Aphids are important pests that cause significant economic losses in agriculture worldwide. Almost all aphids contain endosymbionts, and one of them, Buchnera aphidicola (hereinafter referred to as Buchnera), which is present in almost all aphids, provides essential nutrients to the host aphid and therefore called primary symbiont (Baumann, 2005). Additionally, aphids have a variety of secondary symbionts in their bodies, and the significance of secondary symbionts in enhancing the adaptation of aphids to adverse environments has been widely reported (De Clerck et al., 2015; Manzano-Marín et al., 2016; Li et al., 2018; Li et al., 2021). In recent years, it is worth noting the growing number of studies have shown that the function of Buchnera not only in providing essential amino acids to the host aphid, but also has potential effects in improving the heat tolerance of aphids (Zhang et al., 2019), revealing the differentiation process (Perreau et al., 2021; Zhang S. et al., 2021) and enhancing their resistance to drugs (Guo et al., 2020). Therefore, exploiting the close and mutually beneficial relationship between aphids and Buchnera may produce new ideas for developing green control strategies of aphids.

In China, the grain aphid Sitobion miscanthi is one of the most prevalent wheat pests and causes substantial economic losses in agriculture (Li et al., 2021). As genome sequencing technologies continuous upgrading and costs decrease, a large number of insect genomic information continues to be deciphered. Based on our previously published genome information of wheat aphid S. miscanthi (LF clone) (Jiang et al., 2019) and its primary symbiont Buchnera (Li et al., 2022), making it more convenient to study the nutrient metabolism interaction network between them. Previously, we used genomic information to identify a key relay gene ilvE, linking Buchnera to aphids in the leucine, isoleucine and valine synthesis pathways. Meanwhile, RNA interference (RNAi) experiment reveals a vital function in three essential amino acid synthesis pathways (Li et al., 2022). However, whether exist other metabolic relay genes are present in the aphid and Buchnera nutrient synthesis chains and can be used as candidate target genes for RNAi is still unknown.

Here, we have mined another key gene ilvA in the aphid-Buchnera relay synthesis of isoleucine pathway through the genomic information obtained in our previous work. Sequence and bioinformatics analysis showed that the ilvA gene was highly conserved in different aphid genomes, while the gene expression profile in different developmental stages and tissues of aphids was clarified by qPCR assay. Subsequently, the effects of ilvA on aphid life parameters were measured by RNAi experiments. Our results indicate that the ilvA gene, which links the aphid and Buchnera amino acid synthesis pathways, has an important effect on aphid weight and offspring, all of which suggest that ilvA gene can be used as candidate target for RNAi against aphids.

Aphids and their primary symbiont Buchnera aphidicola have formed a long-term and stable symbiotic relationship. At the same time, they are considered to be typical cases in studying the coevolution relationship between insects and endosymbionts (Clark et al., 2000). Numerous studies have shown that Buchnera provides aphids with essential nutrients that are missing from the phloem sap of feeding host plants (Shigenobu et al., 2000). There is persuasive experimental evidence that the Buchnera genome provides aphids with almost all of the key genes in the essential amino acid synthesis pathway in the model insect pea aphid A. pisum, however, little is known about the consistency of this tight nutrient supply chain model in other aphids. Given that almost all aphids contain Buchnera, which plays a vital role in aphids, therefore, taking Buchnera as the starting point, developing a strategy to break the stable nutrient supply chain between them may become an effective new idea for green prevention and control of aphids. At present, RNA interference technology is widely used in insect gene function verification, is considered to be a new direction for the development of green biological pesticides (Bautista et al., 2009; Belles, 2010; Wuriyanghan et al., 2011; Liu et al., 2020). However, RNA interference technology cannot be effectively implemented in prokaryotes, resulting in direct silencing of aphid primary symbiont Buchnera gene is difficult to achieve. Therefore, it has become a new research direction to excavate and identify the relay genes between host aphid and Buchnera in the synthesis pathway of essential nutrients and using them as RNA interference target sites. It is gratifying that the availability of the genomes of wheat aphid S. miscanthi and its primary symbiont Buchnera makes this work feasible.

It has been known for decades that Buchnera lacks some genes encoding essential amino acid biosynthesis enzymes and compensates via the host pea aphid A. pisum (Shigenobu et al., 2000; Wilson et al., 2010). Meanwhile, in our previous study, we found that a metabolic relay gene ilvE, a branched-chain amino acid transferase gene required for the final step in the synthesis of the three essential amino acids valine, leucine and isoleucine, was absent in the Buchnera genome but was present in the aphid genome (Li et al., 2022). The result was consistent with the previous studies on pea aphid genome (Wilson et al., 2010). In addition, RNAi of the ilvE gene significantly increased aphid mortality. Therefore, nutrient synthesis relay genes between aphids and Buchnera can be used as candidate targets for RNA interference. However, whether there are other metabolic relay genes in the aphid genome and have potential effect on the aphid development is still rarely reported.

In this study, combined with our previously reported genome data, we identified a threonine dehydratase gene called ilvA in the upstream process of isoleucine synthesis, which is absent in the Buchnera genome but exists in the aphid genome, and the function of the gene is to hydrolyze threonine to intermediate product 2-Oxobutanoate ( Figure 1 ). Therefore, to further verify the function of the gene, we cloned the ilvA gene and obtained a complete CDS sequence. Bioinformatics analysis showed that the gene was highly conserved in sequence and location in different aphids ( Figures 3 , 4 ). Meanwhile, it is worth noting that this gene is located on the sex chromosome of aphids ( Figure 2A ). In recent years, with the continuous advancement of genome sequencing technology, more and more aphid genomes have been resolved (Chen et al., 2019; Roberto et al., 2020; Zhang S. et al., 2021). Related studies have reported that in different aphid genomes, sex chromosomes are highly conserved, and the selection pressure of genes on sex chromosomes of A. pisum is significantly higher than that of autosomes (Li et al., 2020). Moreover, the average expression level of genes on sex chromosomes of pea aphid was significantly lower than that on autosomes, which may be the reason for the significant increase of selection pressure (Jaquiery et al., 2018). In our study, we performed the transcriptome analysis of winged and wingless adult aphids and determined that the expression levels of genes on sex chromosomes were significantly lower than those on autosomes, the results are consistent with previous studies. Moreover, the selective pressure on sex chromosomes was also significantly higher than that on autosomes, whether it was paralogous gene pairs within chromosomes or orthologous gene pairs between different aphids ( Figure 5 ). However, the expression level of ilvA and ilvE gene is much higher than the average value of other genes on sex chromosomes ( Supplementary Table S1 ). Meanwhile, ilvA and ilvE are under low selection pressure in different aphids, which means that the function of this kind of gene in aphid is stable. In general, a lower level of gene expression may imply less important to phenotypes (Nabholz et al., 2013), and previous study implicate that the aphid sex chromosome as a less preferred location for highly expressed genes (Li et al., 2020). However, in this study, we found that although the expression level of genes on sex chromosomes is low overall, there are still some highly expressed genes, and the gene has a potential role in the synthesis of some important nutrients. This result also means that the aphid sex chromosome is still a mysterious region worthy of further study.

To investigate the expression level of the ilvA gene in aphids, we next determined the expression pattern of the ilvA in different developmental stages and tissues of S. miscanthi by qPCR. Our results showed that the expression of the ilvA gene was significantly higher in the 2^rd instars of aphids than in the other instars and exhibited lowest expression level in 1^st instars ( Figure 3 ). Surprisingly, the ilvA gene was highly expressed in the bacteriocytes of aphids, where Buchnera shelters. Interestingly, the specific expression location of this gene is consistent with another synthetic relay gene ilvE (Li et al., 2022). Previous reports suggest that Buchnera plays an important role in maintaining amino acid synthesis and supply homeostasis in aphids (Wilson et al., 2010). Therefore, with the growth and development of aphids, the increasing titer of Buchnera may have a potential role in regulating the expression of ilvA and ilvE. Moreover, considering the specificity of the expression location of ilvA and ilvE genes, we hypothesized that the closer distance to Buchnera may be more convenient for the synthesis and transport of essential amino acids regulated by aphids between bacteriocytes and hemocoel.

To further study the function of ilvA in aphid, the RNAi experiment was performed. Unlike the ilvE gene, the indices of mortality decreased slightly, but were not significantly different, after feeding dsilvA 72 and 96 hs ( Figure 7B ). However, the weight and production of aphids decreased significantly ( Figures 7C, D ). Recent studies have shown that common ancestor of Hymenoptera lose all key genes in the valine, leucine and isoleucine synthesis pathway, but parasitoids are able to control related pathways in their host insect, providing them with missing essential nutrients (Ye et al., 2022). Compared with the commensalism relationship between parasitoids and host insects, aphids form a long-term stable mutualistic relationship with their primary symbiont Buchnera. The relay synthesis process in the nutrient synthesis pathway is important evidence of coevolution. Therefore, the destruction of synthetic relay chain may be the reason for the negative impact on the growth and development of S. miscanthi after knocking down the expression of ilvA and ilvE. However, there are also relevant study showed that the Buchnera protein HisC could functionally replace the missing ilvE, catalyzing the terminal reaction in these pathways (Shigenobu et al., 2000). It is worth noting that the ilvA gene is the upstream gene of the pathway, which is different from the downstream gene ilvE in the synthesis pathway. Therefore, whether reducing the expression of ilvA will cause functional complementation or expression response of some genes in the downstream pathway is still needs further exploration. At the same time, reduced expression of the ilvE gene significantly inhibited Leu, Ile and Val production in aphids (Li et al., 2022). Although in this study, we did not quantify isoleucine production in aphids after ilvA interference, but as a result of our previous studies, we speculated that the decrease in aphid growth fitness may be potentially associated with amino acid production, meanwhile, the decrease in the production of single one essential amino acid may also have a much smaller negative effect on aphids than the three essential amino acids. Such speculation also provides an explanation for why silencing the ilvA gene does not result in a significant increase in mortality.

Moreover, secondary symbiont in insect also has potential functions in compensating for the lack of amino acids (Gómez-Valero et al., 2004; Ju et al., 2017). Whether secondary symbiont rescue the negative effects on aphid growth and development caused by RNA interference remains unknown. It is worth noting that ilvE and ilvA are highly conserved in different aphids, so whether the RNAi targeting them has broad consistency is worthy of further study, which will also provide a theoretical basis for screening broad-spectrum RNAi target sites.

Numerous reports have indicated that almost all insects contain various types of endosymbionts (Baumann, 2005). However, so far, a large number of insect endosymbionts have not been cultured in vitro, making the study of their function dependent on host insects (Moran and Mira, 2001). Moreover, RNAi technology is not available for prokaryotic endosymbiont-associated genes, making it difficult to realize the strategy of using symbionts to control insects, necessitating new ideas. In this study, based on our previous studies, we identified another highly conserved isoleucine synthesis pathway upstream gene ilvA that is absent in the Buchenra genome but is present in the aphid genome, which plays an essential role in influencing the body weight and reproduction of aphids. With the growing popularity of genome sequencing, an increasing number of data resources on the genomes of insects and their endosymbionts have been deciphered. Our study may provide a future direction for targeting important junctions in the endosymbiont-insect metabolic relay process to control agricultural pests.

The data presented in the study are deposited in the NCBI repository, accession number PRJNA908645.

QL, JF and JC conceived and designed the experiments. QL and YC performed the experiments. QL analyzed the data. QL and YC wrote the paper. All of the authors read and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.