MicroRNA-1 promotes the development of and prolongs engorgement time in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks

MicroRNAs act as mRNA posttranscriptional regulators, playing important roles in cell differentiation, transcriptional regulation, growth and development. In this study, microRNA expression profiles of Hyalomma anatolicum anatolicum ticks at different developmental stages were detected by high-throughput sequencing and functionally assessed. In total, 2,585,169, 1,252,678, 1,558,217 and 1,155,283 unique reads were obtained from eggs, larvae, nymphs and adults, respectively, with 42, 46, 45 and 41 conserved microRNAs in these stages, respectively. Using eggs as a control, 48, 43 and 39 microRNAs were upregulated and 3, 10 and 9 downregulated in larvae, nymphs and adults, respectively. microRNA-1 (miR-1) was expressed in high abundance throughout Ha. anatolicum development, with an average of nearly one million transcripts, and it is highly conserved among tick species. Quantitative real-time PCR (qPCR) showed that miR-1 expression gradually increased with tick development, reaching the highest level at engorgement. Differential tissue expression was detected, with significantly higher levels in the salivary glands and epidermis than in the midgut. Inhibition assays showed no significant change in body weight or spawning time or amount between experimental and control groups, but there was a significant difference (p<0.01) in engorgement time. With miR-1 inhibition, ticks displayed obvious deformities during later development. To more fully explain the microRNA mechanism of action, the miR-1 family was analyzed regarding target gene; members acting on Hsp60 include miR-5, miR-994, miR-969, and miR-1011, which jointly play a role. Therefore, microRNAs are critical for normal tick development, and the primary structure of the mature sequence of miR-1 is highly conserved. Nonetheless, different developmental stages and tissues show different expression patterns, with a certain role in prolonging feeding. miR-1, together with other family members, regulates mRNA function and may be used as a molecular marker for species origin and evolution analysis and internal reference gene selection.


72
Hsp60 is a component of the defense mechanism against diabetic myocardial injury. The  miR-1 plays an important regulatory role in mammalian muscles, but its function in ticks remains 95 unclear. Here, the expression level of miR-1 in different developmental stages and tissues of ticks 96 is analyzed to predict its possible biological functions. An inhibitor of miR-1 was injected into the 97 fourth sarcomere, and physiological indicators were assessed at various developmental stages. The 98 molecular mechanisms were also investigated, and the biological function of miR-1 in tick 99 development was explored. This is the first report on the function of miR-1 in ticks, and we 100 present the characteristics of tick development upon abnormal expression of miR-1. This study cluster generation (with the Illumina Cluster Station) and then sequenced using a HiSeq2000 following the manufacturer's instructions.
Small RNA bioinformatics analysis 143 Sequence data (raw data or raw reads) conversion was conducted by base calling. We used 144 software developed by BGI for HiSeq sequencing data processing, eliminating some contaminants 145 and low-quality reads to obtain final clean reads. The data were processed according to the 146 following steps: 1) removing low-quality reads; 2) removing reads with 5' primer contamination; 3) 147 removing reads without a 3' primer; 4) removing reads without the insert tag; 5) removing reads 148 with poly-A; 6) removing reads shorter than 18 nt; 7) summarizing the length distribution of the microRNA level is expressed as the 2 -△△CT mean ± SE. One-way ANOVA was applied to examine 243 the significance of differential expression level in each mature/novel microRNA between eggs and 244 larvae, larvae and adults, eggs and adults, and the difference was considered significant at P<0.05.

245
Clones containing an insert of the correct size from four independent PCRs were sequenced on 246 both strands using an ALF sequencer (Pharmacia Biotech).  ticks, and the groups were given a blood meal on a host at 24 h after microinjection.

Statistical analysis 285
All data were analyzed with GraphPad 5 using Student's t-test. Probability values of less than 0.05 286 were considered significant, and results are shown as the mean ± SEM. 47.00%, 79.42% and 71.64% between nymphs and adults, nymphs and larvae, and nymphs and 307 eggs, respectively; 9.67% miRNAs were common to all larvae, nymphs, and adults ( Figure 2).

308
Known microRNAs and differential expression analysis 309 In this study, known microRNAs from Ha. anatolicum ticks were analyzed by miRBase21.

310
The results showed a total of 232 known microRNAs in the egg stage, 1,051 in larvae, 1,122 in 311 nymphs and 743 in adults. In eggs, miR-4175-3p and miR-4419b were predominately expressed, 312 with more than 100,000 reads, and some microRNAs constituted 17.78% (260,875/1,467,411) of 313 the total sequencing reads, suggesting that they are abundantly expressed during this period. The

314
sequencing frequencies of 916 microRNAs were much lower than the 10 reads in larvae, but 315 miR-184, miR-1 and miR-184b were predominately expressed. This was also observed at the 316 nymph tick stage. However, in adults, miR-1-3p, miR-1, let-7-5p, miR-4486, and miR-84a were 317 the most abundant, each with more than 100,000 reads. A total of 1,368 microRNAs displayed the 318 lowest sequencing frequencies, with no more than 10 reads in larvae, nymphs and adults. In the 319 four libraries, miR-1-3p, miR-1, and miR-4175-3p were detected with high abundance (Additional

347
MicroRNA target gene prediction and GO enrichment 348 We screened microRNA 1 (miR-1), which exhibited a high expression level, as an important at the partially fed female stage (Figure 8a). In addition, expression of Hsp60 was highest at the 408 engorged adult stage but was drastically reduced in eggs (Figure 8a). Analysis of different tissues 409 indicated higher mature miR-1 levels in salivary glands than in other studied tissues in unfed each unfed female tick was microinjected with Ant-mir1 or MsAnt, and after 48 hours, real-time PCR was performed to assess the silencing efficiency of Ant-mir1. The results showed that miR-1 414 expression levels decreased to 64.67% after injection of Ant-mir1 compared with MsAnt and 415 noninjection controls (Figure 9) (t=5.800, df=4). In this experiment, the host blood (rabbit) and 416 male ticks were also examined by qPCR after injection of Ant-mir1, with insignificant levels in 417 the rabbit blood. However, the expression level was higher in males than in females, and the 418 expression level of miR-1 was significantly different in engorged females injected with water and 419 Ant-mir1 ( Figure 10).  (Table 5).   475 between eggs and nymphs, and 694 between eggs and larvae. In addition, using eggs as a control,

476
Bantam and Let-7 miRNA and miR-133 showed large differences in various developmental stages.

477
However, differential expression of miRNAs between adult and nymph and larval ticks was to heart diseases such as myocardial infarction, arrhythmia, and cardiac hypertrophy [26]. 4). Dysregulation of miR-1 may inhibit apoptosis or cause cell proliferation. Although multisequence alignment indicated significant differences in the nucleotide precursor sequences of 491 miR-1 from different species, the mature sequence is conserved with regard to primary structure.

492
Specifically, the "seed region" is identical among different species, though the "B region" and "C 493 region" exhibit a difference of 2-3 bases ( Figure 5). This is because the miRNA binds to target 494 genes via the seed region, allowing specific binding between the miRNA and target gene; variable 495 regions of mature sequences emphasize the diversity of these sequences. Such conservation in 496 miRNAs has been applied for the early diagnosis of cancer, species identification, and pathogen 497 detection as well as other purposes. The results also suggest both specificity and diversity in target 498 mRNAs. In addition, we found showed significant differences in nucleic acid sequences for 499 precursors of miR-1 and related species. However, these species clustered into three main 500 branches in phylogenetic trees (Figure 6), demonstrating large differences among arthropods,

501
Nemathelminthes and primates. For example, ticks, mosquitoes and Drosophila display high 502 identity in the arthropod group. In particular, only the "C" region was identical, with 1-2 base 503 differences in mature sequences of miR-1 from different species; the "seed region" was also 504 identical. Therefore, miR-1 is an ancient regulatory molecule that plays an important role in the 505 evolution of species.

506
In this study, it was shown that miR-1 is expressed at various developmental stages in tick 507 development and in different tissues, and miR-1 expression was upregulated with the growth and 508 development of ticks. In particular, the expression level was significantly higher during 509 engorgement than starvation ( Figure 7A), and miR-1 was mainly expressed in the epidermis and 510 midgut (Figure 7b). These results indicate that miR-1 not only maintains normal physiological 511 function but also has a leading role in tick muscle development. It is possible that with the 512 engorging process, the tick's muscles gradually extend to accommodate the filling of the midgut.

513
To confirm this, miR-1 was inhibited via injection of an inhibitor, and the change in physiological 514 indices was significant (Table 5). As time is required for inhibitors to function in living animals, 515 the expression level of miR-1 in ticks was tracked to determine the optimal feeding time after 516 injection, and miR-1 expression was downregulated significantly after 16 h (Figure 8). Ticks were 517 released on the surface of animals for a long time and then collected, and total RNA was extracted 518 to detect miR-1 expression (Figure 9).

519
Previous studies have shown that heat shock proteins (Hsps) are directly related to the  Table 4 and Figure 10). miR-1 regulates the transcription of Hsp60, participating in 526 the formation of specific tissues and organs during development. In other studies, the Hsp60 gene 527 was associated with the development of the hematopoietic system and hematopoietic stem or 528 progenitor cell proliferation, differentiation, maturation, and tumorigenesis [46-51]. Therefore, it 529 will be helpful to explore the functions of miR-1 to understand the biological characteristics of 530 Hsp60 and its expression (Figure 7). expression was significantly lower than that in the control group in the miR-1 inhibition analysis and the inhibitory effect was significant. Thus, in ticks, miR-1 might regulate Hsp60 to resist 536 damage by foreign agents. The findings also indicate that the binding of animal miRNA to target 537 mRNA depends not only on the seed region but that the "B" and "C" regions determine the 538 specificity for the interaction in different species.

539
Previous studies have confirmed that miRNAs can be specifically expressed in high 540 abundance in animal reproductive tissues, such as miR-449a, miR-465c, miR-202, and miR-547  Table 5). To analyze the reasons for the lack of difference in physiological changes, we predicted 556 other miRNAs possibly regulating Hsp60 and found that miR-5, miR-994, miR-969, miR-927, 557 miR-989, miR-977, miR-1011 and miR-1 are common families that regulate the function of 558 Hsp60 (Figure 9). We speculate that when miR-1 expression is inhibited, these family members 559 will compensate for the loss. To verify this, qPCR was applied to detect expression of other 560 miRNA clusters after miR-1 inhibition, which showed that other miRNAs were significantly 561 upregulated after miR-1 was significantly inhibited. In fact, the expression level of miR-5, which 562 has the same binding site as miR-1, was 4 times the normal level. miR-989 was also significantly 563 upregulated ( Figure 10). These results indicate that miR-5 plays an equally important role as 564 miR-989 in the regulation of Hsp60 by miR-1. When miR-1 was inhibited, the two miRNAs 565 preferentially compensated for the loss of function.

567
In conclusion, miR-1, a small noncoding RNA present in lower to higher animals, is highly 568 conserved that is expressed to varying degrees in different stages of tick development and between 569 tissues, with relatively stable expression. miR-1 plays an important role in tick development.

764
Reference nucleotide sequences were selected by BLAST searches of the NCBI nt database.

803
The sequence and count of microRNA families (no specific species) found in the samples.

804
A is the microRNA name (no specific species); B is the count of microRNAs of a family in the 805 developmental stage; C is the sequence of a microRNA with the highest count in the family.

806
Addition file 3: The long sequence indicates microRNA precursor information in the order of 807 sequence, name and long; The parentheses indicate microRNA precursor information in the order 808 of hairpin structure, structure and MFE; "*** "and a short sequence indicate mature microRNA 809 information in the order of sequence, name and length; "…" and a short sequence indicate 810 information of matched sRNA tags in the order of sequence, ID, length, count. Table 1 Alignment to Genbank and Rfam data librarys.

Tables
Annotate the sRNA tags with rRNA, scRNA, snoRNA, snRNA and tRNA from 13 Genbank by BLAST. For some species, its non-coding RNAs from Genbank need to be supplied as reference for this analysis.  Table 2 Known miRNA alignment and expression profile Align sRNA to the miRNA precursor of corresponding species (if there is no precursor information of that species in miRBase, mature miRNA is ok) to obtain the miRNA count; If there is miRNA information of the species in miRBase, align sRNA tags to the miRNA precursor/mature miRNA of corresponding species in miRBase; if not, align sRNA tags to the miRNA precursor/mature miRNA of all plants/animals in miRBase, see Addition file 1.  Table 3. Map the clean tags back to genome by SOAP2 [1] to analyze the expression and distribution of sRNA tags across the genome. If the genome sequence of the species is not available, the corresponding EST or one specified species with available genome sequence as the reference will also work. The characteristic hairpin structure of miRNA precursor can be used to predict novel miRNA. We developed Mireap (http://sourceforge.net/projects/mireap/), a prediction software to predict novel miRNA by exploring the secondary structure, the Dicer cleavage site and the minimum free energy of the unannotated sRNA tags which could be mapped to genome.