evidence from our work, and that of others, indicates that exome matching may have broader application as protein limitation also occurs at the level of single AAs in other species [ 14 , 17 – 20 ].

Nutrition is one of the most important environmental determinants of evolutionary fitness; it supplies organisms with energy and the building blocks they require for growth, reproduction, and somatic maintenance [ 1 ]. However, the natural availability of food and its nutritional qualities vary and inevitably differ from the consumer’s needs [ 1 – 3 ]. As such, evolutionary fitness is constrained by the divergence between nutrient demand and their availability. Because of this, optimising nutrition to enhance growth, reproduction, and health is of major interest from both a fundamental biology and a commercial perspective.

Results

Calculating transcriptome-weighted, exome-matched dietary aa proportions Our previous research demonstrated that female flies fed food containing exome-matched AA proportions (FLYAA) laid more eggs than flies on food with equivalent amounts of protein comprised of mismatched AA proportions [14]. Although FLYAA demonstrably improved egg-laying, we hypothesised it could be further improved by weighting each gene’s contribution to the overall average by its length and expression level. We reasoned that although there is not a 1:1 association between transcription and translation, the transcriptome would be a good approximation for the expression weightings for two reasons. First, transcriptomics readily yields a more complete set of gene expression values than proteomics [29,30]. And, second, if the availability of dietary AAs constrains organismal protein expression, whole genome proteomics would simply reflect the constraints of diet quality. In contrast gene expression values may indicate protein expression levels that could be achieved if dietary AA availability was not a constraint—i.e. better matched to requirements. We downloaded transcriptome profiles of whole male and whole female flies from FlyAtlas 2 and modENCODE, and from these profiles we averaged the levels of gene expression for each sex [31,32]. To make our new sex-specific, transcriptome-matched profiles, we first counted the number of each AA that is encoded by each protein isoform in the fly genome. We then weighted these AA counts by the average isoform relative transcript abundance (FPKM value; see Materials and Methods) found for male or female flies. For each AA, we then summed the weighted AA counts across all genes and used these values to compute each AA’s proportional representation across all expressed genes. Although we observed some differences between the transcriptome profiles obtained from FlyAtlas 2 and modENCODE, they produced concordant changes in the proportion of each AA when compared with FLYAA. These newly designed AA ratios for the sexes were labelled MALEAA and FEMALEAA, and these became the basis for new dietary AA profiles (Fig 1). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Comparison of the exome matched and transcriptome weighted AA ratios. https://doi.org/10.1371/journal.pgen.1010635.g001 Proportion of each AA in the exome matched [14] and transcriptome-weighted exome matched diets (MALEAA and FEMALEAA). In these diets, the average proportions of AAs have been generated after weighting each protein’s contribution by size and its average gene expression in male and female transcriptomes, respectively. The dotted grey line separates the essential (left) from the non-essential (right) AAs. IUPAC single-letter AA codes are shown. Error bars display the standard deviation from weighting the AA ratios by each of the replicate transcriptome profiles from FlyAtlas 2 and modENCODE that were available for males or females.

Transcriptome-weighted exome matching the dietary aa profile does not improve male fertility over that on an exome matched diet To test the effects of dietary AAs on male fertility, we used an assay in which males were challenged to inseminate females at maximum capacity, as this should deplete the males of sperm and/or seminal fluid and thus require them to be synthesising more from the dietary AAs they have available. To do this, we supplied singly housed males with ten new virgin females per day for seven days and counted how many of these females subsequently produced viable offspring. We found that while on the first day, males could inseminate 8 to 10 of these virgins, during the course of the assay, the number of females that each male could inseminate dropped to at least half of the number found for day one (Fig 2A) indicating that the males were indeed operating at maximum capacity in this assay. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Male fecundity is modified by dietary AA concentration but not ratio. (A). The number of virgin female flies successfully fertilised by individual males during our seven-day assay. Male fecundity was reduced by decreasing dietary AA concentrations. AAs were provided in the exome matched proportion, FLYAA). Error bars represent the standard deviation. (B). The change in the cumulative number of females fertilised by males in response to dietary AA change could be modelled by a sigmoidal dose-response curve (R2 = 0.537; least-squares fit). (*** = P<0.001, in comparison with 0 g/L). Error bars represent the standard deviation. (C). Predicted difference in each essential AA when comparing the male transcriptome matched proportions (MALEAA), and the exome matched proportions (FLYAA). A positive difference indicates that the AA is more abundant in MALEAA than in the FLYAA. MALEAA should cover any essential AA deficiency of FLYAA, and thus, the relative increase in the concentration of the most limiting essential AA (Tryptophan, W, 20%) equals the potential increase in fecundity that could be achieved for flies fed with MALEAA. (D). Males fed with a diet containing a transcriptome (MALEAA) matched AA proportion did not differ from those fed the exome matched (FLYAA) diets for any concentration of AAs tested. Error bars represent the standard deviation. (E). The effect of tryptophan dropout from the diet on the daily capacity of males to fertilise females during a seven-day period. The removal of tryptophan from the diet caused a fast decay in fertilisation that matched that caused by the removal of all AAs. AAs were provided in the male transcriptome matched proportion, MALEAA. Error bars represent the standard deviation. (F). Coverage of the predicted dietary AA requirements by MALEAA and FLYAA when compared to the transcriptome weighted exome match proportion from each tissue in male flies. For each tissue, the x-axis displays the degree to which the limiting AA demand is met by the diets MALEAA (blue) and FLYAA (black). The closer to 100%, the better the diet covers the predicted tissue demand for AAs. For all tissues, except the salivary gland, MALEAA is predicted to be a better match for requirements than FLYAA. The predicted limiting AA for each tissue on each diet is indicated by the three letter AA codes. https://doi.org/10.1371/journal.pgen.1010635.g002 To test if male fecundity changed in response to altered dietary protein levels, we performed the above assay on males that were maintained on food in which the AA proportion was fixed (FLYAA), but the total concentration was diluted from 10.7g/l (positive control; the level in our standard “rich” diet) to 2.1 g/l, 1.1 g/l and 0 g/l. Male fertility was significantly lower than the positive control when the flies were maintained on 0 g/l and 1.1 g/l AAs (P<0.001) (Fig 2A). Surprisingly, when the protein concentration was 2.1 g/l, male fertility increased to the level of flies maintained on the positive control condition (Fig 2A and 2B). Thus, maximum male fertility in our assay responded to dietary AA levels and only relatively small amounts were required to support maximal fertility. The response of male fecundity to dietary AA concentration could be modelled by a sigmoidal dose-response curve with an inflexion point somewhere between 1.1–2.1 g/l AA (Fig 2B). If MALEAA represents the ideal proportion of AAs for male fecundity, we predict that males fed FLYAA would be tryptophan (trp, W) limited, and that for a fixed sum of AAs changing the proportion to MALEAA would yield a 20% increase in AA availability for reproduction (Fig 2C). However, when we compared the fecundity response of male flies kept on MALEAA and FLYAA at each of the dietary AA concentrations, we saw that AA ratio did not alter the number of females that were successfully inseminated, even under conditions where male fertility was clearly AA limited (1.1 g/l: Fig 2D). This lack of effect was not due to an insufficient sampling since a power analysis revealed that a 20% difference in fecundity would have been observable at 2.1 g/l. A possible reason why MALEAA did not improve fecundity is that males might contain sufficient stores of tryptophan in body proteins that they can retrieve and use to overcome the limitation we predicted. If this were the case, our prediction of a 20% improvement in fecundity would be an overestimate. To assess this, we made another diet in which the AA profile resembled FLYAA, but tryptophan only was omitted from the diet altogether.We evaluated the effect of this diet and found that it caused a significant reduction in fecundity compared to the positive control diet (10.7g/l). It was also equally as detrimental for fecundity as a diet without AAs, both in terms of the rate at which fertility fell, and the total number of females successfully fertilised during the assay (Fig 2E). Thus, dietary tryptophan is required to sustain male fecundity in this assay, and its requirement does not appear to be lessened due to the recovery of tryptophan stored in body tissue. Another reason why MALEAA may not have improved fecundity over FLYAA is that the actual set of proteins required for male fecundity are only a subset of those included in our calculation and that MALEAA is actually a worse AA balance for the organs responsible for making the proteins required for fecundity. To investigate this possibility, we calculated transcriptome-weighted AA profiles for each tissue type in male flies using RNAseq data from FlyAtlas [31]. We then compared these tissue profiles to both the unweighted (FLYAA) and transcriptome weighted (MALEAA) dietary AA profiles and predicted each tissue’s limiting AA and the degree to which it is limiting. The data are expressed as a relative match where 0 indicates the complete absence of an essential AA and 100 represents that all dietary AAs are perfectly matched to the tissue-specific profile (Fig 2F). The data show that MALEAA is predicted to be a better match than FLYAA for the expression of the genes in each tissue, except for those in the salivary glands in which FLYAA is predicted to be a slightly better match than MALEAA. Thus, unless AA supply to the salivary glands limits whole organism fecundity we still predict that MALEAA would be an improved AA profile over FLYAA for male reproduction if transcriptome weighting the exome provided a superior prediction of dietary requirements. However, our tissue-specific analysis does reveal that MALEAA is predicted to confer a smaller improvement over FLYAA if only the profile of the testis (9.6% increase) or accessory glands (10% increase) matter for our assay of male fecundity. It is possible that this small degree of enhancement in male fecundity was beyond the sensitivity of our assay to be detected.

Transcriptome-weighted exome matching the dietary aa profile does not improve female fecundity over that on an exome matched diet Our previous data indicate that female egg-laying is a reliable indicator of dietary AA composition, and typically has lower variability and greater sensitivity than the male fecundity assay we performed [14]. We thus tested if FEMALEAA had a higher nutritional value than FLYAA to sustain female fecundity. Two-day-old mated females were placed on chemically defined diets, and the number of eggs they laid over the course of eight days was counted. In line with previous results [14], female egg production responded in a linear manner to increasing AA concentrations until at least 10.7 g/L (Fig 3A). This is consistent with dietary AAs quantitatively limiting female egg-laying, which we have previously shown to be due to the most limiting essential AA [14]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Female fecundity responses to changes in dietary AA ratio and concentration. (A). Total female fecundity showed a linear response to increasing dietary AA concentrations (FLYAA). R2 = 0.999. (B). Relative change in the molar concentration of each essential AA between the female transcriptome matched diet (FEMALEAA) and the exome matched diet (FLYAA). A positive difference indicates that the AA is more abundant in FEMALEAA than in FLYAA. The relative increase in the concentration of the most limiting essential AA (Lysine, K, 20%) equals the potential increase in fecundity that could be achieved for flies fed with FEMALEAA. (C). At each concentration of total AA, there was no difference in egg-laying output of females fed with transcriptome (FEMALEAA) or exome (FLYAA) matched diets. (D). Lysine dilution limits egg production in a linear manner. Percentages of Lysine concentration are relative to the standard Lysine concentration on FLYAA with a total AA concentration of 10.7 g/L. R2 = 0.995. (E). Coverage of the predicted dietary AA requirements of each tissue in female flies by FEMALEAA and FLYAA. Tissue dietary AA requirements are predicted from the tissue-specific transcriptomes. For each tissue, the x-axis displays the percent of the limiting AA demand covered by the diets FEMALEAA (green) and FLYAA (black). The closer to 100%, the better the diet meets the theoretical tissue AA demand. For all tissues, except the salivary gland, FEMALEAA is predicted to be a better match for requirements than FLYAA. The predicted limiting AA for each tissue on each diet is indicated by the three-letter AA codes. https://doi.org/10.1371/journal.pgen.1010635.g003 We predicted that if the transcriptome-weighted diet (FEMALEAA) represented the actual AA requirements for females for egg-laying, lysine (K, Lys) would limit egg production for females feeding on the non-weighted AA ratio (FLYAA) (Fig 3B). By comparing the AA profile of FEMALEAA to that of FLYAA, we also predicted that egg production could be up to 20% higher when incorporating transcriptome weightings into the exome match profile (Fig 3B). However, FEMALEAA did not improve female fecundity output in comparison to FLYAA at either concentration of dietary AAs tested (Fig 3C). This included a concentration at which AAs clearly limited egg production (5.4 g/L) and so should be the most sensitive test of the change in availability of the most limiting AA. This lack of effect was not due to insufficient sampling since a power analysis revealed that a 20% difference in fecundity would have been observable at 5.4 g/l and 10.7 g/l AA. We tested if the reason why female flies produced no more eggs on FEMALEAA than when on FLYAA is that they could supplement limiting dietary lysine by retrieving it from body protein reservoirs. If dietary lysine limits egg-laying, and the flies do not supplement it from body reserves, the flies should exhibit reduced egg production in proportion to the dilution of lysine in the diet. This is exactly what we observed when we reduced lysine only in an otherwise constant nutritional background containing 10.7 g/L FLYAA (Fig 3D). This demonstrates that lysine is both essential and limiting in FLYAA for female fecundity and indicates that lysine limitation is not lessened by flies retrieving it from stored body protein. We also tested if FEMALEAA is not superior to FLYAA to support egg production because the flies use only a subset of the transcriptome to produce eggs. To do this, we assessed the match between each tissue-specific AA profile and FEMALEAA or FLYAA. Similar to the comparison we made for males, FEMALEAA was a better match than FLYAA to the transcriptome weighted AA proportions of every female tissue except for the salivary glands (Fig 3E). Furthermore, if we consider only the tissues most relevant to reproduction, the ovaries and fat body, FEMALEAA represents a better match, and to a similar extent as whole-body samples, to their transcriptome-weighted exome profiles than FLYAA (16% and 15% for ovaries and fat body respectively). These data support our prediction that FEMALEAA should improve dietary AA availability for egg production over that found in FLYAA.

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