Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development

Abstract

Background: Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology.

Results: We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch over to the late embryonic stages in pea aphid development.

Conclusions: Our data show that, in the development of A. pisum, a specific host gene set regulates the biosynthetic pathways of amino acids, demonstrating how the regulation of gene expression enables an insect to control the production of metabolites crucial for its own development and symbiotic metabolism.

Figure 1

Global expression analysis in pea aphid development. A) Micro-photographs of the four stages analysed in this work where the scale bar represents 200 μm in all photographs to allow for size comparison. The microphotographs show just one embryo stage among those belonging to the corresponding groups (see Table 1 for detail). The performed comparisons, early embryos (EE) versus intermediate embryos (IE), intermediate versus late embryos (LE) and late embryos versus first larval stage (L1) are labelled, respectively, in rose, violet and light blue. B) Unsupervised hierarchical clustering (generated by an average linkage method with euclidean distance and no leaf order optimization) of 50% of transcripts (12,005) showing the higher standard deviation among all the samples. The colour chart indicates expression intensities using a base 2 logarithmic scale: blue and red represent, respectively, lower (2.0) and upper (16.0) expression intensities (see bottom panel legend). C) The Venn diagram of the 3945 genes showing significantly differential expression in the three comparisons (comparison colour code as in Figure 1A). Numbers in red identify the genes increasing in expression during development; in dark blue, the genes decreasing in expression during development; and, in black, the genes found to be significant in two or more groups where the expression changes are not varying in the same direction in the different comparisons.

Figure 2

Developmental gene expression analysis in pea aphid. A) Hierarchical clustering (generated by an average linkage method with euclidean distance and no leaf order optimization) on the 368 developmental genes present on the microarrays (out of the 387 annotated by Shigenobu et al.[55]). The colour chart indicates expression intensities using a base 2 logarithmic scale: blue and red represent, respectively, lower (2.0) and upper (16.0) expression intensities (see bottom panel legend). On the right of the cluster, genes showing significant differential expression in at least one of the comparisons are indicated: in green, 27 homeobox-containing genes; in red, 32 signalling pathways genes; and, in black, 59 other developmental genes. B) The Venn diagram, showing the 118 developmental genes with significantly differential expression in the three comparisons (comparison colour code as in Figure 1A) analysed in this study. Numbers in red identify the genes increasing in expression during development; in dark blue, the genes decreasing in expression during development; and, in black, the genes found to be significant in two or more groups where the expression changes are not varying in the same direction in the different comparisons.

Figure 3

Amino acid metabolism gene expression profiling during pea aphid development. A) Hierarchical clustering (generated by an average linkage method with euclidean distance and no leaf order optimization) of the 135 amino acid metabolism genes annotated by Wilson et al.[29]. The colour chart indicates expression intensities using a base 2 logarithmic scale: blue and red represent, respectively, the lower (2.0) and upper (16.0) expression intensities (see bottom panel legend). On the right of the cluster we reported: in black, the genes varying significantly in at least one of the comparisons, with the exception of the ACYPI007803 gene, shown in green, and ACYPI004243, shown in purple. The three other genes coding the 2.6.1.1 enzyme activity are highlighted in grey but do not vary significantly during development. B) Final steps of the pathway for phenylalanine and tyrosine biosynthesis encoded in the pea aphid genome. C) Expression profiles of the genes involved in the pathway for phenylalanine and tyrosine biosynthesis in the pea aphid. Expression intensity is given as log2. Expression intensities of each stage are means of the three biological replicates.

Figure 4

Proportion of free amino acids during pea aphid development. Free amino acid contents of early, intermediate and late embryos, and in the first larval stage taken at two distinct times of development (early L1 ≤ 6 hours and late L1 ≥ 13 hours). Values (n = 4) are expressed as a percentage of the total amount of amino acids in the different samples. Among the 18 amino acids analysed, only those varying the most (P < 0.0001) and phenylalanine (P = 0.0015), the precursor of tyrosine, are represented. In particular among the significant changes (see Table S8 for details) it is worth noting: a decrease of aspartic acid from 16.3% in the early embryos (EE) to 0.7% in L1 larvae; a glutamic acid decrease from IE (35.2%) to the L1 stages (around 17.4%); an accumulation of tyrosine during development from 1.9% in early embryos (EE) to 12.1% in the late embryo group (LE), accumulation that continues in early first larval stage (17.6%) to decrease rapidly to 6.8% for the late larvae L1.

Figure 5

Gene expression regulations acting on the integrated pathway for tyrosine and other precursors of the cuticular tanning biosynthesis in a pea aphid embryo associated with B. aphidicola. A) Schematic representation of the pea aphid embryo (green rectangle) and its symbiotic bacteria (grey rectangle). A summarizing diagram of the Tyr/dopamine pathway during pea aphid development is represented. Black arrows represent reactions, grey arrows transport reactions; solid arrows are annotated events; dotted arrows are possible events; red crosses indicate events not present in the annotation of the genome (see text for details). Only the pea aphid genes whose expression levels change significantly during development are represented. The two B. aphidicola genes did not show significant expression changes during pea aphid development [53]. B) Expression profiles of the genes involved in the Tyr/dopamine pathway in the pea aphid. Expression intensity is given as log2. Expression intensities of each stage are means of the three biological replicates.