Genome assembly and annotation
We estimated a genome size of ~ 3.09 Gb, a heterozygosity rate of 3.54‒3.65% and a repetitive content of 65%. The left and the right peaks implied that the genome may have high levels of heterozygosity and repetition (FigureS1).The final assembly had 6,490 scaffolds/7,059 contigs, a total length of 3.12 Gb and scaffold/contig N50 length of 1.09/1.05 Mb. Assembled size was very close to the estimated one. Genome completeness assessment against arthropod dataset (n = 1,066) (Table 2) revealed our assembled version covered 92.4% complete, 8.9% complete and duplicated, 1.1% fragmented, and 6.5% missing BUSCO genes.
A total of 64.81% (2.02 Gb) repetitive elements were identified in the longhorned tick genome. The top five abundant repeat categories were Unclassified (21.10%), LTR (18.37%), LINE (18.31%), DNA elements (2.99%), and SINE (2.23%) (Table S1). We identified 5,601 ncRNAs: 191 rRNAs, 65 miRNAs, 820small nuclear RNAs (snRNAs), 2 long non-coding RNAs (lncRNAs), 8 small RNAs (sRNAs), 4,271 tRNAs (22 isotypes), and 244 other ncRNAs (Table S2). SnRNAs were classified into 771 spliceosomal RNAs (U1, U2, U4, U5, U6, U11), three minor spliceosomal RNAs (U12, U4atac, U6atac), and six H/ACA box and 40 C/D box snoRNAs. We predicted 21,550 protein-coding genes with the mean length of genes, exons and introns as 22,231.43, 262.16 and 3,451.43 bp, respectively, and 91.8% BUSCO completeness (Table 1). InterproScan and eggNOG functional annotations assigned protein domains of 17,963 (83.35%) genes, 14,464 GO terms, 10,903 KEGG ko terms, 4,148 Enzyme Codes, 6,788 KEGG and 4,083 Reactome pathways, and 15,557 COG categories, respectively.
Table 1
Genome assembly and annotation statistics of the longhorned tick
Elements
|
Current version
|
Genome assembly
|
|
Assembly size (Mb)
|
3,117.76
|
Number of scaffolds
|
6,490
|
Number of contigs
|
7,059
|
Longest scaffold (Mb)
|
8.69
|
Longest contig (Mb)
|
8.69
|
N50 scaffolds length (Mb)
|
1.09
|
N50 contig length (Mb)
|
1.05
|
GC (%)
|
47.50
|
Gaps (%)
|
0.003
|
BUSCO completeness (%)
|
92.4
|
Gene annotation
|
|
Protein-coding genes
|
21,550
|
Mean protein length (aa)
|
437.15
|
Mean gene length (bp)
|
22,231.43
|
Exons per gene
|
7.02
|
Exon (%)
|
0.013
|
Mean exon length
|
262.16
|
Intron (%)
|
14.09
|
Mean intron length
|
3,451.43
|
BUSCO completeness (%)
|
91.8
|
Gene family evolution
A total of 162,773 (88.24%) genes were assigned into 18,611 gene families; among them, 3,482 families were shared by all nine species and 470 are single-copy ones; 143 families and 1,075 orthologs are common to four Parasitiformes species (Figure 1a, Table S3). In H. longicornis, 19,211 (91.91%) genes were clustered into 9,639 gene families; 850 families and 4,008 genes were species-specific. Phylogenetic tree clustered H. longicornis and I. scapularis, both representing Ixodidae originated from early Cretaceous (Figure 1a). It indicated that the emergence of parasitic ixodids may be related to the pervasive reptiles, birds, mammals in Cretaceous.
CAFÉ identified 350 rapidly evolving gene families, 255 and 95 of them experienced significant expansions and contractions, respectively (Figure1a). The largest expanded families were shown in Figure 1b and Table S4. Many of them are related to dietary digestion and detoxification, cuticle synthesis, such as ABC transporter, Cytochrome P450, carboxylesterase, tick histamine binding protein, insect cuticle protein, putative secreted salivary gland peptide, juvenile hormone acid O-methyltransferase, secretin family etc. GO (Figure 1c) and KEGG (Figure 1d) enrichment further confirmed it, involving various biological progress or pathways, for example, ABC transporters, insect hormone biosynthesis, fatty acid elongation and biosynthesis, fat digestion and absorption, and ovarian steroidogenesis (Figure 1d). KEGG pathways of toxoplasmosis and amoebiasis may relate to parasitic life of the longhorned tick. These findings adapted to the parasitic life are very similar to another ixodid tick I. scapularis [49].
Gene families related to detoxification
Digestion and detoxification function are important for parasitic progress unique to ticks, particularly feeding of blood meal, haemoglobin digestion, haem detoxification and prolonged off-host survival. We compared four dietary detoxification-related gene families in three tick and mite species. ABC, P450 and CCE families showed large expansions in the longhorned tick genome (Table 2). Expansions of ABCs occurred in the ABCA and ABCC subfamilies, which includes five and three large (≥ 5 orthologs) clusters on the phylogenetic tree (Fig. 2a). ABCA transporters function in lipid transport and resistance in insects [50–52]. ABCC transporters, also known as multidrug resistance proteins (MRPs), are known to be involved in ion transport, signal transduction, and toxin secretion [53]. Major P450 expansions of two tick species were discovered in clan 3 and clan 4 (Table 2), which had three clan 3 and three clan 4 large clusters on the tree for H. longicornis (Fig. 2b). P450 clan 3 and clan 4 may be linked to xenobiotic metabolism, insecticide resistance, odorant or pheromone metabolism [54]. GST expansions of two ticks mainly occurred in Mu class (Figure S2), which may activate in drug metabolism, particularly in the detoxification of reactive oxygen species (cyclised o-quinones) produced via oxidative metabolism of catecholamines [55, 56]. CCE in H. longicornis showed extreme expansions (Table 2) in neuro/developmental class although classifications of some members were unclear in Chelicerata [57]. These large expansions in the longhorned tick genome greatly enhance abilities in xenobiotic metabolism and insecticide resistance, and thus are considered to contribute to the parasitic adaptation.
Table 2
Comparison of digestion and detoxification gene families among threeAcari species
Families
|
Clan/Group
|
H.longicornis
|
I. scapularis
|
Tetranychusurticae
|
P450
|
Clan 2
|
50
|
65
|
45
|
|
Clan 3
|
99
|
100
|
10
|
|
Clan 4
|
45
|
33
|
25
|
|
Mitochondrial
|
10
|
7
|
5
|
|
Total
|
204
|
205
|
85
|
GST
|
Delta
|
18
|
17
|
19
|
|
Kappa
|
2
|
1
|
1
|
|
Mu
|
17
|
23
|
10
|
|
Sigma
|
1
|
1
|
0
|
|
Zeta
|
1
|
7
|
1
|
|
Omega
|
5
|
5
|
2
|
|
Microsomal
|
5
|
1
|
0
|
|
Total
|
49
|
55
|
33
|
CCE
|
Total
|
155
|
93
|
71
|
ABC
|
A
|
82
|
25
|
8
|
|
B
|
6
|
5
|
2
|
|
C
|
57
|
38
|
37
|
|
D
|
4
|
2
|
2
|
|
E
|
1
|
1
|
1
|
|
F
|
3
|
3
|
4
|
|
G
|
19
|
13
|
33
|
|
Total
|
174
|
87
|
87
|