Diptera: Oestridae) Larvae and Its Adults

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. On each lateral side of the nal (-COOH) of cytochrome oxidase I gene (cox1). PCR am- ventral surface of segments 1–10 there were a fan shaped plifications were carried out in a volume of 25 μl, including structure each provided with 7 long spines. ~100 ng of genomic DNA (except for the non-template control), following the PCR protocol reported by (Otranto et al. Second stage larvae (2nd larvae) of Rhinoestrus spp. 2006; Otranto and Colwel 2008). Amplicons were purified using Exo SAP-IT (Affymetryx, Cleveland, Ohio, U.S.A.) It was provided with posterior spiracles. According to the and then sequenced directly using the Taq DyeDeoxy shape of the peritreme and number of spiracular openings; Terminator Cycle Sequencing Kit (Version 3.1; Applied two types of 2nd stage larvae were reported in Egypt. The Biosystems) in an automated sequencer (ABI-PRISM 377; peritreme in R. usbekistanicus was kidney in shape while in Applied Biosystems). Sequences were determined in both di- R. purpureus was oval. The number of spiracular openings in rections (using the same primers individually as for the PCR) R. usbekistanicus reached up to 20 and in the R. purpureus it and the electrophotograms verified by eye. Molecular analysis reached up to 38 (Fig. 2;Table1). of sequence data was conducted using MEGA version 5.

Third stage larvae (3rd larvae)

Results The fully mature larvae were yellowish to brownish in color with dark brown bands on the dorsal surface of its segments. Morphological description of the larval stages and The R. usbekistanicus had the number of spiracular pores adults of Rhinoestrus spp varying from 370 to 430 (Table 1). The R. purpureus had the number of spiracular pores ranging from 290 to 350. The First stage larvae cuticle surrounding the posterior spiracles was provided with 10 sensorial ciliated papillae. These papillae were distributed They were whitish in color and had 12 segments without as 4 of them regularly situated on dorsal lip; another 4 on spiracular pores. ventral lip while the last two were located below the anal orifice. The cuticle bearing papillae formed a raised circular area in which one papilla originates centrally. Each papilla Dorsal surface was long and conical in shape (Figs. 3 and 4).

There was one row of spines on all segments except 12th Adult stage segment. The posterior end of the 12th segment showed 3 rows of spines. Other spines or hooklets were present around A relatively medium sized fly, eyes broadly separated from the anal opening on the 12th segment forming one row of anal one another in both sexes; the eye length was longer in male spines; they ranged in number from 6 to 10 (Fig. 1;table1). than female.

Table 1 Length of different stages of Rhinoestrus spp. Measurements Length Width

First stage larvae 1.93–2.49 mm (2.2 mm ± 0.14) 665–875 μm(720μm ± 44.2) Second stage larvae 0.6–1.4 cm (1 cm ± 0.2) 0.3–0.5 cm (0.4 cm ± 0.1) Third stage larvae 1.9–2.6 cm (2.2 cm ± 0.1) 0.5–1.2 (0.8 cm ± 0.2) 244 Int J Trop Insect Sci (2021) 41:241–250

Fig. 1 Scanning electron microscopic (SEM) micrographs of first stage larvae of Rhinoestrus spp. Fig. a, b, c) dorsal view d) ventral view. a 1st stage larva notes one row of spines on each of segments 2–11; Scale bar: 100 μm. b Anterior end of the larva: note median location of the dorsal row of spines, Scale bar: 100 μm. d Ventral view, anterior end showing two antennary lobes (an), oral hooks (oh) and 3 irregularly arranged 3 rows of spines on 1st segment, Scale bar: 10 μm

Head capsule ± 0.2) in width. Two globular large compound eyes were located on the dorsolateral sides of the cranium with length The Head of Rhinoestrus spp. was globular in shape and 2.8 mm–3 mm (2.8 mm ± 0.1) in male while in female showed sexual dimorphism, male head was longer. The head 2 mm - 2.5 mm. (2.2 mm ± 0.2). The parafrontalia were pro- of male measures from 3.4–4.6 mm (4 mm ± 0.2) in length vided with large brown tubercles, which were isolated from and 3.3–4.4 mm (4 ± 0.2) in width and female measures from each other in R. usbekistanicus. While in the R. purpureus, 3.3–4.3 mm (3.6 mm ± 0.2) in length and 3.4–4mm(3.6mm these tubercles were closer to each other. Two

Fig. 2 Light microscopic micrographs of 2nd stage larvae of Rhinoestrus shaped structure with larger number of spiracular openings (37), Scale spp. a Cephalopharyngeal skeleton (Oh: oral hooks; Hs: hypostomal bar: 100 μm, d Scanning electron microscopic Scanning electron sclerite; Dc: dorsal cornue; Vc: ventral cornue), Scale bar: 100 μm. b microscopic micrographs of first stage larvae of Rhinoestrus spp. Posterior spiracles note kidney shaped peritreme of the micrographs of 2nd stage larvae of Rhinoestrus spp. note the four rows R. usbekistanicus and the number of spiracular opening (18 in number), of spines between which medioventral sensorial structure that resembles Scale bar: 100 μm. c Posterior spiracles of R. purpureus showing oval sucker Int J Trop Insect Sci (2021) 41:241–250 245

Fig. 3 Scanning electron microscopic micrographs of 3rd stage larvae of Rhinoestrus spp. a Ventral surface, showing cephalopharyngeal skeleton with its two maxillae. Scale bar: 50 μm. b Three complete rows of spines, and in complete fourth row of spines, medioventral sensorial structure that resembles sucker, Scale bar: 50 μm. d High magnification SEM micrographs of medioventral sensorial structure Scale bar: 100 μm. c Light microscope micrographs of the 3rd stage larva showing three complete rows of spines and in complete fourth row of spines, medioventral sensorial structure that resembles sucker; Scale bar: 500 μm

morphologically different sizes of tubercles were reported in reduced protuberances, a median long and two oval segments both types of flies; large elevated (44–63 in number) and small forming the mouth parts (Fig. 5 and 6). non elevated one (20–26 in number). Ocellars were dark brown to black triangular dorsally bearing three shiny bead- Thorax like ocelli. A pair of three segmented aristate antennae was located in the antennal grove posterior to the frons, 1st seg- The prescutum in R. usbekistanicus had a median central ment was oval in shape with its length of 68 μm-72μm broad dark pair of pre-sutural stripes which were fused ante- (70 μm ± 0.1), 2nd segment was oval and had a length of riorly, with narrow space between them posteriorly. These 80 μm-88μm(85μm ± 0.1). It was provided with a cleft stripes terminate just before the transverse suture. A pair of in its middle. The 3rd segment was also oval measuring similar post-sutural median quadrangular and two lateral tri- 120 μm −150 μm(146μm ± 0.2) in length and provided with angular one with its base attached to the transverse suture and several tubercle 35–44 (40 ± 0.1). The arista was bear with a extending just before the second suture (between scutum and length of 360 μm- 380 μm(368μm ± 0.1). The epistome was scutelleum). In R. purpureus thepre-scutumhadamedian found dorsally. It was composed of three nonfunctional pair of pre-sutural stripes which were light grey or black

Fig. 4 Scanning electron microscopic micrographs of 3rd stage larvae of Rhinoestrus spp. a, b R. usbekistanicus the most frequent species showing oval to kidney shaped posterior spiracles and sensory papillae (sp). c Spiracular openings in posterior spiracles of R. usbekistanicus oval to kidney showing the straight slit. d Pointed and ciliated sensorial papillae showing by arrow 246 Int J Trop Insect Sci (2021) 41:241–250 brown in color. They were narrower in their diameter than end of costa. The main stem of radius divided into three those of the R. usbekistanicus, the stripes not fused and had branches, (R1, R2 and R3). Media divides into two branches a wide space between them. A pair of similar post-sutural (M1 and M2) and cubitus was unbranched. The axillary vein quadrangular and two lateral ones of irregular shape and ligh- was very short and unbranched. The cross- veins were: the ter in color were also present on the scutum. The pre-sutural humeral cross-vein between costa and subcostal; radio- ones were usually glossy. The mesonotum showed setiferous medial cross vein between radius and media; upper marginal tubercles increasing in size towards and on the scutellum. The sigmoid cross- vein between radius and media, lower marginal scutellum was relatively small without morphological differ- cross vein between branches of media, Median cross vein and ences between both types of Rhinoestrus spp. The length of medio- cubital cross vein between media and cubital vein. The male thorax was similar to female. The mesothorax was pro- wings were covered with small irregularly distributed black vided with tubercles varying in number from 30 to 59, 35–68 batches (8–10 batches) on its thoracic margin. The dimensions and 30–55 on prescutum, scutum and scutelleum respectively of the two wings were similar in both sexes and the two types (Fig. 7). of Rhinoestrus spp. It ranged from 5.6 mm–6.7 mm (6.3 mm ±0.1)(Fig.9). Abdomen of Rhinoestrus spp.

The abdomen was globular in shape, dark brown in color Legs of Rhinoestrus spp. similar in both species, its dimension varied from 5 to 5.6 mm (5.4 mm ± 0.2) in length and 3–4 mm (3.6 mm ± The legs were yellowish brown in color and covered with long 0.2) in width in male and 5–5.8 mm (5.6 mm ± 0.1) in length hairs. The three pairs of legs were morphologically similar in and 3–4 mm (3.8 mm ± 0.4) in width in female. The abdomen both sexes and the two types of Rhinoestrus spp. The leg was composed of 5 visible segments; it bears irregularly distribut- formed of rectangular coxa, triangular trochanter, cylindrical ed dark tubercles. The last segment had external genitalia. The femur and tibia. Tarsus had five segments with the last tarsal female genitalia consisted of fused dorsal plate, sternal plate, segments was provided with two pulvilli and one empodium. anal plates and ventral plate. External genitalia of male had The empodium was oval, with short terminal process; its lat- two pairs of claspers with a median intermittent organ (Fig. 8). eral sides were striated while its middle was provided with sensory hair like structure. The two pulvilli were elongated Wings of Rhinoestrus spp. covered with longer hairs than those on the empodium. Two claws were elongated with its basal part broad and its terminal The wing venation showed the following: unbranched costa part blunt. The length of the fore, mid and hind legs varied and subcostal which was parallel to and terminates before the from 4.4 mm – 5.8 mm (5 mm ± 0.2),4.8 mm – 5.9 mm

Fig. 5 Light microscopic micrographs of head of the two morphotypes of Rhinoestrus spp. a Female head of the of R. usbekistanicus note separated tubercles on the parafrontalia, O: Ocelli, F: frons, Pt: Ptellinium, Pts: Ptellinium suture, Ag: antennal groove, CE: Compound Eyes, Pfr: Parafrontalia, Pfa: Parafacial, note the length of the female eyes shorter than male. b Head of the male of the R. usbekistanicus with longer eyes. c Female head of the of Rhinoestrus purpureus note attached tubercles, Fs: Frontal suture. d Male head of the R. usbekistanicus note attached tubercles Int J Trop Insect Sci (2021) 41:241–250 247

Fig. 6 Scanning electron microscopic micrographs of the heads of Rhinoestrus spp. a Anterior region of adult fly. b Antenna showing 1st, 2nd, 3rd segment c The 2nd segment of antenna provided with a number of spinulae. d Bare arista

(5.1 mm ± 0.1) and 5.8 mm – 6 mm (5.9 mm ± 0.1) respec- Discussion tively Fig. 10. This study gave a detailed description of the morphology of the 1st stage larvae of Rhinoestrus spp. infesting donkeys for the Molecular identification of the larvae by PCR 1st time. Additional description adds up to the previous description of (Zumpt 1965; Guitton et al. 1996) that segment 2 and 3 In each PCR, the primer combination yielded amplicons of had either complete 3 rows or interrupted medially in 1st row. 689 bp. Each specimen (10 specimens) examined resulted Concerning, an additional description adds up to the 2nd stage nearly 99% homologous to Rhinoestrus usbekistanicus (ac- larvae that the dorsal surface of 1st and 2nd segments were cession number: LC482203 to LC482212). No insertions or covered with two complete uninterrupted rows of spines and deletions were detected in the sequences and none of the se- on the ventral surface (11th&12th) segments with 3 irregular quences exhibited any unusual mutations. rows of spines. These results were disagreed of description who The molecular analysis revealed 18 identical and 22 repre- recorded by Guitton et al., (1997). On the other hand, Zumpt sentative sequences, with an overall intraspecific pairwise di- (1965) stated that the dorsal surface of R. purpureus had lateral vergence ranging from 0.15 to 0.78% (Fig. 10). group of tentacles on a few anterior segments.

Fig. 7 Light microscopic micrographs of thorax of Rhinoestrus spp. adult. a Thorax of R. usbekistanicus note two longitudinal dark broad stripes (st). b Thorax of R. purpureus notes two stripes (st) which were narrow 248 Int J Trop Insect Sci (2021) 41:241–250

Fig. 8 Light microscopic micrographs of adult Rhinoestrus spp. a lateral view of thorax and abdomen. b; c Light and SEM of female genitalia showing dorsal plate (dp), ap: anal plate, vp: ventral plate and stp: Sternal plate. d Posterior end of male showing its genitalia with two pairs of claspers (cls), and intromittent organ (ig)

This study describes in detail the spines which presents in the first time additional exhibited of presence suckers on each each ventral segments in L2 which is not described before; side from 2nd to the 10th segments. while Zumpt (1965) recorded that the second segment shows a The 3rd stage larvae of the two morphotypes had common few irregularly placed conical spines but those on the follow- morphological features which are described in detail. The dis- ing segments to the 12th were wedge- shape and placed in tributions of spines on the dorsal surface of the 1st, 2nd and several dense rows. While our finding disagreed with 11th segments were described for the 1st time. In additions, Guitton et al. (1997) who mentioned that there were 3–4 spines on the ventral surface of the 1st, 5th to 10th and 12th spines rows on segment 3–11 without description of the spines segments were found to be of diagnostic value between on 1st and 2nd segments. R. usbekistanicus and R. purpureus. The 2nd stage larvae of Rhinoestrus spp. in the present Other descriptions were similar to previous reports of study using light and SEM exhibited two suckers like James (1947), Zumpt (1965) Guitton et al. (1996)and sensorial structures to the complete rows of spines on the (Hilali et al. 2015). segment from 4th to 10th. This result disagreed with the The peritreme was the most important features for differ- description of Guitton et al. (1997) who described these entiation of both morphotypes. The length similar to width in suckers from 2nd to 4th and 5th segments. In this study, for R. usbekistanicus and length longer than width in R. purpureus. This finding is found similar to previous description of Guitton et al. (1996) and (Hilali et al. 2015). Concerning the adult stage; our results revealed the full description of head, thorax and abdomen of the imago of two morphotypes of Rhinoestrus spp. Dealing with the head capsule, the dimensions of the head and the length of the eyes of the male and female are recorded. According to these results, we could differentiate between both sexes by the length of the two eyes. Ocelli were described and the antennae were described in detail for the 1st time in this study. The parafrontalia were provided with large brown tubercles which were isolated from each other in R. usbekistanicus while these tubercles were closer to each Fig. 9 Camera Lucida drawings showing parts of adults Rhinoestrus Spp. other in R. purpureus. These results agreed with Zumpt Rhinoestrus A: Wing of adults spp. showing the wing venation; C: Costa, (1965) and Guitton et al. (1996) who described parafrontalia SCo: subcosta, R: Redius, M: Media, AX: Axillary, Cu: Cubitus, M-cu: Rhinoestrus Mediocubitus cross vein, r-m: redio-median cross vein, tap: upper mar- of both spp. ginal cross vein, Tp: lower marginal cross vein and HMC: Humeral cross The description of thorax showed two morphologically vein different features that in R. purpureus; the prescutum had a Int J Trop Insect Sci (2021) 41:241–250 249

Fig. 10 Phylogenetic tree constructed in MEGA by neighbor- joining method for the CO1 region of the tested R.usbekistanicus in Egypt

median central broad pair of presutural stripes which were R. purpureus (8%), R. usbekistanicus (8%) and 84% evi- fused anteriorly, dark in color. A pair of similar postsutural denced intermediate features. In contrast molecular analysis median quadrangular and two lateral triangular ones. In view of COI gene of the larvae confirmed uniformity at genetic of the description of Zumpt (1965) and Guitton et al. (1996), level in the Mediterranean area. In a conclusion, our investi- this adult could be identified as R. usbekistanicus While in gation indicated that Rhinoestrus spp. present in Egypt is Type II the prescutum had a median pair of presutural stripes mainly R. usbekistanicus, which includes two morphotypes, which were light grey or black brown in color, a pair of similar one is R. usbekistanicus like and the other R. purpureus like. postsutural quadrangular and two lateral triangular ones. These features are like previous description of Zumpt (1965) for R. purpureus. This study showed that the wings and legs of both types of Conclusion Rhinoestrus spp. were morphologically similar. Zumpt (1965) mentioned that the legs were red and yellowish- brown with In a conclusion, the molecular analysis used in the present the femora darkened. study have been shown to be successful and reliable for diag- Concerning the description of abdomen of Rhinoestrus nostic and taxonomic studies of Oestridae species. spp. in the present study, the two morphotypes of Additionally, the present study enriches the knowledge of Rhinoestrus spp. had the same morphology. This study gave Rhinoestrus spp. morphology of the larval stages and adults; more detailed description of the abdomen than the previous cephalopharyngeal skeleton, cephalic segment morphology, reports of Zumpt 1965 and Guitton et al. 1996. In addition, posterior spiracles, prothorax morphology, and posterior seg- both male and female genitalia were described for the 1st time ments. This study provides a phylogenetic and molecular in this study. Zumpt (1965) described only the abdomen of characterization of Rhinoestrus spp. larval from donkeys. R. purpureus that was covered by glossy black and brown The present sequence analyses demonstrate that Rhinoestrus setiferous tubercles. While Guitton et al. (1996) stated that spp. larval from donkeys greatly resemble R. usbekistanicus few tubercles were visible on lateral sides of the abdomen of but there are exhibited many micro-morphological between R. usbekistanicus. the two species of Rhinoestrus spp.; R. usbekistanicus and From the present study; we reported two morphologically R. purpureus were described. different Rhinoestrus spp. namely R. usbekistanicus and R. purpureus from the same Provinces in Egypt. However, Author’s contributions Olfat A. Mahdy and Marwa M. attia Conception, M. attia molecular analysis of the COI gene of ten, 3rd stage larvae design of the study. Marwa collection and rearing of the larvae; Marwa M. attia photographed the samples; Olfat A. Mahdy Analysis and confirmed the presence of one species only in Egypt interpretation of the data. Olfat A. Mahdy and Marwa M. attia Drafting (R. usbekistanicus). Similar studies were carried out by and revising the manuscript for important intellectual content. All authors Otranto et al. (2005) who detected 4 morphologically different read and approved the final manuscript. Rhinoestrus spp. (R. purpureus, R. purpureus like, R. usbekistanicus and R. usbekistanicus like) from horses in Funding information No funding received for this work. Italy while molecular examination of the same material confirmed the presence of one unique species. Furthermore Mula Compliance with ethical standards et al. (2013) studied Rhinoestrus spp. infesting horses in Italy, Conflict of interest All Authors declare that there is no conflict of their results indicated that 3 morphotypes were found, interest. 250 Int J Trop Insect Sci (2021) 41:241–250

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