Identification and expression analysis of neuropeptides and their G protein-coupled receptors in the predatory stink bug, Arma custos (Hemiptera: Pentatomidae)

doi:10.21203/rs.3.rs-3218912/v1

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Identification and expression analysis of neuropeptides and their G protein-coupled receptors in the predatory stink bug, Arma custos (Hemiptera: Pentatomidae)

https://doi.org/10.21203/rs.3.rs-3218912/v1

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The predatory stink bug Arma custos has been selected as an effective biological agent and has been successfully massly bred and released into fields for the control of a diverse insect pests. As a zoophytophagous generalist, A. custos relies on a complex neuropeptide signaling system to prey on distinct food and adapt into different environment. However, information about neuropeptide signaling genes in predatory bugs has not been reported to date. In the present study, a total of 57 neuropeptide precursor transcripts and 41 potential neuropeptide GPCR transcripts were found mainly using our sequenced transcriptome data. Furthermore, a set of neuropeptides and their GPCR receptors that were enriched in heads, guts, salivary glands, antennae, and legs of A. custos were identified, which might play critical roles in central nervous regulation, feeding, digestion, odor perception and motor modulation, etc. Our study provides basic information for in-depth understanding of biological and ecological characteristics of the predatory bug and would aid in the development of better pest management strategies based on the effective utilization and protection of beneficial nature enemies.

The predatory stink bug Arma custos (previously named A. chinensis¹) (Hemiptera: Pentatomidae) has been selected as a potential biological agent for the control of a wide range of agricultural and forest insect pests, such as those in the orders Lepidoptera, Coleoptera, Hymenoptera, and Hemiptera². It is widely distributed in China and other Asian regions and frequently found on various trees and crops². A. custos has successfully massly bred by a number of nature enemy factories in China, and has been released in tobacco field to control some important pest insects, such as Spodoptera frugiperda and Helicoverpa assultam³. In-depth understanding of biology, ecology and physiology of A. custos could help us to better utilize these biological control agents.

Neuropeptides are by far the most abundant and diverse signaling molecules in insects and they play a key role in growth, development, reproduction, feeding, metabolism, and many more physiological processes⁴. Most neuropeptides signal via G protein-coupled receptors (GPCRs)⁵. Due to their critical role in insect survival and high specificity, neuropeptides and their receptors represent a promising target for the development of novel “green” pest control strategies that offer improved selectivity and environmental compatibility^6,7. Recent advances in genomic and transcriptome analysis have led to the discovery of neuropeptides and their receptors in a number of important insects including several beneficial insects, which will promote the development of novel target-specific insecticides.

Hemiptera represents the largest and most diverse group of hemimetabolous insects⁸, which includes many species harmful to human, animals and plants and also contains a few important natural enemies such as predatory bugs. Comprehensive analysis of gene sequences of neuropeptides and their GPCRs of Hemiptera has been reported in several hemipteran insects, such as Acyrthosiphon pisum^9,10, Aphis craccivora¹¹, Daktulosphaira vitifoliae¹² of Sternorrhyncha, Nilaparvata lugens¹³ of Auchenorrhyncha, and four Heteropteran species: Rhodnius prolixus^14,15, Lygus hesperus^16,17, Nezara viridula and Halyomorpha halys¹⁸. However, research on the identification of neuropeptides or their GPCRs in predatory bugs is absent to date.

In the present study, we predicted the neuropeptide precursors and their receptors of A. custos using our sequenced transcriptome data and the publicly accessible transcriptome data. Furthermore, we also analyzed the expression of these genes in different tissues based on our custom transcriptome data. Our study provides fundamental information that may further our understanding of various underlying biological characteristics in this nature enemy, which would aid in the development of better pest management strategies.

Identification of Neuropeptide Precursors in A. custos

In the A. custos transcriptome, a total of 57 neuropeptide precursor transcripts were identified, belonging to 43 conserved families: adipokinetic hormone (AKH), AKH/corazonin-relate peptide (ACP), agatoxin-like peptide (ALP), allatostatin (Ast), allatotropin (AT), arginine-vasopressin-like peptide (AVLP), bursicon (Bur), capability/cardio acceleratory peptide 2b (CAPA), Carausius neuropeptide-like precursor (CNP), CCHamide (CCHa), CNMamide (CNMa), corazonin (Crz), crustacean cardioactive peptide (CCAP), diuretic hormone (DH), ecdysis triggering hormone (ETH), eclosion hormone (EH), elevenin (Ele), FMRFamide (FMRFa), glycoprotein hormone (GP), IDLSRF-like (IDLSRF), Insulin-like peptide (ILP), Ion Transport peptide (ITP), ITG-like (ITG), leucokinin (LK), myosuppressin (MS), natalisin (NTL), neuroparsin A (NPA), neuropeptide F (NPF), neuropeptide-like precursor (NPLP), NVP-like (NVP), orcokinin (OK), PaOGS36577, parathyroid hormone (PTH), pigment dispersing factor (PDF), proctolin (Pro), prothoracicotropic hormone (PTTH), pyrokinin (PK), RFLamide (RFLa), RYamide (RYa), short neuropeptide F (sNPF), SIFamide (SIFa), sulfakinins (SK), and tachykinins (TK) (Table 1, Supplementary Data 1). A couple of novel neuropeptides, including CNP, PTH, PaOGS36577, and RFLa were also present in A. custos, as well as in other hemipteran species by homology searching against NCBI nr/nt database (Fig. 1). AST-C and trissin were not found in the A. custos transcriptome, which were also missing in other hemipteran species (Fig. 1). AKH was not found in our custom transcriptome but identified in NCBI SRA data (SRR10098905). 54 out of 57 neuropeptide transcripts have full-length sequences and the remaining three non-full-length sequences include ALP2, IDLSRF-like and PTH (Supplementary Data 1). Most of A. custos mature neuropeptides predicted are identical or highly similar to H. halys or other bugs, whereas a few share low similarities with homologous sequences, such as NPAs, OKB and PTTH (Figs. 2 ~ 4).

Table 1

Putative neuropeptide precursor genes identified from *A. custos*
Neuropeptide	Acronym	ORF (aa)	Homology search against NCBI NR database (blastp)
Neuropeptide	Acronym	ORF (aa)	NR ID	Species	E-value
Adipokinetic hormone	AKH	46	AZK31332.1	Nezara viridula	8.00E-23
AKH/Corazonin-relate peptide	ACP	96	XP_014285630.1	Halyomorpha halys	1.50E-38
Agatoxin-like 1	ALP1	127	XP_014292753.1	Halyomorpha halys	5.30E-61
Agatoxin-like 2	ALP2	85*	XP_014255021.1	Cimex lectularius	8.50E-21
Allatostatin A/FGLamide Allatostatin	AstA	179	BAV78789.1	Plautia stali	1.30E-86
Allatostatin B/Myoinhibitory peptide	AstB	288	BAU88428.1	Plautia stali	9.40E-120
Allatostatin CC	AstCC	110	BAV78791.1	Plautia stali	3.30E-55
Allatostatin CCC	AstCCC	89	AZK31378.1	Nezara viridula	3.60E-30
Allatotropin	AT	97	XP_014274846.1	Halyomorpha halys	1.60E-34
Arginine-vasopressin-like peptide	AVLP	130	XP_014287933.1	Halyomorpha halys	2.80E-57
Bursicon alpha	Burα	147	AZC86174.1	Nezara viridula	8.30E-80
Bursicon beta	Burβ	132	XP_024214523.1	Halyomorpha halys	3.80E-63
Capability/Cardio acceleratory peptide 2b	CAPA	152	AYP97817.1	Halyomorpha halys	1.50E-64
Carausius neuropeptide-like precursor	CNP	440	XP_024214992.1	Halyomorpha halys	2.50E-171
CCHamide 1	CCHa1	147	XP_014293977.1	Halyomorpha halys	2.00E-91
CCHamide 2	CCHa2	144	AZK31334.1	Nezara viridula	1.90E-26
CNMamide	CNMa	115	BAV78799.1	Plautia stali	5.10E-43
Corazonin	Crz	93	XP_014274138.1	Halyomorpha halys	2.90E-27
Crustacean Cardioactive peptide	CCAP	126	AZK31338.1	Nezara viridula	2.00E-87
Diuretic hormone 31	DH31	118	XP_024214033.1	Halyomorpha halys	2.10E-44
Diuretic Hormone 44	DH44	119	BAV78801.1	Plautia stali	8.30E-31
Ecdysis triggering hormone	ETH	151	AZK31339.1	Nezara viridula	1.10E-70
Eclosion hormone 1	EH1	77	XP_024214295.1	Halyomorpha halys	4.40E-26
Eclosion hormone 2	EH2	71	BAV78806.1	Plautia stali	9.90E-25
Elevenin	Ele	93	BAV78807.1	Plautia stali	1.10E-29
FMRFamide	FMRFa	201	XP_024219218.1	Halyomorpha halys	1.60E-104
Glycoprotein hormone alpha 2	GPA2	122	BAV78809.1	Plautia stali	2.80E-65
Glycoprotein hormone beta 5	GPB5	142	BAV78810.1	Plautia stali	7.20E-69
IDLSRF-like	IDLSRF	120*	XP_014290093.2	Halyomorpha halys	7.2E-56
Insulin-like peptide 1	ILP1	125	AZK31343.1	Nezara viridula	2.80E-47
Insulin-like peptide 2	ILP2	270	XP_024216521.1	Halyomorpha halys	8.60E-119
Ion Transport peptide	ITP	126	XP_014274475.1	Halyomorpha halys	1.50E-67
ITG-like	ITG	210	XP_014275756.1	Halyomorpha halys	8.00E-119
Leucokinin	LK	477	BAV78814.1	Plautia stali	7.00E-248
Myosuppressin	MS	93	XP_024214034.1	Halyomorpha halys	9.20E-44
Natalisin	NTL	73	BAV78816.1	Plautia stali	4.60E-26
Neuroparsin A1	NPA1	91	BAV78817.1	Plautia stali	1.50E-34
Neuroparsin A2	NPA2	105	AZK31353.1	Nezara viridula	1.50E-42
Neuroparsin A3	NPA3	107	XP_014279505.1	Halyomorpha halys	1.50E-55
Neuroparsin A4	NPA4	107	XP_014279505.1	Halyomorpha halys	7.60E-34
Neuropeptide F	NPF	102	BAV78819.1	Plautia stali	1.60E-52
Neuropeptide-like precursor	NPLP	487	XP_014276590.1	Halyomorpha halys	4.40E-212
NVP-like	NVP	300	AZK31360.1	Nezara viridula	4.40E-166
Orcokinin A	OKA	113	XP_014280358.1	Halyomorpha halys	2.40E-49
Orcokinin B	OKB	560	XP_049862630.1	Schistocerca gregaria	3.00E-106
PaOGS36577	-	646	XP_014283619.2	Halyomorpha halys	0
Parathyroid hormone	PTH	109*	XP_014293861.1	Halyomorpha halys	1.10E-41
Pigment dispersing factor	PDF	82	AZK31364.1	Nezara viridula	1.40E-32
Proctolin	Pro	78	XP_014283232.1	Halyomorpha halys	2.00E-32
Prothoracicotropic hormone	PTTH	113	XP_014239433.1	Cimex lectularius	6.20E-12
Pyrokinin	PK	107	AYP97818.1	Halyomorpha halys	1.90E-48
RFLamide	RFLa	143	XP_014286502.1	Halyomorpha halys	5.30E-70
RYamide	RYa	111	XP_014276031.1	Halyomorpha halys	1.20E-54
Short Neuropeptide F	sNPF	70	BAV78827.1	Plautia stali	6.50E-29
SIFamide	SIFa	76	AZK31367.1	Nezara viridula	5.70E-35
Sulfakinins	SK	79	XP_014274494.1	Halyomorpha halys	9.70E-30
Tachykinins	TK	192	BAV78830.1	Plautia stali	1.80E-90
* not full length

A total of four A. custos NPA transcripts were found (Figs. 1 ~ 2). Besides, an expansion in the NPA gene family was also found in N. viridula, H. halys and L. hesperus, of 13, 12 and 4 numbers, respectively (Fig. 1), supporting the hypothesis that this could be a particularity of Pentatomidae and Miridae^17,18. The A. custos NPA precursors can encode mature peptides with a length of about 80 amino acid residues and 10 ~ 14 cystine residues that form 5 ~ 7 intrachain disulfide bridges (Fig. 2). Sequence alignment of NPA mature peptides showed that ArmcuNPA2 and ArmcuNPA3 are highly similar to their orthologues in H. halys, HalhaNPA2 and HalhaNPA3, respectively (with about 90% identity), while NPA1 and NPA4 share relative lower identities (no more than 70%) with their orthologues, respectively (Fig. 2).

The A. custos OKB precursor can encode 24 mature peptides including 16 distinct peptides characterized by the reported DXI/LGGG consensus sequence (Figs. 3 ~ 4). OKB precursors from Apolygus lucorum, N. viridula, H. halys and R. prolixus, contain 11, 9, 8 and 7 distinct mature peptides, respectively (Figs. 3 ~ 4). The ArmcuOKB precursor shares very low identity (no more than 30%) with their orthologues from four other bugs (Fig. 3). A. custos and H. halys only share one identical OKB sequence (NLDTIGGGHLV) (Figs. 4).

The A. custos PTTH precursor can encode a mature peptide with a length of 91 amino acid residues and seven cystine residues that form one interchain and three intrachain disulfide bridges (Fig. 5). PTTH is absent in a number of hemipteran species such as H. halys, N. viridula, and R. prolixus, but was found in some hemipterans such as L. hesperus, N. lugens and A. pisum. The A. custos PTTH precursor shares 22%~34% amino acid identity with those of four other hemipterans (Fig. 5).

Identification of Neuropeptide GPCRs in A. custos

Using homology research against our custom transcriptome data, a total of 41 potential neuropeptide GPCR genes were identified (Table 2, Supplementary Data 2), including 33 Family-A GPCRs (rhodopsin-like receptors) and eight Family-B GPCRs (secretin-like receptors). Phylogenetic analysis revealed that all A. custos neuropeptide GPCRs had high identities to their orthologs in A. lucorum and N. lugens, with the UF bootstrap values of more than 99% (Fig. 6).

Table 2

Neuropeptides GPCR genes identified from *A. custos*
GPCR name	Putative ligand	Gene Length	Predicted TMHs	Homology search against NCBI NR database (blastp)
GPCR name	Putative ligand	Gene Length	Predicted TMHs	NR ID	Species	E-value
Armcu_A1	AstC	2031	7	XP_014285192.1	Halyomorpha halys	4.20E-209
Armcu_A2	SIFa	4286	7	XP_024215098.1	Halyomorpha halys	5.30E-209
Armcu_A3	ETH	915	5	XP_024081485.1	Cimex lectularius	9.90E-73
Armcu_A4	sNPF	2250	7	XP_024214325.1	Halyomorpha halys	1.00E-208
Armcu_A5	Proc	1548	7	XP_014279581.1	Halyomorpha halys	5.20E-159
Armcu_A6	SK	362	2	XP_024083285.1	Cimex lectularius	2.20E-39
Armcu_A7	MIP	2022	7	XP_024218766.1	Halyomorpha halys	6.10E-200
Armcu_A8	Orphan	1235	7	XP_024217465.1	Halyomorpha halys	5.00E-205
Armcu_A9	MS	2387	7	XP_014285898.1	Halyomorpha halys	3.00E-190
Armcu_A10	CCHa	384	2	XP_014274580.1	Halyomorpha halys	9.20E-65
Armcu_A11-1	CNMa	456	1	XP_024082326.1	Cimex lectularius	8.20E-28
Armcu_A11-2	CNMa	415	1	XP_024218217.1	Halyomorpha halys	1.20E-17
Armcu_A12	RYa	502	3	XP_014255151.1	Cimex lectularius	2.90E-42
Armcu_A13	LK	1498	7	XP_014277091.1	Halyomorpha halys	6.30E-202
Armcu_A14	TK	471	1	XP_024215512.1	Halyomorpha halys	1.40E-70
Armcu_A15	CAPA	2564	4	XP_024217080.1	Halyomorpha halys	2.40E-185
Armcu_A16	CCAP	1491	7	XP_024216533.1	Halyomorpha halys	5.00E-199
Armcu_A17	CCAP	1858	7	XP_022190770.1	Nilaparvata lugens	5.40E-142
Armcu_A18	CCAP	1820	2	XP_014282181.1	Halyomorpha halys	1.20E-135
Armcu_A19	Crz	1904	7	XP_014282594.1	Halyomorpha halys	3.70E-215
Armcu_A20	AKH	2319	7	XP_014271665.1	Halyomorpha halys	2.40E-184
Armcu_A21	PK	1572	7	XP_014282792.1	Halyomorpha halys	5.80E-190
Armcu_A22	PK	1930	7	XP_014287370.1	Halyomorpha halys	1.10E-177
Armcu_A23	NPF	1583	7	XP_014287015.1	Halyomorpha halys	9.00E-199
Armcu_A24-1	NPF	1304	5	XP_014291375.2	Halyomorpha halys	5.80E-127
Armcu_A24-2	NPF	522	2	XP_024217008.1	Halyomorpha halys	3.10E-71
Armcu_A25	FMRFa	2722	7	XP_014284854.1	Halyomorpha halys	3.50E-182
Armcu_A26	Orphan	3423	7	XP_014285974.1	Halyomorpha halys	1.10E-190
Armcu_A27	Orphan	2110	7	XP_014287372.1	Halyomorpha halys	1.80E-149
Armcu_A28	Orphan	5567	7	XP_014291441.1	Halyomorpha halys	0.00E + 00
Armcu_A29	Orphan	507	3	XP_024218051.1	Halyomorpha halys	7.60E-75
Armcu_A30	GPA2/GPB5	3128	7	XP_014284245.1	Halyomorpha halys	1.60E-194
Armcu_A31	GPA2/GPB5	3442	7	XP_014284756.1	Halyomorpha halys	0.00E + 00
Armcu_A32	ILP	1654	7	XP_024214941.1	Halyomorpha halys	9.60E-244
Armcu_B1	DH31	2163	7	XP_024216452.1	Halyomorpha halys	5.90E-209
Armcu_B2	DH31	1447	7	XP_024216453.1	Halyomorpha halys	7.50E-208
Armcu_B3	PDF	1884	7	XP_024218284.1	Halyomorpha halys	3.20E-195
Armcu_B4	DH31	707	2	XP_024218724.1	Halyomorpha halys	7.80E-102
Armcu_B5	Orphan	1031	4	XP_024218723.1	Halyomorpha halys	6.20E-116
Armcu_B6	DH44	1538	7	XP_024218615.1	Halyomorpha halys	3.50E-216
Armcu_B7	DH44	3133	7	XP_024216524.1	Halyomorpha halys	2.70E-210
Armcu_B8	Orphan	3009	7	XP_024216828.1	Halyomorpha halys	2.50E-250
Armcu_B9	Orphan	4173	7	XP_014287795.1	Halyomorpha halys	1.40E-190

Family-A neuropeptide GPCRs of A. custos can be classed into 25 groups based on their putative ligands: receptors for AstC, SIFa, ETH, sNPF, Proc, SK, MIP, MS, CCHa, CNMa, RYa, LK, TK, CAPA, CCAP, Crz, AKH, PK, NPF, FMRFa, Ele, Bur, GPA2/GPB5, ILP and Orphan (Table 2). Duplications of five receptor genes, CCAP, PK, NPF, GPA2/GPB5 and ILP, were found in A. custos (Table 2). No orthologous gene encoding the receptors for AstA, AT, AVLP and trissin was identified in A. custos. Family-B neuropeptide GPCRs of A. custos can be subdivided into four groups: receptors for DH31, DH44, PDF and PTH. Duplications of DH31 and DH44 receptor genes were found in A. custos (Table 2).

Tissue-specific expression patterns of neuropeptide precursors in A. custos

A heatmap based on FPKM values of 52 neuropeptide genes in heads, antennae, legs, guts and salivary glands was shown in Fig. 7 and Supplementary Table S1. Expression values of five out of 57 neuropeptide genes were not found, because they were not identified from the unigene sequences of our transcriptome. Four genes (CCHa1, CCAP, OKB and Pro) were identified from the trinity sequences of our transcriptome and one gene (AKH) was identified from the public SRA database.

All of these 52 neuropeptide genes were found to be expressed in heads if a criterion of more than 1 FPKM in at least one repeat was adopted (Supplementary Table S1). 39 out of 52 neuropeptide genes showed FPKM values higher than ten in at least one repeat (Supplementary Table S1). The top ten most highly expressed neuropeptide genes in the heads of A. custos were NPLP, NPA3, NVP, ILP1, ETH, ITG, AstCCC, SIFa, NPA4 and PaOGS36577 (Fig. 7; Supplementary Table S1).

Expressions of 26 out of 52 neuropeptide genes were detected in antennae of A. custos, with the FPKM values of more than 1 in at least one repeat (Supplementary Table S1). ILP1, NVP and CNP presented FPKM values higher than ten in at least one repeat (Fig. 7; Table S1).

Thirteen neuropeptide genes were expressed in legs of A. custos (FPKM values > 1 in at least one repeat) (Supplementary Table S1). Only ILP1 and ITP presented FPKM values higher than ten in at least one repeat (Fig. 7; Table S1).

A total of 29 neuropeptide genes showed FPKM values higher than one in at least one gut library (Supplementary Table S1). The top ten most highly expressed neuropeptide genes in the guts of A. custos were DH31, CCHa2, ETH, MS, ILP1, NVP, AstCC, CNMa, DH44 and TK, with the FPKM values of more than 10 in at least one repeat (Fig. 7; Supplementary Table S1).

Only ten out of 52 neuropeptide genes were detected in the salivary glands of A. custos (DH31, ITG, OKA, ETH, TK, NVP, PTH, AstB, DH44, RYa; Table S1). DH31 was the most highly expressed neuropeptide gene in the salivary glands of A. custos, following by ITG that was the other neuropeptide gene with the FPKM values of more than 10 in at least one repeat (Fig. 7; Supplementary Table S1).

After comparing the expression levels of 52 neuropeptide genes among different tissues or parts of A. custos, we found that only one gene (DH31) was significantly less expressed in heads compared to other tissues/parts (salivary glands), with fold < 0.5 and P_adj < 0.05 (Fig. 7).

Tissue-specific expression patterns of neuropeptide GPCR genes in A. custos

A heatmap based on FPKM values of 43 neuropeptide GPCR genes in heads, antennae, legs, guts and salivary glands was shown in Fig. 8 and Supplementary Table S2. A total of 33 out of 41 neuropeptide GPCR genes were expressed in heads of A. custos, with the FPKM values higher than one in at least one repeat (Supplementary Table S2). Three top most highly expressed neuropeptide GPCR genes in heads of A. custos, Armcu_A5_Proc, Armcu_A1_AstC and Armcu_A18_CCAP, presented the FPKM values higher than 10 in at least one repeat (Fig. 8; Supplementary Table S2).

Nine out of 41 neuropeptide GPCR genes were expressed in antennae of A. custos (FPKM > 1 in at least one repeat) (Supplementary Table S2). Armcu_A16_CCAP was the top most highly expressed neuropeptide GPCR genes in antennae of A. custos, showing the FPKM value higher than 10 in one repeat (Fig. 8; Supplementary Table S2).

Only six neuropeptide GPCR genes (Armcu_A5_Proc, Armcu_B1_DH31, Armcu_A18_CCAP, Armcu_A27_Orphan, Armcu_A33_Orphan and Armcu_A20_AKH) were detected in legs of A. custos with the FPKM values higher than one in at least one repeat (Fig. 8; Supplementary Table S2).

Thirteen out of 41 neuropeptide GPCR genes were expressed in guts of A. custos (FPKM > 1 in at least one repeat), among which three receptor genes (Armcu_A15_CAPA, Armcu_A32_ILP and Armcu_A29_GPA2/GPB5) presented the FPKM value higher than 10 in one repeat (Fig. 8; Supplementary Table S2).

Only six neuropeptide GPCR genes (Armcu_B7_DH44, Armcu_A32_ILP, Armcu_A33_Orphan, Armcu_A11-1_CNMa, Armcu_B1_DH31, and Armcu_A26_Ele) were detected in the salivary glands of A. custos (FPKM > 1 in at least one repeat) (Fig. 8; Supplementary Table S2).

Comparison of the expression levels of 41 neuropeptide GPCR genes among different tissues or parts of A. custos showed that the majority (31/41) of them were enriched in heads (Fig. 8). Based on a criterion of Log2 (fold) > 1 and P_adj < 0.05 for significant difference, two GPCRs (Armcu_A16_CCAP and Armcu_A25_FMRFa) were higher expressed in antennae compared to heads; while Armcu_A15_CAPA, Armcu_A30_GPA2/GPB5 and Armcu_A32_ILP were up-regulated in the guts than heads. Armcu_A29_GPA2/GPB5 was up-regulated in the guts and salivary glands than heads (Fig. 8).

In the present study, a total of 57 neuropeptide precursor transcripts and 41 potential neuropeptide GPCR receptor transcripts were identified, with the similar numbers to those found in other bugs such as R. prolixus, N. viridula, H. halys and A. lucorum^15,18,19. Compared with some other neuropeptide signaling systems, A. custos lacked both the trissin precursor and its receptor. Trissin and its receptor have not been identified in other hemipteran species to date^12,18. AST-C was not found in the A. custos transcriptome, however, AST-CC and AST-CCC were identified in A. custos, which is consistent with many insect species which lost AST-C²⁰. No orthologous genes encoding the receptors for AstA, AT and AVLP were identified in A. custos. It is difficult to determine the absence of a gene, especially for the species without genome data.

Comparative analysis of gene sets of neuropeptide signaling system among different species could provide useful information for developing target-specific insecticidal agents. For example, insect kinin was reported to be a potential lead for green pesticides, with high efficiencies against aphids but being safety to the beneficial insect Chrysoperla carnea, based on the hypothesis of kinin lost in C. caernea²¹. In the present study, all kinds of neuropeptide signal system identified in other Hemiptera species were also found in the predatory bug A. custos. Most of A. custos neuropeptides and their GPCR receptors had high identities to their orthologs in other bugs. Thereafter, it seems hard to discover any useful information for developing target-specific insecticidal agents. However, there were still a few neuropeptides in A. custos have low similarities with homologous sequences in other bugs, such as NPAs, OKB and PTTH. Whether these three neuropeptide signal classes could be potential targets for the development of high selective insecticidal agents need to be further determined.

Insect NPA displays a high diversity in the precursor gene numbers, as well as in mature peptide sequences. In most insect genomes sequenced to date, only one NPA gene exists¹⁸. The expansion of NPA genes was found in Pentatomidae species and a red bug C. lectularius¹⁸. Recent studies reported an inverse correlation in the numbers of ILP and NPA paralogues in a given genome^18,22. Our results that A. custos have four NPA genes and two ILP genes supported this observation. NPA was initially isolated from the pars intercerebralis-corpora cardiac complex of the locust Locusta migratoria²³ and was determined to be involved in reproduction and metamorphosis in arthropods²⁴. Both NPA and ILP were revealed to act on similar tyrosine kinase receptors and exhibit similar physiological roles in some insects, such as to stimulate the ovary to produce ecdysteroids and rigger vitellogenin synthesis^25,26, which suggests a complementation between these two hormonal systems²². Insect ILPs play a crucial role in the regulation of metabolism, growth and development, fecundity, stress resistance, and lifespan²⁷. Nevertheless, the function of the NPA in insects is less clear, as its absence from Drosophila melanogaster²⁸. The roles of the expanded NPA paralogues in Pentatomidae, however, remain to be further determined.

Insect orcokinin displays a high diversity in numbers and sequences of mature peptides. In most insects, two types of OK precursors, OKA and OKB, were identified, each encoding more than one mature peptides. OKA mature peptides are highly conserved in A. custos and other Pentatomidae species, whereas OKB of Pentatomidae species are variable in numbers and sequences of mature peptides. OK precursor genes have been identified in all insect genomes sequenced to date; however, the OK receptor in insects awaits deorphanized²⁹. OKs were first characterized as myotropic peptides in the crustacean Orconectes limosus³⁰. Insect OKs were proved to have a variety of effects, e.g. regulation of pigmentation³¹, ecdysis³², vitellogenin transcription³³ and circadian rhythmicity^34,35. Knockdown of orcokinin gene of D. melanogaster caused a disinhibition of male courtship behavior, however, it is unclear whether OKA or OKB is involved in the control of the courtship³⁶. Knowledge of the physiological role of OKB in insects is still limited³⁶.

PTTH was one of the earliest hormones discovered in insects³⁷. PTTH is absent in a number of hemipteran species such as H. halys, N. viridula, and R. prolixus, but was found in some hemipterans such as L. hesperus, N. lugens, A. pisum and A. custos. The sequences of the hemipteran PTTH genes are much less conserved, but all have seven Cys residues that forms a homodimer linked by an interchain disulfide bond and a cystine knot motif in each chain composed of three intrachain disulfied bridges. The primary function of PTTH is to promote biosynthesis of ecdysone in the prothoracic glands³⁷. PTTH and its receptor are absent in all fully-sequenced social bee genomes, supporting the hypothesis of the association with the evolution of sociality in bees³⁸. Whether insect PTTH could be a potential target for developing high selective insecticidal agents is a very worthy study.

Tissue-specific expression profiles of neuropeptides and their GPCRs in A. custos provide basic information for in-depth investigation of the biology and ecology of this important biological control species. All of neuropeptide precursor genes and a majority of their GPCR receptor genes of A. custos were found to be predominately expressed in heads, indicating that most of neuropeptide signal systems could mainly act as neuroregulators in the predatory bugs. A few neuropeptide signaling genes that were also expressed in antennae may play roles in odor perception and zoophytophagous feeding habit. The present study also presented a set of gut-enriched and salivary gland-expressed neuropeptide signal genes, which may have critical roles in the regulation of feeding and digestion.

The expression of neuropeptide genes and their receptor genes in antennae has already been reported in a few insect species^39–42. In R. prolixus, AstCC, AstCCC, ITG, IDLSRF, and OK, and receptors for CCAP-R1, NPF-R1, ITP-R, GPA2/GPB5-R, RFa-R were highly expressed in antennae⁴². In the present study, ILP1, NVP and CNP, and a receptor of CCAP (A16_CCAP) were highly expressed in the antennae of A. custos. Our results added an evidence to support that neuropeptide signaling systems potentially involved in the regulation of antennal sensory function in A. custos.

Neuropeptides in insect guts take part in the regulation of feeding processes including food choice, appetite, digestion, metabolism and excretion⁴³. DH31, CCHa-1, CCHa-2 and NPF are well-known neuropeptides associated with the gut-brain axis, regulating appetite, sleep and even courtship^44–47. As listed in flyAtlas expression database, a few other neuropeptide signal genes were also expressed in Drosophila guts, including AstC, Bura, ILP3, ITP, MS, Limostatin, NPLP2 and TK, and receptors for AstA, CAPA, CCHa-1, DHs, GPA2/GPB5, ILPs, LK, NPF, NPLP1, RYa, and TK²⁹. In the present study, DH31, CCHa2, ETH, MS, ILP1, NVP, AstCC, CNMa, DH44 and TK, and three receptors for CAPA, ILP and GPA2/GPB5 were highly expressed in the guts of A. custos.

Insect salivary glands may also have endocrine roles. Currently, the endocrine function of insect salivary glands is poorly understood and has been only demonstrated in a few studies^48–50. In flyAtlas database, ITP, ILP6, NPLP2, NPLP4, and receptors for Crz, GPA2/GPB5, ILPs and NPLP1 were expressed in Drosophila salivary glands²⁹. DH31, CCAP, CCAPR, AstB, Pro and NPLP1 were expressed in the salivary glands of R. prolixus⁵¹. FMRFa, AstA, SIFa and TK have also been detected in the salivary glands of other insects^48,52,53. Receptors for PDF, AstA, DH44, TK, sNPF and DH31 were expressed at higher levels in the salivary glands of the ectoparasitoid, Habrobracon hebetor⁵³. The present study demonstrated that DH31 was highly expressed in the salivary glands of A. custos whereas a few neuropeptide signal genes were also expressed in the salivary glands of A. custos. The physiological roles of DH31 were well studied in D. melanogaster and other insects, including stimulating secretion of Malpighian tubules, controlling the switch between feeding and courtship behavior, and positively regulating memory formation and sleep^46,54−56. However, there is no report about the function of DH31 in insect salivary glands.

Insect motor neurons are under peptidergic modulation⁴. However, the modulation function of neuropeptides in insect legs is poorly understood and has been only demonstrated in the stick insect C. morosus⁵⁷. Receptors for CCAP, DH31, CCHa-2, ILPs, AstB, Pro, sNPF, TK were detected in the leg muscle transcriptome of C. morosus and AstB was demonstrated to be the only neurally-mediated peptide modulator of stick insect leg muscle⁵⁷. In A. custos leg transcriptome, a few neuropeptide signaling genes such as ILP1, ITP and receptors for Proc, DH31, CCAP, and AKH were highly expressed, however, AstB and its receptor were quite low expressed in A. custos legs.

Predatory stink bugs are zoophytophagous generalists of various insect pests in a wide of plant habitats. This might be the reason why A. custos harbors almost all of neuropeptides identified in other hemipteran species. The predatory bug relies on a complex neuropeptide signal system to modulate various physiology and behavior to adapt to different environment, to catch diverse preys, to utilize food resources effectively, and to cope with all kinds of intracompetition and intercompetition. The present study displayed a set of neuropeptides and their receptors that were enriched in heads, guts, salivary glands, antennae and legs of A. custos, which provides basic information for in-depth study of biology and ecology of the predatory bug and for further research of the development of novel “green” pest control strategies.

Identification of neuropeptides and their GPCRs

The tBLASTn researches were performed to predict the genes of neuropeptide precursors and their GPCR receptors from our custom A. custos transcriptome (BioProject No. PRJNA878928, heads, guts, salivary glands, antennae and Legs from fifth instar nymphs, RNA-seq)³. Amino acid sequences of known neuropeptide precursors from H. halys¹⁸, N. lugens¹³ and R. prolixus¹⁴, and neuropeptide GPCRs from N. viridula¹⁸, A. lucorum¹⁹, R. prolixus¹⁴, and N. lugens¹³ were used as reference queries. In addition, sequences of some potential novel neuropeptide precursors, agatoxin-like peptide (ALP), Carausius neuropeptide-like precursor (CNP), parathyroid hormone (PTH), PaOGS36577, RFLamide (RFLa), and PTH receptors from Periplaneta americana⁵⁸ or Tribolium castaneum⁵⁹ were used for the identification of these novel neuropeptide signaling genes in A. custos. What’s more, the remaining sequences that are absent in our custom A. custos transcriptome database, were identified using a public Sequence Read Archive (SRA) database (SRR10098905) via the NCBI tBLASTn program.

Structural and domain analyses

The structures of mature peptides of A. custos were predicted through the determination of signal peptides using SignalP 5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/) and prohormone cleavage sites based on the rules presented by Veenstra⁶⁰. Structural information for all putative neuropeptide GPCRs was predicted by TMHMM (v2.0) (https://services.healthtech.dtu.dk/services/TMHMM-2.0/).

Sequence alignment and phylogenetic analysis

Multiple alignments of amino acid sequences were performed using Clustal X 2.0⁶¹ and the results were visualized using GeneDoc software⁶². Sequence logos of manually aligned homologous neuropeptide sequences were generated using the online tool WebLogo (http://weblogo.berkeley.edu/logo.cgi)⁶³.

Phylogenetic analysis by Maximum likelihood (ML) was carried out using IQ-TREE⁶⁴, with an ultrafast (UF) bootstrap test with 3000 replicates and default settings for reduction of overestimation of bootstrap support (-bnni)⁶⁵. ModelFinder⁶⁶ was used to choose the best-fit tree model using Bayesian Information Criterion. The phylogenetic trees were visualized by the online tool Interactive Tree Of Life (iTOL) (https://itol.embl.de/)⁶⁷.

Differential expression analysis

Gene expression levels for guts (G), salivary glands (SG), antennae (A), legs (L), and heads without antennae and salivary glands (H) of A. custos 5th-instar nymphs were obtained by the FPKM (fragments per kb per million fragments) method from our custom A. custos transcriptome. Each of the FPKM values was transformed into log10 (FPKM + 1) value, and the expression profiling of the putative genes was generated and visualized by heatmaps. Differentially expressed genes (DEGs) between different tissue samples were defined based on a criterion of |Log2 (fold)| >1 and the adjust P value (P_adj) < 0.05.

Acknowledgments

This research was funded by the grants from Key Project of Guizhou Provincial Science and Technology Foundation (ZK[2023]023) and Guizhou Provincial Tobacco Company Zunyi Branch (Science and Technology Major Project No. 2021XM01).

Author information

Authors and Affiliations

Guizhou Provincial Tobacco Company Zunyi Branch, Zunyi 563000, China

Chunyang Huang, Zhimo Li & Xiang Yang

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Jingmiao Zou, Mingwei Yang, Xinyi Wang & Yueping He

Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China;

Wenhong Li

Contributions

C.H., W.L. and Y.H. wrote the project. Y.H. and C.H. designed and performed the experiments, analyzed the data and wrote the first draft of the manuscript. W.L. supervised the research and contributed to the drafting of the manuscript. J.Z., M.Y. and X.W. contributed to perform the experiments and analyzing the data. Z.L. contributed in designing the experiments and data analysis. X.Y. and C.H. contributed to perform bioinformatics analyses. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Wenhong Li or Yueping He

Ethics declarations

Competing interests

The authors declare no competing interests.

Data availability

Further inquiries on our data can be directed to the corresponding authors (W.L., Y.H.).

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No competing interests reported.

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Identification and expression analysis of neuropeptides and their G protein-coupled receptors in the predatory stink bug, Arma custos (Hemiptera: Pentatomidae)

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