First Report of Spirocerca Lupi Larva in Dung Beetles (Scarabaeus Armeniacus) in Iran: A Morphological and Molecular Identication

Background: Dung beetles are detritivores insects. They naturally use feces of vertebrates as foods and reproduction beds. This leads to frequent contacts between dung beetles and parasitic helminths. The current study was carried out to assess infections of dung beetles with larval stages of helminths in rural areas of Taleqan County, Alborz Province, Iran. In total, 200 dung beetles were randomly collected in June 2017 from the highlands of Taleqan County. Beetles were dissected in normal saline and carefully studied using stereomicroscopy. Morphological characteristics of the recovered larvae were drawn using camera lucida equipped microscope at 400× magnication. Furthermore, genomic DNAs of the recovered larvae were extracted and PCR amplications of the mitochondrial cytochrome c oxidase subunit 1 (cox1) genes were carried out and the amplicons were sequenced. Results: All collected dung beetles were identied as Scarabaeus armeniacus from Scarabaeidae family (55.5% were male and 44.5% female). Three females of the beetles were infected with nematode larva, morphologically identied as the third-stage of Spirocerca lupi larvae. The average length and width of the larvae were 2.95 (2.81–3.15; CI 95%) and 0.12 (0.1–0.15; CI 95%) mm, respectively. The phylogenetic analysis showed that S. lupi belonged to a clade within Spirocercidae family, well separated from Onchocercidae family. Conclusions: In the current study, S. armeniacus was introduced as a potentially biological vector for the transmission of S. lupi to vertebrates the region. To the best of the authors’ knowledge, this is the rst reported on larval stages of S.


), CI: 95%]
and excretory pore from the anterior end [0.09mm (0.07-0.11 mm, CI: 95%], respectively.The average size of buccal capsule was 0.051 × 0.013 mm. the average size of rectum and tail were 0.053 and 0.041 mm, respectively.Most of the larvae moved freely in the normal saline after the beetle dissection; some of them were still in encysted form, which were spontaneously emerged.Although some cysts were found attached to the tracheal tubes, most of them were free in the normal saline.In morphological characteristics of S. lupi larvae, thickness of the larvae was uniform, with transverse ridges.Two cephalic horns were recognized at the interior end of the terminal mouth, leading to a strong, sclerotic cylindrical buccal capsule.The tail was formed with ve closely sets of circular spines.Camera lucida drawings of the anterior and posterior ends of S. lupi 3rd larval stage are illustrated in Fig. 2.

Molecular Findings Of The Recovered Larvae Specimens of S. lupi were successfully ampli ed with nearly 680-bp amplicons for the partial cox1 genes.In this study, a unique nucleotide sequence of the partial cox1 gene from S. lupi was annotated in GenBank database (accession number MT522373).Phylogenetic analysis based on the cox1 gene illustrated that S. lupi belonged to a clade within the Spirocercidae family and was well separated from Onchocercidae family (Fig. 3).In this study, the sequence alignment revealed high levels of sequence homologies between S. lupi isolates and those globally documented in GenBank (Fig. 4).Results showed that the current nucleotide sequence of S. lupi included 100% identity with the recovered isolates of dogs from outh Africa (EF394612.1),Israel (EF394606.1),Austria (EF394605.1)and Iran (EF394608.1)(Fig. 4).Moreover, pairwise distance showed intraspecies genetic variations of 0-0.4% between the current isolate of S. lupi, and those available in GenBank.


Discussion

Regarding prospective roles of dung beetles in transmission of certain helminths such as acanthocephalans and Spirurid nematodes to humans and animals, faunistic investigation of their parasites seems to include great veterinary and medical signi cances [1].The current study rstly demonstrated roles of dung beetles (S. armeniacus), as intermediate hosts of S. lupi, in Iran.Literatures are available on spirocercosis prevalence in dogs in Iran.Oryan et al. reported 19% of S. lupi infection in stray dogs in the southern regions of Iran [26].In Mirzayans et al. study, S. lupi was reported as one of the most common helminths in house-dogs in Tehran Province, Iran [27].Moshfe et al. documented 17.4% of S. lupi in stray dogs in western areas of Iran [28].In Sadighian study, 45.4% of the stray dogs were infected by S. lupi in Northern Iran [29].Moreover, S. lupi infection in Vulpes vulpes (red fox) in Northwestern Iran was reported as 17.9% [30].In some of these studies, high prevalence of spirocercosis is attributed to the presence of dung beetles.However, presence of intermediate hosts (e.g.amphibians, reptiles, lizards, domestic and wild birds) and small mammals (e.g.mice and rabbits) as well as different life and hunting styles are important factors in prevalence

in carnivores [26,31,32].

To the best of
he authors' knowledge, no studies have been carried out on S. lupi infection in dung beetles in Iran.However, dung beetle infection with Gongylonema spp.has been recorded in Northwestern Iran [33].Studies have documented presence of S. armeniacus in Central and Northern Iran [34,35].Dung beetles play critical roles in ecosystem,

anure and nutrient recycling.They rely on feces
for food for themselves and their larvae and lay their eggs in feces.An individual dung beetle can bury feces 250 times larger than that a beetle can in one night.Dung beetles are capable of carrying weights, nearly 50 times heavier than their body weights.Many dung beetles prefer a certain type of animal feces and can y up to 10 miles to nd their desirable food [36,37].Several factors affect dung beetle communities, including nature of the soil substrate, ora of the speci c region, rainfall and temperature [7,23].In Iran, various aspects of S. lupi life cycle is still undescribed.In the present study, beetles were collected in summer.Prevalence of spirocercosis differs over a comparatively brief period [7,38].In a study by Chhabra and Singh [24], prevalence of infection in beetles increased in mid-season of laboratory-infected dung beetles.In another study, detection rates of spirocercosis during cold months have signi cantly been higher [39].This might be explained by the seasonality in the studied country.

Nucleotide BLAST analysis from the current study showed high sequence homologies between S. lupi isolates and those from other studies.The current results showed that nucleotide sequence of S. lupi from this study included 100% identity with recovered isolates of dogs from South Africa (EF394612.1),Israel (EF394606.1),Austria (EF394605.1)and Iran (EF394608.1)[40].Based on the pairwise distance of the isolate from the current study, intraspecies genetic variation with

S. lupi nucleotide sequences included 0-0.
% with an overall average of 0.1% between the current isolate of S. lupi and the sequences available in GenBank.Several studies have been carried out on the molecular characterization of Spirocerca spp.In Traversa et al. study, the cox1 gene of S. lupi collected from ve countries was investigated and only a low genetic variation within the sequences was detected, demonstrating at least 99% nucleotide similarity [40].In contrast, de Waal et al. reported signi cant genetic diversities in Spirocerca isolates collected from a dog [41].A study by Rojas et al. on detection of spirocercosis in dogs using HRM qPCR in fecal samples showed that HRM qPCR of ITS1 included the best performance in limit of detection and absence of cross-ampli cation with other canine parasites [42].Ruggeri et al. detected 18S ribosomal DNA (rDNA) of S. lupi gene in cerebrospinal uid (CSF) of presumptively-affected dogs using PCR.Results revealed that PCR targeting 18S rDNA of S. lup

in CSF was
apable of antemortem diagnosis of canine intraspinal spirocercosis [43].


Conclusion

In the current study, S. armeniacus has been introduced as a potentially biological vector for the transmission of S. lupi to vertebrates in Iran.To the best of the authors' knowledge, this is the rst report on the larval stages of S. lupi in S. armeniacus.Further studies are necessary to investigate better control and prevention of the disease in dogs.Better knowledges of dynamics of the intermediate host-parasite intersections between dung beetles and S. lupi in Iran can establish preventive methods to limit spread of this disease in domestic dogs.


Materials And Methods


Sample collection

Totally, 200 dung beetles were randomly collected in June 2017 from the highlands of Taleqan County, Alborz Province, Iran (36.1748°N, 50.7650°E), a common passage o jackals, foxes and other canids and felids (Fig. 5) [44].Wild boar, dog and livestock dung pitfall traps were used to capture dung beetles, previously described by Du Toit et al. [14].Trapped dung beetles were transferred to the Helminthology Lab of the Department of Medical Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran, using ventilated containers.


Species identi cation of the dung beetles

At the time of the assessment, most of the dung beetles were still alive.Therefore, stored in a refrigerator with autoclaved soil for 2 h to immobilize them.Then, species and sexes of the collected beetles were identi ed taxonomically, using entomology keys [45].


Morphological analysis of the recovered larvae

Beetles dissected in normal saline solution and carefully studied for the presence of parasites with special focuses on larval stages using stereomicroscope.The beetles have been reported individually as positive or negative for larval stages of helminths.For positive beetles, larvae were removed and transferred into normal saline on ice for 1 h to relax and immobilize [33].Removed larvae were transferred into lactophenol and 70% ethanol for morphological and molecular studies.Morphological characteristics of the isolates were recorded carefully using camera lucida equipped microscope at 400× magni cation.Identi cation was comparatively carried out based on taxonomic key references 46].


Molecular analysis of the recovered larvae


DNA extraction

Ethanol preserved larvae were washed three times with sterile distilled wat

by centrif
gation at 5000× g for 5 min to remove ethanol.Then, larvae were subjected to ve cycles of freezing in liquid nitrogen and thawing in boiling water.Approximately 300 mg of glass beads (0.5 mm in diameter) were added to the larvae and shaken intensively for 5 min.Then, genomic DNA was extracted using genomic DNA extraction kit (GeneAll Exgene, South Korea) according to the manufacturer's instructions and stored at -20 °C until use.The DNA concentration was assessed spectrometrically (NanoDrop ND-1000, Thermo Fischer Scienti c, USA) at 260 nm.The 260/280 ab

istilled water) and positive control (extr
cted DNA
 AcknowledgementsThe authors are grateful to Tehran University of Medical Sciences for cooperation during the study.Authors' information 1 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 2 Departm

t of Parasitology and Mycology, School of Medic
ne, Shiraz University of Medical Sciences, Shiraz, Iran. 3 Department of Medical Parasitology and Mycology, School of Medicine, Jiroft University of Medical Sciences, Jiroft, Iran. 4 School of Biology, College of Science, University of Tehran, Tehran, Iran. 5 Department of Environmental Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran.Availability of data and materialsPage 8/11All data generated or analyzed during this study are included in this published article.The original datasets are available upon request to the corresponding author.The generated sequence of S. lupicox1 gene was deposited in the GenBank database under the accession number MT522373.FundingThis study did not receive any speci c grants from funding agencies in the public, commercial or not-forpro t sectors.from S. lupi provided by the Faculty of Veterinary Medicine, University of Tehran) were used in each set.Ampli cation was carried out using P qSTAR Thermal Cycler (PeqLab, Germany) using the following cycling protocol of initial denaturation at 94 °C for 7 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min and extension at 72 °C for 1 min.Final extension was carried out at 72 °C for 10 min.Ampli ed products were electrophoresed on 1.5% agarose gels and visualized using UV transilluminator.The PCR products were sequenced sing Sanger method in both directions (Bioneer, South Korea).Sequence analysisSequences were edited and trimmed using software v.2.6.1 (Chromas, Analysis of the sequencing data was carried out using BLAST program and NCBI databases (http://www.ncbi.nlm.nih.gov/).Multiple sequence alignments were carried out using Clustal W method and BioEdit software v.7.1 (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and results were compared to sequence results from GenBank database.Phylogenetic analysisSequences were edited and compar

to entries from N
BI GenBank for further analysis.The best-t model of nucleotide substitution was statistically selected by the MEGA software v.6.0 (Pennsylvania State University, USA)[47].The phylogenetic tree of Spirocerca spp. was constructed using the Maximum Likelihood (ML) method in agreement with Hasegawa-Kishino-Yano model with uniform rates for transitions and transversions.Bootstraps of 1,000 replicates were used for the as

ssment of topology rel
ability of the trees.Abbreviations stage of larvae; cox1: cytochrome c oxidase subunit 1; mtDNA: mitochondrial DNA; PCR: Polymerase chain reactions; BLAST: basic local alignment search tool; ML method: Maximum Likelihood method; HRM: High Resolution Melting Analysis; qPCR: quantitative PCR; rDNA: ribosomal DNA; CSF: cerebrospinal uid.DeclarationsEthics approval and consent to participate Not applicable.Consent for publicationNot applicable.Competing InterestsThe that they have no competing interests.Authors' contributions SM and GM conceived and design