Molecular identification of Haemaphysalis sulcata (Acari: Ixodidae) larval stages collected using the Berlese funnel in Northern Iran

Ticks are among one of the most notorious and well-known arthropods in terms of bites and transmission of serious pathogens to animals and humans such as viruses, bacteria and protozoa (Marcondes and Dantas-Torres 2017). Genus Haemaphysalis is the second largest ixodid tick genus around the world with numerous species found in Asia and Africa (Estrada-Peña et al. 2017). Finding immature stages of ticks and identifying hosts provides important information about the biology of tick species (Keskin et al. 2013). Some ticks of domestic animals spend their immature stages on wild hosts like Haemaphysalis sulcata whose immature stages have been reported from reptiles (Estrada-Peña et al. 2004). This tick is one of the most important species infected with Spotted Fever Group (SFG) Rickettsia in Iran (Hosseini-Chegeni et al. 2020). Two earlier studies in Iran emphasized the taxonomy of adult stages using morphological and molecular characters (Hosseini-Chegeni et al. 2014, 2017). Molecular detection techniques based on conventional PCR and sequencing of the partial genome of living organisms is considered an important tool for identification (Reller et al. 2007). In this study, we report immature H. sulcata isolated from soil. To our knowledge, this is the first molecular systematic study on larval ticks collected using Berlese funnel and showing usefulness of molecular techniques in Iran. In the present study, two samples of tick larvae were collected in Mirafzal forest area (36° 07' 39.0" N, 53° 35' 45.0" E) located in Mazandaran Province, northern Iran. In this study, samples were randomly selected from different regions of up to 25 cm soil depth so decayed leaves were removed. For isolation of tick samples, a modified Berlese funnel was used according to Krantz and Walter (2009). The collected samples were transferred into a glass jar containing 99% ethanol. Tick samples were initially identified using a generic identification key (Bregetova et al. 1955) and in order to narrow identification, PCR and Sanger sequencing techniques were performed.