International Journal of Tropical Insect Science (2021) 41:241–250 https://doi.org/10.1007/s42690-020-00199-4 ORIGINAL RESEARCH ARTICLE Comparative micro-morphological and phylogenetic analysis between Rhinoestrus purpureus and Rhinoestrus usbekistanicus (Diptera: Oestridae) larvae and its adults Olfat A. Mahdy1 & Marwa M. Attia1 Received: 3 December 2019 /Accepted: 19 June 2020 / Published online: 14 July 2020 # African Association of Insect Scientists 2020 Abstract Rhinoestrus purpureus and Rhinoestrus usbekistanicus (Diptera: Oestridae) cause specific nasal myiases of family equidae and are of importance in the horse medicine since it causes severe respiratory diseases. During an epidemiological survey in donkeys from Beni Suef government, Egypt; some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus spp. 1st, 2nd and 3rd stage larvae based on the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus, R. purpureus and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The genes showed a very low level of sequence variation (COI 0–0.43%, 16S 0–1.45%, 28S 0–0.23%) falling within the intraspecific ranges. Finally, the peritreme features and the spinulation of the third segment of the two morphotypes examined could not be used to differentiate the Rhinoestrus spp. Keywords Rhinoestrus purpureus . Rhinoestrus usbekistanicus . Light and scanning electron microscope . Rhinoestrosis . Molecular identification . PCR Introduction Rhinoestrus spp. may cause different forms of ophthalmo- myiasis and conjunctivitis in humans (Peyeresblanques 1964). Infestation by Rhinoestrus spp. is the main specific nasal my- The monthly prevalence of 1st, 2nd and 3rd stage larvae was iasis that occurs in members of the family Equidae, especially of primary interest as these values indicated the durations of in regions with temperate climate, such as the Mediterranean various stages of the life cycle of Rhinoestrus spp. and the num- countries. The presence of Rhinoestrus spp. in nasal passages ber of generations occurring per year as well as the suitable time and sinuses induces local inflammation, and the infestation is for control and disruption of the life cycle (Hilali et al. 2015). In a characterized by different respiratory signs of varying intensi- previous study occured in Egypt, Zayed et al. (1993) and Hilali ty and severity, ranging from inflammation to dyspnoea, et al. (2015) who, reported the occurrence of two generations of sneezing and cough. Moreover, lesions of the upper respira- Rhinoestrus spp. infesting donkeys in Egypt had two generations tory tract and lungs and damage of the olfactory nerves were throughout the year, with two peaks of infestation for both 1st- reported upon the larval attack of the olfactory nerves. stage larvae and total number of larvae occurring in (March and Restlessness, reduction in athletic yield, and even death June) and (January and June) respectively. On the other hand, (Dong et al. 2018;Yanaetal.2019) may occur due to enceph- Mula et al. (2009) suggested that there are three defined periods alomyelitis caused by penetration of the ethmoid and menin- in the chronobiology of this fly. Several previous studies provid- ges (Zumpt 1965; Otranto et al. 2005). Additionally, ed a full description of the 3rd-stage larvae of the two species present in Egypt and Italy with a molecular analysis of Rhinoestrus sp. L , which revealed two morphotypes; * 3 Marwa M. Attia R. usbekistanicus-like and the other R. purpureus-like; (Otranto [email protected]; [email protected] et al. 2005;Mulaetal.2013). Molecular analysis of cytochrome- 1 Parasitology Department, Faculty of Veterinary Medicine, Cairo oxidase subunit I (COX-1) of L3 in Egypt revealed that 99% University, El-Giza 12211, Egypt homology with R. usbekistanicus (Hilali et al. 2015). 242 Int J Trop Insect Sci (2021) 41:241–250 The turbinates were the preferred sites for L1 and L2 and the soda (NaOH) and left at room temperature for 1–2hforthe1st lamina cribrosa was the most appropriate location for L3, instar and overnight for the second, third instar and adult. The while the nasal passage and pharynx were rarely inhabited larvae were evacuated from its contents, washed with water. by 3rd-stage larvae (Mula et al. 2009 and Hilali et al. 2015). Then dehydrated through ascending serial concentration of The adult fly was partially described previously by Zumpt ethanol 70, 80, 90 and 100% for 1 h each. Finally, they were (1965)andGuittonetal.(1997). Therefore, the aim of this cleared in clove oil then put in xylene for a few minutes. The study is to fully detail L1 and L2 as well as describe the adult larvae mounted in Canada balsam and incubated at 40 °C to fly to complete the specific characterization of this species. dry for 24 h, Hilali et al. 2015. Molecular analysis of 3rd-stage larvae was conducted by Hilali et al. (2015). Preparation of specimens for scanning electron The first-stage larvae (L1) are expelled by adult females microscopy (SEM) around the nostrils of animals and migrate through the upper respiratory tract and reach nasal sinuses, where these larvae Freshly collected 1st, 2nd, 3rd stage larvae and its adults were develop into second stage (L2) and third stage (L3) larvae. The first washed several times with saline. Fresh specimens were third-stage larvae leave the host through the nasal cavities via immersed in 2.5% glutraldehyde according to Hilali et al. sneezing and becoming pupae in the soil, emerging as adult 2015,Attiaetal.2018. Specimens were then dehydrated insects in the environment (Zumpt 1965). through ascending ethanol series, dried in CO2 critical point The present investigation was conducted to cover the fol- drier (Autosamdri-815, Germany); the specimens were glued lowing aspects: collection of Rhinoestrus spp. 1st, 2nd, 3rd over stubs and coated with 20 nm gold in a sputter coater (Spi- stage larvae and identification by light microscope and Module sputter Coater, UK), finally the specimens were ex- scaning electron microscope with phylogenetic analysis of amined and photographed with scanning electron microscope 3rd instar larvae. at a magnification ranging from 35X to 500X (JSM 5200, Electron prob. Microanalyzer Jeol, Japan; at Faculty of Agriculture, Cairo University). Materials and methods Preparation and identification of Rhinoestrus spp. Collection of Rhinoestrus species larvae from donkeys adults The head of each donkey was separated from the rest of the One hundred mature 3rd instar larvae of two morphotypes of body and cut sagittally, the nasal passages and pharynx were the Rhinoestrus spp. were incubated at 32 °C and 30% relative examined directly for the presence of any larvae. The larvae humidity. Every 10 larvae were put in a clean beaker (500 cc) (L2- L3) were collected labelled and placed into vials contain- containing a suitable amount of sterile sand and covered with ing saline. The two turbinate bones (near the brain) were also a gauze fixed with plastic band. The pupae were observed placed in plastic bags and labelled. The samples were exam- daily to collect the dead larvae, the newly emerged adults were ined on the same day of collection in the parasitology labora- killed with chloroform then the adults were examined macro- tory of the Faculty of Veterinary Medicine, Cairo University. scopically using a stereoscopic microscope for morphological examination and differentiation between species (Attia and Examination of the turbinates Salaeh 2020). Then the adults were mounted as previously described. The two turbinates were immediately examined in the labora- tory; each turbinate was washed in a dish of warm normal Identification of Rhinoestrus spp. larvae and adults saline at 37–40 °C several times for migrating L1. After ap- proximately 10 min, the turbinates were removed, and the The identification was carried out following the key and mor- saline was examined under a stereoscopic microscope (×10) phological characters mentioned by Zumpt (1965); Guitton to detect first instar larvae. et al. (1996) and Hilali et al. 2015. The morphological param- eters for 20 specimens from each larval stage and adults were measured under a stereoscopic and light microscope. Morphological studies Molecular study of the larvae for identification of the Preparation of permanent specimens species For detailed morphological studies, the larvae were washed Ten Rhinoestrus spp. 3rd stage larvae were sent to Prof. Dr. several times with saline then placed in enough 5% caustic Otranto (Department of Animal Health and Welfare, School Int J Trop Insect Sci (2021) 41:241–250 243 of Veterinary Medicine, University of Bari, Valenzano, Italy) Ventral surface for molecular identification of the species. The larvae were obtained from Bani-Suef infested donkeys, Egypt. Genomic The head composed of two antennary lobes each lobe varied DNAwasextractedfromeachspecimenwithacommercial in shape from oval to quadrangular. The oral hooks were kit (DNeasy Blood & Tissues, Qiagen, GmbH, Hilden, large, and each hook was broad at its base and pointed at its Germany) according to manifacturer’s protocol. PCR ampli- terminal end. Segment 1 to 3 had 3 rows of spines either fication was performed by using specific primers, UEA7 (5- continuous or interrupted. The 4th to 10th segments had 4 TACAGTTGGAATAGACGTTGATAC-3) (Zhang and complete rows of spines in front of these complete rows short Hewitt 1996) and UEA10 (5-TCCAATGCACTAAT middle row was present. In addition, short rows (1–2rows) CTGCCATATTA-3) (Lunt et al. 1996), designed on the re- were present on both lateral sides behind these 4 rows. The gion spanning from external loop 4 (E4) to carboxylic termi- 11th segment had 7 rows of spines.