Differentiation of Laboratory-Obtained Ixodes ricinus × Ixodes persulcatus Hybrid Ticks: Selection of Suitable Genes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ticks
2.2. DNA Isolation
2.3. PCR
2.4. PCR Product Purification, TA-Cloning, and Plasmid Purification and Sequencing
2.5. qPCR
2.6. Bioinformatics
3. Results
3.1. Testing ITS Primer Sets
3.2. Searching for Prospective Genes for Hybrid Determination
3.3. Testing Lysozyme and Toll_R Amplicons as a Hybrid Differentiator
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Tick Specimen № | Tick Species | Sex | Collection Place | GPS |
---|---|---|---|---|
172-1 | Ixodes persulcatus | ♀ | Russia, Republic of Tuva | 51.9923° 94.0842° |
173 | Ixodes persulcatus | ♀ | 51.6283° 94.4292° | |
338-1 | Ixodes persulcatus | ♂ | Russia, Republic of Karelia | 62.0586° 33.9427° |
338-2 | Ixodes persulcatus | ♂ | ||
338-3 | Ixodes persulcatus | ♂ | ||
338-4 | Ixodes persulcatus | ♂ | ||
707-1 | Ixodes persulcatus | ♀ | Russia, Republic of Karelia | 62.0747° 33.9495° |
707-2 | Ixodes persulcatus | ♀ | ||
707-3 | Ixodes persulcatus | ♀ | ||
414-1 | Ixodes ricinus | ♀ | Russia, Voronezh region | 51.7801° 39.4085° |
420 | Ixodes ricinus | ♀ | ||
509-1 | Ixodes ricinus | ♂ | ||
421a | Ixodes ricinus | ♂ | Russia, Voronezh region | 51.7850° 39.4054° |
417-1 | Ixodes ricinus | ♂ | Russia, Kaliningrad region | 55.1839° 20.8572° |
417-2 | Ixodes ricinus | ♂ | ||
871 | Ixodes ricinus | ♀ | Russia, Kaliningrad region | 55.1585° 20.8437° |
872 | Ixodes ricinus | ♀ |
Primer Set Name | Oligonucleotide | Oligonucleotide Sequence | Primer Set Temperature | Reference | Amplicon Length |
---|---|---|---|---|---|
FITSMR-3SA | FITSMR | 5′-ccagtattcatcggggacgc-3′ | 55 °C | [9,21] | ≈660 |
3SA | 5′-ctaagcggtggatcactcgg-3′ | ||||
JB9A-3SA | JB9A | 5′-gcactatcaagcaacacgactc-3′ | 52 °C | [9] | ≈1100 |
3SA | 5′-ctaagcggtggatcactcgg-3′ | ||||
Lysozyme | Lys_eSNP1F | 5′-tgtctttggcttggatcgtc-3′ | 55 °C | [11] | 512 |
Lys_eSNP1R | 5′-attcttccacctgccctacg-3′ | ||||
Serpin 2 | Serp2Ae1-6_F | 5′-ttacgctcccgacgttattc-3′ | 50–55 °C * | [11] | 651 |
Serp2Ae1-6_R | 5′-ttcgagggatcaaacaggtc-3′ | ||||
Ixoderin | IxodBe2-3F | 5′-acacgtatgcctcaaagtgg-3′ | 50–55 °C * | [11] | 502 |
IxodBe2-3R | 5′-gcactatatccagcgggaag-3′ | ||||
Actin | Act_E6_F | 5′-ggagcacccgctccaggta-3′ | 55 °C | - ** | 268 |
Act_E6_R | 5′-ctgtaattgcagctgccggac-3′ | ||||
OdorR | OdorR_F | 5′-attccccacatcgcgcaa-3′ | 50–55 °C * | - | 536 |
OdoR_R | 5′-ccctcatttctcagattctagcg-3′ | ||||
JH_Met | JH_MT_F | 5′-gtctctaaacagatggtcgact-3′ | 52 °C | - | 308 |
JH_MT_R | 5′-agactcacctcggcgta-3′ | ||||
Toll_full | Toll_F | 5′-ctcgaacgtcatgaagact-3′ | 50 °C | - | 1403 |
Toll_end_R | 5′-acttttgcggctatctgtttat-3′ | ||||
Toll_L | Toll_F | 5′-ctcgaacgtcatgaagact-3′ | 50 °C | - | 811 |
Toll_mid_R | 5′-ttcgatccagttgttacccaggct-3′ | ||||
Toll_R | Toll_mid_F | 5′-gcgattgaatctctggaggg-3′ | 50 °C | - | 739 |
Toll_end_R | 5′-acttttgcggctatctgtttat-3′ |
Tick Specimen No | Sex | Tick Species | Ct I. persulcatus | Ct I. ricinus | Ct Delta | Tick Species (as Determined by Kovalev et al.’s System) |
---|---|---|---|---|---|---|
h604 | ♀ | hybrid ♀ I.persulcatus × ♂ I.ricinus | 15.93 | 15.49 | 0.44 | Hybrid 1:1 |
h605 | ♀ | hybrid ♀ I.persulcatus × ♂ I.ricinus | 17.24 | 18.27 | 1.03 | Hybrid 1:2 |
h606 | ♂ | hybrid ♀ I.persulcatus × ♂ I.ricinus | 23.00 | nd * | - ** | I. persulcatus |
h730 | ♂ | hybrid ♀ I.persulcatus × ♂ I.ricinus | 15.05 | nd | - | I. persulcatus |
h422-1 | ♀ | hybrid ♀ I.ricinus × ♂ I.persulcatus | 20.73 | 22.51 | 1.78 | Hybrid 1:4 |
h422-2 | ♀ | hybrid ♀ I.ricinus × ♂ I.persulcatus | 17.79 | 18.99 | 1.20 | Hybrid 1:2 |
h423-1 | ♂ | hybrid ♀ I.ricinus × ♂ I.persulcatus | 29.25 | 19.17 | 10.08 | Hybrid 1:∞ |
h423-2 | ♂ | hybrid ♀ I.ricinus × ♂ I.persulcatus | 30.62 | 20.10 | 10.52 | Hybrid 1:∞ |
707-2 | ♀ | I. persulcatus | 14.33 | nd | - | I. persulcatus |
707-3 | ♀ | I. persulcatus | 15.05 | nd | - | I. persulcatus |
338-1 | ♂ | I. persulcatus | 15.09 | nd | - | I. persulcatus |
173 | ♀ | I. persulcatus | 14.95 | nd | - | I. persulcatus |
872 | ♀ | I. ricinus | nd | 15.20 | - | I. ricinus |
417-2 | ♂ | I. ricinus | nd | 16.88 | - | I. ricinus |
421a | ♂ | I. ricinus | nd | 17.01 | - | I. ricinus |
414-1 | ♀ | I. ricinus | nd | 14.60 | - | I. ricinus |
Crossing | ♀ I. persulcatus × ♂ I. ricinus | ♀ I. ricinus × ♂ I. persulcatus | |||||||
---|---|---|---|---|---|---|---|---|---|
Individual tick | h604 ♀ | h605 ♀ | h606 ♂ | h730 ♂ | h422-1 ♀ | h422-2 ♀ | h423-1 ♂ | h423-2 ♂ | |
Clones from FITSMR-3SA ratio | Individual | 6:0 * | 4:2 | 6:0 * | 10:0 * | 10:0 * | 7:1 | 0:9 * | 1:8 |
Total by sex | 10:2 | 16:0 * | 17:1 * | 1:17 * | |||||
Clones from JB9A-3SA ratio | Individual | 8:2 | 5:5 | 10:0 * | 6:0 * | 6:0 * | 5:0 | 0:6 * | 0:5 |
Total by sex | 13:7 | 16:0 * | 11:0 * | 0:11 * |
Crossing | Both ♀ I. persulcatus × ♂ I. ricinus and ♀ I. ricinus × ♂ I. persulcatus | |
---|---|---|
FITSMR-3SA ITS in | ||
♀ | ♂ | |
I. persulcatus:I. ricinus ratio | 27:3 * | 17:17 |
maternal:paternal ratio | 11:19 † | 33:1 # |
JB9A-3SA ITS in | ||
♀ | ♂ | |
I. persulcatus:I. ricinus ratio | 24:7 * | 16:11 |
maternal:paternal ratio | 13:18 † | 27:0 # |
Total ITS in | ||
♀ | ♂ | |
maternal:paternal ratio | 24:37 † | 60:1 # |
Crossing | ♀ I. persulcatus × ♂ I. ricinus | ♀ I. ricinus × ♂ I. persulcatus | |||
---|---|---|---|---|---|
Individual tick | h606 ♂ | h730 ♂ | h423-1 ♂ | h423-2 ♂ | |
Clones from Lysozyme ratio | Individual | 1:4 | 0:5 | 0:5 | 0:4 |
Total | 1:18 * | ||||
Lysozyme total maternal:paternal ratio | 10:9 ‡ | ||||
Clones from Toll_R ratio | Individual | 3:2 | 4:1 | 2:3 | 3:2 |
Total | 12:8 | ||||
Toll_R total maternal:paternal ratio | 12:8 ‡ |
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Litov, A.G.; Belova, O.A.; Bugmyrin, S.V.; Kholodilov, I.S.; Romanova, L.I.; Karganova, G.G. Differentiation of Laboratory-Obtained Ixodes ricinus × Ixodes persulcatus Hybrid Ticks: Selection of Suitable Genes. Microorganisms 2022, 10, 1306. https://doi.org/10.3390/microorganisms10071306
Litov AG, Belova OA, Bugmyrin SV, Kholodilov IS, Romanova LI, Karganova GG. Differentiation of Laboratory-Obtained Ixodes ricinus × Ixodes persulcatus Hybrid Ticks: Selection of Suitable Genes. Microorganisms. 2022; 10(7):1306. https://doi.org/10.3390/microorganisms10071306
Chicago/Turabian StyleLitov, Alexander G., Oxana A. Belova, Sergey V. Bugmyrin, Ivan S. Kholodilov, Lidia Iu. Romanova, and Galina G. Karganova. 2022. "Differentiation of Laboratory-Obtained Ixodes ricinus × Ixodes persulcatus Hybrid Ticks: Selection of Suitable Genes" Microorganisms 10, no. 7: 1306. https://doi.org/10.3390/microorganisms10071306
APA StyleLitov, A. G., Belova, O. A., Bugmyrin, S. V., Kholodilov, I. S., Romanova, L. I., & Karganova, G. G. (2022). Differentiation of Laboratory-Obtained Ixodes ricinus × Ixodes persulcatus Hybrid Ticks: Selection of Suitable Genes. Microorganisms, 10(7), 1306. https://doi.org/10.3390/microorganisms10071306