Identification and comparative analysis of G protein-coupled receptors in Pediculus humanus humanus

Genomics 104 (2014) 58–67

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Identification and comparative analysis of G protein-coupled receptors in Pediculus humanus humanus Chengjun Li, Xiaowen Song, Xuhong Chen, Xing Liu, Ming Sang, Wei Wu, Xiaopei Yun, Xingxing Hu, Bin Li ⁎ Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China

a r t i c l e

i n f o

Article history: Received 13 March 2014 Accepted 9 June 2014 Available online 19 June 2014 Keywords: GPCR Biogenic amine Neuropeptide Sulfakinin Corazonin Pediculus humanus humanus

a b s t r a c t The body louse has the smallest genome size among the known genome-sequenced insects. Here, 81 GPCRs were identified in Pediculus humanus humanus, 56, 14, 6 and 5 GPCRs for family-A, -B, -C and -D, respectively. These GPCRs constitute the comparable repertoire of GPCRs with other insects. Moreover, it contains a more complete set of neuropeptide and protein hormone receptors not even than Acyrthosiphon pisum but also Drosophila melanogaster, for example, Sulfakinin, Corazonin, Trissin and PTHRL only presented in P. h. humanus but lost either in A. pisum or D. melanogaster. However, it has less duplication among the sub-families. Meanwhile, ACP, AVPL, HE6 receptors and Boss were also absent from P. h. humanus. These results indicated that the repertoire of GPCRs in P. h. humanus were not affected by its smallest genome size, and further suggested that P. h. humanus has a relatively original and concise GPCR regulation system. © 2014 Elsevier Inc. All rights reserved.

1. Introduction G protein-coupled receptors (GPCRs) play vital roles in the growth, development, lifespan and reproduction of organisms. They usually function as signal transducers, transforming extracellular signals into intracellular signals by activating second messengers in the cells, which leads to a series of cascading reactions [1]. Because of their crucial roles in the regulation of multiple physiological processes, more than 50% of pharmaceutical drugs target these molecules [2,3]. Based on sequence similarities and phylogenetic relationships, they were classified into four families, the rhodopsin family (A), the secretin-receptor family (B), the metabotropic glutamate receptor family (C) and the atypical 7TM protein family (D) [4]. Recent studies indicated that GPCRs are the potential targets of insecticides [5–7]. As an obligatory parasite of humans, the body louse (Pediculus humanus humanus) is an important vector for human diseases, including epidemic typhus, relapsing fever and trench fever. Compared with other genome-sequenced insects, P. h. humanus has the smallest genome size, which spans ~108 Mb and codes for 10,773 genes as well as 57 microRNAs [8]. However, there is no detailed information about the GPCRs in this insect. Additionally, the genome of another hemimetabolous insect, Acyrthosiphon pisum, has been

⁎ Corresponding author. Fax: +86 25 85891763. E-mail addresses: [email protected] (C. Li), [email protected] (X. Song), [email protected] (X. Chen), [email protected] (X. Liu), [email protected] (M. Sang), [email protected] (W. Wu), [email protected] (X. Yun), [email protected] (X. Hu), [email protected] (B. Li).

http://dx.doi.org/10.1016/j.ygeno.2014.06.002 0888-7543/© 2014 Elsevier Inc. All rights reserved.

published in 2010, which is ~464 Mb in size and includes 34,604 gene predictions [9]. Of these genes, 82 genes were identified to code for GPCRs in A. pisum [10]. In contrast, the genome size and predicted gene quantity of A. pisum are much larger than that of P. h. humanus; thus, whether the quantity and diversity of GPCRs were significantly decreased in P. h. humanus due to its smallest genome and how they evolved in this hemimetabolous insect must be addressed. Therefore, we performed genome-wide identification of GPCRs in P. h. humanus. In addition, we carried out comparative genomic analysis of GPCRs of P. h. humanus with the model insects Drosophila melanogaster and A. pisum. 2. Results Using the GPCRs of D. melanogaster and A. pisum as reference sequences, we have identified 81 putative GPCRs from the P. h. humanus genome. These GPCRs were classified into four families, including 56 family-A members, 14 family-B members, 6 family-C members and 5 family-D members (Table 1). 2.1. Family-A GPCRs Insect family A GPCRs include opsins, biogenic amine receptors and neuropeptide and protein hormone receptors. In this study, 56 familyA GPCRs were identified in the genome of P. h. humanus, and these receptors were composed of 3 opsins, 19 biogenic amine receptors and 34 neuropeptide and protein hormone receptors (Table 2).

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Table 1 The number of P. h. humanus GPCRs of each family in comparison with the other five insects.

Family-A Opsin Biogenic amine receptors Neuropeptide and protein hormone receptors Family-B Family-C Family-D Total

P. h. humanus

pisum

melanogaster

T. castaneum

A. gambiae

B. mori

56 3 19 34 14 6 5 81

62 5 18 39 9 6 5 82

74 7 21 46 24 8 6 112

68 2 20 46 20 9 6 103

71 12 18 41 14 8 5 98

69 6 16 47 11 9 4 93

Data for D. melanogaster are cited from [4], data for A. gambiae are from [17,20], data for B. mori are from [13,17], data for T. castaneum are from [17,22], and data for A. pisum are from [10].

2.1.1. Opsins Three groups of opsins have been reported in the insect. One of these groups is related to long-wavelength absorbing invertebrate visual pigments; another group is related to the short-wavelength absorbing visual pigments; and the third is significantly different from the previous two groups. In this study, 3 putative opsins were found from P. h. humanus (Table 2). PHUM009850 shares 52% and 50% sequence identity with CG9668 and CG10888; PHUM310950 has 62% sequence identity with CG5192; BK008815 shows 44% sequence similarity with CG5638 (Table 2). In the phylogenetic tree, PHUM009850 is close to the first group; PHUM310950 is related to the second group; PHUM596710 is orthologous to CG5638 (Figs. 1 and S1). Together, these results provide evidence that these genes encode the opsins in P. h. humanus. 2.1.2. Biogenic amine receptors At present, only five biogenic amines were reported in the insect, containing octopamine, dopamine, tyramine, serotonin and acetylcholine. However, these amines exert their actions through approximately 20 receptors in the insect. Here, we identified 19 biogenic amine receptors in P. h. humanus for the first time (Table 2). The majority of these receptors show one-to-one orthologous relationships with the counterparts of D. melanogaster and A. pisum, providing direct evidence for us to determine which type of GPCRs they belong to. Based on phylogenetic analysis and sequence similarity (Figs. 2 and S2, Table 2), PHUM497230, PHUM549550, PHUM027040 and PHUM457080 are the octopamine-like receptors; PHUM515970, PHUM434420, PHUM286710 and PHUM581080 are the orthologues of the dopamine receptors; PHUM434300 is the specific receptor for tyramine; PHUM559700, PHUM204430 and PHUM220540 were confirmed to be serotonin-like receptors; PHUM238080 is the GPCR that could be stimulated by two structurally related endogenous ligands, octopamine and tyramine; PHUM412420 and PHUM486980 are both receptors for acetylcholine. Additionally, there are 4 orphan receptors (BK008816, PHUM440560, PHUM042150 and BK008817) of this subfamily in P. h. humanus (Figs. 2 and S2, Table 2). 2.1.3. Neuropeptide and protein hormone receptors Different from biogenic amines, there are 23 types of neuropeptides and protein hormones that serve as the ligands of GPCRs in insects; at the same time, there were slightly more neuropeptide receptors (ranging from 39 to 47 members) that have been reported in insects (Table 1) [4,11–13]. In P. h. humanus, 34 genes were identified as the GPCRs for the neuropeptides and protein hormones in this study (Table 2). Of these GPCRs, 26 receptors were shown to have clear orthologues that have deorphanized in D. melanogaster; 13 of them exhibited one-toone orthologous relationships between A. pisum, D. melanogaster and P. h. humanus, including the receptors for tachykinin, ecdysis triggering hormone (ETH), Capa, Proctolin, FMRFamide, sex peptide, short neuropeptide F, neuropeptide F, crustacean cardioactive peptide (CCAP), adipokinetic hormone (AKH), Bursicon and glycoprotein hormone alpha2 (GPA2)/glycoprotein hormone beta5 (GPB5). Unlike

D. melanogaster and A. pisum, there is only one receptor for RYamide, Kinin, Sulfakinin, SIFamide, Allotostatins, CCHamide and GPA2/GPB5 identified from P. h. humanus. The remaining 8 GPCRs (PHUM507060, PHUM538530, BK008818, PHUM337980, PHUM084240, PHUM060140, PHUM007790 and PHUM038260) are orphan receptors without known ligands (Figs. 3 and S3, Table 2). Similar to D. melanogaster and A. pisum, the arginine vasopressin-like peptide (AVPL) and AKH/Corazoninrelated peptide (ACP) receptors were not found in the genome of P. h. humanus. 2.2. Family-B GPCRs In total, 14 family-B GPCRs were identified from the genome of P. h. humanus in this study, which consist of 5 subfamilyB1 members, 1 subfamilyB2 member and 8 subfamilyB3 members (Table 3). Insect subfamilyB1 has four types of GPCRs, including corticotrophinreleasing-factor-related diuretic hormone (CRF-DH) receptor, calcitonin receptor, pigment-dispersing factor (PDF) receptor and parathyroid hormone receptor-like (PTHRL) [14]. In D. melanogaster, two CRF-DH receptors (CG8422 and CG12370) were identified [15,16], while only one CRF-DH receptor (ACYPI54924) was found from A. pisum [17]. Additionally in P. h. humanus, only one receptor (PHUM132710) was confirmed to be the CRF-DH receptor, which not only has an average of 46% sequence identity with the CRF-DH receptors of D. melanogaster and A. pisum but is also evolutionarily close to them. Similar to D. melanogaster, only one calcitonin receptor (PHUM428070) was identified for sequence and phylogenetic analysis. Different from these two receptors, the gene coding for the PDF receptor has one copy in each insect. On average, PHUM127410 shows a 42% sequence identity with the PDF receptors of D. melanogaster and A. pisum, and they constitute a monophyletic group in the phylogenetic tree (Figs. 4 and S4, Table 3), which indicates that PHUM127410 is the PDF receptor of P. h. humanus. Interestingly, PHUM233900 has no orthologues in D. melanogaster and A. pisum but shares an average of 37% sequence identity with two parathyroid hormone receptor-like (PTHRLs) of Tribolium castaneum (Fig. S5) and shows close relationship with pthrl of T. castaneum in the phylogenetic tree (Fig. S6), which strongly supports that PHUM233900 is a putative PTHRL in P. h. humanus. PHUM015970 is also a subfamilyB1 GPCR for its close evolutionary relationship with calcitonin receptors (Figs. 4 and S4). Latrophilin-like and human epididymis6 (HE6) receptor-like are two members of subfamilyB2 in the insect. Investigators have found latrophilin-like in all insects with a sequenced genome. We have identified a Latrophilin-like (PHUM461700) in P. h. humanus that has an approximately 50% sequence identity with Latrophilin-like (CG8639) of D. melanogaster and A. pisum (Table 3) and forms a monophyletic cluster with them (Figs. 4 and S4). However, HE6 receptor-like was absent from the genome of P. h. humanus, which is also observed in A. pisum. There is only one group of receptors in subfamilyB3, i.e., Methuselah (Mth)/Methuselah-like (Mthl). Similar to A. pisum [17], the orthologue of mth (CG6936) has not been found from P. h. humanus, but 8 Mthls are identified in this organism (Table 3). Phylogenetic analysis demonstrated that PHUM063410 and PHUM420330 are two mthl genes, which

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Table 2 Identification of family-A GPCRs in P. h. humanus. P. h. humanus receptor accession no.

CG no. of the D. melanogaster orthologue

Endogenous ligand for the D. melanogaster deorphanized receptor

Gene location

Protein region identified

Opsins PHUM009850 PHUM310950 BK008815

CG9668, CG10888 CG5192 CG5638

Orphan Orphan Orphan

DS234992 DS235313 DS235879

Complete Complete Complete

6 6 3

Biogenic amine receptors PHUM497230 PHUM549550 PHUM027040 PHUM457080 PHUM515970 PHUM434300 PHUM559700 PHUM204430 PHUM238080

CG6989 CG42244 CG6919 CG3856 CG18741 CG7431, CG16766 CG15113, CG16720 CG12073 CG7485

DS235830 DS235858 DS235004 DS235797 DS235845 DS235767 DS235861 DS235165 DS235219

Complete Complete Complete Complete Complete Complete Complete Complete Complete

1 1 3 5 1 11 3 3 1

PHUM434420 BK008816 PHUM286710 PHUM220540 PHUM440560 PHUM042150 PHUM412420 PHUM486980 BK008817 PHUM581080

CG9652 CG18208 CG33517 CG1056 CG42796 CG12796 CG7918 CG4356 CG13579 CG18314

Octopamine Octopamine Octopamine Octopamine Dopamine Tyramine Serotonin Serotonin Tyramine, octopamine Dopamine Orphan Dopamine Serotonin Orphan Orphan Acetylcholine Acetylcholine Orphan Ecdysteroids, domamine

DS235767 DS235806 DS235271 DS235184 DS235776 DS235012 DS235745 DS235822 DS235322 DS235873

TM2–TM7 Complete TM4–TM7 TM3–TM7 TM2–TM4 Complete Complete Complete TM2–TM7 Complete

5 2 7 3 2 2 6 7 4 4

RYamide Kinin Tachykinin Tachykinin Sulfakinin SIFamide ETH Capa Pyrokinin-1 Pyrokinin-1 na Proctolin Orphan FMRFamide Sex peptide Myosuppressin Myosuppressin Allatostatin-C Allatostatin-A Trissin CCHamide sNPF NPF CCAP Corazonin AKH Orphan Orphan na Bursicon GPA2/GPB5 Orphan Orphan Orphan

DS235639 DS235155 DS235088 DS235856 DS235886 DS235150 DS235797 DS235243 DS235780 DS235088 DS235842 DS235250 DS235854 DS235283 DS235073 DS235308 DS235023 DS235068 DS235346 DS235263 DS235873 DS235806 DS235004 DS235111 DS235306 DS235389 DS235271 DS235341 DS235051 DS235030 DS235787 DS234991 DS235028 DS235006

TM2–TM7 TM3–TM7 TM2–TM7 Complete Complete Complete Complete Complete TM2–TM7 Complete TM6–TM7 Complete TM1–TM6 Complete Complete Complete Complete TM2–TM7 Complete TM6–TM7 Complete Complete Complete Complete TM2–TM7 Complete Complete TM1–TM5 Complete Complete Complete TM2–TM7 Complete Complete

2 6 5 4 5 5 8 4 2 2 7 6 1 1 2 4 1 0 4 1 2 1 1 8 2 4 7 12 14 16 6 1 4 7

Neuropeptide and protein hormone receptors PHUM407550 CG5811 PHUM196020 CG10626 PHUM129610 CG6515 PHUM545910 CG7887 PHUM615310 CG42301, CG32540 PHUM189320 CG10823 PHUM456700 CG5911 PHUM262680 CG14575 PHUM446190 CG9918 PHUM128380 CG9918 PHUM507060 na PHUM268560 CG6986 PHUM538530 CG16726 PHUM296990 CG2114 PHUM110750 CG16752 PHUM309270 CG43745, CG8985 PHUM054050 CG43745, CG8985 PHUM101310 CG13702, CG7285 PHUM343230 CG10001, CG2872 PHUM278660 CG34381 PHUM579960 CG14593, CG30106 PHUM460790 CG7395 PHUM025830 CG1147 PHUM157220 CG33344 PHUM307450 CG10698 PHUM370810 CG11325 BK008818 CG4322 PHUM337980 CG31096 PHUM084240 na PHUM061060 CG8930 PHUM452330 CG7665 PHUM007790 na PHUM060140 CG7497 PHUM038260 CG4332

No. of introns of the P. h. humanus gene coding region

Sequence identity between P. h. humanus and the closest D. melanogaster/A. pisum receptor 52%, 50%/48%, 48% 62%/66% 44%/51%, 49%

65%/64% 75%/76% 63%/64% 42%/46% 61%/57% 69%, 60%/60% 60%, 57%/57% 64%/62% 57%/46% 70%/62% 58%/73% 58%/40% 77%/60% 65%/69% 37%/na 82%/57% 49%/51% 67%/53% 61%/52%

49%/55%, 47% 52%/66%, 67% 64%/57% 60%/63% 49%, 45%/na 60%/61% 54%/57% 50%/66% 44%/53%, 50% 46%/51%, 54% na/na 42%/49% 46%/46% 52%/62% 63%/77% 45%, 43%/42%, 46% 46%, 49%/39%, 49% 56%, 53%/62% 44%, 49%/54% 69%/na 46%, 46%/53%, 54% 50%/53% 45%/30% 63%/54% 48%/na 54%/60% 52%/50%, 51% 31%/34% na/na 58%/59% 48%/34%, 50% na/na 40%/na 45%/45%

na, not annotated.

correspond to mthl1 (CG4521) and mthl5 (CG6965) of D. melanogaster, respectively; PHUM300610, PHUM317230, PHUM300710, PHUM274680, PHUM163700 and PHUM514090 are also mthl genes, and these genes have no counterparts in D. melanogaster and A. pisum (Figs. 4 and S4).

2.3. Family-C GPCRs There are two types of GPCRs in family C, the glutamate and γ-amino butyric acid (GABA-B) receptors and the metabotropic glutamate (mGlu) receptor. Until now, 8 family C GPCRs from D. melanogaster

C. Li et al. / Genomics 104 (2014) 58–67

100 97 94

100

100

100 100 94 93

99 92 100

CG9668 CG10888 ACYPI002544 ACYPI004442 PHUM009850 CG5279 CG4550 CG16740 CG5192 ACYPI009332 PHUM310950 CG5638 BK008815 ACYPI005074 ACYPI001006 CG2114

61 68 100

I

100 93

99

97 100 74 100

II

70 100 99

III

100 75 98 98 100

0.05

70 98

60

Fig. 1. Phylogenetic tree of opsins produced with the neighbour-joining method. The D. melanogaster GPCRs are named by their CG number. The P. h. humanus GPCRs are highlighted in red, and A. pisum GPCRs are highlighted in green. The scale bar indicates the p-distance. The tree was rooted by the D. melanogaster FMRFamide receptor CG2114.

100

70 100

98 95 100

77 66

and 6 from A. pisum have been reported. By using these reference sequences, 6 family-C members of P. h. humanus were identified here (Table 3). Similar to A. pisum, there are two GABA-B receptors (PHUM155390 and PHUM479480) in P. h. humanus. PHUM155390 has a 61% sequence identity with CG6706, while PHUM479480 shares a 74% sequence similarity with CG15274 (Table 3). In the phylogenetic tree, PHUM155390 is close to CG6706; additionally, PHUM479480 is orthologous to CG15274 (Figs. 5A and S7), which strongly suggests that they are both the GABA-B like receptors. Four mGlu receptors of P. h. humanus were distributed into four clusters. Among them, PHUM384840 and its orthologues (CG31660 and ACYPI40549) constitute a cluster; next to it, PHUM496910 forms a monophyletic cluster with CG31760 and ACYPI006068; PHUM170240 and PHUM216190 with their corresponding orthologues are distributed into another two clusters, which are also next to each other (Figs. 5A and S7).

100

99

100 100 72

80

65

100 99 100

61

90 100 85 100

100 89 99 100

2.4. Family-D GPCRs 96

Family-D GPCRs, also known as atypical 7TM proteins, include the Frizzled-like receptor, Smoothened, starry night (Stan) and bride of sevenless (Boss). In the genome of P. h. humanus, 5 candidate GPCRs of family-D were reported in this study (Table 3). Three Frizzled-like receptors (CG17697, CG9739 and CG4626) were reported in D. melanogaster [4], and an equal number of Frizzledlike receptors (PHUM188130, PHUM024690 and PHUM440660) were also identified from P. h. humanus (Figs. 5B and S8, Table 3). Phylogenetic analysis revealed that PHUM188130 corresponds to CG9739; PHUM440660 is the orthologue of CG4626; PHUM024690 independently locates in the position between these two clusters (Figs. 5B and S8). In addition, PHUM188130 and PHUM440660 also share a more than 40% sequence identity with their orthologues, respectively (Table 3). The GPCR PHUM190020 shares a 47% and 42% sequence identity with the Smoothened D. melanogaster and A. pisum, respectively (Table 3). Additionally, it is located in one cluster of the phylogenetic tree with the smoothened of other two insects (Figs. 5B and S8), indicating that PHUM190020 is the gene coding for Smoothened in P. h.

100 99

CG6989 (Octopamine) PHUM497230 ACYPI004658 ACYPI010025 CG42244 (Octopamine) PHUM549550 CG6919 (Octopamine) ACYPI007386 PHUM027040 PHUM457080 ACYPI005578 CG3856 (Octopamine) ACYPI009241 PHUM515970 CG18741 (Dopamine) CG16766 PHUM434300 CG7431 (Tyramine) ACYPI004336 CG15113 (Serotonin) CG16720 (Serotonin) XP_001949725 PHUM559700 PHUM204430 XP_003241835 CG12073 (Serotonin) ACYPI007379 PHUM238080 CG7485 (Octopamine/Tyramine) PHUM434420 CG9652 (Dopamine) ACYPI006935 ACYPI010155 BK008816 CG18208 PHUM286710 CG33517 (Dopamine) ACYPI007415 PHUM220540 CG1056 (Serotonin) ACYPI008969 ACYPI50707 PHUM440560 CG42796 PHUM042150 CG12796 PHUM412420 CG7918 (Acetylcholine） ACYPI001255 ACYPI005180 PHUM486980 CG4356 (Acetylcholine) BK008817 CG13579 ACYPI008777 ACYPI005538 PHUM581080 CG18314 (Ecdysteroids, Dopamine)

CG2114 0.1

Fig. 2. Phylogenetic tree of biogenic amine receptors produced by the neighbour-joining method. The D. melanogaster GPCRs are named by their CG number. The P. h. humanus GPCRs are highlighted in red, and A. pisum GPCRs are highlighted in green. The scale bar indicates the p-distance. The tree was rooted by the D. melanogaster FMRFamide receptor CG2114.

humanus. Another family-D member of P. h. humanus (PHUM040650) constitutes a cluster with stan of D. melanogaster and A. pisum in the phylogenetic tree (Figs. 5B and S8). It has a 52% and 49% amino acid sequence identity with them, respectively (Table 3). The information supports that PHUM040650 is a stan-like gene. Different from

62

C. Li et al. / Genomics 104 (2014) 58–67

A

99 100 100

63

100 77

90

92 99 100 100 100 99

62 100 99

100

71

100 94 96 100 100

100

80 62

100

100

99

100 92

81

74 100 65 80 100 79

100 100

100 100 79 100

73 70 100

PHUM407550 CG5811 (RYamide) ACYPI000981 ACYPI002886 CG10626 (Kinin) ACYPI010083 PHUM196020 ACYPI000762 PHUM129610 CG6515 (Tachykinin) ACYPI001103 CG7887 (Tachykinin) PHUM545910 ACYPI002917 PHUM615310 CG42301 (Sulfakinin) CG32540 (Sulfakinin) ACYPI44480 100 ACYPI000675 ACYPI065077 ACYPI008341 PHUM189320 CG10823 (SIFamide) CG5911 (ETH) ACYPI009758 PHUM456700 PHUM262680 ACYPI007245 CG14575 (Capa) CG8784 (Pyrokinin-2) CG8795 (Pyrokinin-2) PHUM446190 ACYPI000735 CG9918 (Pyrokinin-1) PHUM128380 ACYPI005805 PHUM507060 PHUM268560 ACYPI30716 CG6986 (Proctolin) CG16726 PHUM538530 ACYPI008027 PHUM296990 ACYPI006053 CG2114 (FMRFamide) CG5936 PHUM110750 ACYPI003290 CG16752 (sex peptide) CG13229 ACYPI41803 PHUM309270 PHUM054050 ACYPI002393 CG43745 (Myosuppressin) CG8985 (Myosuppressin) CG13995

B 94

87 100 99 61 100 91

100

81 100

94 100 99 87

100 82 100 100

99 88

100 98 100 83 100

97

74 100 85 100 6485

75

100

100

100 100 85

100

100 100

70 100 88

CG13702 (Allatostatins-C) CG7285 (Allatostatins-C) ACYPI002528 PHUM101310 CG10001 (Allatostatins-A) CG2872 (Allatostatins-A) ACYPI008623 PHUM343230 PHUM278660 CG34381 (Trissin) CG14593 (CCHamide) CG30106 (CCHamide) ACYPI004781 PHUM579960 ACYPI004920 ACYPI007659 ACYPI40167 CG30340 CG7395 (short neuropeptide F) ACYPI005474 PHUM460790 ACYPI007664 PHUM025830 CG1147 (neuropeptide F) PHUM157220 CG33344 (CCAP) ACYPI062442 PHUM307450 CG10698 (Corazonin) CG11325 (AKH) PHUM370810 ACYPI002471 CG3171 ACYPI005234 ACYPI006293 BK008818 CG4322 CG12610 ACYPI000671 CG13575 PHUM337980 CG31096 ACYPI008291 CG34411 PHUM084240 PHUM061060 ACYPI000221 CG8930 (Bursicon) ACYPI004597 CG7665 (GPA2/GPB5) PHUM452330 ACYPI060229 ACYPI138121 CG9753 ACYPI24713 PHUM060140 CG7497 CG12290 PHUM007790 ACYPI000822 PHUM038260 CG4332

CG11144

CG11144 0.1

96 100

0.1

Fig. 3. Phylogenetic tree of neuropeptide and protein hormone receptors (A and B) produced with the neighbour-joining method. The D. melanogaster GPCRs are named by the CG number. The P. h. humanus GPCRs are highlighted in red, and A. pisum GPCRs are highlighted in green. The scale bar indicates the p-distance. The tree was rooted by the D. melanogaster metabotropic glutamate receptor CG11144.

D. melanogaster and A. pisum, Boss was not found from the genome of P. h. humanus.

3.1. P. h. humanus has a comparable repertoire of GPCRs that are identified in any sequenced insect genome to date

3. Discussion

The fly D. melanogaster is the first insect to have a sequenced genome, which is ~180 Mb in size, coding for 13,600 genes [18]. Of these genes, 112 genes have been reported to encode GPCRs (Table 1) [4,11, 12]. Another dipteran insect, Anopheles gambiae, has a genome of ~ 278 Mb, including 14,000 protein-encoding transcripts [19], and 98 GPCRs have been identified from its genome (Table 1) [20]. There are

In this study, 81 GPCRs have been identified from the genome of P. h. humanus. The GPCRs were compared and discussed from the quantity and diversity between P. h. humanus and other well-studied insects with GPCR annotations.

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Table 3 Identification of family-B, -C and -D GPCRs in P. h. humanus. P. h. humanus receptor accession no.

CG no. of the D. melanogaster orthologue

Endogenous ligand for the D. melanogaster deorphanized receptor

Gene location

Protein region identified

No. of introns in of the P. h. humanus gene coding region

Sequence identity between P. h. humanus and the closest D. melanogaster/A. pisum receptor

Family-B PHUM132710 PHUM233900 PHUM015970 PHUM428070 PHUM127410 PHUM461700 PHUM300610 PHUM063410 PHUM317230 PHUM300710 PHUM274680 PHUM163700 PHUM514090 PHUM420330

CG8422, CG12370 na na CG32843 CG13758 CG8639 na CG4521 na na na na na CG6965

DH44 na na DH31 PDF Orphan Orphan Orphan Orphan Orphan Orphan Orphan Orphan Orphan

DS235090 DS235207 DS234994 DS235760 DS235088 DS235809 DS235289 DS235032 DS235321 DS235289 DS235255 DS235123 DS235845 DS235751

Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete

8 8 9 10 9 14 1 4 9 8 5 5 6 5

44%,47%/47% na/na na/na 52%/43%, 57% 48%/36% 49%/51% na/na 36%/41% na/na na/na na/na na/na na/na 45%/43%

Family-C PHUM155390 PHUM479480 PHUM170240 PHUM216190 PHUM394840 PHUM496910

CG6706 CG15274 CG30361 CG32447 CG31660 CG31760

Orphan Orphan Orphan Orphan Orphan Orphan

DS235111 DS235819 DS235129 DS235172 DS235459 DS235829

Complete Complete Complete Complete TM2–TM7 Complete

14 13 8 1 4 11

61%/58% 74%/69% 73%/68% 48%/44% 51%/46% 53%/53%

Family-D PHUM188130 PHUM024690 PHUM440660 PHUM190020 PHUM040650

CG9739 na CG4626 CG11561 CG11895

Orphan na Orphan Orphan Orphan

DS235149 DS235002 DS235777 DS235150 DS235008

Complete Complete Complete Complete Complete

1 2 1 10 46

57%/57% na/na 40%/na 47%/42% 52%/49%

na, not annotated.

93 genes that code for GPCRs from the ~432 Mb genome size of Bombyx mori [13,17,21]. Thus far, a total of 103 GPCRs have been reported from the ~160 Mb genome sequence of T. castaneum (Table 1) [12,17,22]. In 2010, when the genome of the first hemimetabolous insect A. pisum was sequenced [9], we mined out 82 genes coding for GPCRs from its ~ 464 Mb genome sequence (Table 1) [10]. The genome of P. h. humanus is ~ 108 Mb in size and is the smallest genome size among genome-sequenced insects [8]. However, 81 GPCRs were found from its genome here (Tables 1–3). The number of GPCRs showed no significant difference with the other insects (Table 1). Of course, some GPCRs have not been found in P. h. humanus, including the ACP receptor, AVPL receptor, HE6 receptor-like and Boss. However, the ACP receptor was also absent from D. melanogaster, Apis mellifera and A. pisum [23]; the AVPL receptor was absent from the genomes of D. melanogaster, A. gambiae, B. mori, A. mellifera and A. pisum [10,12,24]; HE6 receptorlike was reported to be lost from A. mellifera, Nasonia vitripennis, Rhodnius prolixus and A. pisum [17]; Boss was abandoned in B. mori, A. mellifera, N. vitripennis and R. prolixus [10]. Therefore, P. h. humanus has a comparable repertoire of GPCRs identified from any sequenced genome even though its genome is smaller than any of these insects. 3.2. P. h. humanus possesses an original and concise GPCR regulation system Of 23 neuropeptides and protein hormones that have served as the ligands of GPCRs in known genome sequenced insects, 20 types of neuropeptides and protein hormones were detected from the genome of P. h. humanus [8], while there are 21, 19 and 16 types of neuropeptides and protein hormones that have been reported from D. melanogaster, T. castaneum and A. pisum to date [11,25–29], respectively. On the other hand, there are only 34 neuropeptide and protein hormone receptors identified from P. h. humanus (Tables 1 and 2), whereas there are 46, 41, 47, 46 and 39 receptors for neuropeptides and protein hormones in D. melanogaster, A. gambiae, B. mori, T. castaneum and

A. pisum, respectively (Table 1). Thus, the majority of neuropeptide and protein hormone/receptor pairs exist in P. h. humanus. This finding indicates that P. h. humanus possesses a relatively complete set of neuropeptides and protein hormones but has a slightly smaller set of their receptors than other insects (Table 1). The divergence of receptors in the quantities found among the insects is likely caused by the speciesspecific duplication of some neuropeptide and protein hormone receptors. For example, the receptors for Sulfakinin, Allotostatins and CCHamide were reported to be duplicated in D. melanogaster [4,11], whereas the RYamide, Kinin, SIFamide, CCHamide and GPA2/GPB5 receptors are duplicated in A. pisum (Figs. 3 and S3) [10]. Additionally, the duplications of these receptors were also found in T. castaneum and B. mori [12,13]. In contrast, only single copies of these receptors were identified from P. h. humanus (Fig. 3), which results in the slightly smaller number of neuropeptides and protein hormone receptors for this insect. Furthermore, the ACP and AVPL receptors were not present in P. h. humanus, but they were also absent in other insects. These results suggest that the basic sub-family of GPCRs does not decrease in P. h. humanus. Moreover, P. h. humanus is well-known for an obligatory parasite of humans, and they live in a relatively stable environment that did not have exposure to such environmental stresses as other insects; thus, these insects have less gene duplication to adapt to multiple and subtle regulation. Additionally, the gene families that are associated with environmental sensing and responses were also notably less duplicated in P. h. humanus [8]. In addition, P. h. humanus locates the basal position among the insects in the phylogentic tree. Therefore, P. h. humanus kept the relatively original and concise GPCR regulation system. 3.3. P. h. humanus has a more complete set of neuropeptide and protein hormone receptors than A. pisum This study revealed that nearly all of the GPCRs of D. melanogaster were identified from the genome of P. h. humanus; for example, the

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PHUM132710 ACYPI54924 CG8422 (DH44) CG12370 (DH44) PHUM233900 PHUM015970 CG4395 ACYPI009569 ACYPI001361 CG32843 (DH31) PHUM428070 ACYPI007222 ACYPI46431 CG13758 (PDF) PHUM127410 PHUM461700 CG8639 ACYPI005705 CG15556 CG11318 PHUM300610 PHUM063410 ACYPI008388 CG4521 PHUM317230 PHUM300710 ACYPI000729 CG31720 PHUM274680 PHUM163700 PHUM514090 CG32476 ACYPI003439 PHUM420330 CG6965 CG32475 CG17084 CG17061 CG32853 CG7476 CG16992 CG30018 CG31147 CG6530 CG6536 CG17795 CG6936 (Sun)

subfamilyB1

subfamilyB2

subfamilyB3

CG1056 0.1

Fig. 4. Phylogenetic tree of family-B GPCRs produced with the neighbour-joining method. D. melanogaster GPCRs are named by their CG number. The P. h. humanus GPCRs are highlighted in red, and A. pisum GPCRs are highlighted in green. The scale bar indicates the p-distance. The tree was rooted by the D. melanogaster serotonin receptor CG1056.

receptors for Sulfakinin, Trissin and Corazonin and PTHRL existed in P. h. humanus, but they were lost in A. pisum [10], while PTHRL was also lost in D. melanogaster [17]. Removal of sulfakinin stimulated food intake in the Mediterranean field cricket Gryllus bimaculatus [30]. Conversely, injection of Sulfakinin and analogues of Sulfakinin-related peptides reduced food intake in Schistocerca gregaria [31] and T. castaneum [32], respectively. Knockdown of both sulfakinin and its receptor stimulated food intake in the larvae of T. castaneum; in parallel, injection with a Sulfakinin analogue reduced the food intake [33]. As is known, P. h. humanus depends on sucking human blood to survive. The occurrence of Sulfakinin [8] and its receptor in P. h. humanus likely involved this special parasitic lifestyle. As shown in Fig. 3B, CCAP, Corazonin and AKH receptors originated from one ancestor in the insect. However, Corazonin and its receptor were confirmed to be lost in A. pisum [10,25]. One of A. pisum CCAP and AKH receptors likely overtook the role that Corazonin receptor has in other insects. This situation is different in P. h. humanus. All of CCAP, Corazonin and AKH receptors were identified in P. h. humanus (Fig. 3B, Table 2) [8]. As the basal insect, P. h. humanus represents a

relatively original being after the ancestor of CCAP, Corazonin and AKH receptors were duplicated in the insect. In mammals, PTHRs were shown to be involved in bone growth and development [34,35] and calcium and phosphate balance [34]. Recently, PTHRL was reported in the insect for the first time: two copies in T. castaneum, one copy in A. mellifera [17], one from Tetranychus urticae and two from Nilaparvata lugens [36]. They were assumed to be associated with water balance due to their close relationship with the CRF-DH receptor and calcitonin receptor in the phylogenetic tree [17]. When P. h. humanus sucked human blood, a mass of calcium and phosphate was also introduced into the body of P. h. humanus along with the blood, which resulted in temporary excessive accumulation of them in vivo. The PTHRL in P. h. humanus is likely to sustain calcium and phosphate balance by regulating water metabolism. However, the Trissin receptor was newly identified from D. melanogaster [27], and the function of the Trissin/receptor couple is unclear; thus, it is not discussed here. P. h. humanus has a repertoire of GPCRs that is comparable to those identified in any genome-sequenced insect to date. However, it has less duplication of sub-family members of neuropeptide and protein hormone receptors but not sub-families in comparison with other insects. At the same time, P. h. humanus contains a relatively complete set of neuropeptide and protein hormone receptors, which constitutes more even than A. pisum and also more than D. melanogaster; for example, Sulfakinin, Corazonin, Trissin and PTHRL only existed in P. h. humanus but were lost in either A. pisum or D. melanogaster. These results suggest that P. h. humanus has a relatively original and concise GPCR regulation system. 3.4. Family-B is divergent among insects due to the loss or duplication of its member As described in Table 1, three families (A, C and D) of GPCRs show no significant differences in the quantity among six insects. But the number of family-B GPCRs is significantly different among insects, ranged from 9 to 24 (Table 1). Within family-B, subfamily-B1 is more conserved than subfamilies-B2 and -B3 in the insect [17], and thus it is inferred that loss or duplication of GPCRs in these two subfamilies mainly attributed to the diversity of family-B. As expected, HE6 receptor-like was also lost from P. h. humanus and A. pisum; only 1 HE6 receptor-like was separately identified in A. gambiae and B. mori by using 2 Drosophila members [4]; HE6 receptor-like occurred species-specific duplications and produced 6 copies in T. castaneum [17]. Furthermore, mthl was found to be duplicated in the insect to a different degree, and generated 3 copies in A. pisum, 4 both in A. mellifera and B. mori, and 5 in T. castaneum, 7 in A. gambiae, 8 in P. h. humanus and 16 in D. melanogaster [4,13,17,20,22]. Therefore, family-B of GPCRs is divergent in the insect due to the loss or duplication events. 4. Materials and methods 4.1. Identification of P. h. humanus GPCRs Using D. melanogaster and A. pisum GPCRs as references, TBLASTN and BLASTP searches were performed for GPCR candidates in the genome database of P. h. humanus (https://www.vectorbase.org/index. php). The full sequences of candidate proteins were identified with the Invitrogen Vector NTI Advance 9.0 package (InforMax). Data from the National Center for Biotechnology Information (NCBI, http://www. ncbi.nlm.nih.gov/) was also used as supplementary data. These candidates were confirmed by reciprocal BLASTP against non-redundant protein sequences from NCBI without species limits and with a cut-off e-value of e − 5 [37]. Furthermore, the sequence identity of the whole protein sequence calculated by BLASTP, 7TM domains, and phylogenetic analysis methods were also adopted as criteria for this study [12].

C. Li et al. / Genomics 104 (2014) 58–67

A

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PHUM155390 ACYPI003265 CG6706 CG15274 PHUM479480 ACYPI003729 CG3022 PHUM394840 CG31660 ACYPI40549 ACYPI006068 PHUM496910 CG31760 CG11144 ACYPI001079 PHUM170240 CG30361 ACYPI008512 CG32447 PHUM216190 CG6919

65

B CG17697 ACYPI007768 CG9739

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GABA-B receptor

100 78

100 92

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Frizzled

PHUM440660 CG4626 ACYPI002697 PHUM190020 Smoothened CG11561 CG8285 Boss ACYPI002001 ACYPI001529

100

100 100

mGlu receptor

ACYPI003976 PHUM188130 PHUM024690

100 74 100 84

PHUM040650 CG11895

Stan

CG6919 0.1

0.1

Fig. 5. Phylogenetic tree of family-C (A) and -D (B) GPCRs produced with the neighbour-joining method. D. melanogaster GPCRs are named by their CG number. The P. h. humanus GPCRs are highlighted in red, and A. pisum GPCRs are highlighted in green. The scale bar indicates the p-distance. The tree was rooted by the D. melanogaster Octopamine receptor CG6919.

Pre-phylogenetic analysis was used to remove non-GPCRs from candidate pools that were identified in previous steps. 4.2. Structural analyses and gene locations of the GPCRs Structural information for all of the candidate GPCRs was predicted with the server TMHMM (v2.0) from the Centre for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/TMHMM/) and the SMART programme (http://smart.embl-heidelberg.de/). In addition, gene locations within the genome were identified by running TBLASTN on the genome of P. h. humanus using the protein sequences. 4.3. Phylogenetic analysis By using TMHMM (v2.0) and SMART programme (http://smart. embl-heidelberg.de/), the 7TM domain sequences of these GPCRs were adopted, and then the sequences were aligned with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). Phylogeny tests were accomplished using the bootstrap method with 1000 replications to reconstruct the neighbour-joining trees (Figs. 1–5) by the programme MEGA5. Pairwise deletion of gaps/missing data was employed, and uniform rates among sites and similar patterns among lineages were selected for the neighbour-joining (NJ) trees. Additionally, maximum likelihood (ML) trees (Figs. S1–S4 and S7–S8) using the bootstrap method with 1000 replications, a Poisson model, a gamma distribution among sites and partial deletion were reconstructed to examine the topology of the NJ trees. The topologies of the two phylogenetic trees produced by the NJ and ML methods were similar. Therefore, the NJ trees are represented in the main text (Figs. 1–5), and the ML trees are provided in the supplementary material (Figs. S1–S4, S7 and S8). Bootstrap values below 60% are not shown (Figs. 1–5, S1–S4 and S7–S8). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ygeno.2014.06.002. Abbreviations GPCRs G protein-coupled receptors 7TM 7 transmembrane ETH ecdysis triggering hormone CCAP crustacean cardioactive peptide AVPL arginine vasopressin-like peptide

AKH ACP GPA2 GPB5 PDF CRF PTHRL HE6 Mth Mthl GABA Stan Boss

adipokinetic hormone AKH/Corazonin-related peptide glycoprotein hormone alpha2 glycoprotein hormone beta5 pigment-dispersing factor corticotrophin-releasing factor parathyroid hormone receptor-like human epididymis6 Methuselah Methuselah-like glutamate and gamma-amino butyric acid starry night bride of sevenless

Competing interests The authors declare that they have no competing interests.

Authors' contributions The majority of the work here described was finished by Chengjun Li. This work was also assisted by Xiaowen Song, Xuhong Chen, Xing Liu, Ming Sang, Wei Wu, Xiaopei Yun and Xingxing Hu. Bin Li designed the study and crucially revised the manuscript for important intellectual content and data analysis. All authors have read and approved the final manuscript.

Acknowledgments This work was supported by the National Natural Science Foundation of China (no. 31172146), the Key Project of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (no. 10KJA180023), the Natural Science Foundation of Jiangsu Province, China (BK2011785), the PAPD of Jiangsu Higher Education Institutions, the Excellent Talent Project of Nanjing Normal University of China, and the Graduate Innovation Research Projects of Jiangsu Colleges and Universities (CXZZ11_0884 and CXZZ13_0412).

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