Molecular Characterization, Evolution and Expression Analysis of Ammonium Transporter from Four Closely Related Bactrocera Species (Tephritidae)

Abstract

Numerous insects are attracted to low levels of ammonia, utilizing it as a cue to locate food sources. The Ammonium Transporter (Amt), a highly conserved, atypical olfactory receptor, has been shown to mediate the detection of ammonia in insects. While the attraction of Tephritidae to ammonia is well established, knowledge about the Amt in this family is limited. The species Bactrocera dorsalis (Hendel 1912), Bactrocera cucurbitae (Coquillett 1899), Bactrocera correcta Bezzi 1916 and Bactrocera tau (Walker 1849), which are common agricultural pests within Tephritidae, exhibit numerous ecological similarities, offering a solid foundation for studying Amt characteristics in this family. In this study, we elucidated the sequences, evolutionary relationships, and expression patterns of Amt in these four species. The results indicated that these Amts share the same open reading frame, containing 1770 bp that encode a protein of 589 amino acid residues. These Amt proteins exhibit the typical structural characteristics of Amts, including an 11-transmembrane domain with an extracellular N-terminus and an intracellular C-terminus. They also have the ability to form trimers in the membrane. Additionally, they contain three conserved amino acid residues essential for ammonia transport: A189, H195, and H352. Phylogenetic and expression pattern analyses showed that they are highly conserved in Diptera and are significantly expressed in antennae. This study is the first report characterizing the Amt gene in four Tephritidae species. These findings provide a foundation for further exploration into the roles of these genes in their particular biological contexts.

1. Introduction

The Tephritid fruit fly (Diptera: Tephritidae) is a group of insects that severely damages fruits and vegetables [1]. Comprising over 4600 species, this family is widely distributed across tropical, subtropical, and temperate regions [2]. The primary source of damage from Tephritid fruit flies is attributed to the females. Females lay their eggs within fruits or vegetables, causing mechanical damage and promoting microbial infections. The emerging larvae then feed internally, leading to the premature drop and decay of the fruits and vegetables. Consequently, the affected agricultural products become inedible, and stringent quarantine measures further impede the export of fruits and vegetables, resulting in significant economic losses [3]. Among these fruit flies, the genus Bactrocera is notably destructive, primarily found in the tropical and subtropical regions of Asia [4]. Common species within this genus include Bactrocera dorsalis (Hendel 1912), Bactrocera cucurbitae (Coquillett 1899), Bactrocera correctan Bezzi 1916, and Bactrocera tau (Walker 1849).

Protein is a crucial nutrient throughout the growth and development of Tephritid fruit flies. To support the development of their reproductive organs, adult females require a substantial amount of this nutrient [5,6,7,8,9,10]. Without sufficient protein, females are unable to lay eggs [11]. In their natural habitats, fruit flies obtain protein from host plants, bacteria, and secretions of bacteria [12]. However, these minor proteins and their hydrolyzed amino acids lack strong volatility, hindering insects from pinpointing these protein sources across vast distances. Consequently, fruit flies have developed sophisticated localization strategies to secure an adequate supply of protein-rich food; they depend on the volatile scents emitted from the decomposition of proteins as indicators to find protein-abundant food sources [12,13]. Ammonia and its derivatives serve as typical indicative scents. Leveraging this trait, early agricultural workers conceived ammonia-derivative-based lures that have proven effective in managing Tephritidae populations [14,15,16]. Therefore, ammonia compounds hold considerable ecological significance for fruit flies.

In the natural environment, insects predominantly rely on their highly sensitive olfactory systems to detect volatiles released by hosts or food sources [17]. Identifying olfactory molecular targets for key compounds is crucial in the development of olfaction-based behavioral modifiers, as it forms the fundamental basis for such advancements [18]. Despite numerous accounts of the attractant effects of amine compounds on flies, a regrettable gap in research exists regarding their olfactory recognition mechanisms. Fortunately, studies have elucidated the olfactory recognition pathways for ammonia in mosquitoes and Drosophila. Research has revealed that Ammonium Transporter (Amt), an atypical olfactory receptor, significantly influences the transport of ammonia in antennae [19,20,21]. Amt is a ubiquitous transmembrane protein found across various organisms, including bacteria, fungi, plants, and animals [22,23,24,25]. It features 11 transmembrane domains and forms a functional trimeric structure on the membrane. These proteins display a pronounced affinity for ammonium ions (NH₄⁺) and are instrumental in mediating their transmembrane transport. In both Drosophila melanogaster Meigen 1830 and Anopheles gambiae Giles 1902, the Amt gene exhibits high expression in the antennae and directly facilitates the response of specific neurons within the antennal sensors to ammonia. This represents the first known instance of a transport protein functioning as an olfactory receptor in animals [19,20,21]. Although Amt is also expressed in the taste organ labellum of Drosophila, it does not appear to mediate the gustatory perception of amines [26]. Introducing the A. gambiae Amt into Drosophila mutants deficient in Amt effectively reinstates the electrophysiological response of neurons to ammonia, and orthologs of Amt have been found in many insect species [19,20,21]. Notably, for larger molecular amines produced by protein degradation, such as putrescine and cadaverine, there are no reports indicating the involvement of Amt in their perception. Based on this evidence, we hypothesize that the function of Amt may be conserved among insect species. Nevertheless, there are currently no reports on the function of the Amt gene in Tephritid fruit flies.

In this study, utilizing the peripheral transcriptomes of B. dorsalis, B. cucurbitae, B. correcta, and B. tau, we analyzed the amino acid domain structure of their Amt, coupled with an evolutionary comparison with closely related species. We also delineated the expression profiles of these genes across various olfactory organs and body regions. Our aim is to provide a foundational basis for future research into the functional role of the Amt gene in olfactory recognition.

2. Materials and Methods

2.1. Insect Rearing and Sample Collection

The wild-type laboratory strains of B. dorsalis, B. cucurbitae, B. correcta, and B. tau used in this study were provided by the Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences. These flies were reared on an artificial diet at 26 ± 1 ℃ and 60 ± 10% relative humidity under a 14 L/10 D photoperiod. The larval diet comprised banana, yeast, sucrose, corn flour, and cellulose paper. Upon maturity, larvae were transferred to wet sand for pupation. After pupation completion and sand drying, pupae were collected and placed in insect cages (18 cm × 24 cm × 14 cm) for emergence. Three days before emergence, males and females were separated and housed in smaller cages (18 cm × 12.5 cm × 14 cm). Adults were fed an artificial diet of yeast and sugar (1:1) with an additional water source.

At 12 days post-emergence, peripheral sensory tissues (antennae, mouthparts, maxillary palps, legs, and external genitalia) from both sexes were collected. Each replicate included 100 individuals, with a total of three replicates. Samples were immediately cryopreserved in liquid nitrogen and stored at −80 °C until further use.

2.2. RNA Isolation and cDNA Synthesis

Total RNA was extracted from various tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and its integrity and concentration were assessed by agarose gel electrophoresis and a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Approximately 1 µg of total RNA was used as a template for cDNA synthesis with the HiScript^® III 1st Strand cDNA Synthesis (+gDNA wiper) Kit (Vazyme, Nanjing, China). The resulting cDNA was diluted and stored at −20 °C for subsequent PCR use.

2.3. Homology Search and Molecular Cloning

Utilizing the previously acquired peripheral transcriptome data of B. dorsalis (China National GeneBank Database: PRJCA020830), B. cucurbitae (unpublished), B. correcta (unpublished), and B. tau (unpublished) from our laboratory, we independently assembled transcriptomes for each Bactrocera with Trinity-v2.8.5 [27] and identified candidate coding regions using TransDecoder v5.5.0. We downloaded the D. melanogaster complete genome protein sequence from NCBI and performed homology alignment with Orthofinder v2.5.4 [28] against the transcript protein sequences of four Bactrocera species. From these alignments, we selected candidate Amts homologous to D. melanogaster Amt (DmelAmt) for each species. We performed a multiple sequence alignment using DNAMAN and predicted the secondary structures of these candidate Amts with DeepTMHMM (https://services.healthtech.dtu.dk/services/DeepTMHMM-1.0/, accessed on 16 June 2024). We compared the nucleic acid sequences of the candidate Amts from the four Bactrocera species to confirm the completeness of all candidate Amt structures and to eliminate intron retention.

We designed specific primers based on the predicted gene sequences for amplifying the ORFs of four Amts, as detailed in Appendix A. Following PCR, we purified the products using the EasyPure Quick Gel Extraction Kit (TransGen Biotech, Beijing, China) and cloned them into a blunt vector. Sequencing was conducted in both the 5′- and 3′-directions by Sangon Biotech, Shanghai, China.

2.4. Bioinformatic Analyses

We used the ProtParam tool from ExPASy (https://www.expasy.org/resources/protparam, accessed from 15 June 2024, to 23 June 2024) to calculate the theoretical isoelectric points, molecular weights, instability indices, and aliphatic indices of the proteins, and we used the ProtScale tool from ExPASy (https://www.expasy.org/resources/protparam, accessed from 15 June 2024, to 23 June 2024) to calculate the average hydropathy indices of the proteins. The tertiary structures were predicted using the AlphaFold Server (https://alphafoldserver.com/, accessed from 15 June 2024, to 23 June 2024), which is based on the advanced AlphaFold 3 model [29]. The accuracy of these predictions was assessed via Ramachandran plot analysis in UCLA-DOE LAB-SAVES v6.0 (https://saves.mbi.ucla.edu/, accessed from 15 June 2024, to 23 June 2024). Protein structure images were generated using PyMOL v2.5 [30].

2.5. The Phylogenetic Analysis of the Amt Family

In order to explore the phylogenetic relationships between four Amts and documented Amts from various Diptera species, we acquired the Amt amino acid sequences of 16 Diptera species. These species include Bactrocera tryoni (Froggatt 1897), Bactrocera neohumeralis Hardy 1950, Bactrocera latifrons (Hendel 1915), Bactrocera oleae (Rossi 1790), Anastrepha ludens (Loew 1873), Ceratitis capitata (Wiedemann 1824), Anastrepha obliqua (Macquart 1835), Rhagoletis pomonella (Walsh 1867), Teleopsis dalmanni (Wiedemann 1830), Musca vetustissima Walker 1849, Musca domestica Linnaeus 1758, Lucilia cuprina (Wiedemann 1830), D. melanogaster, Aedes aegypti (Linnaeus 1762), A. gambiae, and Anopheles coluzzii Coetzee & Wilkerson 2013, all of which were retrieved from the Uniprot database (https://www.uniprot.org/, accessed on 15 April 2024; the accession numbers of the sequences used are listed in Appendix B). Following this, multiple sequence alignment was performed using MAFFT v7.515 [31]. The optimal nucleotide substitution model was determined with ModelFinder in IQ-TREE v2.2.0.3 [32]. A phylogenetic tree was then constructed using the maximum likelihood method.

2.6. Analysis of Expression Patterns of Amt in Different Peripheral Tissues

We realigned clean reads from each sample to the Amt using HISAT v0.1.6-beta (Hierarchical Indexing for Spliced Alignment of Transcripts) and assessed Amt expression in peripheral tissues via FPKM quantification [33,34,35,36]. Differential expression analysis was conducted with R package DESeq2 [37], identifying significant gene expression changes with a p-value ≤ 0.05 and an absolute log2Ratio ≥ 1 [38]. PCR conditions and primer sequences are detailed in Appendix A. The PCR reaction mixture totaled 10 µL, consisting of 5 µL of 2 × Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China), 0.5 µL of each 10 µM primer, 1 µL of cDNA template, and 3 µL of RNase-free water. The amplification process was executed on a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Specificity was confirmed by dissociation curve analysis, and gene expression was quantified by the 2^−ΔΔCT method [39].

2.7. Statistical Analyses

All replicated data are presented as mean ± standard error. The normality of the raw data was initially assessed using the Shapiro–Wilk test. Non-normally distributed data were transformed using log (x + 1). Differences in the data were analyzed using an unpaired t-test, with significance considered at p < 0.05. All statistical analyses were performed using GraphPad Prism (Version 8.0.1).

3. Results

3.1. Identification and Sequence Characteristics of Amts

Using the transcriptome datasets from four related fruit fly species, we successfully identified the genes BdorAmt, BcucAmt, BcorAmt, and BtauAmt by aligning with DmelAmt sequence (

Table 1. Candidate Amt genes of B. dorsalis, B. cucurbitae, B. correcta, and B. tau.

Gene Name	Blastx Best Hit (Reference/Name/Species)	E-Value	Score
BdorAmt	XP_049304235.1 putative ammonium transporter 2 [Bactrocera dorsalis]	0	1211
BcorAmt	XP_049304235.1 putative ammonium transporter 2 [Bactrocera dorsalis]	0	1204
BcucAmt	XP_054081809.1 putative ammonium transporter 2 [Zeugodacus cucurbitae]	0	1209
BtauAmt	XP_054081809.1 putative ammonium transporter 2 [Zeugodacus cucurbitae]	0	1097

). Using their antennae cDNA as templates, we designed specific primers for PCR amplification of these genes (Appendix A). The electrophoresis showed that the amplified target fragment was consistent with the length of the target genes (Figure 1A). These genes are the same length, containing a 1770 bp ORF encoding an Amt protein consisting of 589 amino acid residues (Figure 1A; Appendix C).

The theoretical isoelectric points of BdorAmt, BcucAmt, BtauAmt, and BcorAmt range from 6.39 to 6.50, and their molecular weights range from 63.9 to 64 kDa. Their instability indices are 24.76, 26.76, 27.31, and 23.44; aliphatic indices are 99.03, 98.37, 98.37, and 99.86; and average hydropathy indices are 0.280, 0.255, 0.254, and 0.282, respectively (Figure 1A; Appendix D). To ascertain the completeness of the ORF lengths obtained, we performed multiple sequence alignments and secondary structure predictions. The results of the multiple sequence alignment revealed extensive conserved regions within these proteins (Figure 1B), while secondary structure predictions indicated that all four Amts encompass 11 transmembrane domains, with an apoplastic N-terminus and a cytosolic C-terminus (Figure 1C). These findings confirmed the integrity of the identified Amt ORFs.

3.2. Phylogenetic Analysis of Amts

We analyzed the conservation of Amt genes and constructed a phylogenetic tree based on amino acid sequences from Diptera available in public databases. This analysis revealed significant homology among Amt proteins across various Diptera species. The four identified Amt proteins show the highest homology with those from the Tephritidae family, followed in descending order by proteins from several species within the Muscidae, Calliphoridae, Diopsidae, Drosophilidae, and Culicidae families (Figure 2A). Specifically, BdorAmt and BltaAmt clustered together, while BcucAmt and BtauAmt formed another branch (Figure 2A). Detailed amino acid sequence analysis indicated that the Tephritidae Amts shared >90% similarity; the four Amt proteins exhibited sequence similarity exceeding 82% with Amts from M. domestica, T. dalmanni, M. vetustissima, and L. cuprina; and each demonstrated a sequence similarity of up to 77% with DmelAmt and up to 66% with AgamAmt2 (Figure 2B). It is noteworthy that DmelAmt and AgamAmt2 have been implicated in the olfactory recognition of ammonia [19,20,21]. Additionally, other Amts from fruit flies exhibited over 66% homology with both DmelAmt and AgamAmt2 (Figure 2B). Such a high level of sequence similarity suggests a conserved functional role for this gene family.

3.3. Structural Prediction of Amt and Analysis of Conserved Functional Sites

Amt’s role in the transport of NH₄⁺ across various organisms, including bacteria, fungi, plants, and insects, has been well established [22,23,24,25]. To predict the functional roles of BdorAmt, BcucAmt, BtauAmt, and BcorAmt, we analyzed their structural features and conserved functional sites. In Escherichia coli (Migula, 1895), extensive research has elucidated the functional residues of EcAmt [22,23]. Building on this knowledge, we conducted a comprehensive multiple sequence alignment focused on EcAmt to identify highly conserved residues critical for the functionality of the four Amts. Our analysis revealed three conserved positions corresponding to previously documented functional residues in EcAmt: Ec Asp160, Ec H168, and Ec H318. Specifically, Ec Asp160 aligns with A189 in the four Amts, Ec H168 with H195, and Ec H318 with H352 (Figure 3A). Notably, Ec Asp160, Ec H168, and Ec H318 from the E. coli homolog, EcAmtB, have been confirmed to play a crucial role in ammonium ion transport [22,23].

To delve deeper into the structural features of the four Amt proteins, we used AlphaFold 3 to predict their tertiary structures (Figure 3B–E). The Ramachandran favored values were all above 90% (Appendix D), indicating the validity of the predicted three-dimensional structures. The characteristics of their 2D structures align with those of the 3D structures. Another important functional characteristic of the Amt proteins is their ability to form trimers on the cell membrane [22,23,24,25], and we predicted a similar trimeric structure (Figure 3B–E, Appendix D).

3.4. Expression Profiles of Amts in Different Peripheral Tissues

We conducted a transcriptome analysis to examine the expression of four Amt genes in various peripheral tissues and olfactory organs. We then confirmed these findings with qPCR. The results confirmed the presence of Amt transcripts in adult tissues and revealed a highly consistent expression pattern of the Amt genes across peripheral tissues in the four fly species. Specifically, high levels of transcription were observed in the antennae, whereas almost no expression was detected in the mouthparts, maxillary palps, legs, and external genitalia (Figure 4A–D). Furthermore, there were no significant differences in the expression of Amt genes between males and females in these flies (Figure 4A–D).

4. Discussion

Ammonia serves as a mediator for insects to locate protein-rich food sources [12,13]. The function of Amt as an ammonia receptor was first explored in Drosophila [19,21]. Subsequently, AgamAmt2 was reported to have similar functionality in A. gambiae [20]. The function of Amt in the olfactory detection of ammonia is hypothesized to be conserved across insects. However, in the economically significant pest genus Bactrocera, research on Amts remains absent. Therefore, this study reports the sequence information and expression patterns of BdorAmt, BcucAmt, BtauAmt, and BcorAmt based on the peripheral transcriptomes from four closely related species, B. dorsalis, B. cucurbitae, B. correcta, and B. tau, and predicts the structural characteristics of the amino acids. These findings will allow future study of the physiological functions of these genes.

The structure and functional characteristics of Amt genes have been extensively studied in microorganisms and plants [22,23,24,25]. In E. coli, the crystal structure of EcAmtB has been reported, showing that it is a protein with 11 transmembrane regions that forms trimers in the membrane. Each monomer contains a hydrophilic channel located between the relatively polar cytoplasmic and periplasmic vestibules, directly involved in the transport of NH₄⁺ [22,23,24]. Similar reports have also been found in studies on Amt in fungi and plants [22,25]. The structural and functional characteristics of this protein are similar to those of an ammonia transporter protein, RhCG (SLC43A3), in humans, which is a member of the 386 human SLC superfamily members. It also forms a trimeric functional structure and participates in the transport of ammonia in the human kidney. However, it possesses an additional transmembrane domain compared to EcAmtB [40,41]. In this study, based on transcriptome data from four species, we screened and cloned their respective Amt genes: BdorAmt, BcucAmt, BtauAmt, and BcorAmt. These genes have identical ORF lengths, each comprising 1770 bp and encoding a protein of 589 amino acid residues. Structural predictions revealed that all four Amts possess a typical 11-transmembrane domain structure with an N-terminus outside the membrane and a C-terminus inside and are capable of forming trimers in the plasma membrane. They also contain three conserved amino acid residues, A189, H195, and H352, which are known to be key residues in ammonia transport in E. coli [22,23]. These findings suggest a high accuracy in the sequences of the four Amts identified, and the predicted structural features suggest potential for transporting NH₄⁺. The complete sequence information will provide a foundation for future in vitro functional studies.

Through phylogenetic analysis, we found that the four Amt proteins are highly homologous within Diptera, containing numerous conserved segments. Among the Tephritidae species used in this study, sequence similarity between these Amts exceeds 80%. Notably, BdorAmt, BcucAmt, BtauAmt, and BcorAmt show sequence similarity of up to 77% with DmelAmt and 66% with AgamAmt2. Additionally, peripheral olfactory expression profiling showed that all four Amts are predominantly expressed in the antennae. Among these homologous genes, DmelAmt and AgamAmt2 have been demonstrated to be significantly expressed in the antennae and participate in ammonia recognition as atypical olfactory receptors [19,20,21]. It was hypothesized that Amt may directly facilitate the rapid and efficient transport of ammonia/ammonium ions into cells, leading to changes in membrane potential, which may trigger action potentials through an unknown signaling mechanism. In the antennae, highly expressed olfactory proteins often play crucial roles in olfaction [42,43], and highly conserved genes in insects often have similar functions [44,45,46]. The four Amts identified in this study are highly conserved within Diptera and significantly expressed in the antennae, suggesting they may have similar functions to those reported for mosquitoes and Drosophila, i.e., participating in ammonia recognition in the antennae. Additionally, previous studies have indicated low Amt expression in the labellum of Drosophila, yet we found no significant Amt levels in the mouthpart transcriptome [26]. This discrepancy likely arises from sampling the entire mouthparts, diluting the labellum signal. Future studies should specifically sample the labellum to obtain accurate measurements. Future research should explore in vitro functional studies using the Drosophila empty neuron system [47,48], and in vivo functional validation through knockout experiments.

5. Conclusions

In summary, this study has investigated the sequence information, structural characteristics, phylogenetic relationships, and expression patterns of Amt genes from B. dorsalis, B. cucurbitae, B. correcta, and B. tau. The findings indicate that all four Amt proteins possess the typical Amt transmembrane domain structural features, including three conserved amino acid sites previously reported to be involved in ammonia transport. These residues are highly conserved across Diptera and significantly expressed in the antennae of the four Tephritid fruit flies. We postulate that these Amt proteins might play a role in the antennal recognition of ammonia. Future research is required to validate the in vivo and in vitro functions of these genes.