Protein feeding mediates sex pheromone biosynthesis in an insect
Abstract
Protein feeding is critical for male reproductive success in many insect species. However, how protein affects the reproduction remains largely unknown. Using Bactrocera dorsalis as the study model, we investigated how protein feeding regulated sex pheromone synthesis. We show that protein ingestion is essential for sex pheromone synthesis in male. While protein feeding or deprivation did not affect Bacillus abundance, transcriptome analysis revealed that sarcosine dehydrogenase (Sardh) in protein-fed males regulates the biosynthesis of sex pheromones by increasing glycine and threonine (sex pheromone precursors) contents. RNAi-mediated loss-of-function of Sardh decreases glycine, threonine, and sex pheromone contents and results in decreased mating ability in males. The study links male feeding behavior with discrete patterns of gene expression that plays role in sex pheromone synthesis, which in turn translates to successful copulatory behavior of the males.
Editor's evaluation
The manuscript describes the effects of dietary yeast and sugars on male Bactrocera dorsalis sex pheromone biosynthesis and other mating-related traits. This is an important study showing that yeast feeding stimulates the production of specific sex pheromones and promotes fly mating ability. The data are solid and will be of interest to the fields of chemical ecology and pest management.
https://doi.org/10.7554/eLife.83469.sa0Introduction
Females in many animal species are ‘investment breeders’, foraging for reproductive resources during adulthood, which are directed into offspring production (Stearns, 1989; Stephens et al., 2009; Stephens et al., 2014). Intriguingly, males of many species may be categorized in a similar manner (Soulsbury, 2019), depending on foraging success to secure copulations and manipulate female behavior, while prioritizing different resources than the females (Gwynne, 2008; Ng et al., 2019). Indeed, a number of studies on males from various insect groups suggest a strong link between adult foraging and reproductive success (e.g. Lepidoptera [Boggs, 1981]; Orthoptera [Gwynne, 2008]; Mecoptera [Sauer et al., 1998]; Diptera [Yuval et al., 2007]).
The dipteran family Tephritidae contains over 4000 species, and almost all tephritid adults need post-teneral carbohydrate and protein nutrition to realize their fitness potential (Pereira et al., 2013; Taylor et al., 2013). Post-teneral protein feeding affects the reproduction of male tephritid flies in multiple ways, including the following: (1) sexual organ development. For example, the reproductive organs (testes, accessory glands, ejaculatory duct, and apodemes) of Bactrocera dorsalis males can significantly enlarge after protein consumption (Reyes-Hernández et al., 2019). In Bactrocera tryoni, protein feeding accelerates the development of the male copulatory apodemes (Weldon and Taylor, 2011; Taylor et al., 2013; Reyes-Hernández et al., 2019); (2) pheromone release, which is a key step in reproduction. In Ceratitis capitata, protein feeding increases male intensity of sex pheromone release (Yuval et al., 2002). B. tryoni males release an incomplete mixture of sex pheromones when deprived of protein (Weldon and Taylor, 2011) (3) mating behavior. In many species of Tephritidae, mating competitiveness, mating probability, and mating duration are significantly increased in flies supplied with protein (Yuval et al., 2002; Prabhu et al., 2008; Taylor et al., 2013; Reyes-Hernández et al., 2019); (4) sperm storage and egg fertilization. Protein supplementation for males increases the amount of sperm stored by mated females (Taylor et al., 2013; Reyes-Hernández et al., 2019) and the probability of egg fertilization (Blay and Yuval, 1997; Shelly et al., 2002; Yuval et al., 2007); (5) inhibition of female receptivity. Ejaculates of protein-fed males inhibit the remating behavior of females (Radhakrishnan and Taylor, 2007). Conversely, the remating rate in females increases significantly when males lack access to protein (Yuval et al., 2002; Taylor et al., 2013).
Males of the oriental fruit fly, B. dorsalis, one of the most economically important tephritid species, attract females by emitting a pheromone produced by bacteria in the rectal glands (Ren et al., 2021). The pheromone has been identified as a cocktail of trimethylpyrazine (TMP) and tetramethylpyrazine (TTMP), and the pathway of sex pheromone biosynthesis has been proposed in a previous study (Zhang et al., 2019, Ren et al., 2021). Although sex pheromone-producing Bacillus in B. dorsalis have been identified, the source of the precursors has not been determined. Given that protein feeding has a significant influence on the reproductive performance of B. dorsalis males (Shelly and Edu, 2007), we hypothesized that protein feeding in B. dorsalis mediates sex pheromone biosynthesis by affecting the precursor substance content in the rectum. Accordingly, in this study, we investigated the mechanism by which protein feeding influences the biosynthesis of sex pheromones in B. dorsalis. The results indicate that protein feeding is the key factor that controls sex pheromone biosynthesis in the male rectum. Ingested protein supplies the glycine and threonine pathway and provides substrates for sex pheromone production.
Results
Protein feeding is required for sex pheromone biosynthesis and successful mating
To determine whether protein feeding is required for reproductive performance, we tested the effects of protein feeding on male survival, rectum width, sex pheromones, and mating ability (Figure 1A). As in previous studies (Orankanok et al., 2013; Shelly, 2017), yeast hydrolysate (YH) was used as the protein source. Since rectal Bacillus also need to use glucose to synthesize sex pheromone (Ren et al., 2021), we fed male flies three different types of sugar (sucrose, fructose, and glucose) to investigate the influence of glucose feeding or not on sex pheromone synthesis and reproductive performance of male B. dorsalis (Figure 1A). The results showed that different types of sugars had no effect on survival, rectum width, sex pheromone biosynthesis, and mating ability of mature male (12 d old; Figure 1B–G). Though protein feeding also did not affect the survival and rectum width of the males (Figure 1B and C), major products with the same retention time were only detected in YH-supplemented male rectums when rectal extracts were analyzed by gas chromatography‒mass spectrometry (GC‒MS; Figure 1D). These products were tentatively identified as TMP and TTMP in all three YH-supplemented cases based on their mass spectra (Figure 1E and F). And the mating ability of the males deprived of YH was significantly lower than that of the males fed with YH (Figure 1G). These results indicate that protein feeding instead of sugar type can significantly affect the male reproductive performance (sex pheromone synthesis and mating ability). Given that sex pheromones are synthesized by rectal Bacillus using glucose and amino acids as precursors (Ren et al., 2021), we infer that dietary proteins may influence sex pheromone synthesis and mating ability by influencing the abundance of Bacillus in the rectum or the amount of the precursors.

Influence of post-teneral protein and sugar feeding on male flies.
(A) A schematic showing how the male flies were reared and the biological parameters compared. (B) Effect of post-teneral protein and sugar on survival (n=200 individuals, Kaplan‒Meier survival analysis was used, and NS: no significance). (C) Rectum size comparisons between yeast hydrolysate (YH)-deprived (YH−) and YH-fed (YH+) males (n=40 individuals, Student’s t test, and NS: no significance). (D) Gas chromatography‒mass spectrometry (GC‒MS) ion chromatograms of rectum extracts of males fed different types of food. Traces for the flies fed with YH expressing trimethylpyrazine (TMP; red arrow) and tetramethylpyrazine (TTMP; blue arrow) are shown. (E) and (F) GC‒MS mass spectra of TMP and TTMP. (G) Mating ability comparisons between YH− and YH+ males (n=5 replicates, Wilcoxon matched-pairs signed rank test, and *** p<0.001). In violin plots, where the violin encompass the first to the third quartiles, inside the violin the horizontal line shows the median.
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Figure 1—source data 1
Raw data used for analysis in Figure 1A, B and F.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig1-data1-v2.xlsx
Precursor amino acids of sex pheromones are affected by protein feeding
To examine the above hypotheses, the absolute abundance and composition of the mature male (12 d old) rectum microbial communities were inferred by 16S rRNA gene quantification and amplicon sequencing (Supplementary file 1). The results showed that there is no significant difference for total bacteria contents between YH-supplemented and YH-deprived male rectum (Figure 2A). 16S rRNA amplicon sequencing results showed that microbial communities at the class level in YH-supplemented male rectum were similar with those in YH-deprived male rectum, especially the abundance of Bacilli was very similar (Figure 2B and C). Alpha diversity in 16S rRNA amplicon sequencing also indicated that protein feeding had no influence on diversity except the males feeding on sucrose (Supplementary file 2, Figure 2—figure supplement 1). These results indicate that protein intake may not affect the abundance of Bacillus synthesizing pheromones in the rectum, and sex pheromone loss in YH-deprived males may not be associated with Bacillus.

Influence of post-teneral protein on rectal bacteria and sex pheromone precursors.
(A) Boxplot showing total bacteria in the male rectum estimated from 16S rRNA gene quantitative PCR (qPCR; n=6 replicates, Student’s t test, and NS: no significance). (B) Principal coordinate analysis of the microbial community structure (beta diversity and class level) measured by the Bray‒Curtis distance matrix of 16S rRNA gene amplicon sequences. (C) Class-level relative abundance of 16S rRNA gene amplicon sequences. Values are averaged according to yeast hydrolysate (YH)-deprived and YH-supplied males. (D), (E), and (F) Influence of YH supply on rectum glucose (n=15 replicates), threonine (n=5 replicates), and glycine (n=5 replicates) contents (Student’s t test, NS: no significance, * p<0.05, and *** p<0.0001). In violin plots, where the violin encompass the first to the third quartiles, inside the violin the horizontal line shows the median.
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Figure 2—source data 1
Raw data used for analysis in Figure 2A, D, E and F.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig2-data1-v2.xlsx
Since rectal Bacillus, glucose, and threonine/glycine are three essential factors for sex pheromone synthesis, we further measured glucose and threonine/glycine contents to confirm whether protein supplementation regulates sex pheromone synthesis by influencing rectal glucose or threonine/glycine contents. The results showed that YH feeding only increased glucose contents in males in the sucrose and fructose groups (Figure 2D), which indicates that increased rectal glucose content caused by protein supplementation may not be the main factor affecting sex pheromones synthesis. On the other hand, YH feeding significantly increased threonine and glycine contents in males in all sugar groups (Figure 2E and F), which indicates that the decreased precursor amino acid contents may be the main factor affecting sex pheromone producing in rectum of YH-deprived males.
Glycine and threonine pathway involved in protein metabolism
If YH supplementation is necessary for threonine and glycine synthesis, we reasoned that molecular pathways in the rectum mediating threonine and glycine synthesis may show different expression patterns between YH-fed individuals and YH-deprived individuals. We first carried out RNA-seq in the rectum of YH-fed males and YH-deprived males (12 d old). Principal component analysis (PCA) using the expression profiles of the identified genes indicated that YH-deprived males were significantly different from the YH-fed ones (Figure 3A, Supplementary file 3). Pearson correlation coefficients, which were generated by the expression profiles, between samples also indicated that YH-fed males had higher similarity than YH-deprived individuals (Figure 3B). Pairwise differential expression (DE) analysis identified 770, 914, and 746 DE genes in the sucrose, glucose, and fructose groups, respectively (Supplementary file 4, Figure 3—figure supplement 1). To identify the DE genes involved in synthesizing glycine or threonine, a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed. The glycine and threonine pathway was significantly enriched in the sucrose, glucose, and fructose groups, with the sarcosine dehydrogenase gene (Sardh) and alanine-glyoxylate transaminase (AGXT2) being the significantly differentially expressed genes (DEGs) in all groups, and the expression of Sardh was significantly induced in YH-fed males (Figure 3C, Figure 3—figure supplement 2, Supplementary file 5). Quantitative PCR (qPCR) also confirmed that Sardh expression was significantly enhanced by YH feeding, yet unaffected by sugar identity (Figure 3D). In the glycine and threonine metabolism pathway in insects, Sardh and L-threonine aldolase (ltaE) are responsible for converting sarcosine into glycine (Frisell and Mackenzie, 1962) and threonine (Liu et al., 1998), respectively (Figure 3E). Together, the results suggest that Sardh might be involved in sex pheromone biosynthesis by controlling glycine and threonine synthesis.

Transcriptome comparisons between yeast hydrolysate (YH)-fed and YH-deprived males.
(A) Principal component analysis (PCA) obtained from gene expression profiles showing differences between YH-fed and YH-deprived males. Flies are clustered according to YH fed or not. (B) Pearson correlation coefficient showing the similarity between the samples. Higher similarity of the transcriptome is shown by a darker blue color (higher correlation coefficient). (C) Table showing the number of genes found in any given category and the genes involved in the threonine metabolism pathway between comparisons. (D) Quantitative PCR (qPCR) verifying the expression of Sardh in YH-fed and YH-deprived males (n=5 replicates, Student’s t test, and *** p<0.001). In violin plots, where the violin encompass the first to the third quartiles, inside the violin the horizontal line shows the median. (E) Proposed model of the threonine metabolism pathway in insects.
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Figure 3—source data 1
Raw data used for analysis in Figure 3C.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig3-data1-v2.xlsx
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Figure 3—source data 2
Raw data used for analysis in Figure 3A and D.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig3-data2-v2.xlsx
In normally reared (sucrose and protein were both provided) male B. dorsalis, sex pheromones can only be produced 9 d after emergence (Ren et al., 2021). Thus, we measured the threonine and glycine contents and generated RNA-seq datasets of the male rectum from YH and sucrose fed males at different development times (0 d, 3 d, 6 d, 9 d, and 12 d) to further verify whether the amino acid contents and Sardh expression were associated with sex pheromone biosynthesis. Consistent with the idea that the glycine and threonine metabolism pathway was involved in sex pheromone biosynthesis, we found that the glycine content was significantly higher in older males (6 d, 9 d, and 12 d) (although there was no difference in the threonine content; Figure 4A and B). RNA-seq data indicated that the older males had higher similarity for gene expression profiles (Figure 4C, Figure 4—figure supplement 1, Supplementary file 6). DEG analysis also indicated that a larger number of DE genes occurred in males with greater age differences (Figure 4—figure supplement 1, Supplementary file 7). KEGG analysis indicated that more DE genes were enriched in the glycine and threonine pathway between males with greater age differences (Supplementary file 8). Consistently, Sardh in the glycine and threonine pathway was significantly highly expressed in the rectum of older males (Figure 4D and E). The high expression of Sardh in the rectum suggests that Sardh plays a role in converting YH into glycine and threonine to produce sex pheromones.

Amino acid contents and transcriptome investigation of male rectums at different developmental stages.
(A and B) Threonine (n=5 replicates) and glycine (n=5 replicates) contents in the rectum at different developmental stages (different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test). (C) Principal component analysis (PCA) using differential expression (DE) genes obtained from pairwise comparisons between different developmental stages. (D and E) Expression profiles of Sardh obtained by RNA-seq and quantitative PCR (qPCR; n=5 replicates, different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test). In violin plots, where the violin encompass the first to the third quartiles, inside the violin the horizontal line shows the median.
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Figure 4—source data 1
Raw data used for analysis in Figure 4A, B, D and E.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig4-data1-v2.xlsx
Functional Sardh is necessary for sex pheromone biosynthesis
We next focused on genetically testing whether Sardh was necessary for sex pheromone biosynthesis. To this end, we first measured the relative expression level of Sardh in the head, thorax, and rectum of mature males (12 d old) fed with YH and sucrose to further confirm that Sardh plays a role in sex pheromone biosynthesis in tissue. The qPCR results showed that Sardh was indeed highly expressed in the rectum (Figure 5A). We then performed RNAi in Sardh by injecting dsRNA into the male (12 d old) abdomen and checked the influence on precursor contents and reproductive performance (Figure 5—figure supplement 1). Similar to YH-deprived males, Sardh knockdown males showed significantly decreased rectal threonine and glycine contents (Figure 5B and C). Sex pheromone quantification results indicated that TMP content in the rectum decreased significantly in Sardh knockdown males (Figure 5D) and that Sardh knockdown males showed significantly decreased mating competition ability (Figure 5F). These results show that Sardh plays role in converting the rectal threonine and glycine. Together, the findings provide a functional demonstration that Sardh, which can be induced by protein feeding and plays a role in synthesizing glycine and threonine, is necessary to regulate sex pheromone biosynthesis in male B. dorsalis.

Functional verification of Sardh in sex pheromone biosynthesis.
(A) Expression of Sardh in different tissues with quantitative PCR (qPCR; n=5 replicates, different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test). (B and C) Threonine (n=5 replicates) and glycine (n=5 replicates) contents in the rectum with Sardh knockdown (different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test). (D and E) Sex pheromone (trimethylpyrazine [TMP] and tetramethylpyrazine [TTMP]) quantification in the rectum with Sardh knockdown (n=4 replicates, different letters above the error bars indicate significant differences at the 0.05 level analyzed by ANOVA followed by Tukey’s test). (F) Mating ability comparisons between males with Sardh knockdown and controls (n=5 replicates, different letters above the error bars indicate significant differences at the 0.05 level analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test). In violin plots, where the violin encompass the first to the third quartiles, inside the violin the horizontal line shows the median.
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Figure 5—source data 1
Raw data used for analysis in Figure 5A–F.
- https://cdn.elifesciences.org/articles/83469/elife-83469-fig5-data1-v2.xlsx
Discussion
In recent decades, a large number of studies have reported that protein feeding is critical for male insect reproductive success. In the study, how ingested proteins supply the precursors of sex pheromones to male B. dorsalis was indicated. Highly expressed Sardh can convert protein into threonine and glycine, which can be used by the rectum Bacillus to synthesize the sex pheromone (Figure 6). The study clarifies the molecular mechanism by which host protein feeding regulates sex pheromone biosynthesis.

Schematic illustrating the sex pheromone biosynthesis hypothesis that B. dorsalis rectum.
Bacillus uses threonine and glycine, which are converted by Sardh with post-teneral protein feeding by B. dorsalis, as precursor substances to synthesize the sex pheromone.
A large number of studies have indicated that pyrazines are widely used as pheromones in insects (Bohman et al., 2016; Calcagnile et al., 2019). However, how pyrazines are synthesized in these insects has not yet been revealed. With a series of chemical analysis and molecular biology experiments, we discovered that protein fed by insects contributes to provide precursor substances for pyrazines synthesis. Specifically, we confirmed that the Sardh in the glycine and threonine pathway can convert protein into pyrazine precursor substances-threonine and glycine. Given that the glycine and threonine pathway is conserved in insects (Crawford et al., 2010; Nallu et al., 2018; Sonn et al., 2018), our findings may be relevant for all insects that use protein to synthesize pyrazines.
Previous studies suggest that the influence of protein feeding on the mating success of Tephritidae is caused by affecting the development of testes and accessory glands (Weldon and Taylor, 2011; Taylor et al., 2013; Reyes-Hernández et al., 2019) and increased levels of courtship activity (Pereira et al., 2013). Although researchers have speculated that there is a positive correlation between protein feeding and sex pheromones (Yuval et al., 2007), this relationship has been hard to pin down. Certain plant chemicals, such as methyl eugenol, gingerone, and raspberry ketone, which strongly attract tephritidae males of some species, are thought to be the precursors of sex pheromones (Tan and Nishida, 2012; Kumaran et al., 2014a, Kumaran et al., 2014b, Segura et al., 2018), and a variety of chemicals have been identified and proposed as sex pheromone components in fruit flies (Chuman et al., 1987; Baker and Heath, 1993; Wicker-Thomas, 2007; Robacker et al., 2009; Hiap et al., 2019; Levi-Zada et al., 2020; Ono et al., 2020). However, the biosynthetic pathways of only some suspected pheromones have been elucidated. We have proposed here that protein ingested by B. dorsalis is converted into threonine and glycine, which are precursor substances of the sex pheromone. The positive relationship between protein feeding and reproductive performance in Tephritidae is elucidated in this case by showing that proteins play a role in supplying precursor substances for sex pheromone biosynthesis. One thing should be noted is that glycine and threonine levels may be elevated partly because the flies fed on YH. To answer such question, we need to further determine the composition and content of amino acids in the YH. We need to determine if the YH contains large amounts of glycine and threonine that can enter into the rectum and be used by the rectal Bacillus. In addition to investigating the influence of protein on sex pheromones, the roles of sugars were also tested by feeding males different types of sugars. The results indicate that the amount of sex pheromone produced is significantly affected by the type of sugar (Figure 1D). Males fed fructose produced much higher amounts of sex pheromones than glucose-fed males. However, glucose has been described as the precursor substance for generating TMP and TTMP. We speculate that fructose may also be used in the sex pheromone production process, and the utilization efficiency of fructose may be much higher. In glycolysis, glucose is first converted into fructose 6-phosphate and then regenerated into pyruvate, which is involved in the synthesis of TMP and TTMP (Xiao et al., 2014; Xu et al., 2018; Zhang et al., 2019). However, fructose can be catalyzed directly by hexokinase to form fructose 6-phosphate in the glycolysis process. Such a step can omit the step of glucose to fructose 6-phosphate, which may increase pyruvate conversion efficiency and then generate more TMP and TTMP. This may also be related to the fact that B. dorsalis has a preference for hosts that are fructose-rich fruits. As males congregate on these hosts to attract mates, opportunities for feeding on fruit juices and fruit exudates may abound.
Previous studies have shown that in the glycine and threonine pathway of insects and bacteria, Sardh converts sarcosine into glycine (Frisell and Mackenzie, 1962), and ltaE converts glycine into threonine (Liu et al., 1998). However, how protein ingested by B. dorsalis affects the production of sarcosine remains to be investigated. Does protein supplementation provide sarcosine directly to the fly? Or does protein supplementation provide the fly with other substances that can be converted into sarcosine? If so, what are these substances? How are they converted to sarcosine? These questions are very complex, and more experimental evidence is needed to uncover them. Both glycine and threonine were elevated in the YH-fed flies. However, only Sardh (that converts sarcosine to glycine) was upregulated and ltaE (that converts glycine to threonine) was not differentially expressed. Does YH provide threonine directly to the flies, or are there other ways to synthesize threonine? These questions also need to be confirmed by further experiments. Moreover, we found that Sardh knockdown males showed significantly decreased rectal threonine and glycine contents, but TTMP level was not significantly reduced (Figure 5E). In the pyrazine synthesis pathway of Bacillus, two molecules of glucose can be converted into TTMP, while one molecule of glucose and one molecule of threonine (or glycine) can be converted into TMP (Zhang et al., 2019). Therefore, we speculate that the reason why TTMP level is not affected is that glucose content in the rectum is not regulated by Sardh. Nevertheless, the study links male feeding behavior with discrete patterns of gene expression that lead to pheromone production.
Materials and methods
Insect rearing
Request a detailed protocolThe B. dorsalis strain collected from a carambola (Averrhoa carambola) orchard in Guangzhou, Guangdong Province, was reared under laboratory conditions (27 ± 1°C, 12:12 hr light:dark cycle, 70–80% RH(Relative humidity)). A maize-based artificial diet containing 150 g of corn flour, 150 g of banana, 0.6 g of sodium benzoate, 30 g of yeast, 30 g of sucrose, 30 g of paper towel, 1.2 ml of hydrochloric acid, and 300 ml of water was used to feed the larvae. Adults were fed a solid diet (consisting of 50 g YH [protein food] and 50 g sugar) and 50 ml sterile water. Approximately, 200 adults were held in a 35 cm × 35 cm × 35 cm wooden cage. To test the effect of protein supplementation on sex pheromone synthesis, flies fed only sugar and sterile water were also prepared. To test the effect of different type of sugar on sex pheromone synthesis, flies fed with sucrose, glucose, or fructose as sugar were also prepared.
Sex pheromone identification in the B. dorsalis rectum
Request a detailed protocol60 rectums of 12 d old males fed different types of diets were dissected at 20:00 P.M. Sex pheromones in the rectum were extracted with 500 µl n-hexane by shaking (180 rpm) in a 30°C incubator for 24 hr. Then, GC‒MS with an Agilent 7890B Series GC system coupled to a quadrupole-type-mass-selective detector (Agilent 5977B; transfer line temperature: 230°C, source temperature: 230°C, and ionization potential: 70 eV) was used to identify sex pheromones in the rectum extraction according to our previous method (Ren et al., 2021).
Effect of protein feeding on biological parameters
Request a detailed protocolFlies that were fed with and without YH were prepared to determine the effect of protein on biological parameters (adult survival, rectum width, rectum glucose content, rectum threonine content, rectum glycine content, and mating ability). To study survival, the studies were initiated with six groups of newly emerged males (200 males). Each group was maintained separately and was provided different types of food (sucrose, sucrose + YH, glucose, glucose + YH, fructose, and fructose +YH). The mortality of the males was recorded each day until the males matured (12 d later). The rectum width of the mature males was measured.
Glucose content measurement
Request a detailed protocolThe glucose content in the rectum of the mature males was measured with a glucometer. To determine glucose content, the rectums of 12 mature males were collected and placed in a 1.5 ml microcentrifuge tube containing 10 μl of sterile Milli-Q water. Then, the samples were ground with a grinding machine. The samples were centrifuged for 15 min at 12,000 rpm. Then, the supernatants were collected and analyzed with a glucometer (ONETOUCH, Verio Flex). Then, glucose contents were normalized to rectum weight and compared between different treatments.
Amino acid content measurement
Request a detailed protocolFor threonine and glycine identification, sample preparation for free amino acid analysis was performed as described by Shahzad et al., 2019. Briefly, the rectums of 15 mature males were collected and placed in a 1.5 ml microcentrifuge tube containing 500 μl of sulfosalicylic acid solution (5%, diluted in water). Then, the samples were ground with a grinding machine. The samples were centrifuged for 15 min at 12,000 rpm. Then, the supernatants were collected in another centrifuge tube, and 1 ml of sulfosalicylic acid solution was added. Then, the threonine content in the samples was quantified with an amino acid analyzer (Hitachi L-8900, Japan) according to the standard method. Then glycine/threonine contents were normalized to rectum weight and compared between different treatments.
Mating competition assays
Request a detailed protocolMating competition between YH-deprived males and control males was performed in a 35 cm × 35 cm × 35 cm wooden cage. Briefly, 60 mature males with colored pronota (30 YH-deprived males [red] and 30 control males [green]) were placed in one cage, and then 30 mature unmated females were placed in the cage at 8:00 P.M. Mating behavior was observed for 2 hr, and the number of mated males was recorded and compared. Five replicates were conducted for each diet pair.
Effect of protein feeding on rectal bacterial diversity
Request a detailed protocolTo analyze bacterial diversity in the male rectum, the rectums of five males fed different foods were collected (five replicate samples were prepared). Then, bacterial DNA was extracted from the rectum samples using the Bacterial Genomic DNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. qPCR (16S-338F and 16S-518R primers were used [Supplementary file 9]) was used to estimate the absolute abundance of bacteria in the rectum according to our previous method (Ren et al., 2021). The 16S rRNA V3–V4 region was amplified by PCR (16S-341F and 16S-806R primers were used [Supplementary file 9]). Then, the amplicons were purified and sequenced (2×250) on an Illumina HiSeq 2500 platform. The software Mothur was used to cluster tags of more than 97% identity into OTUs(Operational Taxonomic Unit), and then the abundances of the OTUs were calculated. The taxonomic classification of OTUs was based on the annotation result of contained tags according to the mode principle; that is, the taxonomic rank that contained more than 66% of tags was considered the taxonomic rank of a specific OTU. The bacterial diversity was analyzed by principal coordinate analysis.
Transcriptome sequencing and gene identification
Request a detailed protocolTo identify the genes that contribute to converting protein into threonine, the transcriptome sequencing results of males fed different foods (sucrose, sucrose + YH, glucose, glucose + YH, fructose, and fructose + YH) were compared. For each group, five rectums were dissected for RNA extraction. In addition, five replicates were included for each group. In the next step, paired-end RNA-seq libraries were prepared by following Illumina’s library construction protocol. The libraries were sequenced on an Illumina HiSeq2000 platform (Illumina, USA). FASTQ files of raw reads were produced and sorted by barcodes for further analysis. Prior to assembly, paired-end raw reads from each cDNA library were processed to remove adaptors, low-quality sequences (Q<20), and reads contaminated with microbes. The clean reads were de novo assembled to produce contigs. An index of the reference genome of B. dorsalis was built, and paired-end clean reads were mapped to the reference genome using HISAT2. 2.4 with ‘-rna-strandness RF’ and other parameters set as a default (Kim et al., 2015). To evaluate transcript expression abundances, StringTie software was applied to calculate the normalized gene expression value FPKM (Pertea et al., 2016). Then, gene DE analysis was performed with DESeq2 software (Love et al., 2014). Genes/transcripts with a false discovery rate below 0.01 and absolute fold change ≥2 were considered DEGs/transcripts. Correlation analysis of the samples was performed by R. The correlation coefficient between two samples was calculated to evaluate similarity between samples. The closer the correlation coefficient is to 1, the higher the similarity between the two samples. To reveal the structure/relationship of the samples, PCA was performed with the R package gmodels. To further understand gene biological functions, pathway enrichment analysis was performed to identify the significantly enriched metabolic pathways or signal transduction pathways in DEGs compared with the whole genome. Moreover, the transcriptome of the normally reared (both sugar and protein were provided) male rectum at different developmental times (0 d, 3 d, 6 d, 9 d, and 12 d) was also sequenced and compared according to the above methods.
Expression validation of the identified genes
Request a detailed protocolqRT-PCR analysis was used to validate gene expression in the rectum, head, thorax, and abdomen of the males. Total RNA was extracted. Then, cDNA was synthesized with a One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China) using the extracted RNA. Then, a PerfectStarTM Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) was used to perform quantitative real-time PCR to compare the gene expression levels. Gene-specific primers (Supplementary file 9) were designed on NCBI with primer blast. The α-tubulin and actin genes were used as reference genes (Shen et al., 2010). The PCR procedure was set according to the manufacturers’ instructions. Five biological replicates were performed.
RNA interference
Request a detailed protocoldsRNA primers (Supplementary file 9) tailed with the T7 promoter sequence were designed using the CDSs(Coding DNA Sequence) of Sardh as templates. A MEGAscript RNAi Kit (Thermo Fisher Scientific, USA) was used to synthesize and purify dsRNA according to the manufacturer’s instructions. The GFP gene (GenBank accession number: AHE38523) was used as the RNAi negative control. To knockdown the target gene in males, 0.5 μl (500 ng/μl) dsRNA was injected into the abdomen of 12 d old males. Flies injected with dsGFP(double strain RNA of green fluorescent protein) were prepared as a negative control. After 24 hr, the knockdown efficiency of the genes was checked with qRT-PCR following the method used for validating the expression of the gene above. Then, the threonine and glycine contents, sex pheromone abundance, and mating ability were measured and tested in flies in which Sardh was silenced.
Data analysis
Request a detailed protocolStatistical analysis methods used in the study were indicated in the figure legends. Differences were considered significant when p<0.05. All data were analyzed using the GraphPad Prism version 8, GraphPad Software, La Jolla, CA, USA, https://www.graphpad.com/.
Data availability
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. RNA-sequencing and 16S rRNA amplicon sequencing data have been deposited in the Genome Sequence Read Archive Database of the National Genomics Data Center (BioProject PRJCA010569, PRJCA010560 and PRJCA010555).
References
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Separation and purification of sarcosine dehydrogenase and dimethylglycine dehydrogenaseJournal of Biological Chemistry 237:94–98.https://doi.org/10.1016/S0021-9258(18)81367-9
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Predominant accumulation of a 3-hydroxy-γ-decalactone in the male rectal gland complex of the Japanese orange fly, Bactrocera tsuneonisBioscience, Biotechnology, and Biochemistry 84:25–30.https://doi.org/10.1080/09168451.2019.1664892
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Decision letter
-
Sonia SenReviewing Editor; Tata Institute for Genetics and Society, India
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K VijayRaghavanSenior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
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Sonia SenReviewer; Tata Institute for Genetics and Society, India
In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.
Decision letter after peer review:
Thank you for submitting your article "Protein feeding mediates sex pheromone biosynthesis in an insect" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, including Sonia Sen as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by K VijayRaghavan as the Senior Editor.
The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.
Essential revisions:
1. To strengthen the claim that rectal bacilli use the fly's dietary amino acid to make TMP/TTMP, we recommend that the authors demonstrate that in the absence of bacilli, the dietary amino acids do not make a difference to TMP/TTMP level or mating success.
2. Increase in levels of glycine and threonine: There is a possibility that these levels are elevated because the flies fed on YH and not because a specific threonine and glycine biosynthesis pathway was elevated. Could the authors please explicitly discuss this?
3. Figures: The figures could use considerable improvement. Please see the detailed reviews below for specifics.
4. Text: We request that the authors explain their experimental setups in more detail. Could they also please describe their results before they infer from them?
5. Please normalise the glycine/threonine content to tissue weight or total protein content rather than the sample volume.
In addition to this, please read the detailed reviews provided below and address them wherever possible.
Reviewer #1 (Recommendations for the authors):
The link between dietary protein and reproductive success is well documented in females of various species. Here, the authors investigate this link in male Oriental Fruitflies, which are a major agricultural pest.
In their previous work, the authors demonstrated that bacteria in the male rectum produce sex pheromones to attract females. In this study, the authors demonstrate that dietary protein triggers the expression of the gene, sarcosine dehydrogenase (sardh) in the male rectum. This, in turn, results in an increase in the levels of glycine and threonine, which are the precursors used by bacteria to produce the sex pheromones TMP and TTMP. Consequently, they demonstrate that protein feeding in males is necessary for mating success.
We found the manuscript interesting, and the claims generally well supported, particularly in the context of their previous work. Our recommendations are largely for clarity and presentation of data.
1. Figures: The figures could use considerable improvement. In some cases the graphs are too small to decipher the various conditions, occasionally the resolution is low, and 3D graphs are not ideal. In general, we recommend the use of schematics depicting the experiments and their conditions, and a final schematic to demonstrate their working model will be nice.
2. Text: It would help to have the experimental conditions better explained. For example, the considerations of the various sugar conditions are not really addressed in the text except in a single sentence. The interpretations from these different sugar conditions are also not really discussed, leaving one wondering why they were introduced in the first place. It would also help to have the experimental design briefly discussed in the Results section so that the results are easily interpretable by the reader without having to go to the methods.
3. The link between rectal microbial communities and TMP: The authors make the claim that yeast feeding does not affect bacterial communities. I'm not sure if this claim can be made based on the data, or at least this level of analysis of the data. I am not very familiar with community ecology analyses, but the indices used here are relative abundances of bacterial classes and overall richness. It's suggestive of their claim, but there is a possibility that species composition might be different in these conditions. If the authors agree, perhaps they could bring this point out and temper their claims.
4. The sentence in 107-108: "Yeast feeding only positively influenced glucose contents in males in the sucrose and fructose groups (Figure 2D)." The comparison seems to be being made between YH+ and YH- in each sugar group. The assessment to be made is whether the amount of glucose is affected by yeast feeding or not. So, should the comparison not be made across sugar groups? Glucose will naturally be higher in a glucose-fed fly, hence the apparent lack of difference between YH- and YH+.
5. It wasn't clear to me whether the authors were making the claim that Sardh levels were increased with age irrespective of the protein feeding. This would be interesting! The experimental setup of this section was particularly unclear to me.
6. The methods, for example, the bioinformatics pipelines, could be described better, particularly since this is a non-traditional model organism. Was the functional enrichment analysis done keeping the whole genome as the background? This won't help in determining the enrichment of genes within a specific tissue.
Reviewer #2 (Recommendations for the authors):
Gui et al. investigated the effects of dietary yeast and sugars on male Bactrocera dorsalis sex pheromone biosynthesis and other mating-related traits (rectum width, rectal glucose, threonine, glycine content, and mating ability). The authors first compared male B. dorsalis fed on diets with or without yeast hydrolase ("YH-supplemented" vs. "YH-deprived") and on three different sugars (glucose, fructose, and sucrose), showing that the sex pheromone compounds (particularly TMP) were detected in YH-supplemented male fly rectums but are absent in YH-deprived counterparts. The mating ability was also significantly lower in the YH-deprived males. Following the initial observations, the team conducted a series of experiments, including 16S amplicon seq, RNAseq, and gene knockdown by RNAi to identify the mechanism underlying these effects.
The manuscript is overall well-written, and the figures are nicely presented. Results demonstrating the importance of the sarcosine dehydrogenase (Sardh) gene in sex pheromone biosynthesis and mating are novel. However, I have reservations about some of the statements and data interpretation.
1. Previous work from the group suggests that the rectal bacteria can produce sex pheromones in male B. dorsalis (Ren, Ma et al. 2021). However, the involvement of bacteria in the dietary yeast effects on sex pheromone biosynthesis and mating is not clear in this study. The microbiome data suggest that the dietary yeast treatments did not affect the dominant rectal bacterium Bacillus abundance. The only indirect evidence was that rectal threonine and glycine appeared to be elevated in YH-fed flies, and Bacillus can use these amino acids to produce sex pheromones. In my opinion, the data present are not sufficient to support statements like "We show that male flies rely on rectal Bacillus to produce a complex sex pheromone. (L11-12)", "This study clearly links male feeding behavior with discrete patterns of gene expression that lead to pheromone production by rectal Bacillus… (L18-20)", "This is the first report that clarifies the molecular mechanism by which host feeding and rectal microbes co-regulate sex pheromone biosynthesis. (L173-174)". For these statements to be valid, one would need to use flies deprived of Bacillus or the microbiome to confirm that there is no difference between YH-fed and YH-deprived flies.
2. Some sections of the manuscript would benefit from clarification, for instance,
a. The authors reasoned that YH feeding would stimulate threonine and glycine synthesis pathways (L116-117) when they justified focusing on glycine/threonine metabolism DEGs. This is an interesting prediction but presumably, yeast can provide these amino acids directly as a protein source. Knowing how much threonine and glycine are available in the YH might be helpful.
b. The RNAseq data identified two DEGs, Sardh and AGXT2, associated with glycine or threonine synthesis. As shown in Figure 3E, Sardh can convert Sarcosine to glycine, but not much information was provided regarding where Sarcosine may come from. Does the YH provision Sarcosine to the fly? What is the alternative hypothesis for the Sardh upregulation by YH feeding if not?
3. Based on the fold changes and p values, the positive effect of YH feeding on rectal threonine level is more significant than on glycine (Figure 2E and 2F). Sardh upregulation can explain the elevated glycine level in YH-fed flies, but ltaE (that converts glycine to threonine) was not differentially expressed. The genetic basis for the elevated threonine seems unresolved and is worthy of more in-depth discussion.
4. Figure 1C shows that TTMP (blue arrow) is only detected in the glucose + YH treatment but not in the sucrose + YH or fructose + YH treatments. What are the possible explanations?
5. In Figure 5, Sardh knockdown males showed significantly decreased rectal threonine and glycine contents, but TTMP level was not significantly reduced. What are the possible explanations?
6. Please indicate the sample time (day 12?) of rectal glycine/threonine for figures 2 and 5.
7. For RNAseq analysis, the FPKM normalization method is generally not recommended for between-sample comparisons for DE analysis. Suggest using the median of ratios method in DESeq2 or TMM in EdgeR.
8. L203: "Our results indicate that the amount of sex pheromone produced is significantly affected by the type of sugar (Figure 2c)." – I think the authors were referring to Figure 1c, not 2c.
9. To measure rectal glycine/threonine content, the team collected 15 male recta per replicate of each treatment group. While rectal width did not differ between the YH-supplemented and YH-deprived flies, it's unclear whether the total protein content or mass was different between the groups. My concern is that the lower glycine/threonine level could be due to the lower amount of tissue collected from the YH-deprived flies. I also noticed that the scale of glycine content varied quite a bit among experiments (e.g., 1-2 μg/ml in Figure 2E, 0.4-0.6 μg/ml in Figure 4B, and 2-3 μg/ml in Figure 5C control group). Normalizing the glycine/threonine content to tissue weight or total protein content will be more appropriate than the sample volume.
https://doi.org/10.7554/eLife.83469.sa1Author response
Essential revisions:
1. To strengthen the claim that rectal bacilli use the fly's dietary amino acid to make TMP/TTMP, we recommend that the authors demonstrate that in the absence of bacilli, the dietary amino acids do not make a difference to TMP/TTMP level or mating success.
Thanks for your recommendation. In previous study, we have found that flies can't synthesize sex pheromones when recta bacilli were removed by antibiotics treatment (Ren, Ma et al. 2021). Therefore, it is difficult to assess the effect of dietary amino acids on sex pheromones in the absence of bacilli (With or without dietary amino acids, there was no sex pheromone synthesis). To make the claim clearer, we have mentioned this in the manuscript. See line 79-84. We hope the explanation will meet with approval.
2. Increase in levels of glycine and threonine: There is a possibility that these levels are elevated because the flies fed on YH and not because a specific threonine and glycine biosynthesis pathway was elevated. Could the authors please explicitly discuss this?
Thanks for your comments. We agree with your opinion that glycine and threonine levels may be elevated because the flies fed on YH. We have discussed such possibility and the ways to make it clear in discussion. See line 196-199. We hope the correction will meet with approval.
3. Figures: The figures could use considerable improvement. Please see the detailed reviews below for specifics.
Thank you very much for you valuable suggestions. We have made corrections to the figures.
4. Text: We request that the authors explain their experimental setups in more detail. Could they also please describe their results before they infer from them?
Thanks for your suggestion. Experimental setups were described in more detail with more statements and schematics. And we have described the results in the result part. We hope the corrections will meet with approval.
5. Please normalise the glycine/threonine content to tissue weight or total protein content rather than the sample volume.
We have done this for both glycine/threonine contents and glucose contents.
In addition to this, please read the detailed reviews provided below and address them wherever possible.
Reviewer #1 (Recommendations for the authors):
The link between dietary protein and reproductive success is well documented in females of various species. Here, the authors investigate this link in male Oriental Fruitflies, which are a major agricultural pest.
In their previous work, the authors demonstrated that bacteria in the male rectum produce sex pheromones to attract females. In this study, the authors demonstrate that dietary protein triggers the expression of the gene, sarcosine dehydrogenase (sardh) in the male rectum. This, in turn, results in an increase in the levels of glycine and threonine, which are the precursors used by bacteria to produce the sex pheromones TMP and TTMP. Consequently, they demonstrate that protein feeding in males is necessary for mating success.
We found the manuscript interesting, and the claims generally well supported, particularly in the context of their previous work. Our recommendations are largely for clarity and presentation of data.
Thank you very much for your positive comments.
1. Figures: The figures could use considerable improvement. In some cases the graphs are too small to decipher the various conditions, occasionally the resolution is low, and 3D graphs are not ideal. In general, we recommend the use of schematics depicting the experiments and their conditions, and a final schematic to demonstrate their working model will be nice.
Thanks for your suggestion. We have improved the figures by increasing the resolution. And we have added schematics to demonstrate the working model. We hope the corrections will meet with approval.
2. Text: It would help to have the experimental conditions better explained. For example, the considerations of the various sugar conditions are not really addressed in the text except in a single sentence. The interpretations from these different sugar conditions are also not really discussed, leaving one wondering why they were introduced in the first place. It would also help to have the experimental design briefly discussed in the Results section so that the results are easily interpretable by the reader without having to go to the methods.
We are sorry for the confusion caused. We have described the experimental conditions in detail in the result and method part. See line 68-72, line 242-243. We hope the corrections will meet with approval.
3. The link between rectal microbial communities and TMP: The authors make the claim that yeast feeding does not affect bacterial communities. I'm not sure if this claim can be made based on the data, or at least this level of analysis of the data. I am not very familiar with community ecology analyses, but the indices used here are relative abundances of bacterial classes and overall richness. It's suggestive of their claim, but there is a possibility that species composition might be different in these conditions. If the authors agree, perhaps they could bring this point out and temper their claims.
We agree with you very much. In order to clarify this issue, we have rewritten this part. We have only highlighted the effect of protein supplementation on microbes at the class level. And we have tempered our claims by saying that the results indicate that protein intake may not affect the abundance of Bacillus synthesizing pheromones in the rectum and sex pheromone loss in YH-deprived males may not be associated with Bacillus. See line 88-97. Thanks again for your important suggestions. We hope the revision will meet with approval.
4. The sentence in 107-108: "Yeast feeding only positively influenced glucose contents in males in the sucrose and fructose groups (Figure 2D)." The comparison seems to be being made between YH+ and YH- in each sugar group. The assessment to be made is whether the amount of glucose is affected by yeast feeding or not. So, should the comparison not be made across sugar groups? Glucose will naturally be higher in a glucose-fed fly, hence the apparent lack of difference between YH- and YH+.
Thanks for your comments. In our study, we found that protein supplementation can significantly affect the synthesis of sex pheromones. Therefore, we wanted to further confirm which part (Bacillus, glucose and amino acids) in sex pheromone synthesis was affected by protein supplementation. In the issue you have mentioned, we want to clarify whether protein supplementation affects sex pheromone synthesis by influencing the rectal glucose contents. We are sorry that we didn't make this issue clear. We have restated the purpose of such experiments in the manuscript. See line 98-104. We hope the explanation will meet with approval.
5. It wasn't clear to me whether the authors were making the claim that Sardh levels were increased with age irrespective of the protein feeding. This would be interesting! The experimental setup of this section was particularly unclear to me.
We are sorry for the unclear description of the experimental setup. In our previous study, we have found that sex pheromones can only be produced 9 days after emergence in normally reared (sugar and protein were both provided) male B. dorsalis. Thus, we infer the reason may be that Sardh levels were increased in mature stage to synthesize amino acid for sex pheromone synthesis. And we can set such experiment to verify the potential role of Sardh in influencing sex pheromone synthesis. We have made correction in the results and method part. Sorry again for the confusion caused. We hope the corrections and explanation will meet with approval.
6. The methods, for example, the bioinformatics pipelines, could be described better, particularly since this is a non-traditional model organism. Was the functional enrichment analysis done keeping the whole genome as the background? This won't help in determining the enrichment of genes within a specific tissue.
Thanks for your comments. Before we did pathway enrichment, we already knew that threonine/glycine contents were affected by protein supplementation. Therefore, the purpose of pathway enrichment analysis is to further screen genes involved in synthesizing threonine/glycine. Doing functional enrichment analysis with the whole genome as the background is commonly used in many literatures. We certainly agree with you that using genes expressed in the rectum as background will make the results more credible. And we re-did the enrichment analysis with the method you recommended, and the results also showed that the glycine and threonine metabolism pathway was significantly enriched. Thanks again for your valuable comments. We hope our explanation will meet with approval.
Reviewer #2 (Recommendations for the authors):
Gui et al. investigated the effects of dietary yeast and sugars on male Bactrocera dorsalis sex pheromone biosynthesis and other mating-related traits (rectum width, rectal glucose, threonine, glycine content, and mating ability). The authors first compared male B. dorsalis fed on diets with or without yeast hydrolase ("YH-supplemented" vs. "YH-deprived") and on three different sugars (glucose, fructose, and sucrose), showing that the sex pheromone compounds (particularly TMP) were detected in YH-supplemented male fly rectums but are absent in YH-deprived counterparts. The mating ability was also significantly lower in the YH-deprived males. Following the initial observations, the team conducted a series of experiments, including 16S amplicon seq, RNAseq, and gene knockdown by RNAi to identify the mechanism underlying these effects.
The manuscript is overall well-written, and the figures are nicely presented. Results demonstrating the importance of the sarcosine dehydrogenase (Sardh) gene in sex pheromone biosynthesis and mating are novel. However, I have reservations about some of the statements and data interpretation.
Thanks for your positive comments.
1. Previous work from the group suggests that the rectal bacteria can produce sex pheromones in male B. dorsalis (Ren, Ma et al. 2021). However, the involvement of bacteria in the dietary yeast effects on sex pheromone biosynthesis and mating is not clear in this study. The microbiome data suggest that the dietary yeast treatments did not affect the dominant rectal bacterium Bacillus abundance. The only indirect evidence was that rectal threonine and glycine appeared to be elevated in YH-fed flies, and Bacillus can use these amino acids to produce sex pheromones. In my opinion, the data present are not sufficient to support statements like "We show that male flies rely on rectal Bacillus to produce a complex sex pheromone. (L11-12)", "This study clearly links male feeding behavior with discrete patterns of gene expression that lead to pheromone production by rectal Bacillus… (L18-20)", "This is the first report that clarifies the molecular mechanism by which host feeding and rectal microbes co-regulate sex pheromone biosynthesis. (L173-174)". For these statements to be valid, one would need to use flies deprived of Bacillus or the microbiome to confirm that there is no difference between YH-fed and YH-deprived flies.
Thanks for your important comments. Although we have shown in previous studies that Bacillus can synthesize sex pheromones, these conclusions are not part of this study. In order to be more precise, we have rephrased or deleted the statements in the manuscript. Thanks again for your valuable comments. We hope the corrections will meet with approval.
2. Some sections of the manuscript would benefit from clarification, for instance,
a. The authors reasoned that YH feeding would stimulate threonine and glycine synthesis pathways (L116-117) when they justified focusing on glycine/threonine metabolism DEGs. This is an interesting prediction but presumably, yeast can provide these amino acids directly as a protein source. Knowing how much threonine and glycine are available in the YH might be helpful.
Thanks for your important comments. We agree with your opinion that glycine and threonine levels may be elevated because the flies fed on YH. And the editor also points out that we need to discuss such possibility. However, our results at least indicate that glycine/threonine metabolism pathway is one of the factors regulating glycine/threonine and sex pheromone synthesis in rectum. Taking the editor's advice into consideration, we have discussed such possibility and the ways to make it clear in discussion. See line 196-199. We hope the corrections will meet with approval.
b. The RNAseq data identified two DEGs, Sardh and AGXT2, associated with glycine or threonine synthesis. As shown in Figure 3E, Sardh can convert Sarcosine to glycine, but not much information was provided regarding where Sarcosine may come from. Does the YH provision Sarcosine to the fly? What is the alternative hypothesis for the Sardh upregulation by YH feeding if not?
Thank you very much for your important comments. Indeed, the issues you have raised are critical for understanding the mechanism that protein supplementary regulated sex pheromone synthesis. We need a series of experiments to clarify these issues, which is almost a whole other topic. However, we agree with you very much. Considering the suggestions of the editor, we have carried out an in-depth discussion in the discussion part, and proposed further research directions. See line 216-230. Thanks again for your comments. We hope our corrections will meet with approval.
3. Based on the fold changes and p values, the positive effect of YH feeding on rectal threonine level is more significant than on glycine (Figure 2E and 2F). Sardh upregulation can explain the elevated glycine level in YH-fed flies, but ltaE (that converts glycine to threonine) was not differentially expressed. The genetic basis for the elevated threonine seems unresolved and is worthy of more in-depth discussion.
We agree with your opinion. We inferred elevated threonine may come from two sources. One is provided directly by YH, another one is converted by other genes. However, we need more date to confirm such speculation. According to your suggestions and the editor’s, we have mentioned this in the discussion part. See line 216-230. We hope the correction will meet with approval.
4. Figure 1C shows that TTMP (blue arrow) is only detected in the glucose + YH treatment but not in the sucrose + YH or fructose + YH treatments. What are the possible explanations?
We are very sorry for our unclear description. TTMP has been detected in the glucose + YH, sucrose + YH and fructose + YH treatments. In order to show the results clearly, we added some extra marks in the Figure. Sorry again for our negligence. We hope the correction will meet with approval.
5. In Figure 5, Sardh knockdown males showed significantly decreased rectal threonine and glycine contents, but TTMP level was not significantly reduced. What are the possible explanations?
Thanks for your important comments. In the pyrazine synthesis pathway of Bacillus, two molecules of glucose can be converted into TTMP, while one molecule of glucose and one molecule of threonine (or glycine) can be converted into TMP (Zhang et al., 2019 Applied and Environmental Microbiology). Therefore, we speculate that the reason why TTMP level is not affected is that glucose content in the rectum is not regulated by Sardh. Thanks for your question. We are sorry that we didn’t discuss this in the manuscript. In the revised manuscript, we have added the explanations in discussion. See line 216-230. We hope the revisions will meet with approval.
6. Please indicate the sample time (day 12?) of rectal glycine/threonine for figures 2 and 5.
Thank you very much for your reminding. The flies used were mature males (12-day-old). We have mentioned this in the manuscript.
7. For RNAseq analysis, the FPKM normalization method is generally not recommended for between-sample comparisons for DE analysis. Suggest using the median of ratios method in DESeq2 or TMM in EdgeR.
Thanks for your suggestions. We have found that both FPKM and TPM are used for quantitative analysis in transcriptome in many literatures. Although the quantitative methods are different, the results obtained are highly consistent. And FPKM is still the main reference transcriptome quantitative method at present. We analyzed the date again with the method you recommended and found it was highly consistent with our previous results. So we think there is no problem in using FPKM normalization method. Thanks again for your recommendation. We hope our explanation will meet with approval.
8. L203: "Our results indicate that the amount of sex pheromone produced is significantly affected by the type of sugar (Figure 2c)." – I think the authors were referring to Figure 1c, not 2c.
We are sorry for our mistake. We have corrected it.
9. To measure rectal glycine/threonine content, the team collected 15 male recta per replicate of each treatment group. While rectal width did not differ between the YH-supplemented and YH-deprived flies, it's unclear whether the total protein content or mass was different between the groups. My concern is that the lower glycine/threonine level could be due to the lower amount of tissue collected from the YH-deprived flies. I also noticed that the scale of glycine content varied quite a bit among experiments (e.g., 1-2 μg/ml in Figure 2E, 0.4-0.6 μg/ml in Figure 4B, and 2-3 μg/ml in Figure 5C control group). Normalizing the glycine/threonine content to tissue weight or total protein content will be more appropriate than the sample volume.
Thank you very much for your valuable suggestions. We have normalized both glycine/threonine content and glucose content to tissue weight in the revised manuscript.
https://doi.org/10.7554/eLife.83469.sa2Article and author information
Author details
Funding
The national natural science foundation of China (3212200346)
- Daifeng Cheng
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements
We are grateful to the national natural science foundation of China (No. 3212200346).
Senior Editor
- K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
Reviewing Editor
- Sonia Sen, Tata Institute for Genetics and Society, India
Reviewer
- Sonia Sen, Tata Institute for Genetics and Society, India
Version history
- Received: September 15, 2022
- Accepted: January 18, 2023
- Accepted Manuscript published: January 19, 2023 (version 1)
- Version of Record published: February 8, 2023 (version 2)
Copyright
© 2023, Gui et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Ecology
- Plant Biology
Global agro-biodiversity has resulted from processes of plant migration and agricultural adoption. Although critically affecting current diversity, crop diffusion from Classical antiquity to the Middle Ages is poorly researched, overshadowed by studies on that of prehistoric periods. A new archaeobotanical dataset from three Negev Highland desert sites demonstrates the first millennium CE&'s significance for long-term agricultural change in southwest Asia. This enables evaluation of the 'Islamic Green Revolution' (IGR) thesis compared to 'Roman Agricultural Diffusion' (RAD), and both versus crop diffusion during and since the Neolithic. Among the finds, some of the earliest aubergine (Solanum melongena) seeds in the Levant represent the proposed IGR. Several other identified economic plants, including two unprecedented in Levantine archaeobotany-jujube (Ziziphus jujuba/mauritiana) and white lupine (Lupinus albus)-implicate RAD as the greater force for crop migrations. Altogether the evidence supports a gradualist model for Holocene-wide crop diffusion, within which the first millennium CE contributed more to global agricultural diversity than any earlier period.
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- Ecology
- Evolutionary Biology
Temperature determines the geographical distribution of organisms and affects the outbreak and damage of pests. Insects seasonal polyphenism is a successful strategy adopted by some species to adapt the changeable external environment. Cacopsylla chinensis (Yang & Li) showed two seasonal morphotypes, summer-form and winter-form, with significant differences in morphological characteristics. Low temperature is the key environmental factor to induce its transition from summer-form to winter-form. However, the detailed molecular mechanism remains unknown. Here, we firstly confirmed that low temperature of 10 °C induced the transition from summer-form to winter-form by affecting the cuticle thickness and chitin content. Subsequently, we demonstrated that CcTRPM functions as a temperature receptor to regulate this transition. In addition, miR-252 was identified to mediate the expression of CcTRPM to involve in this morphological transition. Finally, we found CcTre1 and CcCHS1, two rate-limiting enzymes of insect chitin biosyntheis, act as the critical down-stream signal of CcTRPM in mediating this behavioral transition. Taken together, our results revealed that a signal transduction cascade mediates the seasonal polyphenism in C. chinensis. These findings not only lay a solid foundation for fully clarifying the ecological adaptation mechanism of C. chinensis outbreak, but also broaden our understanding about insect polymorphism.