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CPR63 promotes pyrethroid resistance by increasing cuticle thickness in Culex pipiens pallens

CPR63 promotes pyrethroid resistance by increasing cuticle thickness in Culex pipiens pallens The cuticle protein (CP) encoded by CPR63 plays a role in deltamethrin resistance in Culex pipiens pallens. Herein, we investigated the distribution of CPR63 transcripts in this organism and observed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains was 2.17-fold higher than in deltamethrin-susceptible (DS) strains. Cuticle analysis of small interfering RNA (siRNA) groups by scanning electron microscopy (SEM) revealed a significantly thinner cuticle of the tarsi in the siCPR63 group than in the siNC (negative control siRNA) group. Transmission electron microscopy ( TEM) revealed that the exocuticle and endocuticle thickness of the tarsi were significantly thinner, which contributes the thinner procuticle of tarsi in the siCPR63 group than in the siNC group. Our results suggested that CPR63 might contribute to the resistance phenotype by thickening the cuticle and thereby possibly increasing the tolerance of mosquitoes to deltamethrin. Keywords: CPRs, Deltamethrin, Mosquito, Culex pipiens pallens, Cuticular resistance, Cuticle thickening Introduction Research on the mechanism of mosquito vector resist- Targeting insect vectors has proven to be the most effec - ance is of great significance for mosquito vector control. tive means for preventing the spread of mosquito-borne Generally, insecticide resistance in insects is caused by diseases [1, 2]. Chemical insecticides are the most impor- three major mechanisms: (i) reduced sensitivity of the tant component in this effort. However, the spread of target site, (ii) increased activity and/or abundance of insecticide resistance seriously threatens the success and detoxification enzymes and (iii) reduced penetration sustainability of control interventions [3]. According to of insecticides due to altered cuticles [5]. The cuticle is the latest  World malaria report, 73 countries reported believed to function in insecticide resistance by reduc- mosquito resistance to at least one of the four commonly ing or slowing insecticide uptake. Various CPs (CPR, used insecticide classes during 2010−2019, and 28 coun- CPAPn, CPG, CPF and CPLCG) belonging to different tries reported mosquito resistance to all major insecticide protein families have been identified [6, 7]. Some cuti- classes [4]. Therefore, it is critical to develop and apply cle proteins (CPs) play major roles in insecticide resist- effective insecticide resistance management strategies. ance in mosquitoes. For example, previously, CPLCG3, CPLCG4 and CPLCG5 were involved in a putative cuticle thickening mechanism. CPR124, CPR127, CPR129 and CPR131 were found expressed at higher levels in pyre- throid-resistant compared to susceptible mosquitoes [14, *Correspondence: sunyan@njmu.edu.cn Department of Pathogen Biology, Nanjing Medical University, Nanjing, 24, 25, 27]. Most CPs belong to the CPR family and pos- China sess characteristic Rebers and Riddiford (R&R) consensus Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xu et al. Parasites & Vectors (2022) 15:54 Page 2 of 7 sequences (RR-1 and RR-2) that in an extended form control samples were high Ct values (Ct > 35). When confer chitin-binding properties [8–11]. In a recent the primers were used at the first time, qPCR prod - study, the location of RR-1  s and RR-2  s was found to ucts were sequenced for confirmation. The correla - be more dependent on the properties of individual pro- tion coefficients of the calibration curves in each test teins than had been reported in previous work [12]. A were > 0.99. The relative expression levels were nor - previous study in our laboratory investigated expression malised to the internal control  β-actin  by using the −ΔΔCt ΔCt of the CPR63 gene (GenBank: MF095856.1), encoding 2   method [15–18]: target gene/β-actin = 2 , an RR-2 family member in Cx. pipiens pallens, and this ΔCt = Ct − Ct . Three technical and biolog - β-actin target gene was more abundant in deltamethrin-resistant (DR) than ical replicates were performed for qPCR analyses. deltamethrin-susceptible (DS) strains [13]. Furthermore, the mosquito mortality rate was altered by silencing the CPR63 gene [13]. However, the detailed resistance mech- Gene silencing anism of CPR63 related to the mosquito cuticle remains Mosquitoes for RNA interference (RNAi) experiments unknown. In this study, we revealed that CPR63 might were derived from DR and DS female strains microin- participate in pyrethroid resistance by thickening the jected at 12  h PE, with three tubes for each group (10 cuticle and thereby possibly increasing the tolerance of RNAi mosquitoes per tube). A small interfering RNA mosquitoes to deltamethrin. (siRNA) targeting CPR63 (siCPR63) and a siNC (nega- tive control siRNA) were synthesised by GenePharma (Shanghai, China; Additional file  3: Table S1). The siNC Materials and methods does not cause any gene silencing and has no homolo- Mosquito strains gous genes in the mosquito gene bank. About 364 ng of In this study, we collected Cx. pipiens pallens from Tang- siCPR63 and 350  ng of siNC were separately injected kou (Shandong Province, China) as DS strains, and the into the thorax of female mosquitoes. Other details of LC for deltamethrin for these DS strains was 0.03 mg/l. the gene silencing method have been described previ- DR strains were isolated from DS strains with an LC of ously [15]. After 3 days, qPCR was performed to deter- 7.5 mg/l by repeatedly selecting 84 generations at the lar- mine the interference efficiency of the target gene. val stage. Other details were as described in a previous study [14]. Scanning electron microscopy (SEM) RNA extraction and cDNA synthesis To avoid the influence of mosquito size on cuticle We collected heads, thorax, abdomen, and all legs and thickness, we measured the wing length of all female wings of the DS and DR female mosquitoes at 72 h post- mosquitoes in the experiment [31]. There were 11 eclosion (PE) in three tubes for each tissue (20 mosqui- female mosquitoes in each group (siNC, siCPR63), and toes per tube). Extraction of mosquito total RNA was we selected one right front leg from each female mos- performed according to the RNAiso Plus instructions quito. All microinjected mosquito legs were washed (Takara, Shiga, Japan), and the RNA was then converted twice in 70% ethanol to clean them thoroughly. Alco- to cDNA using the PrimeScriptRT Reagent Kit (TaKaRa, hol was dripped onto tarsomere I of the right front leg Tokyo, Japan). at the midpoint, and the leg was cut with a new plat- inum-coated blade and washed again to remove any Quantitative real‑time PCR (qPCR) debris. Legs were then fixed in 2.5% glutaraldehyde cDNA samples were diluted properly with RNase-free (Sigma, St Louis, MO, USA) for 12 h and incubated for water before use as templates in the quantitative PCR 10  min each in a graded ethanol series (30, 50, 70, 80, process using SYBR Green (Applied Biosystems, Foster 90, 95 and 100%). Legs were dried in an EM CPD300 City, CA, USA) according to the manufacturer’s proto- critical point dryer (Leica, Wetzlar, Germany) using an col. The reaction volume (20 μl) contained the Power automated process for 15 exchanges. A K550 X sput- SYBR Green PCR Master Mix, specific forward and ter coater (Electron Microscopy Sciences, Hatfield, reverse primers (Additional file  3: Table S1) and diluted PA, USA) was used for coating samples. A Quanta 250 cDNA. The PCR conditions were as follows: 50  °C for FEI scanning electron microscope was employed, and 2  min and 95  °C for 10  min, followed by 40 cycles at images were recorded at a 3-kV acceleration voltage. 95  °C for 15  s and 60  °C for 1  min. For qPCR valida- The thickness of the cuticle was examined using image tion, the melting curve program was run immediately J software (http:// imagej. net/ Welco me). The average after the qPCR program showed a single-peaked curve. cuticle thickness of each leg was calculated by measur- Amplification signals in the no template or primer ing the distance at 23 randomly selected points. Xu  et al. Parasites & Vectors (2022) 15:54 Page 3 of 7 Transmission electron microscopy (TEM) indicated that CPR63 might play an important role in leg Six female mosquitoes were included in each group resistance. (siNC, siCPR63), and we selected one right front leg from each female mosquito (i.e. six legs per group). Tarsi were divided into four equal parts, and 2–3 images were cap- Expression of CPR63 following gene silencing tured for each part, resulting in 9–10 images for each Expression of CPR63 was evaluated by qPCR in DR leg and 58 images in total. The thickness of the cuticle strains at 12  h PE after injection of siRNA targeting the was examined using image J software. According to the CPR63 gene. Expression of CPR63 was significantly obtained pictures, we also counted the number of pores decreased by 40.9% (t-test, t = 2.935, p = 0.0426) in (4) in tarsi. The other detailed steps of the TEM experiment the whole bodies of mosquitoes and by 37.6% (t-test, have been described in previous studies [14, 19]. t = 3.146, p = 0.0347) in the legs at 72  h PE after (4) siCPR63 injection compared to injection with siNC (Fig.  2a, b). We also found that interference efficiency of Statistical analysis CPR63 in DS strains is not statistically significant com - Experimental data between two groups were analysed pared with siNC group of DS strains, which could be using Student’s t-test. The expression levels of CPR63 in used as a control (Additional file 2: Figure S2). different tissues were calculated using an ANOVA test. All data are presented as the mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. SEM analysis of cuticle thickness All experiments were performed using at least three To probe the changes in the overall cuticle structure, the independent cohorts. region of the tarsus segment was analysed by SEM. The mosquito leg is composed of the femur, tibia and tar- sus, the tarsus is divided into T1, T2, T3, T4 and T5, and Results the cross section was at T1 (Fig.  3a) [19, 20]. The same CPR63 transcripts are abundant in DR mosquito legs number of specimens was assessed in siCPR63 and siNC To explore the function of CPR63, we examined localisa- groups (n = 11). Mosquito leg tarsi were assessed by com- tion of CPR63 expression in multiple tissues at 72  h PE paring the size of the mosquito wing and measuring the by qPCR in DS and DR strains, including the heads, tho- area of the inner and outer circles of the mosquito leg rax, abdomen, legs and wings. The results showed that cuticle. The results showed that the tarsi were a similar CPR63 was highly expressed in legs. Expression of CPR63 size in the two groups, but they were significantly thinner in the legs of DR strains was 2.17-fold higher (ANOVA, in the siCPR63 than in the siNC group (Fig.  3b, c). The p < 0.0001) than those in DS strains (Fig.  1). The result cuticle thickness was measured at no fewer than 23 ran- showed that CPR63 was highly expressed in legs and dom points to obtain the average cuticle thickness of the tarsus. The measurement results showed that the mean cuticle thickness of the siCPR63 group (1.354 ± 0.23  μm) was thinner than that of the siNC group (2.006 ± 0.73 μm; Fig. 4; Table 1; t-test, t = 3.981, p = 0.0011). (44) Fig. 1 Expression profiles of CPR63 in different mosquito tissues. Constitutive expression of CPR63 in DS and DR strains; mRNA expression levels were measured in the head, thorax, abdomen, legs and wings in DS and DR strain mosquitos. Head of DS strain Fig. 2 Relative expression levels of CPR63 after RNAi silencing. Levels was ascribed an arbitrary value of 1. Results are presented as the of CPR63 expression in whole mosquito bodies (a) and legs (b) after mean ± standard deviation (SD) of three biological replicates. silencing of CPR63 were measured by qPCR. Results are shown as the ****p ≤ 0.0001; ns, not signifcant, p > 0.05 mean ± SD of three biological replicates. *p ≤ 0.05; **p ≤ 0.01 Xu et al. Parasites & Vectors (2022) 15:54 Page 4 of 7 Fig. 3 SEM analysis of the effects of siNC and siCPR63. a Illustration of the position of sectioning on the Cx. pipiens pallens tarsomere 1 (t1–t5 = five tarsal segments) [20]. The red line indicates in which leg part the sections were taken. The SEM images show a front view of a sectioned leg for the siNC group (b) and the siCPR63 group (c) The results showed that the procuticle thickness of the siCPR63 group (2.219 ± 0.86  μm) was thinner than that of the siNC group (3.23 ± 0.74  μm; Fig.  6a; Table  1; t = 6.756, p < 0.0001). The exocuticle and endocuticle (114) thickness of the leg tarsus in the siCPR63 was thinner than in the siNC group (Fig.  6b, c; Table  1). Compared with the siNC group, the chitinous parallel laminae, number and size of pores in the siCPR63 group were comparable to the siNC group (Figs. 5a, b; 6d). Discussion Cuticle proteins play an important role in insect cuti- cle resistance, by thickening the cuticle to prevent the Fig. 4 SEM analysis of cuticle thickness. Measurements were made at 23 different points per individual, allowing for the calculation penetration of insecticides and changing the density, of mean cuticle thickness. Results are shown as the mean ± SD; thickness and insect morphological development of the n = the number of measurements carried out on each group of 11 cuticle. There is increasing evidence that alteration of mosquitoes. **p = 0.0011 the cuticle plays a role in insecticide resistance, based on analysis of CP transcripts and measurement of cuticle thickness [19, 21–25]. For example, CYP4G16, Ultrastructure analysis of tarsi segment cuticles in siCPR63 CPLCG3, CPLCG5 and CPLC8 have been implicated in and siNC mosquitoes by TEM insecticide resistance by contributing to a thicker cuti- To explore differences in the cuticle ultrastructure of cle and thereby slowing penetration of insecticides [14, mosquitoes, the region of the tarsus segment was ana- 21, 24–26]. Three CPR genes (CPR124, CPR129 and lysed by TEM. Additional file  1: Figure S1 shows a sche- CPR127) were found to be constitutively overexpressed matic diagram of how images were captured. The leg in resistant Anopheles gambiae [25]. In another study, cuticle is mainly composed of the procuticle, which is 31 cuticle proteins were differentially regulated in the divided into the exocuticle and the endocuticle (Fig.  5). leg proteome, of which 29 including CPR106, CPR126, Table 1 Average cuticle thickness of each component Cuticle Procuticle thickness Procuticle thickness Edocuticle thickness Exocuticle by SEM (μm) by TEM (μm) by TEM (μm) thickness by TEM (μm) Group siNC 2.006 ± 0.73 3.239 ± 0.74 1.489 ± 0.30 1.609 ± 0.22 siCPR63 1.354 ± 0.23 2.219 ± 0.86 0.924 ± 0.48 1.337 ± 0.36 Xu  et al. Parasites & Vectors (2022) 15:54 Page 5 of 7 Fig. 5 TEM analysis of the effects of siNC and siCPR63. SEM images show a front view of a sectioned leg for siNC (a) and siCPR63 (b) groups. The curve represents the chitin parallel laminae, and the circle represents the pores Fig. 6 TEM analysis of cuticle thickness. Measurements were performed at 58 points per individual, allowing for the calculation of mean cuticle thickness. Results are shown as the mean ± SD; n = the number of measurements carried out on each batch of six mosquitoes. ****p ≤ 0.0001; not signifcant, p > 0.05 CPR121 and CPR151 were overexpressed, and only 2 Overexpression of CPRs in resistant mosquitoes were downregulated [22]. Furthermore, > 65% of dif- has been widely reported, but their cuticle resistance ferentially expressed CPs belonged to the CPR family. mechanisms remain poorly understood. Our previ- Strong overexpression of cuticle protein CPR131 was ous study found that silencing the CPR63 gene made also reported in multi-insecticide-resistant A. gambiae mosquitoes more susceptible to deltamethrin, suggest- [27], and CPR63, CPR47, CPR48, CPR45 and CPR44 are ing that CPR63 participates in pyrethroid resistance highly expressed in DR strains of Cx. pipiens pallens [13]. In the present study, our results led us to specu- [13]. late about the resistance mechanism by which CPR63 Xu et al. Parasites & Vectors (2022) 15:54 Page 6 of 7 might contribute to the resistance phenotype; CPR63 In summary, our results revealed that CPR63 might is involved in thickening of the cuticle, and thereby participate in pyrethroid resistance by thickening the possibly increasing the tolerance of mosquitoes to cuticle and thereby possibly increasing the tolerance deltamethrin. of mosquitoes to deltamethrin. This is the first report Insect CPs are diverse and expressed in the head, tho- linking CPRs to insecticide resistance in mosquito legs. rax and abdomen. Some CPs are also highly expressed in insect legs. For example, CPLCG5 is highly expressed in Abbreviations the legs of Cx. pipiens pallens [14], and members of the DR: Deltamethrin-resistant; DS: Deltamethrin-susceptible; PE: Post-eclosion; CPCFC CP family in A. gambiae are mainly distributed CPs: Cuticle proteins; R&R: Rebers and Riddiford; qPCR: Quantitative real-time PCR; siRNA: Small interfering RNA; siCPR63: Small interfering RNA for silencing in legs [28]. Noh et al. found that cuticle protein TcCPR4 the CPR63 gene; NC: Negative control; siNC: Negative control siRNA; SEM: in Tribolium castaneum was mainly enriched in the legs Scanning electron microscopy; TEM: Transmission electron microscopy. and participated in the formation of pore canals in the rigid cuticle [8]. CPF3, CPLCG3, CPLCG4 and CPLCG5 Supplementary Information mRNA transcripts were mainly located in append- The online version contains supplementary material available at https:// doi. ages (legs and wings) [14, 24]. Similarly, in the present org/ 10. 1186/ s13071- 022- 05175-0. study, CPR63 mRNAs were mainly located in mosquito legs and wings. Since these appendages are associated Additional file 1: Figure S1. Schematic diagram showing image capture. with motion, CPR63 might be related to flight. Addi - Additional file 2: Figure S2. Relative expression levels of CPR63 after RNAi silencing in DS strains. tionally, CPR63 was expressed more highly in the legs of DR strains, indicating that it might help mosquitoes Additional file 3: Table S1. Primers used for qPCR analysis and siRNA synthesis of CPR63. avoid areas treated with insecticides, but this hypothesis requires further exploration. Acknowledgements Different CPs play different roles in cuticular resist - Not applicable. ance. Huang et al. reported that CPLCG5 acts as a major CP and is highly expressed in the legs in Cx. pipiens pal- Authors’ contributions YX, XJW, YZ, XXL and YFM performed experiments. YX and YS wrote the lens [14]. Our current results showed that expression of manuscript and prepared the figures. YS, LM, DZ, BS and CLZ conceived the CPR63 was increased in insecticide-resistant Cx. pipiens study and coordinated the project. All authors read and approved the final pallens and also highly expressed in the legs. In addition, manuscript. silencing of CPLCG5 resulted in larger pore canals, indis- Funding tinct chitinous parallel laminae and thinner endocuti- This work was supported by the National Natural Science Foundation of cle in the leg structures. Specifically, silencing of CPR63 China (grant numbers 81672056, 81772227 and 81672058) and the National Critical Project for S & T on Infectious diseases P.R. of China (grant number resulted in thinner endocuticle and exocuticle, but the 2017ZX10303404-002–006). chitinous parallel laminae and number and size of pores are not significantly altered, indicating that different CPs Availability of data and materials All data are fully available without restriction. perform distinct functions to contribute to cuticular resistance, and CPR63 participates in cuticular resistance Declarations mainly by increasing the cuticle thickness. Early studies suggest that RR-1 and RR-2 proteins Ethics approval and consent to participate are present in different regions within the cuticle itself; All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University for the use of laboratory RR-2 proteins contribute to exocuticle, and RR-1 pro- animals (protocol no. 582/2017). teins are found in the endocuticle [29, 30]. However, a more recent study showed that the location of RR-1  s Consent for publication Not applicable. and RR-2  s depends more on the properties of individ- ual proteins [12]. Our current study showed that silenc- Competing interests ing CPR63 led to thinner endocuticle and exocuticle. We The authors declare that they have no competing interests. therefore speculate that CPR63 may be distributed in Author details both the endocuticle and exocuticle, but this hypothesis Department of Pathogen Biology, Nanjing Medical University, Nanjing, China. needs further verification. In addition, our previous study School of Medicine & Holistic Integrative Medicine, Nanjing University of Chi- nese Medicine, Nanjing, China. found that another cuticle protein, CPR47, is also related to insecticide resistance. There may be an interaction Received: 1 August 2021 Accepted: 22 January 2022 between cuticle proteins, but how this affects resistance and whether it is related to CPR63 remain unknown. The work needs further study. Xu  et al. Parasites & Vectors (2022) 15:54 Page 7 of 7 References 25. Yahouédo GA, Chandre F, Rossignol M, Ginibre C, Balabanidou V, Mendez 1. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. NGA, et al. 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Proc R Soc Lond B support for research data, including large and complex data types Biol Sci. 2019;286:1. • gold Open Access which fosters wider collaboration and increased citations 23. Wood O, Hanrahan S, Coetzee M, Koekemoer L, Brooke B. Cuticle thicken- ing associated with pyrethroid resistance in the major malaria vector maximum visibility for your research: over 100M website views per year Anopheles funestus. Parasit Vectors. 2010;3:67. 24. Vannini L, Reed TW, Willis JH. Temporal and spatial expression of cuticular At BMC, research is always in progress. proteins of Anopheles gambiae implicated in insecticide resistance or Learn more biomedcentral.com/submissions differentiation of M/S incipient species. Parasit Vectors. 2014;7:24. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Parasites & Vectors Springer Journals

CPR63 promotes pyrethroid resistance by increasing cuticle thickness in Culex pipiens pallens

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Abstract

The cuticle protein (CP) encoded by CPR63 plays a role in deltamethrin resistance in Culex pipiens pallens. Herein, we investigated the distribution of CPR63 transcripts in this organism and observed high expression levels in legs and wings. Furthermore, expression of CPR63 in the legs of deltamethrin-resistant (DR) strains was 2.17-fold higher than in deltamethrin-susceptible (DS) strains. Cuticle analysis of small interfering RNA (siRNA) groups by scanning electron microscopy (SEM) revealed a significantly thinner cuticle of the tarsi in the siCPR63 group than in the siNC (negative control siRNA) group. Transmission electron microscopy ( TEM) revealed that the exocuticle and endocuticle thickness of the tarsi were significantly thinner, which contributes the thinner procuticle of tarsi in the siCPR63 group than in the siNC group. Our results suggested that CPR63 might contribute to the resistance phenotype by thickening the cuticle and thereby possibly increasing the tolerance of mosquitoes to deltamethrin. Keywords: CPRs, Deltamethrin, Mosquito, Culex pipiens pallens, Cuticular resistance, Cuticle thickening Introduction Research on the mechanism of mosquito vector resist- Targeting insect vectors has proven to be the most effec - ance is of great significance for mosquito vector control. tive means for preventing the spread of mosquito-borne Generally, insecticide resistance in insects is caused by diseases [1, 2]. Chemical insecticides are the most impor- three major mechanisms: (i) reduced sensitivity of the tant component in this effort. However, the spread of target site, (ii) increased activity and/or abundance of insecticide resistance seriously threatens the success and detoxification enzymes and (iii) reduced penetration sustainability of control interventions [3]. According to of insecticides due to altered cuticles [5]. The cuticle is the latest  World malaria report, 73 countries reported believed to function in insecticide resistance by reduc- mosquito resistance to at least one of the four commonly ing or slowing insecticide uptake. Various CPs (CPR, used insecticide classes during 2010−2019, and 28 coun- CPAPn, CPG, CPF and CPLCG) belonging to different tries reported mosquito resistance to all major insecticide protein families have been identified [6, 7]. Some cuti- classes [4]. Therefore, it is critical to develop and apply cle proteins (CPs) play major roles in insecticide resist- effective insecticide resistance management strategies. ance in mosquitoes. For example, previously, CPLCG3, CPLCG4 and CPLCG5 were involved in a putative cuticle thickening mechanism. CPR124, CPR127, CPR129 and CPR131 were found expressed at higher levels in pyre- throid-resistant compared to susceptible mosquitoes [14, *Correspondence: sunyan@njmu.edu.cn Department of Pathogen Biology, Nanjing Medical University, Nanjing, 24, 25, 27]. Most CPs belong to the CPR family and pos- China sess characteristic Rebers and Riddiford (R&R) consensus Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xu et al. Parasites & Vectors (2022) 15:54 Page 2 of 7 sequences (RR-1 and RR-2) that in an extended form control samples were high Ct values (Ct > 35). When confer chitin-binding properties [8–11]. In a recent the primers were used at the first time, qPCR prod - study, the location of RR-1  s and RR-2  s was found to ucts were sequenced for confirmation. The correla - be more dependent on the properties of individual pro- tion coefficients of the calibration curves in each test teins than had been reported in previous work [12]. A were > 0.99. The relative expression levels were nor - previous study in our laboratory investigated expression malised to the internal control  β-actin  by using the −ΔΔCt ΔCt of the CPR63 gene (GenBank: MF095856.1), encoding 2   method [15–18]: target gene/β-actin = 2 , an RR-2 family member in Cx. pipiens pallens, and this ΔCt = Ct − Ct . Three technical and biolog - β-actin target gene was more abundant in deltamethrin-resistant (DR) than ical replicates were performed for qPCR analyses. deltamethrin-susceptible (DS) strains [13]. Furthermore, the mosquito mortality rate was altered by silencing the CPR63 gene [13]. However, the detailed resistance mech- Gene silencing anism of CPR63 related to the mosquito cuticle remains Mosquitoes for RNA interference (RNAi) experiments unknown. In this study, we revealed that CPR63 might were derived from DR and DS female strains microin- participate in pyrethroid resistance by thickening the jected at 12  h PE, with three tubes for each group (10 cuticle and thereby possibly increasing the tolerance of RNAi mosquitoes per tube). A small interfering RNA mosquitoes to deltamethrin. (siRNA) targeting CPR63 (siCPR63) and a siNC (nega- tive control siRNA) were synthesised by GenePharma (Shanghai, China; Additional file  3: Table S1). The siNC Materials and methods does not cause any gene silencing and has no homolo- Mosquito strains gous genes in the mosquito gene bank. About 364 ng of In this study, we collected Cx. pipiens pallens from Tang- siCPR63 and 350  ng of siNC were separately injected kou (Shandong Province, China) as DS strains, and the into the thorax of female mosquitoes. Other details of LC for deltamethrin for these DS strains was 0.03 mg/l. the gene silencing method have been described previ- DR strains were isolated from DS strains with an LC of ously [15]. After 3 days, qPCR was performed to deter- 7.5 mg/l by repeatedly selecting 84 generations at the lar- mine the interference efficiency of the target gene. val stage. Other details were as described in a previous study [14]. Scanning electron microscopy (SEM) RNA extraction and cDNA synthesis To avoid the influence of mosquito size on cuticle We collected heads, thorax, abdomen, and all legs and thickness, we measured the wing length of all female wings of the DS and DR female mosquitoes at 72 h post- mosquitoes in the experiment [31]. There were 11 eclosion (PE) in three tubes for each tissue (20 mosqui- female mosquitoes in each group (siNC, siCPR63), and toes per tube). Extraction of mosquito total RNA was we selected one right front leg from each female mos- performed according to the RNAiso Plus instructions quito. All microinjected mosquito legs were washed (Takara, Shiga, Japan), and the RNA was then converted twice in 70% ethanol to clean them thoroughly. Alco- to cDNA using the PrimeScriptRT Reagent Kit (TaKaRa, hol was dripped onto tarsomere I of the right front leg Tokyo, Japan). at the midpoint, and the leg was cut with a new plat- inum-coated blade and washed again to remove any Quantitative real‑time PCR (qPCR) debris. Legs were then fixed in 2.5% glutaraldehyde cDNA samples were diluted properly with RNase-free (Sigma, St Louis, MO, USA) for 12 h and incubated for water before use as templates in the quantitative PCR 10  min each in a graded ethanol series (30, 50, 70, 80, process using SYBR Green (Applied Biosystems, Foster 90, 95 and 100%). Legs were dried in an EM CPD300 City, CA, USA) according to the manufacturer’s proto- critical point dryer (Leica, Wetzlar, Germany) using an col. The reaction volume (20 μl) contained the Power automated process for 15 exchanges. A K550 X sput- SYBR Green PCR Master Mix, specific forward and ter coater (Electron Microscopy Sciences, Hatfield, reverse primers (Additional file  3: Table S1) and diluted PA, USA) was used for coating samples. A Quanta 250 cDNA. The PCR conditions were as follows: 50  °C for FEI scanning electron microscope was employed, and 2  min and 95  °C for 10  min, followed by 40 cycles at images were recorded at a 3-kV acceleration voltage. 95  °C for 15  s and 60  °C for 1  min. For qPCR valida- The thickness of the cuticle was examined using image tion, the melting curve program was run immediately J software (http:// imagej. net/ Welco me). The average after the qPCR program showed a single-peaked curve. cuticle thickness of each leg was calculated by measur- Amplification signals in the no template or primer ing the distance at 23 randomly selected points. Xu  et al. Parasites & Vectors (2022) 15:54 Page 3 of 7 Transmission electron microscopy (TEM) indicated that CPR63 might play an important role in leg Six female mosquitoes were included in each group resistance. (siNC, siCPR63), and we selected one right front leg from each female mosquito (i.e. six legs per group). Tarsi were divided into four equal parts, and 2–3 images were cap- Expression of CPR63 following gene silencing tured for each part, resulting in 9–10 images for each Expression of CPR63 was evaluated by qPCR in DR leg and 58 images in total. The thickness of the cuticle strains at 12  h PE after injection of siRNA targeting the was examined using image J software. According to the CPR63 gene. Expression of CPR63 was significantly obtained pictures, we also counted the number of pores decreased by 40.9% (t-test, t = 2.935, p = 0.0426) in (4) in tarsi. The other detailed steps of the TEM experiment the whole bodies of mosquitoes and by 37.6% (t-test, have been described in previous studies [14, 19]. t = 3.146, p = 0.0347) in the legs at 72  h PE after (4) siCPR63 injection compared to injection with siNC (Fig.  2a, b). We also found that interference efficiency of Statistical analysis CPR63 in DS strains is not statistically significant com - Experimental data between two groups were analysed pared with siNC group of DS strains, which could be using Student’s t-test. The expression levels of CPR63 in used as a control (Additional file 2: Figure S2). different tissues were calculated using an ANOVA test. All data are presented as the mean ± standard deviation (SD), and p < 0.05 was considered statistically significant. SEM analysis of cuticle thickness All experiments were performed using at least three To probe the changes in the overall cuticle structure, the independent cohorts. region of the tarsus segment was analysed by SEM. The mosquito leg is composed of the femur, tibia and tar- sus, the tarsus is divided into T1, T2, T3, T4 and T5, and Results the cross section was at T1 (Fig.  3a) [19, 20]. The same CPR63 transcripts are abundant in DR mosquito legs number of specimens was assessed in siCPR63 and siNC To explore the function of CPR63, we examined localisa- groups (n = 11). Mosquito leg tarsi were assessed by com- tion of CPR63 expression in multiple tissues at 72  h PE paring the size of the mosquito wing and measuring the by qPCR in DS and DR strains, including the heads, tho- area of the inner and outer circles of the mosquito leg rax, abdomen, legs and wings. The results showed that cuticle. The results showed that the tarsi were a similar CPR63 was highly expressed in legs. Expression of CPR63 size in the two groups, but they were significantly thinner in the legs of DR strains was 2.17-fold higher (ANOVA, in the siCPR63 than in the siNC group (Fig.  3b, c). The p < 0.0001) than those in DS strains (Fig.  1). The result cuticle thickness was measured at no fewer than 23 ran- showed that CPR63 was highly expressed in legs and dom points to obtain the average cuticle thickness of the tarsus. The measurement results showed that the mean cuticle thickness of the siCPR63 group (1.354 ± 0.23  μm) was thinner than that of the siNC group (2.006 ± 0.73 μm; Fig. 4; Table 1; t-test, t = 3.981, p = 0.0011). (44) Fig. 1 Expression profiles of CPR63 in different mosquito tissues. Constitutive expression of CPR63 in DS and DR strains; mRNA expression levels were measured in the head, thorax, abdomen, legs and wings in DS and DR strain mosquitos. Head of DS strain Fig. 2 Relative expression levels of CPR63 after RNAi silencing. Levels was ascribed an arbitrary value of 1. Results are presented as the of CPR63 expression in whole mosquito bodies (a) and legs (b) after mean ± standard deviation (SD) of three biological replicates. silencing of CPR63 were measured by qPCR. Results are shown as the ****p ≤ 0.0001; ns, not signifcant, p > 0.05 mean ± SD of three biological replicates. *p ≤ 0.05; **p ≤ 0.01 Xu et al. Parasites & Vectors (2022) 15:54 Page 4 of 7 Fig. 3 SEM analysis of the effects of siNC and siCPR63. a Illustration of the position of sectioning on the Cx. pipiens pallens tarsomere 1 (t1–t5 = five tarsal segments) [20]. The red line indicates in which leg part the sections were taken. The SEM images show a front view of a sectioned leg for the siNC group (b) and the siCPR63 group (c) The results showed that the procuticle thickness of the siCPR63 group (2.219 ± 0.86  μm) was thinner than that of the siNC group (3.23 ± 0.74  μm; Fig.  6a; Table  1; t = 6.756, p < 0.0001). The exocuticle and endocuticle (114) thickness of the leg tarsus in the siCPR63 was thinner than in the siNC group (Fig.  6b, c; Table  1). Compared with the siNC group, the chitinous parallel laminae, number and size of pores in the siCPR63 group were comparable to the siNC group (Figs. 5a, b; 6d). Discussion Cuticle proteins play an important role in insect cuti- cle resistance, by thickening the cuticle to prevent the Fig. 4 SEM analysis of cuticle thickness. Measurements were made at 23 different points per individual, allowing for the calculation penetration of insecticides and changing the density, of mean cuticle thickness. Results are shown as the mean ± SD; thickness and insect morphological development of the n = the number of measurements carried out on each group of 11 cuticle. There is increasing evidence that alteration of mosquitoes. **p = 0.0011 the cuticle plays a role in insecticide resistance, based on analysis of CP transcripts and measurement of cuticle thickness [19, 21–25]. For example, CYP4G16, Ultrastructure analysis of tarsi segment cuticles in siCPR63 CPLCG3, CPLCG5 and CPLC8 have been implicated in and siNC mosquitoes by TEM insecticide resistance by contributing to a thicker cuti- To explore differences in the cuticle ultrastructure of cle and thereby slowing penetration of insecticides [14, mosquitoes, the region of the tarsus segment was ana- 21, 24–26]. Three CPR genes (CPR124, CPR129 and lysed by TEM. Additional file  1: Figure S1 shows a sche- CPR127) were found to be constitutively overexpressed matic diagram of how images were captured. The leg in resistant Anopheles gambiae [25]. In another study, cuticle is mainly composed of the procuticle, which is 31 cuticle proteins were differentially regulated in the divided into the exocuticle and the endocuticle (Fig.  5). leg proteome, of which 29 including CPR106, CPR126, Table 1 Average cuticle thickness of each component Cuticle Procuticle thickness Procuticle thickness Edocuticle thickness Exocuticle by SEM (μm) by TEM (μm) by TEM (μm) thickness by TEM (μm) Group siNC 2.006 ± 0.73 3.239 ± 0.74 1.489 ± 0.30 1.609 ± 0.22 siCPR63 1.354 ± 0.23 2.219 ± 0.86 0.924 ± 0.48 1.337 ± 0.36 Xu  et al. Parasites & Vectors (2022) 15:54 Page 5 of 7 Fig. 5 TEM analysis of the effects of siNC and siCPR63. SEM images show a front view of a sectioned leg for siNC (a) and siCPR63 (b) groups. The curve represents the chitin parallel laminae, and the circle represents the pores Fig. 6 TEM analysis of cuticle thickness. Measurements were performed at 58 points per individual, allowing for the calculation of mean cuticle thickness. Results are shown as the mean ± SD; n = the number of measurements carried out on each batch of six mosquitoes. ****p ≤ 0.0001; not signifcant, p > 0.05 CPR121 and CPR151 were overexpressed, and only 2 Overexpression of CPRs in resistant mosquitoes were downregulated [22]. Furthermore, > 65% of dif- has been widely reported, but their cuticle resistance ferentially expressed CPs belonged to the CPR family. mechanisms remain poorly understood. Our previ- Strong overexpression of cuticle protein CPR131 was ous study found that silencing the CPR63 gene made also reported in multi-insecticide-resistant A. gambiae mosquitoes more susceptible to deltamethrin, suggest- [27], and CPR63, CPR47, CPR48, CPR45 and CPR44 are ing that CPR63 participates in pyrethroid resistance highly expressed in DR strains of Cx. pipiens pallens [13]. In the present study, our results led us to specu- [13]. late about the resistance mechanism by which CPR63 Xu et al. Parasites & Vectors (2022) 15:54 Page 6 of 7 might contribute to the resistance phenotype; CPR63 In summary, our results revealed that CPR63 might is involved in thickening of the cuticle, and thereby participate in pyrethroid resistance by thickening the possibly increasing the tolerance of mosquitoes to cuticle and thereby possibly increasing the tolerance deltamethrin. of mosquitoes to deltamethrin. This is the first report Insect CPs are diverse and expressed in the head, tho- linking CPRs to insecticide resistance in mosquito legs. rax and abdomen. Some CPs are also highly expressed in insect legs. For example, CPLCG5 is highly expressed in Abbreviations the legs of Cx. pipiens pallens [14], and members of the DR: Deltamethrin-resistant; DS: Deltamethrin-susceptible; PE: Post-eclosion; CPCFC CP family in A. gambiae are mainly distributed CPs: Cuticle proteins; R&R: Rebers and Riddiford; qPCR: Quantitative real-time PCR; siRNA: Small interfering RNA; siCPR63: Small interfering RNA for silencing in legs [28]. Noh et al. found that cuticle protein TcCPR4 the CPR63 gene; NC: Negative control; siNC: Negative control siRNA; SEM: in Tribolium castaneum was mainly enriched in the legs Scanning electron microscopy; TEM: Transmission electron microscopy. and participated in the formation of pore canals in the rigid cuticle [8]. CPF3, CPLCG3, CPLCG4 and CPLCG5 Supplementary Information mRNA transcripts were mainly located in append- The online version contains supplementary material available at https:// doi. ages (legs and wings) [14, 24]. Similarly, in the present org/ 10. 1186/ s13071- 022- 05175-0. study, CPR63 mRNAs were mainly located in mosquito legs and wings. Since these appendages are associated Additional file 1: Figure S1. Schematic diagram showing image capture. with motion, CPR63 might be related to flight. Addi - Additional file 2: Figure S2. Relative expression levels of CPR63 after RNAi silencing in DS strains. tionally, CPR63 was expressed more highly in the legs of DR strains, indicating that it might help mosquitoes Additional file 3: Table S1. Primers used for qPCR analysis and siRNA synthesis of CPR63. avoid areas treated with insecticides, but this hypothesis requires further exploration. Acknowledgements Different CPs play different roles in cuticular resist - Not applicable. ance. Huang et al. reported that CPLCG5 acts as a major CP and is highly expressed in the legs in Cx. pipiens pal- Authors’ contributions YX, XJW, YZ, XXL and YFM performed experiments. YX and YS wrote the lens [14]. Our current results showed that expression of manuscript and prepared the figures. YS, LM, DZ, BS and CLZ conceived the CPR63 was increased in insecticide-resistant Cx. pipiens study and coordinated the project. All authors read and approved the final pallens and also highly expressed in the legs. In addition, manuscript. silencing of CPLCG5 resulted in larger pore canals, indis- Funding tinct chitinous parallel laminae and thinner endocuti- This work was supported by the National Natural Science Foundation of cle in the leg structures. Specifically, silencing of CPR63 China (grant numbers 81672056, 81772227 and 81672058) and the National Critical Project for S & T on Infectious diseases P.R. of China (grant number resulted in thinner endocuticle and exocuticle, but the 2017ZX10303404-002–006). chitinous parallel laminae and number and size of pores are not significantly altered, indicating that different CPs Availability of data and materials All data are fully available without restriction. perform distinct functions to contribute to cuticular resistance, and CPR63 participates in cuticular resistance Declarations mainly by increasing the cuticle thickness. Early studies suggest that RR-1 and RR-2 proteins Ethics approval and consent to participate are present in different regions within the cuticle itself; All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University for the use of laboratory RR-2 proteins contribute to exocuticle, and RR-1 pro- animals (protocol no. 582/2017). teins are found in the endocuticle [29, 30]. However, a more recent study showed that the location of RR-1  s Consent for publication Not applicable. and RR-2  s depends more on the properties of individ- ual proteins [12]. Our current study showed that silenc- Competing interests ing CPR63 led to thinner endocuticle and exocuticle. We The authors declare that they have no competing interests. therefore speculate that CPR63 may be distributed in Author details both the endocuticle and exocuticle, but this hypothesis Department of Pathogen Biology, Nanjing Medical University, Nanjing, China. needs further verification. In addition, our previous study School of Medicine & Holistic Integrative Medicine, Nanjing University of Chi- nese Medicine, Nanjing, China. found that another cuticle protein, CPR47, is also related to insecticide resistance. There may be an interaction Received: 1 August 2021 Accepted: 22 January 2022 between cuticle proteins, but how this affects resistance and whether it is related to CPR63 remain unknown. The work needs further study. Xu  et al. Parasites & Vectors (2022) 15:54 Page 7 of 7 References 25. Yahouédo GA, Chandre F, Rossignol M, Ginibre C, Balabanidou V, Mendez 1. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. NGA, et al. 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Journal

Parasites & VectorsSpringer Journals

Published: Feb 14, 2022

Keywords: CPRs; Deltamethrin; Mosquito; Culex pipiens pallens; Cuticular resistance; Cuticle thickening

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