INTRODUCTION
The cattle tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite of major importance for the livestock industry. Feeding by large numbers of ticks causes reduction in live weight gain and milk production as well as deterioration of the skin on parasitized cattle. Further, R. microplus transmits diseases of economic importance such as babesiosis and anaplasmosis (Sharma et al. Reference Sharma, Kumar, Kumar, Nagar, Singh, Rawat, Dhakad, Rawat, Ray and Ghosh2012). Its remarkable ability to survive without feeding over long periods makes its control and eradication difficult.
Under optimal environmental conditions R. microplus is able to complete its life cycle in 3–4 weeks; however, in cooler temperatures larvae may survive without feeding for up to 24 weeks (Spickler, Reference Spickler2007). The mechanisms used to survive long-term starvation are poorly understood in ticks. However, it is believed that autophagy may play a significant role, as a strategy to confront adverse environmental conditions (Umemiya-Shirafuji et al. Reference Umemiya-Shirafuji, Matsuo, Liao, Boldbaatar, Battur, Suzuki and Fujisaki2010).
Macroautophagy (hereafter referred to as autophagy) is a cytoplasmic degradation process where organelles and proteins undergo proteolysis to obtain polypeptides in order to maintain amino acid pools and energy balance in the cell. Autophagy is regulated by a set of evolutionarily conserved gene products, known as the ATG proteins (Mizushima et al. Reference Mizushima, Yoshimori and Levine2010). Among ATG genes one subset, composed of 17 genes, is required for efficient autophagosome formation. The corresponding gene products are referred to as the core machinery for autophagosome formation (Xie and Klionsky, Reference Xie and Klionsky2007).
In a previous work, we reported the first molecular characterization of two autophagy-related genes, RmATG8a and RmATG8b, homologues of mammalian GABARAP and MAP1LC3, respectively, in R. microplus. We found that transcripts of both RmATG8a and RmATG8b were up-regulated during starvation, suggesting that autophagy was active in R. microplus (Flores et al. Reference Flores Fernández, Gutiérrez Ortega, Rosario Cruz, Padilla Camberos, Alvarez and Martínez Velázquez2014). To advance our understanding of autophagy in this tick, in the present study we report the molecular characterization of RmATG3, RmATG4 and RmATG6 as well as their expression profiles in different developmental stages and organs of the parasite. The products of all of these genes are part of the core machinery for the autophagosome formation.
The autophagy process seen as a survival strategy of ticks could also be important for the survival of pathogens they transmit. Therefore, targeting autophagy mediators could lead to the development of novel control strategies for ticks and associated pathogens.
MATERIALS AND METHODS
Ticks and tissues
Rhipicephalus microplus ticks were obtained from a susceptible strain reared under laboratory conditions at CENID-PAVET-INIFAP, located in the State of Morelos, Mexico. Ovarian and intestinal tissues were obtained from engorged female ticks the following day after the spontaneous detachment from calves, as described by Tsuda et al. (Reference Tsuda, Mulenga, Sugimoto, Nakajima, Ohashi and Onuma2001). Some engorged female ticks were maintained at 28 °C and 80% relative humidity (RH) to collect eggs 8 days after oviposition and larvae 8 days after hatching.
Treatment of eggs
100 mg of eggs were treated with 5 volumes of 3·8% NaClO for 2 min and centrifuged at 370 g for 2 min. The supernatant was removed and eggs were resuspended in 5 volumes of 3·8% NaClO and incubated for 10 min. Floating eggs were removed and the remaining eggs were rinsed 5 times with 5 volumes of distilled water.
RNA isolation and cDNA synthesis
Total RNA was extracted from eggs, larvae and adult tissues using RNeasy Mini Kit (Qiagen, Germany). Briefly, midgut and ovary tissues were dissected and pooled from 15–20 engorged females; tissues were maintained on ice-cold phosphate-buffered saline immediately after dissection and then stored at −80 °C until RNA extraction. Pools of 100 mg eggs (approximately 2000 eggs) and larvae (approximately 1000 larvae) were collected. Eggs were treated as mentioned previously before RNA extraction. Samples were mixed and crushed with liquid nitrogen. RNA integrity was assessed by denaturing electrophoresis on 1% agarose gels and quantified by a ND-1000 NanoDrop spectrophotometer (NanoDrop Products, Wilmington, DE, USA). cDNA was synthesized from 2 µg of RNA using the SuperScript III® First-Strand Synthesis System (Invitrogen) according to manufacturer's instructions.
Characterization of the complete coding sequences
To obtain the complete coding sequence of RmATG3, RmATG4 and RmATG6 we started from the predicted putative sequences reported by Flores-Fernández et al. (Reference Flores Fernández, Gutiérrez Ortega, Rosario Cruz, Padilla Camberos, Alvarez and Martínez Velázquez2014). Briefly, the nucleotide sequences of HlATG3, HlATG4 and HlATG6 genes from Haemaphysalis longicornis tick (GenBank accession numbers AB513349, AB513350 and AB601889, respectively) were queried against the GenBank R. microplus Expressed Sequence Tags (EST) database, using the Basic Local Alignment Search Tool (BLAST) tool. We retrieved the nucleotide sequences CK182322 (EST771642) and CV442344 (EST896257) for ATG3; CK181180 (EST770500) and CK181181 (EST770501) for ATG4; CV442171 (EST896084) and CV442170 (EST896083) for ATG6 and proceeded with their characterization. Adopting the nomenclature followed by Umemiya-Shirafuji et al. (Reference Umemiya-Shirafuji, Matsuo, Liao, Boldbaatar, Battur, Suzuki and Fujisaki2010) to name tick ATG genes, we designated the characterized genes as RmATG3, RmATG4 and RmATG6, respectively. Specific primers were designed to the above sequences (online Table S1). The forward RmATG4 primer was designed from a conserved region of the sequence of HlATG4 of H. longicornis and two Expressed Sequence Tag (EST) sequences of Ixodes scapularis and Amblyomma americanum (GenBank accession no. AB513350, EW946975 and JZ176775, respectively). End-point polymerase chain reaction (PCR) reactions were performed using cDNA synthesized from larvae as a template. PCR conditions were 94 °C for 5 min followed by 35 cycles, each consisting of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s. A final extension step at 72 °C for 10 min was included. The amplified products were visualized on 1% agarose gels on a UV transilluminator (Bio-Rad Laboratories, Philadelphia, PA, USA).
Cloning and sequencing
To confirm positive PCR products, amplicons were purified and then cloned into the pCR®2.1-TOPO T/A plasmid vector (Invitrogen, Carlsbad, CA, USA) which was used to transform electro-competent Escherichia coli TOP10 cells. Escherichia coli cells were then incubated overnight at 37 °C on LB plates containing ampicillin (50 µg mL−1) and 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal; 40 mg mL−1). Five white colonies were isolated and cultured overnight in LB medium containing 50 µg mL−1 ampicillin. Plasmid DNA was extracted using QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. To confirm the positive transformants the plasmids were analyzed by PCR using forward and reverse M13 primers. Once the correct clones were identified, the plasmids were submitted for sequencing at Macrogen Corp. (Rockville, Maryland, USA). The new sequences of RmATG3, RmATG4 and RmATG6 were deposited in GenBank with accession number KP317124, KR822806 and KR822807, respectively.
Gene expression by quantitative PCR
To determine the expression profiles of RmATG3, RmATG4 and RmATG6 genes in eggs, larvae and adult tissues of R. microplus, specific primers were designed (online Table S1). Elongation factor 1-α (ELFIA) and β-actin (ACTB) genes were used as reference genes (Nijhof et al. Reference Nijhof, Balk, Postigo and Jongejan2009). Real-time PCR amplifications were performed in 96-well-plates using SYBR Green detection chemistry in a StepOnePlus™ Real-time PCR System (Applied Biosystems/Ambion, Austin, TX, USA). Reactions were prepared in a total volume of 15 µL containing 0·4 µL of first strand cDNA template (100 ng µL−1), 0·45 µL of forward and reverse primers (10 µ m), 7·5 µL of SYBR® Select Master Mix (Applied Biosystems) and 6·2 µL of sterile deionized water. The cycling conditions were set as follows: initial denaturation step at 95 °C for 10 min to activate the AmpliTaq® DNA Polymerase, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s. At the end of PCR amplification, a melting curve analysis was performed immediately to confirm the specificity of the reactions. Baseline and threshold cycles (Ct) were automatically determined using Real-Time PCR System software. PCR reactions were run in triplicate for each sample in three independent experiments. Relative expression was calculated using the comparative cycle threshold method (Livak and Schmittgen, Reference Livak and Schmittgen2001). The transcript abundance data were normalized by the average transcript abundance of two endogenous control genes, ELFIA and ACTB, between each sample.
Bioinformatic analysis
Primers were designed with the Primer3 software v.4.0.0 (http://primer3.wi.mit.edu/; Rozen and Skaletsky, Reference Rozen, Skaletsky, Krawetz and Misener2000). The search for similarity among sequences was conducted by the BLAST service from the National Center for Biotechnology Information (NCBI, USA) (http://blast.ncbi.nlm.nih.gov/Blast.cgi; Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990). The nucleotide to aminoacid sequence translation was made using the Translate Tool (http://web.expasy.org/translate/; Gasteiger et al. Reference Gasteiger, Gattiker, Hoogland, Ivanyi, Appel and Bairoch2003). Multiple alignments of the nucleotide and amino acid sequences were performed using the multiple sequence alignment program ClustalW (http://www.genome.jp/tools/clustalw/; Larkin et al. Reference Larkin, Blackshields, Brown, Chenna, McGettigan, McWilliam, Valentin, Wallace, Wilm, Lopez, Thompson, Gibson and Higgins2007). The molecular weight and theoretical isoelectric point were predicted by ProtParam tool (http://web.expasy.org/protparam/; Gasteiger et al. Reference Gasteiger, Hoogland, Gattiker, Duvaud, Wilkins, Appel, Bairoch and Walker John2005).
Statistical analysis
Expression data of RmATG3, RmATG4 and RmATG6 were analyzed by one-way ANOVA using Statgraphics™ 5.1 software. Tukey's test was used to determine significant differences of each gene at different developmental stages and adult tissues in R. microplus. The data represent mean ± s.d. from three independent experiments.
RESULTS
RmATG3
We amplified a PCR product of 1125 bp length. The flanked region is 1085 bp, has an ORF at position 106–1092 and codes for a 328-amino acid polypeptide with a predicted molecular mass of 36·29 kDa and a pI of 4·57 (online Fig. S1). The deduced polypeptide appears to have no signal peptide. The protein is similar in length to Atg3 from H. longicornis and I. scapularis. The identified protein was queried against the non-redundant protein database using the BLAST tool. BLAST analysis showed 86·23, 80·49, 65·53, 64·70, 60·67 and 60·06% identities between RmAtg3 and HlAtg3 of H. longicornis, putative Atg3 of I. scapularis and Atg3 of Spodoptera litura, Bombyx mori, Aedes aegypti and Drosophila melanogaster, respectively. All compared sequences contain three predicted domains, the autophagocytosis associated protein (Atg3), N-terminal domain (Autophagy_N), autophagocytosis associated protein, active-site domain (Autophagy_act_C) and the autophagocytosis associated protein C-terminal (Autophagy_Cterm) domain (Fig. 1). The region of active-site cysteine residue is conserved between RmAtg3 and other Atg3s within Autophagy_act_C domain (Fig. 1).
Fig. 1. Alignment of the deduced amino acid sequence of RmAtg3 (accession no. AKH60756), HlAtg3 of Haemaphysalis longicornis (accession no. BAI82575), putative Atg3 of Ixodes scapularis (accession no. XP_002402769), Atg3 of Spodoptera litura (accession no. AFS31123), Atg3 of Bombyx mori (accession no. NP_001135961), Aedes aegypti (accession no. XP_001657463) and Drosophila melanogaster (accession no. NP_649059). Autophagy_N, Autophagy_act_C and Autophagy_Cterm domains are shaded in gray dark, gray light and gray dark, respectively. The region of active-site cysteine residue 17 is boxed. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties.
RmATG4
We amplified a PCR product of 1263 bp length. The flanked region is 1221 bp and it has an ORF at position 13–1176 that codes for a 387-amino acid polypeptide with a predicted molecular mass of 44·30 kDa and a pI of 5·22 (online Fig. S2). No signal peptide was found in the predicted polypeptide. The protein is similar in length to Atg4 s from other species. The deduced sequence was subjected to BLAST analysis. Among sequences that produced significant alignments, RmAtg4 showed an identity of 82·94, 71·20, 56·84, 49·08 and 48·32% with HlAtg4 of H. longicornis, putative Atg4 of I. scapularis, cysteine peptidase 2 family C54 protein of Tityus serrulatus, cysteine protease ATG4A of Coptotermes formosanus and cysteine protease ATG4B of Zootermopsis nevadensis, respectively. All compared sequences contain a Peptidase_C54 domain. Putative active sites, cysteine (C), aspartic acid (D) and histidine (H) at positions 76, 270 and 272 of RmAtg4, respectively, are widely conserved in all Atg4 homologues (Fig. 2).
Fig. 2. Alignment of the deduced amino acid sequence of RmAtg4 (accession no. ALK28521), HlAtg4 of Haemaphysalis longicornis (accession no. BAI82576), putative Atg4 of Ixodes scapularis (accession no. XP_002434192), cysteine peptidase 2 family C54 protein of Tityus serrulatus (accession no. CDJ26740), cysteine protease ATG4A of Coptotermes formosanus (accession no. AGM32346) and Cysteine protease ATG4B of Zootermopsis nevadensis (accession no. KDR21327). Peptidase_C54 domain is shaded in gray. The putative active sites (Cys, Asp and His) are boxed. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties.
RmATG6
We amplified a PCR product of 1575 bp length which was cloned and sequenced. The flanked region is 1537 bp, and it has an ORF at position 133–1470 that codes for a 445-amino acid polypeptide with a predicted molecular mass of 51·44 kDa and a pI of 5·03 (online Fig. S3). No signal peptide was found in the predicted polypeptide. The protein is 19 aa smaller than HlAtg4 from H. longicornis but 2 aa larger than that from I. scapularis. The deduced sequence of RmAtg6, subjected to BLAST analysis, showed 84·94, 71·55, 51·68, 51·74 and 51·50% identities with HlAtg6 of H. longicornis, putative Beclin of I. scapularis and Stegodyphus mimosarum, and Beclin-1-like protein isoform X2 and X1 of Camponotus floridanus and Cerapachys biroi, respectively. All compared sequences contain the autophagy protein Apg6 domain (Fig. 3).
Fig. 3. Alignment of the deduced amino acid sequence of RmAtg6 (accession no. ALK28522), HlAtg6 of Haemaphysalis longicornis (accession no. BAK26532), putative Beclin of Ixodes scapularis and Stegodyphus mimosarum (accession no. XP_002414849 and KFM67317, respectively) and Beclin-1-like protein isoform X2 and X1 of Camponotus floridanus and Cerapachys biroi (accession no. XP_011257590 and XP_011344236, respectively). Autophagy protein Apg6 domain is shaded in gray. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties.
Expression of RmATG genes in different development stages of R. microplus
The expression of RmATG genes showed a different pattern among development stages (Fig. 4). In midgut, RmATG3 expression was 2-fold higher than in ovary, and egg and larva stages. In egg and ovary, RmATG4 was overexpressed ~10-fold and 20-fold, respectively, compared to larva and midgut. Finally, RmATG6 showed the highest expression level in ovary, followed by egg, midgut and larva.
Fig. 4. RmATG3, RmATG4 and RmATG6 mRNA expression in different development stages of Rhipicephalus microplus. Real-time polymerase chain reaction (PCR) reactions were performed of total RNA from egg, larva and adult ovary and midgut tissues. Relative gene expression was calculated using the comparative cycle threshold method. The transcript abundance data were normalized by the average transcript abundance of two endogenous control genes, Elongation factor 1-α (ELFIA) and β-actin (ACTB) between each sample. Data represent the mean ± s.d. from three independent biological replicates. Be aware that each graph on the y-axis has a different scale.
DISCUSSION
In a previous work, we reported the molecular characterization of RmATG8a and RmATG8b genes and predicted the existence of the putative ATG genes ATG3, ATG4 and ATG6 in R. microplus (Flores et al. Reference Flores Fernández, Gutiérrez Ortega, Rosario Cruz, Padilla Camberos, Alvarez and Martínez Velázquez2014). To give continuity on those findings, in this work we reported the complete sequences and expression analyses of RmATG3, RmATG4 and RmATG6, providing new insights into autophagy in R. microplus. It is known that autophagy is activated in many species firstly by starvation conditions. The process involves the participation of multiple ATG proteins. To date, over 30 ATG genes, which are involved in various subtypes of macroautophagy, have been identified in yeast (Xie and Klionsky, Reference Xie and Klionsky2007). Among ATG genes one subset, composed of 17 genes, is required for autophagosome formation. However, few ATG genes have been characterized in ticks. The genes HlATG3, HlATG4, HlATG6, HlATG8 and HlATG12 were recently characterized in H. longicornis ticks, while at least seven putative ATG genes (ATG3, ATG5, ATG6, ATG7, ATG8, ATG13 and ATG16) have been found in I. scapularis ticks (Umemiya-Shirafuji et al. Reference Umemiya-Shirafuji, Matsuo, Liao, Boldbaatar, Battur, Suzuki and Fujisaki2010).
ATG3, ATG4 and ATG6 gene products, among others, are involved in autophagosome formation. Upon autophagosome induction ATG6 is recruited for vesicle nucleation (autophagosome biogenesis), while ATG3 and ATG4 participate in the elongation step (autophagosome maturation).
Even though the autophagy process is regulated by many ATG proteins, several studies have revealed that a complete set of ATGs may not be necessary in all organisms; some ATG proteins are not essential for starvation-induced autophagy, as is the case of the homologs ULK1 and ULK2 in the chicken, or perform a dual function; for example, in B. mori and other lepidoptera insects it is hypothesized that the function of ATG10 could be compensated for by ATG3 (Zhang et al. Reference Zhang, Hu, Li, Li, Deng, Yang, Cao and Zhou2009; Mizushima and Sahani, Reference Mizushima and Sahani2014). Given the central role that ATG3, ATG4 and ATG6 play in the autophagosome formation, the characterization of their genes provides crucial information to better understand the autophagy process in R. microplus.
The autophagy pathway is not only involved in starvation, but can also act in such diverse processes as cell death, immunity, embryogenesis, development, growth and nutrient utilization (McPhee and Baehrecke, Reference McPhee and Baehrecke2009). In this study, the expression levels of RmATG4 and RmATG6 genes were found elevated in egg and ovary tissue, while RmATG3 showed lower expression; these results are consistent with previous studies in H. longicornis tick, excepting the low expression of RmATG3 (Kawano et al. Reference Kawano, Umemiya-Shirafuji, Boldbaatar, Matsuoka, Tanaka and Fujisaki2011; Umemiya-Shirafuji et al. Reference Umemiya-Shirafuji, Galay, Maeda, Kawano, Tanaka, Fukumoto, Suzuki, Tsuji and Fujisaki2014). Our results, showing a similar gene expression pattern, suggest an involvement of the autophagy process in embryogenesis of R. microplus, as previously has been described in H. longicornis, where the silencing of ATG6 mRNA affected the reproductive and ovipositional systems (Kawano et al. Reference Kawano, Umemiya-Shirafuji, Boldbaatar, Matsuoka, Tanaka and Fujisaki2011).
To deepen the study of autophagy in R. microplus, a combination of methods such as real-time PCR, western blotting, immunostaining and transmission electronic microscopy, as well as RNAi-mediated gene silencing to analyze the function of ATG genes, are recommended. Moreover, a R. microplus genome sequencing project is an urgent need to characterize the complete set of genes that regulate autophagy in this tick.
SUPPLEMENTARY MATERIAL
The supplementary material for this article can be found at http://dx.doi.org/10.1017/S0031182016001542.
ACKNOWLEDGEMENTS
The authors thank Kirk Allen for support in drafting and revising this manuscript.
FINANCIAL SUPPORT
This work was supported by Consejo Nacional de Ciencia y Tecnología México (CONACYT; grant numbers 216321 and 246310, both to M.M.V.). JMFF and CPBA received a scholarship from CONACYT.
