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Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR α subunits from Pardosa pseudoannulata
Neuroscience Letters 584 (2015) 123–128
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Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR ␣ subunits from Pardosa pseudoannulata Xiangkun Meng 1 , Yixi Zhang 1 , Beina Guo, Huahua Sun, Chuanjun Liu, Zewen Liu ∗ Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
h i g h l i g h t s • • • •
A novel nAChR subunit Pp␣8 was cloned from Pardosa pseudoannulata. Key amino acid residue differences were found between Pp␣8 and Pp␣1. Some different residues contributed to neonicotinoid sensitivity directly. Some residues influence sensitivity by enhancing direct effects from other residues.
a r t i c l e
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Article history: Received 21 July 2014 Received in revised form 26 September 2014 Accepted 9 October 2014 Available online 22 October 2014 Keywords: Pardosa pseudoannulata Nicotinic acetylcholine receptor ␣ subunit Amino acid differences Neonicotinoid sensitivity
a b s t r a c t Chemical insecticides are still primary methods to control rice planthoppers in China, which not only cause environmental pollution, insecticide residue and insecticide resistance, but also have negative effects on natural enemies, such as Pardosa pseudoannulata (the pond wolf spider), an important predatory enemy of rice planthoppers. Neonicotinoids insecticides, such as imidacloprid and thiacloprid, are insectselective nAChRs agonists that are used extensively in the areas of crop protection and animal health, but have hypotoxicity to P. pseudoannulata. In the present study, two nAChR ␣ subunits, Pp␣1 or Pp␣8, were found to be successfully expressed with r2 in Xenopus oocytes, but with much different sensitivity to imidacloprid and thiacloprid on two recombinant receptors Pp␣1/r2 and Pp␣8/r2. Key amino acid differences were found in and between the important loops for ligand binding. In order to well understand the relationship between the amino acid differences and neonicotinoid sensitivities, different segments in Pp␣8 or Pp␣1 with key amino acid differences were introduced into the corresponding regions of Pp␣1 or Pp␣8 to construct chimeras and then co-expressed with r2 subunit in Xenopus oocytes. The results from chimeras of both Pp␣8 and Pp␣1 showed that segments 5, 6, and 7 contributed to neonicotinoid sensitivities directly between two receptors. Although the segment 4 including all loop B region had no direct influences on neonicotinoid sensitivities, it could more remarkably influence neonicotinoid sensitivities when co-introductions with 5, 6 or 7. So, key amino acid differences in these four segments were important to neonicotinoid sensitivities, but the difference in 4 was likely ignored because of its indirect effects. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Rice is one of the most important food crops in the world. In many countries, rice is suffering from many insect pests, especially
∗ Corresponding author at: Tongwei Road 6, Nanjing 210095, China. Tel.: +86 25 84399051; fax: +86 25 84399051. E-mail address: [email protected] (Z. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.10.013 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
the brown planthopper, Nilaparvata lugens (Stål), a major rice pest in Asia. China has the second largest area of the rice growing and the highest rice yield [1]. The primary method to control rice insect pests is still the application of chemical insecticides, which not only causes environmental pollution but also reduces natural enemies’ populations. Spiders are recognized as important natural enemies to reduce pest populations in rice fields, particularly in early rice season when specialized predators are still not present. Being one of the most abundant and species-rich groups of natural enemies occurring in all agroecosystems, spiders are
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variably affected by pesticide applications, such as the pond wolf spider Pardosa pseudoannulata, an important predatory enemy of rice planthoppers and aphids [2]. Spiders are mainly affected by acaricides and insecticides, especially neurotoxic substances. However, the ecotoxicology of spiders are rarely studied, as only 3% of toxicology papers on natural enemies have been devoted to spiders [2]. Neonicotinoids are used extensively to control rice pests and show the relative safety to P. pseudoannulata [3]. Neonicotinoids act as neurotoxins selectively on insect nicotinic acetylcholine receptors (nAChRs), the ligand-gated ion channels mediating fast cholinergic synaptic transmission in invertebrate and vertebrate nervous systems [4]. The nAChRs agonist-binding site is present at the interface of adjacent subunits and is formed by loops A–C in ␣ subunits together with loops D–F in either non␣ (, ␥ or ␦) subunits or homomer-forming ␣ subunits [5,6]. In previous studies, different amino acids in insect nAChRs loop B of ␣ subunit or loop D, E, F of  subunit were found to contribute to imidacloprid selectivities [7–9]. Studies also showed that the G275E mutation, predicted to lie at the top of the third-helical transmembrane domain of ␣6 subunit, resulted in the high resistance to spinosad in the western flower thrips, Frankliniella occidentalis [10]. In contrast, there are only few studies about P. pseudoannulata nAChRs and neonicotinoids selectively between natural enemies and insect pests. The pharmacological studies of P. pseudoannulata nAChRs are essential to understand the insecticide selectively and the development of selective insecticides [9]. In the present study, a novel nAChR ␣ subunit (Pp␣8) was cloned from P. pseudoannulata. When successful co-expressed Pp␣8 or Pp␣1 (one ␣ subunit previously cloned) with rat r2 in Xenopus oocytes, the receptor Pp␣8/r2 was found with much lower neonicotinoid sensitivity than Pp␣1/r2. By comparison of these two ␣ subunits, key amino acid differences were found in and between the important loops for ligand binding. The effects of these amino acid differences on neonicotinoid sensitivities were examined by constructing different subunit chimeras and co-expression with r2 in Xenopus oocytes.
Table 1 Primers for clone Pp␣8. Primer name
Sequences of primers
Sense degenerate primer for Pp␣8 Anti-sense degenerate primer for Pp␣8 5 -outer GSP for Pp␣8 5 -inner GSP for Pp␣8 3 -outer GSP for Pp␣8 3 -inner GSP for Pp␣8
5 -AARTGGAAYCCNGAYGAYTAYGGNGG-3 5 -TADATNGCNGGNGGYTTCCANACNACYTT-3 5 -CAGTGTCACTTCGTAGTTGCCATCC-3 5 -GCATTGTTGTAGAGTACGATGTCAGG-3 5 -CAGAGCCTCCGAAGTTCACTGTCAG-3 5 -GGGGCTGAAGATGTCGGTCTGGAAT-3
pGH19 as described previously [11]. In the Pp␣1, the fragments of 68–79th (1), 87–93th (2), 120–128th (3), 152–166th (4), 170–181th (5), 199–206th (6), 208–214th (7) residues were replaced by that Pp␣8 to construct the chimeras Pp␣11−7 , as previously described [8]. Similarly, 5, 6 and 7 fragments of Pp␣8 were replaced by the corresponding sequences of Pp␣1, singly or together, to construct several Pp␣8 chimeras. All plasmid and chimera constructs were verified by nucleotide sequencing. 2.5. In vitro transcription, oocyte preparation and electrophysiology The methods of vitro transcription, oocyte preparation and electrophysiology were detailed described previously [8,9]. 3. Results 3.1. Isolation of Pp˛8 cDNA
P. pseudoannulata was collected from a field of hybrid paddy rice in Nanjing (Jiangsu, China) in August 2008 and reared in door by feeding with N. lugens. Spiders were kept indoors at 25 (±1) ◦ C, humidity 70–80% and 16/8 h light/dark.
A novel nAChRs ␣ subunit was cloned from P. pseudoannulata using RT-PCR with degenerate oligonucleotide primers and RACE techniques with gene-specific primers (Table 1). The fulllength cDNA (Genbank accession number, HM017861) has an open reading frame of 1527 bp and 509 deduced amino acids, and the deduced protein had the common features of insect nAChRs ␣ subunit (Fig. 1), such as three loops important to agonist binding and four transmembrane regions. The subunit showed high similarities to insect nAChRs ␣8 subunits, such as 73.0% to Nilaparvata lugens and 72.0% to Apis mellifera, and was denoted as Pp␣8. Pp␣8 also showed high similarity (70%) to Pp␣1 (HM017860), a subunit previously cloned in our laboratory. However, several key amino acid differences were found in and between the important loops (A, B and C) for ligand binding between Pp␣1 and Pp␣8 (Fig. 1). The region with amino acid differences was divided into seven segments (1–7). The segments in Pp␣8 were introduced into the corresponding regions of Pp␣1 singly or combinedly to construct several Pp␣1 chimeras, and some segments in Pp␣1 were also introduced into Pp␣8 to construct Pp␣8 chimeras.
2.3. Amplification of cDNA
3.2. Functional expression of Pp˛1 and Pp˛8 with rat ˇ2
The full length cDNA of P. pseudoannulata nAChRs ␣8 cDNA was obtained through techniques of polymerase chain reaction (PCR) with degenerate primers and rapid-amplification of cDNA ends (RACE) with gene-specific primers (Table 1), as described previously [9].
When co-expression with the vertebrate rat neuronal 2 (r2) subunit in heterologous expression system Xenopus oocytes, both receptor of Pp␣1/r2 and Pp␣8/r2 could be induced large inward currents by different agonists (1 mM) (Fig. 2A). Further dose–response experiments were performed for acetylcholine (ACh) and some neonicotinoid insecticides. There was small but still significant difference in ACh sensitivity between Pp␣1/r2 and Pp␣8/r2, reflecting in EC50 difference, although there was no difference in Imax values between two receptors (Fig. 2B). In contrast, there were big differences in both Imax and EC50 values for tested neonicotinoids between two receptors, for example, the imidacloprid EC50 of Pp␣8/r2 was 8.9 times of that of Pp␣1/r2 (Fig. 2C).
2. Materials and methods 2.1. Chemicals Acetylcholine and neonicotinoids (imidacloprid, acetamiprid, thiacloprid, clothianidin, dinotefuran) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Collection and rearing of spider
2.4. Plasmids and mutagenesis P. pseudoannulata nAChRs ␣ subunit Pp␣1 (GenBank accession number, HM017860), Pp␣8 (Genbank accession number, HM017861) and Rattus norvegicus  subunit r2 (GenBank accession number, L31622) were subcloned into the expression vector
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Fig. 1. Alignment of Pardosa pseudoannulata Pp␣1, Pp␣8 and ␣8 subunits from some insect species. Loops A, B and C, important to agonist binding site in nAChR ␣ subunit, are marked by dot lines. Tansmembrane domains (TM1–4) are marked by double lines. Important amino acid differences within two ␣ subunits are marked by single lines and ranged into several segments (1–7) for subunit chimera construction of Pp␣1 or Pp␣8. Pardosa pseudoannulata, Pp␣1 (HM017860) and Pp␣8 (HM017861); Apis mellifera, Am␣8 (NP001011575); Bombyx mori, Bm␣8 (ABV72690); Nilaparvata lugens, Nl␣8 (ACK75719).
In addition to imidacloprid, four other neonicotinoid compounds were also examined on two receptors. The significantly bigger Imax and EC50 values were found for all neonicotinoid compounds examined in Pp␣8/r2 than in Pp␣1/r2 (Fig. 2D), although the differences of dinotefuran were much less than that of other neonicotinoid insecticides. In Imax values, there was not significant difference for dinotefuran between two receptors, but the values for other neonicotinoid insecticides on Pp␣8/r2 were 1.5 times of that on Pp␣1/r2 at least. Although EC50 value of dinotefuran on Pp␣8/r2 was only 2.2 times of that on Pp␣1/r2, the ratios for other insecticides were above 5.0 (5.7–10.5). Table 2 gives a summary of Imax and EC50 data obtained in two receptors. 3.3. Influence of amino acid differences between Pp˛1 and Pp˛8 on neonicotinoid sensitivity In order to find out the roles of key amino acid differences between two ␣ subunits in neonicotinoid sensitivities between receptors Pp␣1/r2 and Pp␣8/r2, different segments in Pp␣8 or
Pp␣1 were introduced into the corresponding regions of Pp␣1 or Pp␣8 to construct artificial subunit chimeras and then co-expressed with r2 in heterologous expression system Xenopus oocytes. In voltage-clamp electrophysiological studies, receptors with Pp␣1 chimeras co-expressed with r2 in Xenopus oocytes, large inward currents were detected in response to 1 mM imidacloprid and thiacloprid. When different segments in Pp␣8 were introduced into the corresponding regions of Pp␣1 singly, there were no obvious changes in Imax or EC50 for 1, 2, 3 and 4 when compared to Pp␣1 (Table 3). In contrast, significant changes in Imax or/and EC50 values were found for the introduction of 5 (EC50 only), 6 and 7, especially the segments 6 and 7 (Fig. 3A–C). Although the introduction of 4 did not cause a significant change in EC50 values for both imidacloprid and thiacloprid, such segment introduction could further reduced neonicotinoid sensitivities when combined with other segment introduction, such as 5, 6, 7 or 67 (Table 3 and Fig. 3). In particular, the Imax and EC50 values for both imidacloprid and thiacloprid on Pp␣1467 /r2 were very close to that on Pp␣8/r2, which indicated that key amino acid differences in these three segments (4, 6 and 7) between two ␣
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Fig. 2. Representative whole-cell responses and agonist dose–response curves from hybrid nAChRs expressed in Xenopus oocytes, containing Pp␣1/r2 or Pp␣8/r2. (A) Representative responses elicited by acetylcholine and imidacloprid. (B) Dose–response curves for acetylcholine. (C) Dose–response curves for imidacloprid. (D) Ratios of Imax and EC50 between Pp␣1/r2 and Pp␣8/r2. Data are means of at least three independent experiments ± SEM. Table 2 Imax and EC50 values of acetylcholine and neonicotinoids on nAChRs containing Pp␣1/r2 or Pp␣8/r2 expressed in Xenopus oocytes. Agonist
Pp␣1/r2
Pp␣8/r2
Imax (nA) Acetylcholine Imidacloprid Acetamiprid Thiacloprid Clothianidin Dinotefuran
273.59 156.65 131.29 126.54 109.80 123.23
± ± ± ± ± ±
EC50 (M) 31.82 16.88 18.43 13.71 13.66 9.82
34.65 70.09 92.75 57.36 104.24 82.39
± ± ± ± ± ±
Imax (nA) 6.67 8.50 11.24 8.33 13.17 7.68
281.76 239.54 222.27 196.85 187.81 134.63
± ± ± ± ± ±
EC50 (M) 27.54 28.42 34.16 23.38 25.49 17.87
59.36 627.28 535.83 602.29 716.40 183.44
± ± ± ± ± ±
10.08 54.16 60.03 51.62 82.83 16.04
Data are means of at least three independent experiments ± SEM.
subunits contributed mostly to neonicotinoid sensitivities between Pp␣1/r2 and Pp␣8/r2 receptors. In order to confirm the importance of segments 4, 6 and 7 in neonicotinoid sensitivity differences between Pp␣1/r2 and Pp␣8/r2, some segments in Pp␣1 were also introduced
into Pp␣8 singly or together to construct several Pp␣8 chimeras. Similarly, the introduction of 5 (thiacloprid only), 6, 7 or 67 increased sensitivities for both imidacloprid and thiacloprid (Table 4). Although the introduction of 4 did not increase neonicotinoid sensitivities, it could further increase neonicotinoid
Table 3 Imax and EC50 values of imidacloprid and thiacloprid on nAChRs containing Pp␣1, Pp␣8 or Pp␣1 chimeras plus r2 expressed in Xenopus oocytes. Receptors
Imidacloprid Imax (nA)
Pp␣1/r2 Pp␣11 /r2 Pp␣12 /r2 Pp␣13 /r2 Pp␣14 /r2 Pp␣15 /r2 Pp␣16 /r2 Pp␣17 /r2 Pp␣145 /r2 Pp␣146 /r2 Pp␣147 /r2 Pp␣167 /r2 Pp␣1467 /r2 Pp␣8/r2
156.65 168.31 162.44 149.88 151.13 173.85 185.28 204.19 168.46 193.30 221.24 216.59 233.93 239.54
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Thiacloprid EC50 (M)
16.88 a 19.55 ab 23.01 ab 17.48 a 20.23 a 26.47 ab 21.21 b 24.76 bc 21.01 ab 22.23 bc 19.57 c 22.71 c 26.30 c 28.42 c
70.09 62.76 68.11 73.55 64.69 88.14 144.38 132.02 92.25 212.61 340.38 256.63 582.09 627.28
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Imax (nA) 8.50 a 7.53 a 10.12 a 8.94 a 9.47 a 9.23 b 17.51 c 14.64 c 11.38 b 25.80 d 41.23 f 29.80 e 60.48 g 54.16 g
126.54 133.96 127.22 121.07 122.08 135.43 150.67 165.94 135.42 148.96 180.72 174.25 187.97 196.85
± ± ± ± ± ± ± ± ± ± ± ± ± ±
EC50 (M) 13.71 a 9.84 ab 14.28 a 13.19 a 15.04 a 11.71 ab 13.95 b 20.36 b 16.72 ab 13.38 b 23.26 c 19.13 bc 22.20 c 23.38 c
57.36 61.24 54.59 66.63 55.76 82.63 139.56 164.22 84.15 197.85 316.72 278.83 541.90 602.29
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.33 a 8.14 a 6.63 a 7.36 ab 9.01 a 10.39 b 15.25 c 19.84 cd 11.13 b 26.71 d 37.73 e 29.16 e 64.53 f 51.62 f
Data are means of at least three independent experiments ± SEM. The lowercase letters in the column for Imax and EC50 of imidacloprid and thiacloprid indicated the significant differences at 0.05 level.
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Fig. 3. Dose–response curves for imidacloprid on hybrid nAChRs expressed in Xenopus oocytes, containing Pp␣1 or Pp␣1 chimeras and r2 (A–C), and Pp␣8 or Pp␣8 chimeras and r2 (D). Data are means of at least three independent experiments ± SEM.
Table 4 Imax and EC50 values of imidacloprid and thiacloprid on nAChRs containing Pp␣1, Pp␣8 or Pp␣8 chimeras plus r2 expressed in Xenopus oocytes. Receptors
Imidacloprid Imax (nA)
Pp␣8/r2 Pp␣84 /r2 Pp␣85 /r2 Pp␣86 /r2 Pp␣87 /r2 Pp␣867 /r2 Pp␣8467 /r2 Pp␣1/r2
239.54 232.07 216.84 197.02 183.35 173.33 160.38 156.65
± ± ± ± ± ± ± ±
Thiacloprid EC50 (M)
28.42 a 26.55 a 24.29 ab 22.15 b 16.40 bc 18.11 c 21.05 c 16.88 c
627.28 608.13 522.75 333.34 267.37 142.80 83.65 70.09
± ± ± ± ± ± ± ±
Imax (nA) 54.16 a 61.39 a 52.28 a 36.19 b 25.42 c 17.06 d 11.23 e 8.50 e
196.85 202.46 172.20 164.46 163.13 144.33 132.67 126.54
± ± ± ± ± ± ± ±
EC50 (M) 23.38 a 25.10 a 18.74 ab 17.16 b 14.90 b 16.67 bc 15.59 c 13.71 c
602.29 611.01 476.03 301.21 258.30 150.81 71.28 57.36
± ± ± ± ± ± ± ±
51.62 a 58.75 a 53.11 b 37.19 c 31.06 c 22.67 d 12.05 e 8.33 e
Data are means of at least three independent experiments ± SEM. The lowercase letters in the column for Imax and EC50 of imidacloprid and thiacloprid indicated the significant differences at 0.05 level.
sensitivities when combined with the segment introduction of 67 (Fig. 3D). The Imax and EC50 values for both imidacloprid and thiacloprid on Pp␣8467 /r2 were also close to that on Pp␣1/r2, which confirm the importance of key amino acid differences in these three segments (4, 6 and 7) on neonicotinoid sensitivities. 4. Discussion The nAChRs ␣8 subunit was found in many insect species, such as Nilaparvata lugens, Tribolium castaneum and Anopheles gambiae, but the function of ␣8 subunit was rarely studied. In N. lugens, co-expression of ␣8 subunit (Nl␣8) with r2 gave the functional receptors, but which showed much lower agonist potency compared to Nl␣1/r2 or Nl␣2/r2 [12]. In our previous studies,
nAChRs ␣3 subunit (Nl␣3) of N. lugens failed to generate functional nAChRs in oocytes, either when expressed alone or when co-expressed with r2. However, functional nAChRs were detected when co-assembly of Nl␣3 and Nl␣8 with r2 [7,12], which indicated that ␣8 subunit might be apt to co-assemble two or more subunits to the triple, quadruple or quintuple receptors. The special importance of ␣8 subunits in insects makes it interesting to perform study on nAChR ␣8 subunit in Arachnoidea. To the best of our knowledge, Pp␣8 was the first reported nAChR ␣8 subunit in Arachnoidea and no studies were performed on the structure and function of arachnida ␣8 subunit or receptors with this subunit yet. In the present study, functional nAChRs were detected when Pp␣8 was co-expressed with r2 (Pp␣8/r2) and had the similar sensitivity to acetylcholine of Pp␣1/r2, but with significantly different sensitivities to all tested neonicotinoids. Despite evidence
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that the potency of all tested neonicotinoid insecticides was significantly different between two receptors, the difference was less pronounced for dinotefuran, a tertrahydrofuryl compound. This finding was similar to our previous report about the influence of the insecticide resistance associated mutation Y151S on the agonist potency of neonicotinoid insecticides [11]. Based on chemical structures, the neonicotinoids could be divided into three kinds, such as chloropyridyl compounds (acetamiprid, imidacloprid, nitenpyram and thiacloprid), chlorothiazolyl compounds (clothianidin and thiamethoxam) and a tetrahydrofuryl compound (dinotefuran). In contrast to dinotefuran, all of the other neonicotinoid insecticides examined contain a chlorinated heterocyclic (chloropyridyl or chlorothiazolyl) group [11]. The results indicated dinotefuran might have different action modes on insect nAChRs and different selectivity mechanisms among insect species and natural enemies, such as spiders. The nAChRs subunit composition or specific region in one subunit has been identified to influence neonicotinoid insecticide sensitivities on related receptors. In chicken ␣42 nAChRs, replacement of the ␣4 subunit by Drosophila D␣2 subunit resulted in a marked increase of imidacloprid sensitivity and demonstrated that insect ␣ subunit has structural features favorable for interactions with neonicotinoids [13]. The combination of replacing the loop B-C interval region of ␣4 subunit by the corresponding region of D␣2 subunit and E219P mutation in the YECC motif in loop C were found to play important roles in its selective interaction with imidacloprid [14]. Using the similar techniques, loop B to the Nterminus and proline residue in the YPCC motif in loop C from the D␣2 were found important to the imidacloprid selectivity [15]. In present studies, several key amino acid differences were found contributing to imidacloprid sensitivity between P. pseudoannulata two nAChRs ␣ subunits, Pp␣1 and Pp␣8. When the segment 5 (between loop B and C), 6 (between loop B and C) or 7 (within Loop C) in Pp␣8 was introduced into the corresponding regions of Pp␣1, respectively, the sensitivities of Pp␣15−7 /r2 to imidacloprid and thiacloprid were reduced to different extents, which demonstrated that amino acids in segments 5, 6 and 7 had important influences on the neonicotinoids. No obvious changes in neonicotinoid sensitivity were found when only4 (including loop B) was introduced. However, sensitivities to imidacloprid and thiacloprid were more reduced when introducing 4 with 5, 6 or 7 together. The 4 also remarkably increased insensitivity of Pp␣167 /r2 when introduced 467 together, with EC50 of Pp␣1467 /r2 to imidacloprid and thiacloprid close to or nearly identical to Pp␣8/r2. The importance of key amino acid differences in several segments was also confirmed by the opposite introduction from Pp␣1 to Pp␣8. Similarly, the indirect roles of 4 segment were also observed when comparing Pp␣867 /r2 and Pp␣8467 /r2. Although all residues contributing to the six loops were thought to be important for agonist binding, some of them play the roles in a weak manner, such as the segment 4, which included the important agonist binding region loop B. The amino acid differences in 4 segment did not directly cause neonicotinoid sensitivity difference between receptors containing different ␣ subunits, but it could significantly enhance the sensitivity differences caused by other amino acid changes. Because amino acid differences in 4
segment did not contribute to insecticide sensitivity directly, these key amino acid differences were often and rashly ignored. If a specific insecticide-associated mutation occurred incidentally at the site like key amino acid difference in 4 segment, it might be overlooked if the roles of the mutation on insecticide resistances were only examined directly and separately, because such mutation might act only as an enhancer for insecticide resistances. Acknowledgments This work was supported by National Natural Science Foundation of China (31322045, 31130045 and 31171869), Jiangsu Science for Distinguished Young Scholars (BK20130028), National High Technology Research and Development Program of China (863 Program, 2012AA101502), National Key Technology Research and Development Program (2012BAD19B01) and the Special Fund of Industrial (Agriculture) Research for Public Welfare of China (201003031). References [1] Y.G. Lou, G.R. Zhang, W.Q. Zhang, Y. Hu, J. Zhang, Reprint of: Biological control of rice insect pests in China, Biol. Control 68 (2014) 103–116. [2] P. Stano, Spiders (Araneae) in the pesticide world: an ecotoxicological review, Pest Manag. 68 (2012) 1438–1446. [3] L.D. Tang, B.L. Qiu, S.X. Ren, A review of insecticide resistance in the natural enemies of pest insects, Chin. J. Appl. Entomol. 51 (2014) 13–25. [4] K. Matsuda, S.D. Buckingham, D. Kleier, J.J. Rauh, M. Grauso, D.B. Sattelle, Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors, Trends Pharmacol. Sci. 22 (2001) 573–580. [5] T. Grutter, J.P. Changeux, Nicotinic receptors in wonderland, Trends Biochem. Sci. 26 (2001) 459–463. [6] K. Brejc, W.J. Dijk, R.V. Klaassen, M. Schuurmans, J. Oost, A.B. Smit, T.K. Sixma, Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors, Nature 411 (2001) 269–276. [7] Z.W. Liu, M.S. Williamson, S.J. Lansdell, I. Denholm, Z.J. Han, N.S. Millar, A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper), Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 8420–8425. [8] X.M. Yao, F. Song, F.J. Chen, Y.X. Zhang, J.H. Gu, S.H. Liu, Z.W. Liu, Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor  subunits influence neonicotinoid selectivity, Insect Biochem. Mol. Biol. 38 (2008) 834–840. [9] F. Song, Z.Q. You, X.M. Yao, J.G. Cheng, Z.W. Liu, K.J. Lin, Specific loops D, E and F of nicotinic acetylcholine receptor 1 subunit may confer imidacloprid selectivity between Myzus persicae and its predatory enemy Pardosa pseudoannulata, Insect Biochem. Mol. Biol. 39 (2009) 833–841. [10] A.M. Puinean, S.J. Lansdell, T. Collins, P. Bielza, N.S. Millar, A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis, J. Neurochem. 124 (2013) 590–601. [11] Z.W. Liu, M.S. Williamson, S.J. Lansdell, Z.J. Han, I. Denholm, N.S. Millar, A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoids, J. Neurochem. 99 (2006) 1273–1281. [12] Y.X. Zhang, Z.W. Liu, Z.J. Han, F. Song, X.M. Yao, Y. Shao, J. Li, N.S. Millar, Function co-expression of two insect nicotinic acetylcholine receptor subunits (Nl␣3 and Nl␣8) reveals the effects of a resistance-associated mutation (Nl␣3Y151S ) on neonicotinoid insecticides, J. Neurochem. 110 (2009) 1855–1862. [13] Z.W. Liu, X.M. Yao, Y.X. Zhang, Insect nicotinic acetylcholine receptors (nAChRs): important amino acid residues contributing to neonicotinoid insecticides selectivity and resistance, Afr. J. Biotechnol. 7 (2008) 4935–4939. [14] M. Shimomura, M. Yokota, K. Matsuda, D.B. Sattelle, K. Komai, Roles of loop C and the loop B–C interval of the nicotinic receptor subunit in its selective interactions with imidacloprid in insects, Neurosci. Lett. 363 (2004) 195–198. [15] M. Shimomura, H. Satoh, M. Yokota, M. Ihara, K. Matsuda, D.B. Sattelle, Insectvertebrate chimeric nicotinic acetylcholine receptors identify a region, loop B to the N-terminus of the Drosophila D␣2 subunit, which contributes to neonicotinoid sensitivity, Neurosci. Lett. 385 (2005) 168–172.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Identification of key amino acid differences contributing to neonicotinoid sensitivity between two nAChR ␣ subunits from Pardosa pseudoannulata Xiangkun Meng 1 , Yixi Zhang 1 , Beina Guo, Huahua Sun, Chuanjun Liu, Zewen Liu ∗ Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
h i g h l i g h t s • • • •
A novel nAChR subunit Pp␣8 was cloned from Pardosa pseudoannulata. Key amino acid residue differences were found between Pp␣8 and Pp␣1. Some different residues contributed to neonicotinoid sensitivity directly. Some residues influence sensitivity by enhancing direct effects from other residues.
a r t i c l e
i n f o
Article history: Received 21 July 2014 Received in revised form 26 September 2014 Accepted 9 October 2014 Available online 22 October 2014 Keywords: Pardosa pseudoannulata Nicotinic acetylcholine receptor ␣ subunit Amino acid differences Neonicotinoid sensitivity
a b s t r a c t Chemical insecticides are still primary methods to control rice planthoppers in China, which not only cause environmental pollution, insecticide residue and insecticide resistance, but also have negative effects on natural enemies, such as Pardosa pseudoannulata (the pond wolf spider), an important predatory enemy of rice planthoppers. Neonicotinoids insecticides, such as imidacloprid and thiacloprid, are insectselective nAChRs agonists that are used extensively in the areas of crop protection and animal health, but have hypotoxicity to P. pseudoannulata. In the present study, two nAChR ␣ subunits, Pp␣1 or Pp␣8, were found to be successfully expressed with r2 in Xenopus oocytes, but with much different sensitivity to imidacloprid and thiacloprid on two recombinant receptors Pp␣1/r2 and Pp␣8/r2. Key amino acid differences were found in and between the important loops for ligand binding. In order to well understand the relationship between the amino acid differences and neonicotinoid sensitivities, different segments in Pp␣8 or Pp␣1 with key amino acid differences were introduced into the corresponding regions of Pp␣1 or Pp␣8 to construct chimeras and then co-expressed with r2 subunit in Xenopus oocytes. The results from chimeras of both Pp␣8 and Pp␣1 showed that segments 5, 6, and 7 contributed to neonicotinoid sensitivities directly between two receptors. Although the segment 4 including all loop B region had no direct influences on neonicotinoid sensitivities, it could more remarkably influence neonicotinoid sensitivities when co-introductions with 5, 6 or 7. So, key amino acid differences in these four segments were important to neonicotinoid sensitivities, but the difference in 4 was likely ignored because of its indirect effects. © 2014 Published by Elsevier Ireland Ltd.
1. Introduction Rice is one of the most important food crops in the world. In many countries, rice is suffering from many insect pests, especially
∗ Corresponding author at: Tongwei Road 6, Nanjing 210095, China. Tel.: +86 25 84399051; fax: +86 25 84399051. E-mail address: [email protected] (Z. Liu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.neulet.2014.10.013 0304-3940/© 2014 Published by Elsevier Ireland Ltd.
the brown planthopper, Nilaparvata lugens (Stål), a major rice pest in Asia. China has the second largest area of the rice growing and the highest rice yield [1]. The primary method to control rice insect pests is still the application of chemical insecticides, which not only causes environmental pollution but also reduces natural enemies’ populations. Spiders are recognized as important natural enemies to reduce pest populations in rice fields, particularly in early rice season when specialized predators are still not present. Being one of the most abundant and species-rich groups of natural enemies occurring in all agroecosystems, spiders are
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variably affected by pesticide applications, such as the pond wolf spider Pardosa pseudoannulata, an important predatory enemy of rice planthoppers and aphids [2]. Spiders are mainly affected by acaricides and insecticides, especially neurotoxic substances. However, the ecotoxicology of spiders are rarely studied, as only 3% of toxicology papers on natural enemies have been devoted to spiders [2]. Neonicotinoids are used extensively to control rice pests and show the relative safety to P. pseudoannulata [3]. Neonicotinoids act as neurotoxins selectively on insect nicotinic acetylcholine receptors (nAChRs), the ligand-gated ion channels mediating fast cholinergic synaptic transmission in invertebrate and vertebrate nervous systems [4]. The nAChRs agonist-binding site is present at the interface of adjacent subunits and is formed by loops A–C in ␣ subunits together with loops D–F in either non␣ (, ␥ or ␦) subunits or homomer-forming ␣ subunits [5,6]. In previous studies, different amino acids in insect nAChRs loop B of ␣ subunit or loop D, E, F of  subunit were found to contribute to imidacloprid selectivities [7–9]. Studies also showed that the G275E mutation, predicted to lie at the top of the third-helical transmembrane domain of ␣6 subunit, resulted in the high resistance to spinosad in the western flower thrips, Frankliniella occidentalis [10]. In contrast, there are only few studies about P. pseudoannulata nAChRs and neonicotinoids selectively between natural enemies and insect pests. The pharmacological studies of P. pseudoannulata nAChRs are essential to understand the insecticide selectively and the development of selective insecticides [9]. In the present study, a novel nAChR ␣ subunit (Pp␣8) was cloned from P. pseudoannulata. When successful co-expressed Pp␣8 or Pp␣1 (one ␣ subunit previously cloned) with rat r2 in Xenopus oocytes, the receptor Pp␣8/r2 was found with much lower neonicotinoid sensitivity than Pp␣1/r2. By comparison of these two ␣ subunits, key amino acid differences were found in and between the important loops for ligand binding. The effects of these amino acid differences on neonicotinoid sensitivities were examined by constructing different subunit chimeras and co-expression with r2 in Xenopus oocytes.
Table 1 Primers for clone Pp␣8. Primer name
Sequences of primers
Sense degenerate primer for Pp␣8 Anti-sense degenerate primer for Pp␣8 5 -outer GSP for Pp␣8 5 -inner GSP for Pp␣8 3 -outer GSP for Pp␣8 3 -inner GSP for Pp␣8
5 -AARTGGAAYCCNGAYGAYTAYGGNGG-3 5 -TADATNGCNGGNGGYTTCCANACNACYTT-3 5 -CAGTGTCACTTCGTAGTTGCCATCC-3 5 -GCATTGTTGTAGAGTACGATGTCAGG-3 5 -CAGAGCCTCCGAAGTTCACTGTCAG-3 5 -GGGGCTGAAGATGTCGGTCTGGAAT-3
pGH19 as described previously [11]. In the Pp␣1, the fragments of 68–79th (1), 87–93th (2), 120–128th (3), 152–166th (4), 170–181th (5), 199–206th (6), 208–214th (7) residues were replaced by that Pp␣8 to construct the chimeras Pp␣11−7 , as previously described [8]. Similarly, 5, 6 and 7 fragments of Pp␣8 were replaced by the corresponding sequences of Pp␣1, singly or together, to construct several Pp␣8 chimeras. All plasmid and chimera constructs were verified by nucleotide sequencing. 2.5. In vitro transcription, oocyte preparation and electrophysiology The methods of vitro transcription, oocyte preparation and electrophysiology were detailed described previously [8,9]. 3. Results 3.1. Isolation of Pp˛8 cDNA
P. pseudoannulata was collected from a field of hybrid paddy rice in Nanjing (Jiangsu, China) in August 2008 and reared in door by feeding with N. lugens. Spiders were kept indoors at 25 (±1) ◦ C, humidity 70–80% and 16/8 h light/dark.
A novel nAChRs ␣ subunit was cloned from P. pseudoannulata using RT-PCR with degenerate oligonucleotide primers and RACE techniques with gene-specific primers (Table 1). The fulllength cDNA (Genbank accession number, HM017861) has an open reading frame of 1527 bp and 509 deduced amino acids, and the deduced protein had the common features of insect nAChRs ␣ subunit (Fig. 1), such as three loops important to agonist binding and four transmembrane regions. The subunit showed high similarities to insect nAChRs ␣8 subunits, such as 73.0% to Nilaparvata lugens and 72.0% to Apis mellifera, and was denoted as Pp␣8. Pp␣8 also showed high similarity (70%) to Pp␣1 (HM017860), a subunit previously cloned in our laboratory. However, several key amino acid differences were found in and between the important loops (A, B and C) for ligand binding between Pp␣1 and Pp␣8 (Fig. 1). The region with amino acid differences was divided into seven segments (1–7). The segments in Pp␣8 were introduced into the corresponding regions of Pp␣1 singly or combinedly to construct several Pp␣1 chimeras, and some segments in Pp␣1 were also introduced into Pp␣8 to construct Pp␣8 chimeras.
2.3. Amplification of cDNA
3.2. Functional expression of Pp˛1 and Pp˛8 with rat ˇ2
The full length cDNA of P. pseudoannulata nAChRs ␣8 cDNA was obtained through techniques of polymerase chain reaction (PCR) with degenerate primers and rapid-amplification of cDNA ends (RACE) with gene-specific primers (Table 1), as described previously [9].
When co-expression with the vertebrate rat neuronal 2 (r2) subunit in heterologous expression system Xenopus oocytes, both receptor of Pp␣1/r2 and Pp␣8/r2 could be induced large inward currents by different agonists (1 mM) (Fig. 2A). Further dose–response experiments were performed for acetylcholine (ACh) and some neonicotinoid insecticides. There was small but still significant difference in ACh sensitivity between Pp␣1/r2 and Pp␣8/r2, reflecting in EC50 difference, although there was no difference in Imax values between two receptors (Fig. 2B). In contrast, there were big differences in both Imax and EC50 values for tested neonicotinoids between two receptors, for example, the imidacloprid EC50 of Pp␣8/r2 was 8.9 times of that of Pp␣1/r2 (Fig. 2C).
2. Materials and methods 2.1. Chemicals Acetylcholine and neonicotinoids (imidacloprid, acetamiprid, thiacloprid, clothianidin, dinotefuran) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Collection and rearing of spider
2.4. Plasmids and mutagenesis P. pseudoannulata nAChRs ␣ subunit Pp␣1 (GenBank accession number, HM017860), Pp␣8 (Genbank accession number, HM017861) and Rattus norvegicus  subunit r2 (GenBank accession number, L31622) were subcloned into the expression vector
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Fig. 1. Alignment of Pardosa pseudoannulata Pp␣1, Pp␣8 and ␣8 subunits from some insect species. Loops A, B and C, important to agonist binding site in nAChR ␣ subunit, are marked by dot lines. Tansmembrane domains (TM1–4) are marked by double lines. Important amino acid differences within two ␣ subunits are marked by single lines and ranged into several segments (1–7) for subunit chimera construction of Pp␣1 or Pp␣8. Pardosa pseudoannulata, Pp␣1 (HM017860) and Pp␣8 (HM017861); Apis mellifera, Am␣8 (NP001011575); Bombyx mori, Bm␣8 (ABV72690); Nilaparvata lugens, Nl␣8 (ACK75719).
In addition to imidacloprid, four other neonicotinoid compounds were also examined on two receptors. The significantly bigger Imax and EC50 values were found for all neonicotinoid compounds examined in Pp␣8/r2 than in Pp␣1/r2 (Fig. 2D), although the differences of dinotefuran were much less than that of other neonicotinoid insecticides. In Imax values, there was not significant difference for dinotefuran between two receptors, but the values for other neonicotinoid insecticides on Pp␣8/r2 were 1.5 times of that on Pp␣1/r2 at least. Although EC50 value of dinotefuran on Pp␣8/r2 was only 2.2 times of that on Pp␣1/r2, the ratios for other insecticides were above 5.0 (5.7–10.5). Table 2 gives a summary of Imax and EC50 data obtained in two receptors. 3.3. Influence of amino acid differences between Pp˛1 and Pp˛8 on neonicotinoid sensitivity In order to find out the roles of key amino acid differences between two ␣ subunits in neonicotinoid sensitivities between receptors Pp␣1/r2 and Pp␣8/r2, different segments in Pp␣8 or
Pp␣1 were introduced into the corresponding regions of Pp␣1 or Pp␣8 to construct artificial subunit chimeras and then co-expressed with r2 in heterologous expression system Xenopus oocytes. In voltage-clamp electrophysiological studies, receptors with Pp␣1 chimeras co-expressed with r2 in Xenopus oocytes, large inward currents were detected in response to 1 mM imidacloprid and thiacloprid. When different segments in Pp␣8 were introduced into the corresponding regions of Pp␣1 singly, there were no obvious changes in Imax or EC50 for 1, 2, 3 and 4 when compared to Pp␣1 (Table 3). In contrast, significant changes in Imax or/and EC50 values were found for the introduction of 5 (EC50 only), 6 and 7, especially the segments 6 and 7 (Fig. 3A–C). Although the introduction of 4 did not cause a significant change in EC50 values for both imidacloprid and thiacloprid, such segment introduction could further reduced neonicotinoid sensitivities when combined with other segment introduction, such as 5, 6, 7 or 67 (Table 3 and Fig. 3). In particular, the Imax and EC50 values for both imidacloprid and thiacloprid on Pp␣1467 /r2 were very close to that on Pp␣8/r2, which indicated that key amino acid differences in these three segments (4, 6 and 7) between two ␣
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Fig. 2. Representative whole-cell responses and agonist dose–response curves from hybrid nAChRs expressed in Xenopus oocytes, containing Pp␣1/r2 or Pp␣8/r2. (A) Representative responses elicited by acetylcholine and imidacloprid. (B) Dose–response curves for acetylcholine. (C) Dose–response curves for imidacloprid. (D) Ratios of Imax and EC50 between Pp␣1/r2 and Pp␣8/r2. Data are means of at least three independent experiments ± SEM. Table 2 Imax and EC50 values of acetylcholine and neonicotinoids on nAChRs containing Pp␣1/r2 or Pp␣8/r2 expressed in Xenopus oocytes. Agonist
Pp␣1/r2
Pp␣8/r2
Imax (nA) Acetylcholine Imidacloprid Acetamiprid Thiacloprid Clothianidin Dinotefuran
273.59 156.65 131.29 126.54 109.80 123.23
± ± ± ± ± ±
EC50 (M) 31.82 16.88 18.43 13.71 13.66 9.82
34.65 70.09 92.75 57.36 104.24 82.39
± ± ± ± ± ±
Imax (nA) 6.67 8.50 11.24 8.33 13.17 7.68
281.76 239.54 222.27 196.85 187.81 134.63
± ± ± ± ± ±
EC50 (M) 27.54 28.42 34.16 23.38 25.49 17.87
59.36 627.28 535.83 602.29 716.40 183.44
± ± ± ± ± ±
10.08 54.16 60.03 51.62 82.83 16.04
Data are means of at least three independent experiments ± SEM.
subunits contributed mostly to neonicotinoid sensitivities between Pp␣1/r2 and Pp␣8/r2 receptors. In order to confirm the importance of segments 4, 6 and 7 in neonicotinoid sensitivity differences between Pp␣1/r2 and Pp␣8/r2, some segments in Pp␣1 were also introduced
into Pp␣8 singly or together to construct several Pp␣8 chimeras. Similarly, the introduction of 5 (thiacloprid only), 6, 7 or 67 increased sensitivities for both imidacloprid and thiacloprid (Table 4). Although the introduction of 4 did not increase neonicotinoid sensitivities, it could further increase neonicotinoid
Table 3 Imax and EC50 values of imidacloprid and thiacloprid on nAChRs containing Pp␣1, Pp␣8 or Pp␣1 chimeras plus r2 expressed in Xenopus oocytes. Receptors
Imidacloprid Imax (nA)
Pp␣1/r2 Pp␣11 /r2 Pp␣12 /r2 Pp␣13 /r2 Pp␣14 /r2 Pp␣15 /r2 Pp␣16 /r2 Pp␣17 /r2 Pp␣145 /r2 Pp␣146 /r2 Pp␣147 /r2 Pp␣167 /r2 Pp␣1467 /r2 Pp␣8/r2
156.65 168.31 162.44 149.88 151.13 173.85 185.28 204.19 168.46 193.30 221.24 216.59 233.93 239.54
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Thiacloprid EC50 (M)
16.88 a 19.55 ab 23.01 ab 17.48 a 20.23 a 26.47 ab 21.21 b 24.76 bc 21.01 ab 22.23 bc 19.57 c 22.71 c 26.30 c 28.42 c
70.09 62.76 68.11 73.55 64.69 88.14 144.38 132.02 92.25 212.61 340.38 256.63 582.09 627.28
± ± ± ± ± ± ± ± ± ± ± ± ± ±
Imax (nA) 8.50 a 7.53 a 10.12 a 8.94 a 9.47 a 9.23 b 17.51 c 14.64 c 11.38 b 25.80 d 41.23 f 29.80 e 60.48 g 54.16 g
126.54 133.96 127.22 121.07 122.08 135.43 150.67 165.94 135.42 148.96 180.72 174.25 187.97 196.85
± ± ± ± ± ± ± ± ± ± ± ± ± ±
EC50 (M) 13.71 a 9.84 ab 14.28 a 13.19 a 15.04 a 11.71 ab 13.95 b 20.36 b 16.72 ab 13.38 b 23.26 c 19.13 bc 22.20 c 23.38 c
57.36 61.24 54.59 66.63 55.76 82.63 139.56 164.22 84.15 197.85 316.72 278.83 541.90 602.29
± ± ± ± ± ± ± ± ± ± ± ± ± ±
8.33 a 8.14 a 6.63 a 7.36 ab 9.01 a 10.39 b 15.25 c 19.84 cd 11.13 b 26.71 d 37.73 e 29.16 e 64.53 f 51.62 f
Data are means of at least three independent experiments ± SEM. The lowercase letters in the column for Imax and EC50 of imidacloprid and thiacloprid indicated the significant differences at 0.05 level.
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Fig. 3. Dose–response curves for imidacloprid on hybrid nAChRs expressed in Xenopus oocytes, containing Pp␣1 or Pp␣1 chimeras and r2 (A–C), and Pp␣8 or Pp␣8 chimeras and r2 (D). Data are means of at least three independent experiments ± SEM.
Table 4 Imax and EC50 values of imidacloprid and thiacloprid on nAChRs containing Pp␣1, Pp␣8 or Pp␣8 chimeras plus r2 expressed in Xenopus oocytes. Receptors
Imidacloprid Imax (nA)
Pp␣8/r2 Pp␣84 /r2 Pp␣85 /r2 Pp␣86 /r2 Pp␣87 /r2 Pp␣867 /r2 Pp␣8467 /r2 Pp␣1/r2
239.54 232.07 216.84 197.02 183.35 173.33 160.38 156.65
± ± ± ± ± ± ± ±
Thiacloprid EC50 (M)
28.42 a 26.55 a 24.29 ab 22.15 b 16.40 bc 18.11 c 21.05 c 16.88 c
627.28 608.13 522.75 333.34 267.37 142.80 83.65 70.09
± ± ± ± ± ± ± ±
Imax (nA) 54.16 a 61.39 a 52.28 a 36.19 b 25.42 c 17.06 d 11.23 e 8.50 e
196.85 202.46 172.20 164.46 163.13 144.33 132.67 126.54
± ± ± ± ± ± ± ±
EC50 (M) 23.38 a 25.10 a 18.74 ab 17.16 b 14.90 b 16.67 bc 15.59 c 13.71 c
602.29 611.01 476.03 301.21 258.30 150.81 71.28 57.36
± ± ± ± ± ± ± ±
51.62 a 58.75 a 53.11 b 37.19 c 31.06 c 22.67 d 12.05 e 8.33 e
Data are means of at least three independent experiments ± SEM. The lowercase letters in the column for Imax and EC50 of imidacloprid and thiacloprid indicated the significant differences at 0.05 level.
sensitivities when combined with the segment introduction of 67 (Fig. 3D). The Imax and EC50 values for both imidacloprid and thiacloprid on Pp␣8467 /r2 were also close to that on Pp␣1/r2, which confirm the importance of key amino acid differences in these three segments (4, 6 and 7) on neonicotinoid sensitivities. 4. Discussion The nAChRs ␣8 subunit was found in many insect species, such as Nilaparvata lugens, Tribolium castaneum and Anopheles gambiae, but the function of ␣8 subunit was rarely studied. In N. lugens, co-expression of ␣8 subunit (Nl␣8) with r2 gave the functional receptors, but which showed much lower agonist potency compared to Nl␣1/r2 or Nl␣2/r2 [12]. In our previous studies,
nAChRs ␣3 subunit (Nl␣3) of N. lugens failed to generate functional nAChRs in oocytes, either when expressed alone or when co-expressed with r2. However, functional nAChRs were detected when co-assembly of Nl␣3 and Nl␣8 with r2 [7,12], which indicated that ␣8 subunit might be apt to co-assemble two or more subunits to the triple, quadruple or quintuple receptors. The special importance of ␣8 subunits in insects makes it interesting to perform study on nAChR ␣8 subunit in Arachnoidea. To the best of our knowledge, Pp␣8 was the first reported nAChR ␣8 subunit in Arachnoidea and no studies were performed on the structure and function of arachnida ␣8 subunit or receptors with this subunit yet. In the present study, functional nAChRs were detected when Pp␣8 was co-expressed with r2 (Pp␣8/r2) and had the similar sensitivity to acetylcholine of Pp␣1/r2, but with significantly different sensitivities to all tested neonicotinoids. Despite evidence
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that the potency of all tested neonicotinoid insecticides was significantly different between two receptors, the difference was less pronounced for dinotefuran, a tertrahydrofuryl compound. This finding was similar to our previous report about the influence of the insecticide resistance associated mutation Y151S on the agonist potency of neonicotinoid insecticides [11]. Based on chemical structures, the neonicotinoids could be divided into three kinds, such as chloropyridyl compounds (acetamiprid, imidacloprid, nitenpyram and thiacloprid), chlorothiazolyl compounds (clothianidin and thiamethoxam) and a tetrahydrofuryl compound (dinotefuran). In contrast to dinotefuran, all of the other neonicotinoid insecticides examined contain a chlorinated heterocyclic (chloropyridyl or chlorothiazolyl) group [11]. The results indicated dinotefuran might have different action modes on insect nAChRs and different selectivity mechanisms among insect species and natural enemies, such as spiders. The nAChRs subunit composition or specific region in one subunit has been identified to influence neonicotinoid insecticide sensitivities on related receptors. In chicken ␣42 nAChRs, replacement of the ␣4 subunit by Drosophila D␣2 subunit resulted in a marked increase of imidacloprid sensitivity and demonstrated that insect ␣ subunit has structural features favorable for interactions with neonicotinoids [13]. The combination of replacing the loop B-C interval region of ␣4 subunit by the corresponding region of D␣2 subunit and E219P mutation in the YECC motif in loop C were found to play important roles in its selective interaction with imidacloprid [14]. Using the similar techniques, loop B to the Nterminus and proline residue in the YPCC motif in loop C from the D␣2 were found important to the imidacloprid selectivity [15]. In present studies, several key amino acid differences were found contributing to imidacloprid sensitivity between P. pseudoannulata two nAChRs ␣ subunits, Pp␣1 and Pp␣8. When the segment 5 (between loop B and C), 6 (between loop B and C) or 7 (within Loop C) in Pp␣8 was introduced into the corresponding regions of Pp␣1, respectively, the sensitivities of Pp␣15−7 /r2 to imidacloprid and thiacloprid were reduced to different extents, which demonstrated that amino acids in segments 5, 6 and 7 had important influences on the neonicotinoids. No obvious changes in neonicotinoid sensitivity were found when only4 (including loop B) was introduced. However, sensitivities to imidacloprid and thiacloprid were more reduced when introducing 4 with 5, 6 or 7 together. The 4 also remarkably increased insensitivity of Pp␣167 /r2 when introduced 467 together, with EC50 of Pp␣1467 /r2 to imidacloprid and thiacloprid close to or nearly identical to Pp␣8/r2. The importance of key amino acid differences in several segments was also confirmed by the opposite introduction from Pp␣1 to Pp␣8. Similarly, the indirect roles of 4 segment were also observed when comparing Pp␣867 /r2 and Pp␣8467 /r2. Although all residues contributing to the six loops were thought to be important for agonist binding, some of them play the roles in a weak manner, such as the segment 4, which included the important agonist binding region loop B. The amino acid differences in 4 segment did not directly cause neonicotinoid sensitivity difference between receptors containing different ␣ subunits, but it could significantly enhance the sensitivity differences caused by other amino acid changes. Because amino acid differences in 4
segment did not contribute to insecticide sensitivity directly, these key amino acid differences were often and rashly ignored. If a specific insecticide-associated mutation occurred incidentally at the site like key amino acid difference in 4 segment, it might be overlooked if the roles of the mutation on insecticide resistances were only examined directly and separately, because such mutation might act only as an enhancer for insecticide resistances. Acknowledgments This work was supported by National Natural Science Foundation of China (31322045, 31130045 and 31171869), Jiangsu Science for Distinguished Young Scholars (BK20130028), National High Technology Research and Development Program of China (863 Program, 2012AA101502), National Key Technology Research and Development Program (2012BAD19B01) and the Special Fund of Industrial (Agriculture) Research for Public Welfare of China (201003031). References [1] Y.G. Lou, G.R. Zhang, W.Q. Zhang, Y. Hu, J. Zhang, Reprint of: Biological control of rice insect pests in China, Biol. Control 68 (2014) 103–116. [2] P. Stano, Spiders (Araneae) in the pesticide world: an ecotoxicological review, Pest Manag. 68 (2012) 1438–1446. [3] L.D. Tang, B.L. Qiu, S.X. Ren, A review of insecticide resistance in the natural enemies of pest insects, Chin. J. Appl. Entomol. 51 (2014) 13–25. [4] K. Matsuda, S.D. Buckingham, D. Kleier, J.J. Rauh, M. Grauso, D.B. Sattelle, Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors, Trends Pharmacol. Sci. 22 (2001) 573–580. [5] T. Grutter, J.P. Changeux, Nicotinic receptors in wonderland, Trends Biochem. Sci. 26 (2001) 459–463. [6] K. Brejc, W.J. Dijk, R.V. Klaassen, M. Schuurmans, J. Oost, A.B. Smit, T.K. Sixma, Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors, Nature 411 (2001) 269–276. [7] Z.W. Liu, M.S. Williamson, S.J. Lansdell, I. Denholm, Z.J. Han, N.S. Millar, A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper), Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 8420–8425. [8] X.M. Yao, F. Song, F.J. Chen, Y.X. Zhang, J.H. Gu, S.H. Liu, Z.W. Liu, Amino acids within loops D, E and F of insect nicotinic acetylcholine receptor  subunits influence neonicotinoid selectivity, Insect Biochem. Mol. Biol. 38 (2008) 834–840. [9] F. Song, Z.Q. You, X.M. Yao, J.G. Cheng, Z.W. Liu, K.J. Lin, Specific loops D, E and F of nicotinic acetylcholine receptor 1 subunit may confer imidacloprid selectivity between Myzus persicae and its predatory enemy Pardosa pseudoannulata, Insect Biochem. Mol. Biol. 39 (2009) 833–841. [10] A.M. Puinean, S.J. Lansdell, T. Collins, P. Bielza, N.S. Millar, A nicotinic acetylcholine receptor transmembrane point mutation (G275E) associated with resistance to spinosad in Frankliniella occidentalis, J. Neurochem. 124 (2013) 590–601. [11] Z.W. Liu, M.S. Williamson, S.J. Lansdell, Z.J. Han, I. Denholm, N.S. Millar, A nicotinic acetylcholine receptor mutation (Y151S) causes reduced agonist potency to a range of neonicotinoids, J. Neurochem. 99 (2006) 1273–1281. [12] Y.X. Zhang, Z.W. Liu, Z.J. Han, F. Song, X.M. Yao, Y. Shao, J. Li, N.S. Millar, Function co-expression of two insect nicotinic acetylcholine receptor subunits (Nl␣3 and Nl␣8) reveals the effects of a resistance-associated mutation (Nl␣3Y151S ) on neonicotinoid insecticides, J. Neurochem. 110 (2009) 1855–1862. [13] Z.W. Liu, X.M. Yao, Y.X. Zhang, Insect nicotinic acetylcholine receptors (nAChRs): important amino acid residues contributing to neonicotinoid insecticides selectivity and resistance, Afr. J. Biotechnol. 7 (2008) 4935–4939. [14] M. Shimomura, M. Yokota, K. Matsuda, D.B. Sattelle, K. Komai, Roles of loop C and the loop B–C interval of the nicotinic receptor subunit in its selective interactions with imidacloprid in insects, Neurosci. Lett. 363 (2004) 195–198. [15] M. Shimomura, H. Satoh, M. Yokota, M. Ihara, K. Matsuda, D.B. Sattelle, Insectvertebrate chimeric nicotinic acetylcholine receptors identify a region, loop B to the N-terminus of the Drosophila D␣2 subunit, which contributes to neonicotinoid sensitivity, Neurosci. Lett. 385 (2005) 168–172.