Molecular Characterization and Phylogenetic Congruence of Hydropsyche sciligra (Tricoptera: Hydropsychidae) Using Mitochondrial and Nuclear Markers

  • Naseh Maleki-Ravasan Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran (PII), Tehran, Iran
  • Abbas Bahrami Department of Medical Parasitology and Mycology, Faculty of Medicine, Alborz University of Medical Sciences, Alborz Province, Karaj, Iran Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Hassan Vatandoost Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Mansoureh Shayeghi Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Mona Koosha Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
  • Mohammad Ali Oshaghi Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
Keywords: Caddisflies, Hydropsyche sciligra, COI, LSU rDNA, Molecular systematics, Iran


Background: Caddisflies have significant roles in freshwater ecosystems. Morphological identification is the major impediment in accurate species identification of Hydropsychids. Mitochondrial and nuclear markers are suitable for molecular systematics of these group of arthropods.Methods: Trichopteran specimens of Lavasan District in northeastern Tehran, Iran were collected in 2012, and de­scribed using the morphological and molecular characters of mitochondrial cytochrome c oxidase subunit I (mt-COI) and three expansion fragments of large subunit (LSU) nuclear ribosomal DNA (28S rDNA) D1, D2, and D3. The resemblance of the specimen sequences was obtained by conducting BLAST searches against the GenBank database and by using simple maximum likelihood clustering using COI, D1, D2, D3, and combination of D1-D2-D3 se­quence data sets.Results: Based on morphological traits the specimens were resembled to Hydropsyche sciligra however there were no its counterpart sequences in the GenBank. Due to lack of unique group of data set for each gene fragment, the specimens were associated with different taxa on molecular phylograms. The sequence contents of the COI, D1, D2, D3, and D1-D3 regions clustered H. sciligra with H. brevis, H. angustipennis, H. occidentalis, H. hedini, H. gra­hami, and H. longifurca/H. naumanni, respectively.Conclusion: Phylogenies obtained from combination of D1-D3 showed the highest bootstrap values for most of clades suggesting that long LSU-rDNA potentially is more useful for understanding phylogenetic relationships of caddisflies. A large-scale molecular and zoogeographic study on trichopteran species is suggested to revise and to develop the current knowledge of the caddisfly fauna and distributions in the country.


Absavaran A, Rassi Y, Parvizi P, Oshaghi MA, Abaie M, Rafizadeh S, Mohebali M, Zarea Z, Javadian E (2009) Iden- tification of sand flies of the subgenus Larroussius based on molecular and mor- phological characters in North Western Iran. Iran J Arthropod-Borne Dis. 3(2):22–35.

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI- BLAST: a new generation of protein database search programs. Nucleic Ac- ids Res. 25(17): 3389–3402.

Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Enayati AA, Mardani N, Ghoorchian S (2012) Iden- tification of bacterial microflora in the midgut of the larvae and adult of wild caught Anopheles stephensi: a step to- ward finding suitable paratransgenesis candidates. Acta Trop. 121(2): 129–134.

Chavshin AR, Oshaghi MA, Vatandoost H, Pourmand MR, Raeisi A, Terenius O (2014) Isolation and identification of culturable bacteria from wild Anophe- les culicifacies, a first step in a para- transgenesis approach. Parasit Vectors.7: 419–426.

Chavshin AR, Oshaghi MA, Vatandoost H, Yakhchali B, Zarenejad F, Terenius O(2015) Malpighian tubules are important determinants of Pseudomonas transsta- dial transmission and longtime persis- tence in Anopheles stephensi. Parasit Vectors. 8: 36–42.

Chvojka P (2006) Contribution to the knowledge of the caddisfly fauna (Tri- choptera) of Iran: description of new species and new distributional data. Acta Entomol Mus Nat Pragae. 46:245–255.

Corbet PS (1966) A quantitative method of assessing the nuisance caused by non- biting aquatic insects. Can Entomol.93: 683–687.

Dale C, Moran NA (2006) Molecular inter- actions between bacterial symbionts and their hosts. Cell. 126: 453–465.

Espeland M, Johanson K (2010a) The effect of environmental diversification on species diversification in New Caledo- nian Orthopsyche and Caledopsyche caddisflies (Insecta: Trichoptera: Hy- dropsychidae). J Biogeogr. 37: 879–890.

Espeland M, JohansonKA (2010b) The di- versity and radiation of the largest mon- ophyletic animal group on New Cal- edonia (Trichoptera: Ecnomidae: Ag- mina). J Evolution Biol. 2: 2112–2122. Felsenstein J (1985) Phylogenies and the com- parative method. Amer Nat. 125: 1–15.

Floyd MA (1995) Larvae of the caddisfly genus Oecetis (Trichoptera: Leptoceri- dae) in North America, Ohio Biologi- cal Survey, College of Biological Sci- ences, Ohio State University. New Ser- ries 10: 1–85.

Frania HE, Wiggins GB (1997) Analysis of morphological and behavioural evidence for the phylogeny and higher clas- sification of Trichoptera (Insecta), Life Sci Contrib R Ont Mus. 160: 1–62.

Fremling CR (1959) Biology and possible control of economically important Tri- choptera and phemeroptera of the up- per Mississippi River. [PhD disserta-tion]. Iowa State University of Science and Technology, Ames, Iowa.

Geraci CJ, Zhou X, Morse JC, Kjer KM (2010) Defining the genus Hydropsy- che (Trichoptera: Hydropsychidae) based on DNA and morphological evidence. J N Am Benthol Soc. 29: 918–933.

Gillespie J, Cannone J, Gutell R, CognatoA (2004) A secondary structural model of the 28S rRNA expansion segments D2 and D3 from rootworms and related leaf beetles (Coleoptera: Chrysomelidae, Galerucinae). Insect Mol Biol. 13(5):495–518.

Glover JB (1996) Larvae of the caddisfly genera Triaenodes and Ylodes (Trichop- tera: Leptoceridae) in North America, Ohio Biological Survey, College of Bi- ological Sciences, Ohio State Univer- sity New Series 11.

Hall TA (1999) BioEdit: a user-friendly bi- ological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser. 41: 95–98.

Hyliš M, Oborník M, Nebesářová J, VávraJ (2007) Aquatic tetrasporoblastic micro- sporidia from caddis flies (Insecta, Tri- choptera): Characterization, phylogeny and taxonomic reevaluation of the gen- era Episeptum Larsson, 1986, Pyrothe- ca Hesse, 1935 and Cougourdella Hes- se, 1935. Eur J Protistol. 43(3): 205–224.

Ishiwata K, Sasaki G, Ogawa J, Miyata T, Su ZH (2011) Phylogenetic relationships among insect orders based on three nuclear protein-coding gene sequences. Mol Phylogenet Evol. 58: 169–180.

Ivanov VD (2011) Caddisflies of Russia: Fauna and biodiversity. Zoosymposia.5: 171–209.

Johanson KA (2007) Association and de- scription of males, females and larvae of two New Caledonian Xanthochore- ma species (Trichoptera: Hydrobiosidae) based on mitochondrial 16S and COI sequences. J Entomol Sci. 10: 179–199.

Johanson KA, Espeland M (2010) Phylog- eny of the Ecnomidae (Insecta: Tri- choptera). Cladistics. 26: 36–48.

Johanson KA, Kjer K, Malm T (2009) Test- ing the monophyly of the New Zealand and Australian endemic family Conoe- sucidae Ross based on combined mo- lecular and morphological data (Insec- ta: Trichoptera: Sericostomatoidea). Zool Scripta. 38: 563–573.

Johanson KA, Malm T (2010) Testing the monophyly of Calocidae (Insecta: Tri- choptera) based on multiple molecular data. Mol Phylogenet Evol. 54: 535–541.

Johanson KA, Malm T, Espeland M, Weingart- ner E (2012) Phylogeny of the Pol- ycentropodidae (Insecta: Trichoptera) based on protein-coding genes reveal non-monophyletic genera. Mol Phylo- genet Evol. 65: 126–135.

Kjer KM, Blahnik RJ, Holzenthal RW (2001) Phylogeny of Trichoptera (cad- disflies): characterization of signal and noise within multiple datasets. Syst Biol. 50: 781–816.

Kjer KM, Blahnik RJ, Holzenthal RW (2002) Phylogeny of caddisflies (Insecta, Tri- choptera). Zool Scripta. 31: 83–91.

Lenat DR (1993) A biotic index for the southeastern United States: derivation and list of tolerance values, with crite- ria for assigning water-quality ratings. J N Am Benthol Soc. pp. 279–290.

Lenat DR, Resh VH (2001) Taxonomy and stream ecology-the benefits of genus- and species-level identifications. J N Am Benthol Soc. 20(2): 287–298.

Li K, Tian J, Wang Q, Chen Q, Chen M, Wang H, Zhou Y, Peng Y, Xiao J, Ye G (2011) Application of a novel method PCR-ligase detection reaction for track- ing predator-prey trophic links in in- sect-resistant GM rice ecosystem. Eco- toxicol. 20(8): 2090–2100.

Lunt DH, Zhang DX, Szymura JM, Hewitt GM (1996) The insect cytochrome ox-idase I gene: evolutionary patterns and conserved primers for phylogenetic studies. Insect Mol Biol. 5(3): 153–65. Maleki-Ravasan N, Oshaghi MA, Afshar D, Arandian MH, Hajikhani S, Akhavan

AA, Yakhchali B, Shirazi MH, Rassi Y, Jafari R, Aminian K, Fazeli-var- zaneh RA, Durvasulla R (2015) Aero- bic bacterial flora of biotic and abiotic compartments of a hyperendemic Zo- onotic Cutaneous Leishmaniasis (ZCL) focus. Parasit Vectors. 8: 63.

Maleki-Ravasan N, Bahrami A, Shayeghi M, Oshaghi MA, Malek M, Mansoorian AB, Vatandoost H (2013a) Notes on the Iran Caddisflies and Role of Annu- lipalpian Hydropsychid Caddisflies as a Bio-monitoring Agent. J Arthropod- Borne Dis. 7(1): 71–82.

Maleki-Ravasan N, Oshaghi MA, Hajikhani S, Saeidi Z, Akhavan AA, Gerami- Shoar M, Shirazi MH, Yakhchali B, Rassi Y, Afshar D (2013b) Aerobic microbial community of insectary pop- ulation of Phlebotomus papatasi. J Arthropod Borne Dis. 8(1): 69–81.

Maleki-Ravasan N, Shayeghi M, Najibi B,Oshaghi MA (2012) Infantile Noso- comial Myiasis in Iran. J Arthropod- Borne Dis. 6(2): 156–163.

Maleki-Ravasan N, Oshaghi MA, Javadian E, Rassi Y, Sadraei J, Mohtarami F (2009) Blood meal identification in field-cap- tured sand flies: comparison of PCR- RFLP and ELISA assays. Iran J Ar- thropod Borne Dis. 3(1): 8–18.

Malicky H (1986) Die Köcherfliegen (Tri- choptera) des Iran und Afghanistans. Z Arbeitsgem Osterr Entomol.38: 1–16.

Malicky H (2004) Neue Köcherfliegen aus Europa und Asien. Braueria. 31: 36–42. Malm T, Johanson KA (2008) Revision of the New Caledonian endemic genus Gracilipsodes (Trichoptera: Leptoceri-dae: Grumichellini). Zool J Linnean Soc.153: 425–452.

Malm T, Johanson KA (2011) A new clas- sification of the long-horned caddisflies (Trichoptera: Leptoceridae) based on molecular data. BMC Evol Biol. 11(1):10–26.

Mehravaran A, Oshaghi MA, Vatandoost H, Abai M, Ebrahimzadeh A, Roodi AM , Grouhi A (2011) First report on Anophe- les fluviatilis U in southeastern Iran. Acta Trop. 117(2): 76–81.

Mey W (2004) Beitrag zur Trichoptera-Fauna Armeniens und des Iran (Trichoptera). Entomol Nachr Ber. 48: 81–87.

Milne MJ (1938) The "Metamorphotype Meth- od" in Trichoptera. J N Y Entomol Soc.46: 435–437.

Mirmoayedi A, Malicky H (2002) An up- dated checklist of caddisflies (Insecta, Trichoptera) from Iran, with new rec- ords. Zool Middle East. 26: 163–168.

Moin-Vaziri V, Depaquit J, Yaghoobi-Ershadi MR, Oshaghi MA, Derakhshandeh-Pey- kar P, Ferte H, Kaltenbach M, Bargues MD, Leger N, Nadim A (2007) Intra- specific variation within Phlebotomus sergenti (Diptera: Psychodidae) based on mtDNA sequences in Islamic Re- public of Iran. Acta Trop. 102(1): 29–37.

Morales ME, Wesson DM, Sutherland IW, Impoinvil DE, Mbogo CM, Githure JI, Beier JC (2003) Determination of Anoph- eles gambiae larval DNA in the gut of insectivorous dragonfly (Libellulidae) nymphs by polymerase chain reaction.

J Am Mosq Control Assoc. 19: 163–165. Morse JC (ed.) (2015) Trichoptera World Checklist. Available at:[Accessed 26 October 2015].

Morse JC, Bae YJ, Munkhjargal G, Sangpra- dub N, Tanida K, Vshivkova TS, Wang B, Yang L, Yule CM (2007) Freshwa- ter biomonitoring with macroinverte- brates in East Asia. Front Ecol Envi- ron. 5(1): 33–42.

Myers MJ, Sperling F, Resh V (2001) Dispersal of two species of Trichoptera from desert springs: Conservation implications for isolated vs connected populations. J Insect Conserv. 5: 207–215.

Oshaghi MA, Sedaghat M, Vatandoost H (2003) Molecular characterization of the Anopheles maculipennis complex in the Islamic Republic of Iran. East Mediterr Health J. 9(4): 659–666.

Oshaghi MA, Shemshad K, Yaghobi-Ershadi M, Pedram M, Vatandoost H, Abaie M, Akbarzadeh K, Mohtarami F (2007) Genetic structure of the malaria vector Anopheles superpictus in Iran using mi- tochondrial cytochrome oxidase (COI and COII) and morphologic markers: a new species complex? Acta Trop. 101 (3): 241–248.

Oshaghi MA, Vatandoost H, Gorouhi A, Abai M, Madjidpour A, Arshi S, Sadeghi H, Nazari M, Mehravaran A (2011) Anopheline species composition in borderline of Iran-Azerbaijan. Acta Trop. 119(1): 44–49.

Oshaghi MA, Yaaghoobi F, Abaie M (2006a) Pattern of mitochondrial DNA varia- tion between and within Anopheles ste- phensi (Diptera: Culicidae) biological forms suggests extensive gene flow. Acta Trop. 99(2–3): 226–233.

Oshaghi MA, Chavshin AR, Vatandoost H (2006b) Analysis of mosquito blood- meals using RFLP markers. Exp Para- sitol. 114(4): 259–264.

Oshaghi M, Yaghobi-Ershadi M, Shemshad K, Pedram M, AmaniH (2008) The Anopheles superpictus complex: intro- duction of a new malaria vector com- plex in Iran. Bull Soc Pathol Exot. 101: 429–434.

Oshaghi MA, Rasolian M, Shirzadi MR, Moh- tarami F, Doosti S (2010) First report on isolation of Leishmania tropica from sandflies of a classical urban Cutaneous leishmaniasis focus in southern Iran. Exp Parasitol. 126(4): 445–450.

Oshaghi MA, Ravasan NM, Hide M, Ja- vadian EA, Rassi Y, Sadraei J, Mohe- bali M, Sedaghat MM, Hajjaran H, Zarei Z (2009a) P hlebotomus perfil- iewi transcaucasicus is circulating both Leishmania donovani and L. infantum in northwest Iran. Exp Parasitol. 123 (3): 218–225.

Oshaghi MA, Ravasan NM, Javadian EA, Mohebali M, Hajjaran H, Zare Z, Mohtarami F, Rassi Y (2009b) Vector incrimination of sand flies in the most important visceral leishmaniasis focus in Iran. Am J Trop Med Hyg. 81(4):572–577.

Pauls SU, Graf W, Haase P, Lumbsch HT, Waringer J (2008) Grazers, shredders and filtering carnivores-the evolution of feeding ecology in Drusinae (Tri- choptera: Limnephilidae): insights from a molecular phylogeny. Mol Phyloge- net Evol. 46: 776–791.

Pescador ML, Rasmussen AK, Harris SC (1995) Identification manual for the caddisfly (Trichoptera) larvae of Flor- ida. Fla Dept Environ Prot, Tallahas- see, FL.

Previšić A, Walton C, Kučinić M, Mitrikeski PT, Kerovec M (2009) Pleistocene di- vergence of Dinaric Drusus endemics (Trichoptera, Limnephilidae) in multiple microrefugia within the Balkan Penin- sula. Mol Ecol. 18: 634–647.

Resh VH (1972) A technique for rearing caddisflies (Trichoptera). Can Entomol.

: 1959–1961.

Resh VH, Unzicker JD (1975) Water quality monitoring and aquatic organisms: the importance of species identification. J Water Pollut Control Fed. 47: 9–19.

Ruiter DE, Boyle EE, Zhou X (2013) DNA barcoding facilitates associations and diagnoses for Trichoptera larvae of the Churchill (Manitoba, Canada) area. BMC Ecol. 13(1): 5–43.

Russell JA, Funaro CF, Giraldo YM, Goldman-Huertas B, Suh D, Kronauer DJ, Moreau CS, Pierce NE (2012) A ver- itable menagerie of heritable bacteria from ants, butterflies, and beyond: broad molecular surveys and a system- atic review. PloS One. 7(12): e51027.

Schmid F (1959) Trichoptères d'Iran. Akad- emie-Verlag.Sheppard S, Bell J, Sunderland K, Fenlon J, Skervin D, Symondson W (2005) De- tection of secondary predation by PCR analyses of the gut contents of inverte- brate generalist predators. Mol Ecol. (14): 4461–4468.

Seshadri AR (1955) An extraordinary out- break of caddis flies (Trichoptera) in the Meltrudam township area of Salem district, South India. S Indian J En- tomol. 3: 337–340.

Sint D, Raso L, Kaufmann R, Traugott M (2011) Optimizing methods for PCR- based analysis of predation. Mol Ecol Resour. 11(5): 795–801.

Sonnenberg R, Nolte AW, Tautz D (2007) An evaluation of LSU rDNA D1-D2 sequences for their use in species iden- tification. Front Zool. 4(1): 6–17.

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analy- sis using maximum likelihood, evolu- tionary distance, and maximum parsi- mony methods. Mol Biol Evol. 28(10):2731–2739.

Whiting MF, Carpenter JC, Wheeler QD, Wheeler WC (1997) The Strepsiptera problem: phylogeny of the holometab- olous insect orders inferred from 18S and 28S ribosomal DNA sequences and morphology. Syst Biol. 46: 1–68.

Wiggins GB (1996) Larvae of the North American caddisfly genera (Trichop- tera), University of Toronto Press Toronto, p. 457.

Zhang DX, Hewitt GM (1997) Insect mito- chondrial control region: a review of its structure, evolution and usefulness in evolutionary studies. Biochem Sys Ecol. 25: 99–120.

Zhou X, Adamowicz SJ, Jacobus LM, Dewalt RE, Hebert PD (2009) Towards a comprehensive barcode library for arc- tic life-Ephemeroptera, Plecoptera, and Trichoptera of Churchill, Manitoba, Can- ada. Front Zool. 6: 30–39.

Zhou X, Kjer KM, Morse JC (2007) Asso- ciating larvae and adults of Chinese Hydropsychidae caddisflies (Insecta: Trichoptera) using DNA sequences. J N Am Benthol Soc. 26(4): 719–742.

How to Cite
Maleki-Ravasan N, Bahrami A, Vatandoost H, Shayeghi M, Koosha M, Oshaghi MA. Molecular Characterization and Phylogenetic Congruence of Hydropsyche sciligra (Tricoptera: Hydropsychidae) Using Mitochondrial and Nuclear Markers. J Arthropod Borne Dis. 11(1):60-77.
Original Article