Transcriptoma do intestino de fêmeas de <i>Ornithodoros mimon</i> (Acari: Argasidae) e inferência filogenética de carrapatos

Landulfo, Gabriel Alves; Patané, José Salvatore Leister; Silva, Dalton Giovanni Nogueira da; Junqueira-de-Azevedo, Inácio Loiola Meirelles; Mendonca, Ronaldo Zucatelli; Simons, Simone Michaela; Carvalho, Eneas de; Barros-Battesti, Darci Moraes

doi:10.1590/S1984-29612017027

Gabriel Alves Landulfo

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil

Programa de Pós-graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Universidade Federal Rural Rio de Janeiro – UFRRJ, Seropédica, RJ, Brasil

José Salvatore Leister Patané

Laboratório de Bioinformática, Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo – USP, São Paulo, SP, Brasil

Dalton Giovanni Nogueira da Silva

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil

Inácio Loiola Meirelles Junqueira-de-Azevedo

Laboratório Especial de Toxinologia Aplicada, Instituto Butantan, São Paulo, SP, Brasil

Ronaldo Zucatelli Mendonca

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil

Simone Michaela Simons

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil

Eneas de Carvalho

Laboratório de Biotecnologia, Instituto Butantan, São Paulo, SP, Brasil

Darci Moraes Barros-Battesti ^**Corresponding author: Darci Moraes Barros-Battesti. Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil, 150l, CEP 05503-900, São Paulo, SP, Brasil. e-mail: darci.battesti@butantan.gov.br

Laboratório de Parasitologia, Instituto Butantan, São Paulo, SP, Brasil

Programa de Pós-graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Universidade Federal Rural Rio de Janeiro – UFRRJ, Seropédica, RJ, Brasil

*Corresponding author: Darci Moraes Barros-Battesti. Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil, 150l, CEP 05503-900, São Paulo, SP, Brasil. e-mail: darci.battesti@butantan.gov.br

Figuras (5)
Tabelas (2)

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Figure 1
Summary results from BLAST (BLASTX hits) and annotation in the BLAST2GO (B2G). The sequences that presented BLASTX hit were represented in the With BLAST hit group. Annotated complete comprises the group of sequences that showed relevant similarity hits in BLAST and in Gene Ontology (GO). Without GO comprises the sequences that were identified by means of BLASTX, but they did not present GO hits in relation to the functional mapping terms. Without BLAST hit is the group of sequences that did not present similarity with any database sequences.

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Figure 2
Principal functional activities assigned to the contigs. The annotated transcripts (contigs) were grouped in different functional activities in accordance with their biological function assignment.

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Figure 3
Multiple alignment to compare the cathepsin O-like of O. mimon with selected cathepsin O sequences that showed hits in the BLAST search, highlighting (black rectangle) the conserved motif of eukaryotic thiol (cysteine) protease active site of cathepsin O. These conserved motifs of the protein coded have 12 amino acid residues, and the asterisk (*) represents the conserved peptides residues between the protein coded sequences. Alignments were conducted using MAFFT algorithm with default parameters.

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Figure 4
Phylogenetic tree of cathepsin peptidases. Phylogenetic comparison of O. mimon peptidases with selected proteinases that showed hits in the BLAST search. The tree was constructed using the maximum likelihood (ML) method and multiple alignment of peptidase amino acid sequences. Some branches were collapsed to reduce the size of the tree. The complete phylogenetic tree of the peptidases can be observed on the Supplementary Material (Figure S1). The O. mimon peptidase are highlighted in blue. Aspartic peptidases group was used as outgroup to rooting the phylogenetic tree.

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Figure 5
Phylogenetic relationships of ticks, based on 425 ML trees from transcripts of tick transcriptomes, constructed in MulRF. The blue and red bars represent the Ornithodorinae and Argasinae subfamilies, respectively. Ornithodorinae is divided in two groups, Ornithodoros s.s (brown bar) and Neotropical Ornithodorinae (green bar). Metaseiulus occidentalis mite specie was used as outgroup in the phylogenetic relationships of tick species.

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Table 1
Categorization of the transcripts of female gut of O. mimon, according to functional and biological assignment. In bold are highlighted the categories of higher-level expression.

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Table 2
Categories of transcripts potentially involved in blood meal digestion and nutrient uptake.

Categories	Number of contigs	%	Level of RPKM	Total of Reads	%
Amino acid metabolism	7	0.31	3,475.77	223	0.62
Apoptosis	12	0.54	4,000.15	136	0.38
Carbohydrate metabolism	19	0.85	6,578.08	399	1.10
Catalytic activity	390	17.45	115,983.49	5256	14.54
Cell cycle regulatory mechanism (mitosis)	23	1.03	10,003.50	395	1.09
Cytoskeleton	50	2.24	25,052.17	1137	3.15
Energy metabolism	63	2.82	58,631.91	3096	8.57
Enzyme inhibitor	3	0.13	1,322.80	88	0.24
Extracellular matrix and adhesion	25	1.12	8,284.17	478	1.32
Immunity	4	0.18	2,480.54	41	0.11
Lipid metabolism	17	0.76	8,966.18	395	1.09
Nuclear regulation	53	2.37	26,845.04	925	2.56
Oxidant metabolism/detox	70	3.13	27,302.06	1101	3.05
Peptidase inhibitor	17	0.76	11,049.58	395	1.09
Peptidases	67	3.00	42,802.16	1808	5.00
Proteasome machinery	19	0.85	4,869.57	157	0.43
Protein export machinery	3	0.13	1,037.33	26	0.07
Protein modification mechanism	42	1.88	19,357.70	1056	2.92
Protein synthesis	4	0.18	3,538.37	118	0.33
Replication machinery	20	0.89	9,499.16	344	0.95
Ribosomal proteins	27	1.21	10,587.92	374	1.03
Signal transduction	33	1.48	9,390.29	322	0.89
Signaling protein	8	0.36	2,034.22	83	0.23
Storage	6	0.27	5,618.01	175	0.48
Ticks Salivary proteins	12	0.54	4,729.74	141	0.39
Transcription factor	41	1.83	18,161.76	728	2.01
Transcription mechanism	64	2.86	22,813.75	793	2.19
Translation mechanism	60	2.68	31,904.46	1589	4.40
Transporter channel	246	11.01	90,811.27	4518	12.50
Transposable element	5	0.22	1,507.35	44	0.12
Unknown	825	36.91	331,830.85	9804	27.12
Total	2235	100	920,469.35	36145	100

Contig names/Categories	Sequence Description	Putative functions	RPKM	Accession Number	BLASTX-best match NR database
Peptidases
Cysteine
Contig5313, Contig5218	cathepsin B	cysteine-type endopeptidase activity	3,006.84;1,473.44	GEIB01001153.1;GEIB01001273.1	cathepsin B endopeptidase, putative [I. scapularis]
Contig4241, Contig4886	cathepsin L	cysteine-type endopeptidase activity	1,086.54;998.78	GEIB01000021.1;GEIB01000047.1	predicted: cathepsin L-like [S. purpuratus]; cathepsin L-like cysteine protease [I. ricinus]
Contig653	cathepsin C	cysteine-type peptidase activity	686.42	GEIB01001207.1	cathepsin C precursor [I. ricinus]
Contig2830	cathepsin O	cysteine-type peptidase activity	559.2	GEIB01000303.1	cysteine protease and A protease inhibitor, putative [I. scapularis]
Contig957	midgut cysteine	cysteine-type peptidase activity	1,258.64	GEIB01000015.1	midgut cysteine proteinase, putative [I. scapularis]
Contig5200, Contig4866, Contig1966, Contig5258	legumain-like protein	peptidase activity	811.89;721.65; 702.19;510.71	GEIB01001220.1;GEIB01001229.1; GEIB01000064.1; GEIB01000075.1	legumain-like protease precursor [I. ricinus]
Serine
Contig4026, Contig4983	chymotrypsin-like elastase member 3b-like	serine-type endopeptidase activity	8,454.53;1,223.48	GEIB01001133.1; GEIB01000056.1	serine protease-like protein precursor [O. moubata]
Contig5086	tick serine proteinase	serine-type endopeptidase activity	1,491.85	GEIB01000042.1	serine protease-like protein precursor [O. moubata]
Contig3496, Contig4814	probable serine carboxypeptidase cpvl-like	carboxypeptidase activity	378.88;125.76;	GEIB01000176.1;GEIB01002008.1	putative serine carboxypeptidase, partial [A. aureolatum]
Metallo
Contig621, Contig5085	dipeptidase	aminopeptidase activity	421.73;321.65	GEIB01000425.1;GEIB01000292.1	putative xaa-pro dipeptidase-like protein [A. aureolatum]
Contig5173, Contig4027, Contig5249	aminopeptidase	aminopeptidase activity	396.41;202.68- 157.45	GEIB01001324.1;GEIB01001988.1;GEIB01002015.1	conserved hypothetical protein, partial [I. scapularis]
Contig3475	mam-containing astacin-like metalloprotease	metalloendopeptidase activity	401.61	GEIB01000240.1	secreted protein, putative [I. scapularis]
Contig2224	endothelin-converting enzyme	metalloendopeptidase activity	302.04	GEIB01000674.1	putative m13 family peptidase [A. aureolatum]
Contig2690, Contig3707	dipeptidyl peptidase 3-like isoform 2	dipeptidyl-peptidase activity	403.37;168.86	GEIB01000301.1;GEIB01002128.1	putative dipeptidyl peptidase iii [A. aureolatum]
Aspartic
Contig3140, Contig4774, Contig3706	cathepsin D	aspartic peptidase activity	1,912.04;977.20;769.09	GEIB01001141.1;GEIB01001183.1;GEIB01001344.1	cathepsin D-like aspartic protease [I. ricinus]
Carbohydrate metabolism
Contig4231, Contig4692	chitinase	chitinase activity	846.71;212.64	GEIB01000065.1;GEIB01000144.1	chitinase, putative [I. scapularis]; predicted: endochitinase-like [Tetranychus urticae]
Contig571, Contig4163, Contig3170	maltase	hydrolase activity	508.41;506.43;173.88	GEIB01000076.1;GEIB01001334.1;GEIB01002097.1	putative amino acid transporter, partial [A. aureolatum]; PREDICTED: maltase-glucoamylase, intestinal-like, partial [Meleagris gallopavo]; putative sucrase-isomaltase-like protein, partial [A. sculptum]
Contig79, Contig3343	fructose -bisphosphate aldolase	fructose-bisphosphate aldolase activity	548.19;286.33	GEIB01001240.1;GEIB01000239.1	fructose 1,6-bisphosphate aldolase [I. scapularis]
Contig4153, Contig3020	α-glycosidase	α-glucosidase activity	237.32;150.55	GEIB01000542.1;GEIB01000699.1	glucosidase II, putative, partial [I. scapularis]; glucosidase catalytic [A. aureolatum]
Contig39	glycogen phosphorylase	phosphorylase activity	274.7	GEIB01001233.1	glycogen phosphorylase, putative [I. scapularis]
Contig103, Contig5179	α -l-fucosidase	hydrolase activity	138.33;130.22	GEIB01002031.1;GEIB01000885.1	predicted: alpha-L-fucosidase-like [Octopus bimaculoides]; putative alpha-l-fucosidase [A. aureolatum]
Lipid metabolism
Contig1795	phospholipase b	lipase activity	394.32	GEIB01001359.1	predicted: putative phospholipase B-like 2 [Limulus polyphemus]
Contig2581	lipase	lipase activity	294.38	GEIB01000804.1	putative hormone-sensitive lipase hsl, partial [A. aureolatum]
Peptidase inhibitor
Serine proteinase inhibitor
Contig4823	serine proteinase inhibitor serpin	serine-type endopeptidase inhibitor activity	990.82	GEIB01001172.1	serine protease inhibitor 5 RmS5 [R. microplus]
Contig3495, Contig3457	kunitz-type protease inhibitor	serine-type endopeptidase inhibitor activity	407.87;204.22	GEIB01000363.1;GEIB01001015.1	full=Boophilin-G2 putative [R. microplus]
Contig3309	venom protease inhibitor precursor	serine-type endopeptidase inhibitor activity	328.78	GEIB01001820.1	kunitz-type protease inhibitor 4 [Micrurus fulvius]
Contig3184	alpha-2-macroglobulin precursor	endopeptidase inhibitor activity	457.08	GEIB01001287.1	alpha-2-macroglobulin precursor splice variant 1 [O. moubata]
Cysteine proteinase inhibitor
Contig4119, Contig3897	secreted cystatin	cysteine-type endopeptidase inhibitor activity	446.61;271.95	GEIB01001544.1;GEIB01001844.1	cystatin precursor [O. moubata]
Contig5375, Contig5044	thyropin precursor	cysteine-type endopeptidase inhibitor activity	3,454.82;2,269.30	GEIB01000013.1;GEIB01001163.1	putative thyropin precursor [O. moubata]
Immunity
Contig5007	defensin a	defense response	1,115.41	GEIB01000200.1	defensin a [O. moubata]
Contig2696	lysozyme precursor	lysozyme activity	312.63	GEIB01000589.1	lysozyme precursor [O. moubata]
Oxidant metabolism
Contig5141, Contig5040, Contig5426, Contig3924	phospholipid-hydroperoxide glutathione peroxidase	glutathione peroxidase activity	1,710.24;991.96; 871.34;259.29	GEIB01001198.1;GEIB01001224.1; GEIB01000051.1;GEIB01000721.1	phospholipid-hydroperoxide glutathione peroxidase [R. microplus]
Contig5082, Contig5045, Contig5342, Contig3084	glutathione s-transferase	glutathione transferase activity	1,900.88;1,153.5; 1,040.55;341.27	GEIB01001162.1;GEIB01000083.1; GEIB01001199.1;GEIB01000333.1	glutationa-s-transferase, putative [I. scapularis]
Contig131	microsomal glutathione s-transferase 3	glutathione transferase activity	166.75	GEIB01001909.1	microsomal glutathione S-transferase, putative [I. scapularis]
Transporter channels
Contig5201	hemelipoglycoprotein	lipid transporter activity	3,078.88	GEIB01000004.1	vitellogenin-1 [H. longicornis]
Contig4288	fatty acid-binding protein	lipid transporter activity	2,504.31	GEIB01000054.1	fatty acid-binding protein FABP, putative [I. scapularis]
Contig4030	beta 2c	lipid binding	2,025.46	GEIB01001139.1	beta tubulin [I. scapularis]
Contig4250	adp atp translocase 1	transporter activity	1916.54	GEIB01000011.1	ADP/ATP translocase 1-like [Parasteatoda tepidariorum].
Contig4329	vitellogenin-B	lipid transporter activity	713.31	GEIB01001142.1	vitellogenin-B [H. longicornis]
Storage
Contig4611	ferritin	ferroxidase activity	3,846.24	GEIB01001151.1	ferritin [O. moubata]

[1] *Corresponding author: Darci Moraes Barros-Battesti. Laboratório de Parasitologia, Instituto Butantan, Av. Vital Brasil, 150l, CEP 05503-900, São Paulo, SP, Brasil. e-mail: darci.battesti@butantan.gov.br

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Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

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