Identification and Pharmacological Characterization of Two Serotonin Type 7 Receptor Isoforms from Mythimna separata

Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive molecule, as neurotransmitters regulate various biological functions in vertebrates and invertebrates by binding and activating specific 5-HT receptors. The pharmacology and tissue distribution of 5-HT receptors have been investigated in several model insects, and these receptors are recognized as potential insecticide targets. However, little is known about the pharmacological characterization of the 5-HT receptors in important agricultural pests. In this study, we investigated the sequence, pharmacology, and tissue distribution of 5-HT7 receptors from oriental armyworm Mythimna separata (Walker) (Lepidoptera: Noctuidae), an important migratory and polyphagous pest species. We found that the 5-HT7 receptor gene encodes two molecularly distinct transcripts, Msep5-HT7L and Msep5-HT7S, by the mechanism of alternative splicing in M. separata. Msep5-HT7S differs from Msep5-HT7L based on the deletion of 95 amino acids within the third intracellular loop. Two Msep5-HT7 receptor isoforms were activated by 5-HT and synthetic agonists α-methylserotonin, 8-hydroxy-DPAT, and 5-methoxytryptamine, resulting in increased intracellular cAMP levels in a dose-dependent manner, although these agonists showed much poorer potency and efficacy than 5-HT. The maximum efficacy of 5-HT compared to the two 5-HT isoforms was equivalent, but 5-HT exhibited 2.63-fold higher potency against the Msep5-HT7S than the Msep5-HT7L receptor. These two isoforms were also blocked by the non-selective antagonist methiothepin and the selective antagonists WAY-100635, ketanserin, SB-258719, and SB-269970. Moreover, two distinct mRNA transcripts were expressed preferentially in the brain and chemosensory organs of M. separata adults, as determined by qPCR assay. This study is the first comprehensive characterization of two splicing isoforms of 5-HT7 receptors in M. separata, and the first to demonstrate that alternative splicing is also the mechanism for producing multiple 5-HT7 isoforms in insects. Pharmacological and gene expression profiles offer important information that could facilitate further exploration of their function in the central nervous system and peripheral chemosensory organs, and may even contribute to the development of new selective pesticides.

In vertebrates, the effects of serotonin are mediated by its binding to two specific types of receptors: ionotropic and metabotropic receptors. All metabotropic receptors belong to G protein-coupled receptors (GPCRs), with seven transmembrane domains that are divided into six distinct classes based on amino acid sequence homology, pharmacological properties, and downstream second messenger pathways (5-HT1, 2, 4, 5, 6, 7), while the remaining 5-HT3 receptor is an ionotropic receptor [1][2][3]. In most insects, three classes of 5-HT receptors have been found (5-HT1, 5-HT2, and 5-HT7 type receptors), based on sequence similarities and activated second-messenger pathways with their counterparts in vertebrates [3,4,17,18]. Within the same receptor class, the signaling properties seem to be very well maintained between insects and vertebrates [3]. The 5-HT1 type receptors couple preferentially to G i/o proteins, and thus inhibit cyclic adenosine monophosphate (cAMP) synthesis [4,17]. The 5-HT2 receptors couple to G q/11 proteins, causing an increase in cytosolic Ca 2+ concentration [12,19], while 5-HT7 receptors couple to G s proteins, activating adenylyl cyclase (AC) activity and hence increasing intracellular cAMP formation [3,4]. Moreover, Qi et al. [20] recognized a novel 5-HT8 receptor in Pieris rapae (Linnaeus) (Lepidoptera: Pieridae) that mediated increased intracellular calcium concentration, which may be an invertebrate-specific receptor without a counterpart in mammals. The pharmacological characteristics of the same type of 5-HT receptors vary significantly among different insect species [3,21]; therefore, 5-HT receptors are recognized as potential insecticide targets [3,4]. In fact, Cai et al. [22] demonstrated that the 5-HT1A receptor agonist PAPP and its derivatives in Haemonchus contortus (Rudolphi) Cobb (Rhabditida: Trichostrongylidae) could inhibit the growth or kill the larvae of Mythimna separata (Walker) (Lepidoptera: Noctuidae).
The 5-HT7 receptor genes have been cloned in many vertebrates. In humans, mice, and rats, the coding region of this gene contains three introns, and alternative splicing at the second and third introns is a general mechanism that results in a number of splice variants. No major differences in their pharmacology or functionality have been reported so far [23], and all 5-HT7 receptor isoforms are coupled to G s proteins and stimulate cAMP formation [2]. On the contrary, only one 5-HT7 receptor gene transcript has been cloned in insects thus far [3]. Tissue distribution and functional studies have confirmed that 5-HT7 plays a vital role in circadian rhythm, rapid eye movement sleep, thermoregulation, depression, anxiety, schizophrenia, nociception, epilepsy, and memory learning in mammals [23][24][25]. The 5-HT7 receptor has been characterized in several model insects, and has been shown to be involved in normal courtship and mating behaviors in D. melanogaster [26], learning and memory in A. mellifera [27], and saliva secretion in Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) [28]. The pharmacological properties and physiological functions of the 5-HT7 receptor in important agricultural pests remain largely unknown.
In the present study, we cloned the 5-HT7 receptor gene from M. separata, one of the important polyphagous insect pests in China, costing more than ¥ 146 million ($18 million) in insecticide management on wheat alone in 1997 [29]. Different from 5-HT7 receptors gene transcription patterns in other insects [26][27][28], this gene encodes two molecularly distinct transcripts by the mechanism of alternative splicing in M. separata. To our knowledge, this is the first report showing that alternative splicing is the mechanism for producing multiple 5-HT7 isoforms in insects. Furthermore, we examined the pharmacological characteristics and expression profiles in different tissues of M. separata larvae and adults. These results provide a foundation for understanding the actions of serotonin in behavior and physiological function, and for exploring novel pesticides to control this pest in the future.

Molecular Features and Phylogenetic Analysis
The full length of genomic DNA and cDNA of Msep5-HT7 from adult M. separata was amplified by using transcriptome analysis with a PCR-based strategy. The Msep5-HT7 gene contains an intron and produces two molecularly distinct transcripts (Msep5-HT7L and Msep5-HT7S) by the mechanism of alternative splicing ( Figure 1A). The open reading frame of Msep5-HT7L comprises 1695 bp and codes for 564 amino acid residues, with a calculated molecular weight of 63.25 kDa. Msep5-HT7S differs from Msep5-HT7L based on the deleted of 95 amino acids within the third intracellular loop ( Figure 1B). The Msep5-HT7 receptors were predicted to be GPCRs, with seven transmembrane domains (TM1-7) connected by intra-and extracellular loops ( Figure 1C). Sequence motifs in 5-HT receptors that are essential for ligand binding and signal transduction are well conserved in two Msep5-HT7 isoforms, including the highly conserved D-R-Y motif at the N-terminus of the second intracellular loop, a conserved aspartic acid in TM3, the sequence F-X-X-XW-X-P followed by a pair of phenylalanine residues within TM6, and two cystein residues in the intracellular C-terminus which are putative palmitoylation sites. In addition, the Msep5-HT7S receptor has a shorter third intracellular loop, and lacks two consensus sites for phosphorylation by protein kinase C ([S/T-X-[R/K]) compared with the Msep5-HT7L receptor ( Figure 1C).
The dose-response relationship for synthetic agonists was also measured using the same method, with dose concentrations ranging from 0.1 µM to 1 mM. cAMP responses produced by the highest concentration (1 mM) of synthetic agonists were also consistently poorer than the same concentration of 5-HT induced responses, and the efficacy of synthetic agonists was different between the two receptor isoforms. The highest concentration of αm-5-HT and 5-MT (1 mM) resulted in about 69% activation compared with the maximum amount of cAMP stimulation obtained with the Msep5-HT7L receptor with the same concentration of 5-HT. However, these two agonists resulted in about 35% activation of the maximum amount of cAMP obtained in the Msep5-HT7S receptor. The highest efficacy of 8-OH-DPAT was below 30% of the maximum response of 5-HT in the two isoforms ( Figure 3C,D). All tested synthetic agonists exhibited agonistic potency, and the effects were dose-dependent. The EC 50 values of the synthetic agonists were more than 100-fold higher than those of 5-HT, implying that they were considerably less potent than 5-HT ( Table 1). The agonist potency of 8-OH-DPAT against Msep5-HT7L was 2.4-fold higher than against the Msep5-HT7S receptor, while αm-5-HT showed agonist potency against Msep5-HT7S 2.3-fold higher than Msep5-HT7L. The agonist potency of 5-MT was roughly equivalent to the two Msep5-HT7 isoforms (Table 1).  . Dots (•) indicate putative N-glycosylation sites above sequences in the N-terminal region; stars (★) and concentric circles (○) indicate potential phosphorylation sites for protein kinase C and putative palmitoylation sites, respectively; triangles (▼) indicate aspartic acid residue predicted to be involved in agonist binding; diamonds (♦) indicate the pairs of phenylalanine after FxxxWxP motif in TM6, a unique feature of aminergic receptors; rectangles (▄) indicate the highly conserved DRY motif, which is believed to have a key role in receptor activation.
A comparison of the amino acid sequences of two Msep5-HT7 isoforms with other insect 5-HT7 receptor orthologues revealed high sequence similarities. The similarity between Msep5-HT7L and Msep5-HT7S is 83.2%. The similarity between Msep5-HT7L and lepidopteran insect 5-HT7 orthologues is 93.3% for M. sexta, 90.1% for P. rapae, 91.4% for ) indicate the highly conserved DRY motif, which is believed to have a key role in receptor activation.     Potential synthetic antagonists were tested by simultaneously applying 5 µM of 5-HT and an 8-fold dose of antagonists to stable transfected cell lines expressing Msep5-HT7L or Msep5-HT7L receptors. All of the tested mammalian 5-HT receptor antagonists (methiothepin, ketanserin, WAY-100635, SB-258719, and SB-269970) decreased the 5-HT induced response in Msep5-HT7-expressing cell lines by about 33-60% ( Figure 4A,B). The values for the induced receptor response of 5 µM of 5-HT were almost completely blocked by the highest concentration of antagonists (0.64 mM), and the blocking effect of all antagonists on Msep5-HT7 receptor was dose-dependent ( Figure 4C,D). The halfmaximum inhibitory concentration (IC 50 ) values are listed in Table 2. There were no significant differences in the potency or efficacy of the antagonists methithepin, SB-258719, and SB-269970 to inhibit 5-HT-induced cAMP production between Msep5-HT7L and Msep5-HT7S receptors. Meanwhile, higher antagonistic activity of ketanserin and WAY-100635 was measured for the Msep5-HT7L than the Msep5-HT7S receptor based on non-overlap of the 95% fiducial limits.

Expression Profile Analysis of Msep5-HT7 Genes
Msep5-HT7 gene has two distinct transcripts, Msep5-HT7L and Msep5-HT7S, which are generated by alternative splicing. The specific primers that amplify the Msep5-HT7 gene contain these two transcripts ( Figure 1A). The expression profiles of Msep5-HT7S and Msep5-HT7L in different tissues of larvae and adults were quantified by qPCR (relative expression level of Msep5-HT7S = relative expression level of Msep5-HT7 − relative expression level of Msep5-HT7L). The two transcripts were co-expressed in all tested tissues ( Figure 5). In fifth instar larvae, Msep5-HT7 expression was higher in the gut and Malpighian tubules than in other tissues ( Figure 5A). It also showed higher expression in the brain, antennae, and labial palps compared to the wing, leg, thorax, and abdomen at the adult stage. This gene had 10-fold higher expression in highly expressed tissue of adult male antennae than in highly expressed tissue of larval gut. In addition, the expression of Msep5-HT7 was much higher in male than female antennae and labial palps ( Figure 5B). We also noticed that the expression ratio of Msep5-HT7S in adult wing, leg, thorax, and abdomen tissue was 23-43%. However, this expression ratio in highly expressed adult brain, labial palps, and male antennae was increased to 52-67%. The expression ratio of Msep5-HT7S were significantly higher in male than female antennae and labial palps, and was significantly higher in female than male abdomens (p < 0.05) ( Table 3).

Discussion
We cloned one 5-HT7 gene in M. separata that codes two molecularly distinct transcripts of 5-HT7 by the mechanism of alternative splicing, complying with the typical GU-AG rule. Msep5-HT7S differs from Msep5-HT7L based on an extra 3 -terminal and 285 nucleotides cleaved off during autocatalytic excision of the intron. The alternative splicing forms of 5-HT7 receptors are widely found in humans, mice, and rats, and alternative splicing occurs extensively at the second and third of three introns of the 5-HT7 gene [2,[30][31][32]. Moreover, 5-HT7 receptor genes have been cloned and characterized from D. melanogaster [33], Aedes aegypti (Linnaeus) (Diptera: Culicidae) [34], A. mellifera [27], C. vicina [28], M. sexta [35], Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) [36], P. rapae [4], and Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae) [37]. Only one type of the receptor gene transcript has been identified in these insects. With the development of genome sequencing technology, many insect genomes have recently been published. This provides an opportunity to analyze the 5-HT7 genomic sequence in different insect species. We checked the coding region of the 5-HT7 gene from B. mori, T. castaneum, M. sexta, P. rapae, P. xylostella, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), and Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) according to the genomic and coding sequence data. Only one intron was found in these insect 5-HT7 genes, which was located in the sequence encoding the putative third intracellular loop. We need to check whether this kind of alternative splicing is a common mechanism in insect 5-HT7 genes in the future. Two Msep5-HT7 receptor isoforms also contain key sequence motifs that are important for ligand binding and signal transduction for biogenic amine receptors, compared with previous reports [27,[38][39][40][41][42]. The length of the third intracellular loop varies greatly among insect species (Figure 1C), which may be the reason for the different pharmacological properties of the same agonists and antagonists against the same type of receptors in different insect species.
The 5-HT7 receptor in animals is known to stimulate AC activity, which converts ATP to cAMP, and its coupling with specific G-proteins is brought about by amino acids in close proximity to the plasma membrane of the second and third intracellular loops and the cytoplasmic tail of the receptor proteins [2,27]. This feature is conserved in the two 5-HT7 receptor isoforms of M. separata, indicating that these receptor isoforms might have similar signaling pathways. The pharmacological properties of the two receptor isoforms were further explored by testing their downstream signal responses to synthetic agonists and antagonists. The efficacy of 5-HT on the two M. separata 5HT7 receptor isoforms was equal, but the potency of 5-HT was nearly three-fold higher against the Msep5-HT7S receptor than the Msep5-HT7L receptor. A similar potency of 5-HT against the 5-HT7 receptor has also been observed in other insects, such as M. sexta (EC 50 = 10.5 nM), P. rapae (EC 50 = 15 nM), T. castaneum (EC 50 = 27.3 nM), and D. melanogaster (EC 50 = 60 nM) [33,35,36]. The synthetic agonists of 8-OH-DPAT, αm-5-HT, and 5-MT were much less efficient and potent than 5-HT in stimulating the two M. separata 5-HT7 receptor isoforms.
Few studies have investigated the effects of SB-258719 on insect 5-HT receptors to date. The antagonistic potency of these two selective 5-HT7 receptor antagonists was statistically equal in terms of the half maximal inhibitory concentration against Msep5-HT7L and Msep5-HT7S. In our study, when the concentration of five antagonists increased 128-fold over 5-HT, they completely blocked 5-HT7 receptor-mediated cAMP production. These antagonists could be used as candidate agents for functional studies. The differences in the potency and efficacy of agonists and antagonists against the 5-HT7 receptor between mammals and insects, and even between different insects, imply that these types of GPCRs could act as potential targets for the development of selective insecticides.
High transcription levels of 5-HT7 receptor genes were detected in the Malpighian tube, gut, salivary gland, brain, and/or antennae of A. mellifera, G. bimaculatus, M. sexta, A. aegypti, C. vicina, T. castaneum, and Polyrhachis vicina Roger (Hymenoptera: Formicidae) [27,28,[34][35][36]51,52]. Our results also reveal that the 5-HT7 receptor genes are highly expressed in larval Malpighian tubes and guts, indicating that the 5-HT7 receptor potentially has a role in digestion and metabolism. Yang et al. [52] reported that the 5-HT7 receptor's mRNA and protein expres-sion were slightly higher in the pupal than the egg and larval stages, and they deduced that this receptor may be involved in modulating adult formation and caste differentiation in P. vicina. Moreover, the 5-HT7 gene showed strong expression in the brains of adult D. melanogaster, and it mediated normal courtship and mating behaviors [26]. We also noticed that higher transcript numbers of 5-HT7 were found in adult males than females of P. vicina and P. xylostella, as well as in our detected male M. separata antennae and labial palps. In addition, 5-HT-immunoreactive neurons were found to be distributed in the antennal lobe of M. sexta. It was then confirmed that 5-HT changes K+ conductance in the cells and increases the excitability and responsiveness of central olfactory neurons to sex pheromones [14,53], although it is unclear which type of 5-HT receptor mediates intracellular signal transduction. High expression of 5-HT7 receptor genes in male antennae and labial palps should receive careful consideration, as it may contribute to modulating sensitivity and specificity to carbon dioxide and plant volatiles, perception of sex pheromones, and regulating courtship and mating behaviors. High expression of Msep5-HT7S in male antennae and labial palps could be attributed to its higher sensitivity to endogenous 5-HT than Msep5-HT7L receptor, and organisms that express Msep5-HT7S transcript can improve energy efficiency.
The oriental armyworm M. separata is a typical seasonal, long-distance migratory polyphagous pest that can attack nearly 100 families of more than 300 kinds of food and industrial crops, resulting in serious yield losses in China and other parts of Asia and Oceania [54][55][56]. The use of chemical insecticides is the main strategy to control this pest. However, the long-term use of insecticides leads to the development of resistance to traditional insecticides [57]. Thus, there is an urgent need to develop novel insecticides. The potential of 5-HT receptors as insecticide targets has frequently been considered, and some progress has been made toward insecticide discovery through the study of their pharmacological characterization and the design of chemical compounds, with the 5-HT receptor agonist as the lead compound [21,22]. With the present study, detailed knowledge of the pharmacological properties of the Msep5-HT7 receptor and established cell lines that stably express this receptor should facilitate designing, synthesizing, and screening novel insecticides.

Insects Rearing, Tissue Collection and Reagents
The larvae of M. separata were reared on an artificial diet in the climate incubator and maintained under a photoperiod of 14:10 h (light:dark) at 27 • C and 75% relative humidity [58]. Adult moths were provided with 10 % (weight by volume) sucrose solution. Various tissues from fifth instar larvae, including heads, epidermis, fat bodies, Malpighian tubules, and guts, were immediately dissected, and hemocytes were collected by cutting the thoracic legs. Various tissues from three day-old unmated female or male moths including brains, thoraxes, abdomens, antennae, labial palps, wings, and legs were collected, and all samples were used for RNA extraction. The reagents used in the pharmacological ligands, including serotonin hydrochloride

Cloning of M. separata 5-HT7 Receptor Gene
Genomic DNA and total RNA were extracted from adult M. separata using an EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China) and TRIzol reagent (Invitrogen, Carlsbad, CA, USA), respectively. Genomic DNA and single-stranded cDNA synthesized from the RNA using the Fast King gDNA Dispelling RT SuperMix (TianGen, Beijing, China) were used as the template for PCRs to amplify the genomic DNA sequences and open reading frame (ORF) of Msep5-HT7, respectively. Full-length sequences of putative Msep5-HT7 were obtained from our previous brain transcriptomes of M. separata [59]. The gene-specific primers (Table 4) used in our studies were designed by Primer Premier 5.0 software. The amplified DNA fragments were cloned into the pMD19-T Simple Vector (Takara, Dalian, China). Positive clones were identified and sequenced by Sangon Biotech (Shanghai, China). The obtained cDNA sequences were submitted to NCBI as Msep5-HT7L (GenBank Acc. OM025087) and Msep5-HT7S (GenBank Acc. OM025088). Table 4. Primers used in this study.

Multiple Sequence and Phylogenetic Analysis
Alignments of 5-HT7 receptor amino acid sequences were performed by MAFFT online, and the results were displayed by ESPRIPT [60]. The transmembrane segments were predicted by TMHMM 2.0 online. To identify the potential orthology of the identified M. separata 5-HT7 receptor, the phylogenetic trees of 5-HT receptors from other insects were constructed using MEGA 6.0 software. The maximum-likelihood method was used with a bootstrap analysis of 1000 replicates, based on the JTT matrix-based model [61]. The Drosophila melanogaster FMRF amide receptor was used as outgroup. The amino acid sequences used in the phylogenetic analyses are listed in Supplementary Materials.

Construction of Expression Plasmids
The construction of expression plasmids of the Msep5-HT7L and Msep5-HT7S cDNA containing the Kozak consensus sequence (GCCACC) [62] were obtained by PCR amplification with specific primers ( Table 4). The DNA fragments were double digested with KpnI and XhoI (Sangon Biotech, Shanghai, China), and after purification, they were cloned into a pcDNA3.0 vector (Invitrogen, Carlsbad, CA, USA). DNA sequencing was used to confirm the correct recombinant plasmid. Louis, MO, USA) was added to the medium to select for cells that were stably expressed the Msep5-HT7 receptors. After fourteen days of G418 selection, three G418-resistant colonies were trypsinized in cloning cylinders and transferred to 12-well plastic plates for expansion [20].

cAMP Assays
Intracellular cAMP concentrations were measured as previously described [4,63,64]. Briefly, when cells were cultured for 2 days to the concentration of 1 × 10 6 cells/mL, culture media were removed and the cells were washed two times with cold PBS (pH 7.4), then the cells were preincubated in 450 µL of Dulbecco's phosphate-buffered solution (DPBS) containing 100 µM phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) for 20 min at room temperature. After the preincubation, 50 µL of DPBS, containing various concentrations of reagents, was added into the stable transfected cells for incubation for 30 min at 37 • C. The cells were washed three times with cold PBS (pH 7.4) and then lysed in 250 µL of cold cell lysis buffer by re-freezing. The cell debris was removed by centrifugation at 600× g for 10 min at 4 • C. The amount of intracellular cAMP extracted from harvested supernatant was determined using an ELISA reagent (R&D Systems, Minneapolis, MN, USA). Each measurement was performed in duplicate, and three independent assays were carried out for each reagent and concentration tested.

Expression Profiles Analysis by qPCR
Real-time PCR was used to confirm the expression profiles of the Msep5-HT7 gene in larvae and various adult tissues. cDNA was synthesized using total RNA that was extracted from above collected samples with the FastKing gDNA Dispelling RT SuperMix (TianGen, Beijing, China). The specific qPCR primers used to amplify Msep5-HT7 and the reference genes are listed in Table 4. The reference genes β-actin (GenBank Acc. GQ856238.1) and glyceraldehyde-3-phosphatedehydrogenase (gapdh) (GenBank Acc. HM055756.1) were used to normalize Msep5-HT7 gene expression. Reactions for each sample (20 µL) consisted of 10 µL of 2 × SuperReal PreMix Plus (TianGen, Beijing, China), 7.6 µL of sterilized H 2 O, 0.5 µL of each primer (10 µM), 0.4 µL of Rox reference dye, and 1 µL of cDNA. qPCR was carried out with an initial denaturation at 95 • C for 3 min, followed by 40 cycles at 95 • C for 15 s and 60 • C for 30 s. The qPCR reaction of each sample was performed in three technical replicates and three biological replicates. Then, we used a relative quantitation method (2 −∆∆CT ) [65] to evaluate quantitative variation. Transcript amounts were standardized to 1 with the sample from the heads of fifth-instar larvae.

Statistical Analysis
GraphPad Prism 7.0 software was used for curve fitting and statistical analysis. We used one-way analysis of variance (ANOVA) with Tukey's multiple comparison test (p < 0.05) to analyze the expression differences of the Msep5-HT7 gene among different larvae and adult tissues. Then, we used Student's t-test to test for statistically significant differences in Msep5-HT7 expression levels and expression ratio of 5-HT7S between the same tissues from male and female adults, respectively (p < 0.05).

Acknowledgments:
We are grateful to Lin-lin Chen, College of Plant Protection, Henan Agricultural University for her insightful suggestions for drafting this manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.