J Arthropod-Borne Dis, June 2017, 11(2): 338–343 M S Makki et al.: Identification of Hymenolepis … 338 http://jad.tums.ac.ir Published Online: May 27, 2017 Short Communication Identification of Hymenolepis diminuta Cysticercoid Larvae in Tribolium castaneum (Coleoptera: Tenebrionidae) Beetles from Iran Mahsa Sadat Makki 1, Gholamreza Mowlavi 1, Farideh Shahbazi 1, Mohammad Reza Abai 2, Faezeh Najafi 1, Bibi Razieh Hosseini-Farash 3, Salma Teimoori 1,4, Hamid Ha- sanpour 1, *Saied Reza Naddaf 5 1Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medi- cal Sciences, Tehran, Iran 2Department of Medical Entomology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Parasitology and Mycology, Research Center for Skin Disease and Cutaneous Leishmani- asis, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 4Centre of Excellence for Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine, Siriraj Hospital, Bangkok, Thailand 5Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran (Received 15 Jan 2015; accepted 8 Aug 2016) Abstract Background: Hymenolepis diminuta is a cestod of rodents and rarely infects humans. Infection in humans is via ingestion of infected insects. This study was aimed to detect H. diminuta cysticercoids in red flour beetles, Tribolium castaneum, and cockroaches originated from different regions of Iran. Methods: The red flour beetles and cockroaches were collected from local bakeries in five cities including Tehran, Ahvaz, Kazerun, and Sabzevar during 2010–2011. Some beetles and cockroaches were colonized in insectary and adults from F1 generation were fed on H. diminuta eggs. Both laboratory-infected and field-collected samples were dissected and examined for cysticercoids. Detection of H. diminuta DNA in T. castaneum beetles was performed by targeting a partial sequence of Ribosomal gene. Results: Except the beetles from Ahvaz, all specimens were negative for cysticercoid by microscopy. Of the four dissected beetles from Ahvaz, one harbored 12 cysticercoids. Also, 110 (52%) of laboratory-infected beetles showed infection with an average of 12–14 larvae. None of the cockroaches was infected. Two beetles from Ahvaz, includ- ing the remainder of the microscopic positive specimen, yielded the expected amplicon in PCR assay. The H. diminuta DNA sequences generated in this study were identical and matched 97–100% with similar sequences from GenBank database. Conclusion: Lack of infection in the majority of beetles may reflect a low rat infestation rate in those areas, alterna- tively, the examined specimens might not have been the representative samples of the T. castaneum populations. Keywords: Hymenolepis diminuta, Cysticercoid, Tribolium castaneum, Iran Introduction Human hymenolepiasis is caused by two cestodes, Hymenolepis nana, and H. diminuta (Wiwanitkit 2004, Magalhaes et al. 2013). There is a single report on mixed in- fection of the third species H. microstoma with H. nana in remote communities in the northwest of Western Australia (Macnish et al. 2003). The tapeworm, H. nana is the primary cause of human infection, whereas H. diminuta rarely infect humans, and so far, only a few hundred cases have been reported (Tena et al. 1998, Wiwanitkit 2004). However, with in- creased awareness of the disease and im- *Corresponding author: Dr Saied Reza Naddaf, E-mail: snaddaf_2001@yahoo.com J Arthropod-Borne Dis, June 2017, 11(2): 338–343 M S Makki et al.: Identification of Hymenolepis … 339 http://jad.tums.ac.ir Published Online: May 27, 2017 provement of laboratory techniques, more cases become apparent. Although the identified cases are mostly children, the disease can be seen in every age group (Cohen 1989, Tena et al. 1998, Marangi et al. 2003). Unlike, H. nana, transmission of H. diminuta requires an arthropod intermediate host to complete its life cycle. When H. diminuta fertilized eggs are excreted in the stool of an infected definitive host, they are ingested by various arthropods. Once inside the insect body, the oncospheres are released from the eggs, pen- etrate the intestinal wall of the host and de- velop into cysticercoid larvae. Soon after the ingestion of infected arthropod by the mam- malian host, the cysticercoid larvae are re- leased in the stomach, and small intestine and their life cycle is completed. The maturation occurs within 20–25d, and the adult worms can reach an average of 30cm in length. Hu- mans are accidentally infected via ingesting the arthropods carrying cysticercoids. Various arthropod species including flour beetles, moths, earwigs, and flea larvae may serve as the intermediate host. The red flour beetle, Tribolium castaneum, is an efficient in- termediate host of this cestode (Bisseru 2013). It is a worldwide stored food pest and attacks grain products including flour, cereals, pasta, biscuits, beans, and nuts. The adults are long- lived and may live for more than three years. This study was aimed to detect H. diminu- ta cysticercoids in T. castaneum beetles and cockroaches collected from local bakeries in five cities of Iran by microscopy and PCR assay. We also discuss the susceptibility of these beetles along cockroaches to H. diminuta infection under laboratory condition. Materials and Methods Collection of Tribolium castaneum beetles Bakeries in different cities including Teh- ran in upper center, Ahvaz in the southwest, Iranshahr in the southeast, Kazerun in south, and Sabzevar in the northeast of the country were searched for red flour beetles during 2010–2011. The collected samples were kept in tubes containing flour at 28–30 °C and humidity of 20–40%. The collected insects were identified to the species based on mor- phological features using diagnostic keys (Bos- quet 1990). Similarly, some cockroaches were collected from the same bakeries in Tehran. Hymenolepis diminuta eggs Rattus norvegicus rats were captured from rat-infested areas in Tehran. The animals were euthanized, sacrificed, and mature worms were collected from the intestine. Posterior gravid proglottids of the worms were removed and dissected in normal saline to free the eggs. Laboratory-infected beetles Flour beetles, T. castaneum, and American cockroaches, Periplaneta americana, were reared in the insectary at 28–30 °C with rel- ative humidity of 20–40% and light and dark cycle of 12h/12h. The adult progenies from the F1 generations were allowed to feed on dough contaminated with H. diminuta eggs. After three weeks, the cockroaches and bee- tles were dissected in normal saline and ex- amined for cysticercoids under a stereomi- croscope with a magnification of 40X (Mak- ki et al. 2011). Identification of cysticercoids in beetles by microscopy We used 154 field-collected flour beetles (30 specimens from Tehran, 60 from Iranshahr, 30 from Kazeroun and Sabzevar each, and four from Ahvaz), 50 field-collected Ameri- can cockroaches from Tehran, 210 labora- tory-infected beetles, and 40 American and 70 German laboratory- infected cockroaches in this study. All the insects were dissected in normal saline and examined for cysticer- coid larvae under a stereomicroscope with a magnification of 40X. PCR and sequencing DNA extraction was performed on individ- J Arthropod-Borne Dis, June 2017, 11(2): 338–343 M S Makki et al.: Identification of Hymenolepis … 340 http://jad.tums.ac.ir Published Online: May 27, 2017 ual and pools of 20–50 beetles of laboratory- infected beetles as well as field-collected sam- ples by phenol-chloroform method followed by ethanol precipitation as described by others (Ballinger-Crabtree et al. 1992). A partial ri- bosomal DNA sequence of H. diminuta (span- ning the 3’ end of the 18S rRNA gene, inter- nal transcribed spacer 1 (ITS1), 5.8S, ITS2 and the 5’end of the 28S rRNA gene) was targeted using the primers HF1 5’-gcggaa- ggatcattacacgttc-3’ and HR1 5’-gctcgactctt- catcgatccacg-3’ designed by others (Macnish et al. 2002). The 25µ l reaction mixture con- tained 20pmol of each primer, 2.5mM MgCl2, 10mM Tris-HCl, 50mM KCl, 200μM of dNTPs, 1U of Taq polymerase, and 3μl of DNA. All amplifications were programmed for an initial denaturation step for 5 min at 94 °C, followed by 30 cycles of 94 °C for 30s, 63 °C for 40s, and 72 °C for 45s, with a final step of 10min at 72 °C. For field- collected samples, the annealing temperature was reduced to 61 °C and the cycles in- creased to 32. In all amplifications, DNA from H. diminuta and H. nana adult worms were included as positive controls and DNA of adult Taenia sp. as the negative control. The PCR products were run on 1% agarose gel, stained with ethidium bromide and visu- alized under UV. Amplicons from one field- collected beetle and H. diminuta and H. nana adult worms were sequenced in both directions using the same primers used for amplification and compared with similar se- quences from GenBank database. The data for sequences were submitted to GenBank database with accession numbers KJ917784 and KJ917785-7 for H. nana and H. diminuta, respectively. Results Microscopy Except for T. castaneum beetles from Ah- vaz, all the specimens were negative for H. diminuta cysticercoids by microscopy. Out of four dissected beetles from Ahvaz, one harbored 12 H. diminuta cysticercoids (Fig. 1). Also, of the 210 T. castaneum beetles fed on food contaminated with the tapeworm eggs, 110 developed cysticercoids with an average of 12–14 larvae in insects. None of the la- boratory-reared cockroaches fed in similar fashion turned positive. PCR and sequencing PCR amplification of rDNA gene from H. nana and H. diminuta adult worms yielded the expected bands of 645bp and 750bp, re- spectively. The 750bp band diagnostic for H. diminuta was observed in the pools of labor- atory-infected T. castaneum beetles. Our PCR method could detect the DNAs equivalent to one and three cysticercoids in pools of 20 and 50 non-infected beetles, respectively. DNAs from 35 individual and ten pools of 20-50 beetles collected from different areas were negative for H. diminuta. However, DNA samples of two individual beetles from Ahvaz including the remainder of the micro- scopic positive specimen yielded the expected 750bp amplicon, implying the presence of H. diminuta in the insects (Fig. 2). The H. diminu- ta ribosomal DNA sequences generated in this study were identical and showed 97%- 100% homology with three H. diminuta gene sequences from GenBank database (acces- sion numbers, AF461125, JN258039, and JN258038) over 94%–100% of nucleotides. Fig. 1. An H. diminuta cysticercoid derived from a T. castaneum beetle collected from Ahvaz, Iran. J Arthropod-Borne Dis, June 2017, 11(2): 338–343 M S Makki et al.: Identification of Hymenolepis … 341 http://jad.tums.ac.ir Published Online: May 27, 2017 Fig. 2. Amplification of a partial ribosomal DNA sequence (spanning the 3’ end of the 18S rRNA gene to the 5’end of the 28S rRNA gene) from Hymenole- pis spp. 1, H. nana adult worm, M, 100bp marker, 2, H. diminuta adult worm, 3, infected beetle, 4, cysti- cercoid larvae, 5, Taenia sp, 6, negative control. Discussion The rat tapeworm, H. diminuta, is a cestode of rodents. In Iran, it is commonly found in rats and mice (Kia et al. 2001, Meshkekar et al. 2014, Yousefi et al. 2014). Recently, it was found in Tatera indica from southeast of the country (Nateghpour et al. 2015). This species may also affect pet animals like squirrels (Callosciurus prevosti) (d’Ovidio et al. 2015) and laboratory rats (Sreedevi et al. 2015). Hymenolepis diminuta infection in hu- mans is very rare, but with increased aware- ness of the disease and the ability to identify different species of the genus Hymenolepis, more cases are becoming apparent. Infection of H. diminuta has recently been reported from various countries including Turkey, Tamil Nadu, Sri Lanka and India (Kalaivani et al. 2014, Sinhabahu et al. 2014, Tiwari et al. 2014, Kılınçel et al. 2015). In Iran, The last human infection was identified in a child in 2008 (Mowlavi et al. 2008). Infection with this worm is commonly light and self-limit- ing and deworming can be achieved with praziquantel as the drug of choice (Karuna and Khadanga 2013). The flour beetles belonging to the genera Tribolium and Tenebrio are among the known intermediate hosts of this worm. They are adapted to survive in arid environments and are highly resistant to insecticides. The sus- ceptibility to H. diminuta infection may vary among the members of these two genera, and with their life stage. In samples collected from a riding stable in Quebec, the intensity of cysticercoids in Tenebrio obscurus was higher than T. molitor and adults were more susceptible to infection than larvae (Rau 1979). Also, T. castaneum was more vulner- able to the infection than T. confusum (Yan and Norman 1995). In the present study, all the T. castaneum beetles from different cities, except Ahvaz, were negative for H. diminuta cysticercoid by microscopy and PCR assay. Examination of limited specimens from Ahvaz revealed infection in two beetles. This finding sug- gests a high infection rate among the beetles, which can maintain the enzootic cycle. An earlier study in this city revealed high hel- minthic infection rates, particularly with Spi- rurids, among R. norvegicus rats. Out of 72 dissected rats, 8 (11.1%) were infected with H. diminuta (Kia et al. 2001). In the present study, 52% of the T. castaneum beetles fed on dough contaminated with H. diminuta eggs developed cysticercoids, this reflects their high susceptibility to hymenolepiasis, and their po- tency to serve as an efficient intermediate host. Unlike T. castaneum beetles, in none of the cockroaches fed in similar manner cysti- cercoids developed. Cockroaches have shown to be refractory to H. diminuta infection due to encapsulation of larvae by the insect he- mocytes (Tu and Lai 2006). Prior infection of cockroaches with Moniliformis moniliformis may predispose them to H. diminuta infec- tion. In cockroaches, a membranous envelope engulfs the acanthocephalan M. moniliformis and protects it from the hemocyte attack in the arthropod host (Holt 1989). The hatched oncospheres of H. diminuta may penetrate this envelope and, once inside, utilize its protec- tive function to develop. J Arthropod-Borne Dis, June 2017, 11(2): 338–343 M S Makki et al.: Identification of Hymenolepis … 342 http://jad.tums.ac.ir Published Online: May 27, 2017 Conclusion Lack of infection in the majority of bee- tles may reflect a low rat infestation rate in those areas, alternatively, the examined spec- imens might not have been the representative samples of the T. castaneum populations. Further studies with inclusion of more samples and other potential intermediate arthropod hosts are required to elucidate the biology and life cycle of H. diminuta in urban rats. Acknowledgements The authors are very grateful to animal husbandry workers, Neda Mirsepahi and Ali Rahimi from School of Public Health, Teh- ran University of Medical Sciences, for their kind assistances during this research. 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