Discrimination of Phlebotomus perfiliewi transcaucasicus, Ph. major sensu lato and Ph. tobbi (Diptera: Psychodidae) Using Morphometric and DNA Barcoding Methods in the Endemic Foci of Visceral Leishmaniasis in Ardabil Province, North West of Iran
-
Alireza Barmaki
,
Yavar Rassi
,
Azad Absavaran
,
Amir Ahmad Akhavan
,
Eslam Moradi-Asl
,
Alireza Zahraei-Ramazani
,
Sayena Rafizadeh
Abstract
Background: Visceral leishmaniasis, commonly known as kala-azar, and prevalent in more than 70 countries and several regions of Iran. It is one of the main diseases transmitted by sand flies.
In this work, geometric morphometrics and DNA barcoding were employed as novel techniques to enhance the diagnostic tools used in this study.
Methods: Phlebotomus perfiliewi transcaucasicus, Phlebotomus major s.l., and Phlebotomus tobbi caught from three districts in the Ardabil Province, northwest of Iran. The right wings of 286 female sand flies were analyzed using geometric morphometric (GM) tools. Additionally, the COI gene was isolated from each of the three species, amplified using universal primers, and sequenced through the DNA barcoding method for classification. This sequencing data was then formatted to generate morphometric analyses.
Results: The landmarks with the most variations were found in sets 10, 12, 13, and 14, whereas the first set's landmarks at 1 and 11, along with those from the second set at positions 2, 3, and 5 exhibited the greatest variations. Analysis of the size and shape variations in the wings indicates the presence of distinct populations (P< 0.05). Furthermore, the DNA barcoding results not only confirmed the findings from the geometric morphometric analysis but also revealed both interspecific and intraspecific distances.
Conclusion: This study was the first attempt to assess whether wing geometry morphometrics, combined with DNA barcode techniques, can effectively distinguish the three mentioned species in the studied areas. Furthermore, the identification of Phlebotomus neglectus in this area prompted recommendations for additional research.
1. Alvar J, Velez ID, Bern C, Herrero M, Desjeux
P, Cano J, Jannin J, den Boer M (2012) Leishmaniasis worldwide and global es-timates of its incidence. PLOS One. 7 (5): e35671.
2. Mohebali M (2013) Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol. 8(3):348¬¬–358.
3. Rassi Y, Javadian E, Nadim A (2005) Phleboto¬mus (Larroussius) kandelakii the principal and proven vector of visceral leishmaniasis in Northwest of Iran. Pak J Biol Sci. 8(12): 1802–1806.
4. Rassi Y, Javadian E, Nadim A (2009) Phlebotomus perfiliewi transcaucasicus, a vector of Leishmania infantum in north¬western Iran. J Med Entomol. 46(5): 1094–1098.
5. Rassi Y, Abai MR, Oshaghi MA, Javadian E, Sanei A, Rafidzadeh S, Arzamani K (2012) First detection of Leishmania in-fantum in Phlebotomus kandelakii using molecular methods in north-eastern Is-lamic Republic of Iran. EMHJ. 18(4): 387–392.
6. Rassi Y, Sanei Dehkordi A, Oshaghi MA, Abai MR, Mohtarami F, Enayati A, Ja-vadian, E (2012) First report on natural infection of the Phlebotomus tobbi by Leishmania infantum in northwestern Iran. Exp Parasitol. 131(3): 344–349.
7. Azizi K, Rassi Y, Javadian E, Motazedian MH, Asgari Q, Yaghoobi-Ershadi MR (2008) First detection of Leishmania in-fantum in Phlebotomus (Larroussius) ma¬jor (Diptera: Psychodidae) from Iran. J Med Entomol. 45(4): 726–731.
8 .Yaghoobi-Ershadi M (2012) Phlebotomine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania transmis¬sion. J Arthropod-Borne Dis. 6(1): 1–17.
9. Rostamian M, Bashiri H, Yousefinejad V, Bozorgomid A, Sohrabi N, Raeghi S, Re¬zaeian S (2021) Prevalence of human vis¬ceral leishmaniasis in Iran: A system¬atic review and meta-analysis. Comp Im¬mu¬nol Microb. 75: 1–10.
10. Adham D, Moradi-Asl E, Dorosti A, Khaiatzadeh S (2020) Spatial auto¬cor¬re-lation and epidemiological survey of vis¬ceral leishmaniasis in an endemic area of Azerbaijan region, the northwest of Iran. PLoS One 15(8): e0236414.
11. Moradi-Asl E, Rassi Y, Adham D, Hanafi-Bojd AA, Saghafipour A, Rafizadeh S )2018) Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran. Asian Pac J Trop Biomed. 8(9): 425–430.
12. Oshaghi MA, Rassi Y, Hazratian T, Fal-lah E, Rafizadeh S (2013) Natural infec¬tion of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbai¬jan Province, northwestern Iran. J Ar¬thro¬pod-Borne Dis. 50(1): 9–24.
13. Oshaghi MA, Ravasan NM, Hide M, Ja-vadian EA, Rassi Y, Sadraei J, Mohe-bali M, Sedaghat MM, Hajjaran H, Za-rei Z, Mohtarami F (2009) Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infan-tum in northwest Iran. Exp Parasitol. 123(3): 218–225.
14. Killick-kendrick R (1990) Phlebotomine vectors of leishmaniasis: a review. Med Vet Entomol. 4(1): 1–24.
15. Absavaran A, Rassi Y, Parvizi P, Oshaghi M, Abaie M, Rafizadeh S, Mohebali M, Zarea Z, Javadian E (2009) Identifica-tion of Sand flies of the Subgenus Lar-roussius based on Molecular and Mor-phological Characters in North Western Iran. J Arthropod-Borne Dis. 3(2): 22–35.
16. Daly HV (1985) Insect Morphometrics. Annual Review of Entomology. 30(1): 415–438.
17. Bookstein FL (1982) Foundations of mor-phometrics. Annu Rev Entomol. 13(1): 451–470.
18. Pentinsaari M, Salmela H, Mutanen M, Roslin T (2016) Molecular evolution of a widely adopted taxonomic marker (COI) across the animal tree of life. Sci Rep. 6(1): 352–375.
19. Belen A, Alten B, Aytekin AM (2004) Altitudinal variation in morphometric and molecular characteristics of Phleboto¬mus papatasi populations. Med Vet Entomol. 18(4): 343–350.
20. Folmer O, Black M, Hoeh W, Lutz R, Vri¬jemnhoek R (1994) DNA primers for amplification of mitochondrial cyto¬chrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3(5): 294–299.
21. Seyedi-Rashti MA, Nadim A (1992) The genus Phlebotomus (Diptera: Psychodi-dae: Phlebotominae) of the countries of the Eastern Mediterranean Region. Iran J Public Health. 21(1–4): 11–50.
22. Lewis DJ (1982) A taxonomic review of the genus Phlebotomus (Diptera: Psycho¬didae). Bulletin of the British Museum (Natural History) Zoology. (Ent). 45(2): 121–209.
23. Badakhshan M, Sadraei J, Moin‐Vaziri V (2011) Morphometric and morphologi-cal variation between two different popula¬tions of Phlebotomus major sl from en¬demic and non‐endemic foci of visceral leishmaniasis in Iran. J Vector Ecol. 36 (1): 153–158.
24. Badakhshan M, Moin-Vaziri V, Sadraei J (2022) Phlebotomus neglectus (Diptera: Psychodidae): new insights on its pres-ence in Iran based on three independent genetic loci. J Med Entomol. 59(1): 233–239.
25. Giordani BF, Andrade AJ, Galati EAB, Gurgel‐Goncalves R (2017) The role of wing geometric morphometrics in the identification of sandflies within the sub¬genus Lutzomyia. Med Vet Entomol. 31(4): 373–380.
26. Kasap OE, Linton YM, Karakus M, Ozbel Y, Alten B (2019) Revision of the spe-cies composition and distribution of Turk¬ish sand flies using DNA barcodes. Parasit Vectors. 12(1): 1–20.
27. Zelditch ML, Swiderski DL, Sheets HD (2012) Geometric morphometrics for bi¬ologists: a primer. Second edition. Aca¬demic Press. USA.
28. Giordani BF, Andrade A.J D, Galati EAB, Gurgel‐GonÇalves R (2017) The role of wing geometric morphometrics in the iden¬tification of sandflies within the sub¬genus Lutzomyia. Med Vet Entomol. 31 (4): 373–380.
29. Absavaran A, Mohebali M, Moin-Vaziri V, Zahraei-Ramazani A, Akhavan AA, Mozaffarian F, Rafizadeh S, Rassi Y (2019) Morphometric discrimination be¬tween females of two isomorphic sand fly species, Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Phlebotom¬inae) in endemic and non-en¬demic foci of zoonotic cutaneous leish¬maniasis in Iran. Asian Pac J Trop Bio¬med. 12(4): 153–162.
30. Abou-Elaaz FZ, Sereno D, Himmi O, Ghamizi M, Guernaoui S (2023) De-scriptive and Geometric Morphometry of the Wings of Phlebotomus sergenti Pop¬ulations in Central Morocco. J Arthro¬pod-Borne Dis. 17(1): 36–50.
31. Mikery OF, Rebollar-Téllez EA, Cruz-López LC, Marina CF, Castillo A (2019) Traditional and geometric morphometry analyses of Lutzomyia cruciata (Diptera: Psychodidae: Phlebotominae) popula¬tions of Chiapas, Mexico. J Med Entomol. 56 (3): 697–707.
P, Cano J, Jannin J, den Boer M (2012) Leishmaniasis worldwide and global es-timates of its incidence. PLOS One. 7 (5): e35671.
2. Mohebali M (2013) Visceral leishmaniasis in Iran: Review of the Epidemiological and Clinical Features. Iran J Parasitol. 8(3):348¬¬–358.
3. Rassi Y, Javadian E, Nadim A (2005) Phleboto¬mus (Larroussius) kandelakii the principal and proven vector of visceral leishmaniasis in Northwest of Iran. Pak J Biol Sci. 8(12): 1802–1806.
4. Rassi Y, Javadian E, Nadim A (2009) Phlebotomus perfiliewi transcaucasicus, a vector of Leishmania infantum in north¬western Iran. J Med Entomol. 46(5): 1094–1098.
5. Rassi Y, Abai MR, Oshaghi MA, Javadian E, Sanei A, Rafidzadeh S, Arzamani K (2012) First detection of Leishmania in-fantum in Phlebotomus kandelakii using molecular methods in north-eastern Is-lamic Republic of Iran. EMHJ. 18(4): 387–392.
6. Rassi Y, Sanei Dehkordi A, Oshaghi MA, Abai MR, Mohtarami F, Enayati A, Ja-vadian, E (2012) First report on natural infection of the Phlebotomus tobbi by Leishmania infantum in northwestern Iran. Exp Parasitol. 131(3): 344–349.
7. Azizi K, Rassi Y, Javadian E, Motazedian MH, Asgari Q, Yaghoobi-Ershadi MR (2008) First detection of Leishmania in-fantum in Phlebotomus (Larroussius) ma¬jor (Diptera: Psychodidae) from Iran. J Med Entomol. 45(4): 726–731.
8 .Yaghoobi-Ershadi M (2012) Phlebotomine sand flies (Diptera: Psychodidae) in Iran and their role on Leishmania transmis¬sion. J Arthropod-Borne Dis. 6(1): 1–17.
9. Rostamian M, Bashiri H, Yousefinejad V, Bozorgomid A, Sohrabi N, Raeghi S, Re¬zaeian S (2021) Prevalence of human vis¬ceral leishmaniasis in Iran: A system¬atic review and meta-analysis. Comp Im¬mu¬nol Microb. 75: 1–10.
10. Adham D, Moradi-Asl E, Dorosti A, Khaiatzadeh S (2020) Spatial auto¬cor¬re-lation and epidemiological survey of vis¬ceral leishmaniasis in an endemic area of Azerbaijan region, the northwest of Iran. PLoS One 15(8): e0236414.
11. Moradi-Asl E, Rassi Y, Adham D, Hanafi-Bojd AA, Saghafipour A, Rafizadeh S )2018) Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran. Asian Pac J Trop Biomed. 8(9): 425–430.
12. Oshaghi MA, Rassi Y, Hazratian T, Fal-lah E, Rafizadeh S (2013) Natural infec¬tion of wild caught Phlebotomus tobbi to Leishmania infantum in East Azerbai¬jan Province, northwestern Iran. J Ar¬thro¬pod-Borne Dis. 50(1): 9–24.
13. Oshaghi MA, Ravasan NM, Hide M, Ja-vadian EA, Rassi Y, Sadraei J, Mohe-bali M, Sedaghat MM, Hajjaran H, Za-rei Z, Mohtarami F (2009) Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infan-tum in northwest Iran. Exp Parasitol. 123(3): 218–225.
14. Killick-kendrick R (1990) Phlebotomine vectors of leishmaniasis: a review. Med Vet Entomol. 4(1): 1–24.
15. Absavaran A, Rassi Y, Parvizi P, Oshaghi M, Abaie M, Rafizadeh S, Mohebali M, Zarea Z, Javadian E (2009) Identifica-tion of Sand flies of the Subgenus Lar-roussius based on Molecular and Mor-phological Characters in North Western Iran. J Arthropod-Borne Dis. 3(2): 22–35.
16. Daly HV (1985) Insect Morphometrics. Annual Review of Entomology. 30(1): 415–438.
17. Bookstein FL (1982) Foundations of mor-phometrics. Annu Rev Entomol. 13(1): 451–470.
18. Pentinsaari M, Salmela H, Mutanen M, Roslin T (2016) Molecular evolution of a widely adopted taxonomic marker (COI) across the animal tree of life. Sci Rep. 6(1): 352–375.
19. Belen A, Alten B, Aytekin AM (2004) Altitudinal variation in morphometric and molecular characteristics of Phleboto¬mus papatasi populations. Med Vet Entomol. 18(4): 343–350.
20. Folmer O, Black M, Hoeh W, Lutz R, Vri¬jemnhoek R (1994) DNA primers for amplification of mitochondrial cyto¬chrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 3(5): 294–299.
21. Seyedi-Rashti MA, Nadim A (1992) The genus Phlebotomus (Diptera: Psychodi-dae: Phlebotominae) of the countries of the Eastern Mediterranean Region. Iran J Public Health. 21(1–4): 11–50.
22. Lewis DJ (1982) A taxonomic review of the genus Phlebotomus (Diptera: Psycho¬didae). Bulletin of the British Museum (Natural History) Zoology. (Ent). 45(2): 121–209.
23. Badakhshan M, Sadraei J, Moin‐Vaziri V (2011) Morphometric and morphologi-cal variation between two different popula¬tions of Phlebotomus major sl from en¬demic and non‐endemic foci of visceral leishmaniasis in Iran. J Vector Ecol. 36 (1): 153–158.
24. Badakhshan M, Moin-Vaziri V, Sadraei J (2022) Phlebotomus neglectus (Diptera: Psychodidae): new insights on its pres-ence in Iran based on three independent genetic loci. J Med Entomol. 59(1): 233–239.
25. Giordani BF, Andrade AJ, Galati EAB, Gurgel‐Goncalves R (2017) The role of wing geometric morphometrics in the identification of sandflies within the sub¬genus Lutzomyia. Med Vet Entomol. 31(4): 373–380.
26. Kasap OE, Linton YM, Karakus M, Ozbel Y, Alten B (2019) Revision of the spe-cies composition and distribution of Turk¬ish sand flies using DNA barcodes. Parasit Vectors. 12(1): 1–20.
27. Zelditch ML, Swiderski DL, Sheets HD (2012) Geometric morphometrics for bi¬ologists: a primer. Second edition. Aca¬demic Press. USA.
28. Giordani BF, Andrade A.J D, Galati EAB, Gurgel‐GonÇalves R (2017) The role of wing geometric morphometrics in the iden¬tification of sandflies within the sub¬genus Lutzomyia. Med Vet Entomol. 31 (4): 373–380.
29. Absavaran A, Mohebali M, Moin-Vaziri V, Zahraei-Ramazani A, Akhavan AA, Mozaffarian F, Rafizadeh S, Rassi Y (2019) Morphometric discrimination be¬tween females of two isomorphic sand fly species, Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Phlebotom¬inae) in endemic and non-en¬demic foci of zoonotic cutaneous leish¬maniasis in Iran. Asian Pac J Trop Bio¬med. 12(4): 153–162.
30. Abou-Elaaz FZ, Sereno D, Himmi O, Ghamizi M, Guernaoui S (2023) De-scriptive and Geometric Morphometry of the Wings of Phlebotomus sergenti Pop¬ulations in Central Morocco. J Arthro¬pod-Borne Dis. 17(1): 36–50.
31. Mikery OF, Rebollar-Téllez EA, Cruz-López LC, Marina CF, Castillo A (2019) Traditional and geometric morphometry analyses of Lutzomyia cruciata (Diptera: Psychodidae: Phlebotominae) popula¬tions of Chiapas, Mexico. J Med Entomol. 56 (3): 697–707.
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| Keywords | ||
| Visceral leishmaniasis; Phlebotomus perfiliewi transcaucasicus; DNA barcoding; Iran | ||
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How to Cite
1.
Barmaki A, Rassi Y, Absavaran A, Akhavan AA, Moradi-Asl E, Zahraei-Ramazani A, Rafizadeh S. Discrimination of Phlebotomus perfiliewi transcaucasicus, Ph. major sensu lato and Ph. tobbi (Diptera: Psychodidae) Using Morphometric and DNA Barcoding Methods in the Endemic Foci of Visceral Leishmaniasis in Ardabil Province, North West of Iran. J Arthropod Borne Dis. 2025;.
