Prevalence of nasopharyngeal myiasis in roe deer (Capreolus capreolus) from an area with high sympatry between wild and domestic ungulates in Central Spain

Infection by the sheep bot �yOestrus ovis was �rstly reported in a single roe deer from Central Spain in 2023. In order to con�rm whether that �nding was an isolated event or a spillover of O. ovis to a new host, nasopharyngeal cavities of 184 roe deer hunted in Central Spain between January-June 2023 were examined. All larvae were recovered and morphologically identi�ed; in addition, species identi�cation was molecularly con�rmed in a subset of specimens. Forty-four roe deer (23.9%; CI 95 17.95-30.74) were positive for different Oestrinae larval stages. Twenty-six animals (14.1%; CI 95 9.44-20.02%) were infested by the roe deer nasal bot �y (Cephenemyia stimulator) with a mean intensity of 35.2 (SD 49.71) larvae/infested animal,and eighteen (9.8%; CI 95 5.90-15.02%) roe deer harboured the sheep bot �y (O. ovis), with a mean intensity of 2.0 (SD 1.33) larvae/infested animal. No mixed infestations by both Oestrinae were found in a single animal. All larval instars (L1, L2 and L3) of both species were identi�ed. Most C. stimulator specimens were located at the nasal turbinates, and a small percentage (3.2%) at the pharynx; however, all O. ovislarvae were found at the nasal turbinates. Since O. ovis is highly prevalent in sheep and goats from Central Spain, the high sympatry between roe deer populations and small ruminant �ocks in the studied area may have increased the risk of cross-infection. Moreover, the �nding of mature third stage larvae of O. ovis suggests that this species can complete its life cycle in roe deer. Therefore, monitoring bot �ies in sheep and goat �ocks as well as in sympatric wild ruminants is strongly recommended for achieving an optimum control of nasal myiasis.


Introduction
Nose bot ies (Diptera: Oestridae) include nine genera that infest members of the orders Artiodactyla, Perissodactyla and Proboscidea (Scholl et al. 2019).They all belong to the subfamily Oestrinae which differ from other oestrids in that they are larviparous; gravid ies eject rst instars into the host nostrils which migrate to sinusal and/or nasopharyngeal cavities (Colwell 2001;Scholl et al. 2019) causing rhinitis, nasal discharge, and respiratory complications (Dorchies et al. 1998).
The most widely distributed and economically important nasal bot is Oestrus L. which develops within the nasal cavities and frontal sinuses of domestic and wild Bovidae (Zumpt 1965).Four Oestrus species are currently recognized: O. ovis, O. variolosus, O. aureoargentatus and O. caucasicus (Colwell et al. 2006).Oestrus ovis known as the sheep bot y has a worldwide distribution in sheep and goats.In Spain, O. ovis prevalence rates in domestic sheep range from 70 to 84% (Alcaide et al. 2003(Alcaide et al. , 2005;;Gracia et al. 2010) and it has also been reported in wild sheep and goats such as the European mou on (Ovis aries musimon) (Moreno et al. 1999) and Barbary sheep (Ammotragus lervia) (Barroso et al. 2017).In addition, a new species has been detected in the Iberian ibex (Capra pyrenaica) that was tentatively identi ed as O. caucasicus (Pérez et al. 1996 Since morphological features are quite similar between different dipteran species, especially among rst instar larvae, molecular techniques are increasingly employed for diagnostic and taxonomic investigations of specimens belonging to this order (Otranto and Stevens 2002).In this regard, the mitochondrial cytochrome oxidase subunit I (COI) has been used in several studies for assessing the host-speci city, species identi cation, phylogeny, and diagnosis of oestrid larvae (Otranto and Stevens 2006;Moreno et al. 2015).
Oestrids tend to be highly host-speci c, so that when they are introduced to a host that is widely different from the natural host, then frequently the parasite will not develop properly (Colwell 2006).Recently, O. ovis was rstly reported in Cervidae, speci cally in a roe deer from the North of Guadalajara province in Central Spain (Martínez-Calabuig et al. 2023).It is worth noting that sympatry between wild and domestic ruminants is high in this area.After this rst report, a broad survey was designed for providing updated information on the situation of nasal myiasis in roe deer from this area and for con rming whether that nding was an isolated event or a spillover of O. ovis to a new host.

Area of study and animals
The study was conducted in 35 game reserves located in ve provinces of Central Spain (Guadalajara, Burgos, Soria, Segovia, and Zaragoza) from January to June of 2023 (Fig. 1; Table 1).This area has a Continental climate characterized by cold winters and hot and dry summers; rainfall is generally low throughout the year and occurs mainly in spring and early summer (Charraza et al. 2018).Temperatures in summer usually exceed 35ºC, favouring the lifecycle of oestrids, whose ies are active with temperatures above 12-18ºC (Breev et al. 1980 According to the last o cial census, the study area comprises the Spanish regions (Castilla-La Mancha, Castilla-León, and Aragón) with the highest ovine populations (MAPA, 2022).In addition, the abundance of roe deer in Central Spain is also among the highest of the country, as re ected by the high number of captures (average 7,000 roe deer) authorized in these provinces in the 2021/2022 hunting season (Centenera, unpublished data).
A total of 184 roe deer heads (170 females and 14 males) were collected in the eld after hunting and stored at -20ºC.Most animals were killed during the o cial female selective hunting period in order to control roe deer populations.The age of roe deer was determined by teeth features according to Høye (2006), and three host age categories were considered (< 1 year; 1-5 year; > 6 year).

Larval collection and identi cation
Roe deer heads were thoroughly examined for nasopharyngeal myiasis according to Martínez-Calabuig (2020).All larvae found in the nasopharyngeal cavities were recovered, rinsed in physiological saline, and preserved in 70% ethanol.The number, location and stage of the larvae were recorded (Table 2).
Morphological identi cation was performed according to Zumpt (1965).First instars were identi ed according to the shape of the anterior cephalopharyngeal skeleton and the spinulation pattern (Fig. 2a1, b1); identi cation of L2 and L3 was carried out on the basis of the shape of posterior spiracular plates (Fig. 2a2, b2), antennal lobes disposition and spinulation pattern (Fig. 2a3, b3).Morphological identi cation of both species was molecularly con rmed by the analysis of a subset of larvae including specimens of the three larval instars.DNA was extracted using a commercial kit (High Pure PCR Template Preparation Kit, Roche Diagnostics GmbH®, Mannheim, Germany) following the manufacturers' instructions.DNA samples were analysed using a PCR targeting the partial COI gene of Oestridae as previously described (Otranto et al., 2000); DNA of Hypoderma actaeon and nuclease free water were included as positive and negative controls, respectively.
PCR products were separated by electrophoresis on 1% agarose gels stained with RedSafe (iNtRON Biotechnology®, South Korea) and visualized using a GelDoc Go Imagen System (Bio-Rad Laboratories®, California, USA).Selected fragments were puri ed and sequenced at STAB VIDA´s laboratories of the Universidade Nova de Lisboa, Portugal.Sequences were aligned and edited using ChromasPro (Technelysium, Brisbane, Australia) and consensus sequences were then scanned against the GenBank database using BLAST.

Phylogenetic analysis
A phylogenetic analysis was carried out using MrBayes 3.

Statistical analysis
Statistical analyses were performed with R statistical package (R v.4.3.2; 3).Variables were grouped and categorized for statistical analysis as represented in Table 1.
The possible in uence of different factors (age, sex, province, and month of capture) on the prevalence and the intensity of infestation was assessed using the Fisher´s exact test and the ANOVA test, respectively.Since larval counts were not normal distributed, they were log normalized (natural log + 1) previous to the analysis.Only positive animals were introduced in the ANOVA test.
Morphological examination allowed the identi cation of two Oestrinae species: C. stimulator and O. ovis (Fig. 2); these ndings were molecularly con rmed.Regarding O. ovis, COI sequences showed a percentage of identity between 98.9-99.8%when compared to the deposited sequences NC_059851.Regarding to the location of the larvae, all O. ovis specimens (n = 43) were located at the nasal turbinates; those of C. stimulator (n = 916) were also mainly found at the nasal turbinates (n = 884; 96.8%), although a small number was detected at the pharynx (n = 32; 3.2%).
Prevalence and intensity of infestation values considering the different factors analysed are summarized in Table 1.Oestrus ovis was detected in the provinces of Guadalajara, Burgos and Soria with percentages ranging from 6.12-14.29%.Moreover, C. stimulator was found in roe deer from Burgos, Soria and Zaragoza, showing higher prevalences (35.7-57.1%)than O. ovis.It is worth noting that both Oestrinae were detected in roe deer from Burgos and Soria, although no mixed infestations were found in any animal.On the contrary, both species were absent in the province of Segovia.No signi cant differences were detected for O. ovis and C. stimulator prevalence and intensity of infestation when considering the province of origin (p > 0.05).
Regarding the sex of the animals, the prevalence for both species was higher in males than in females, but signi cant differences were only found for C. stimulator (p = 0.031).In general, old animals (> 6 year) showed higher prevalences than young adults and calves; however, those differences were not signi cant for any species (p > 0.05).The in uence of the month when roe deer were hunted was only signi cant for the intensity of infestation by C. stimulator (p = 0.004), with higher larval burdens in February than in April.).Considering those recent ndings, and the supposed speci city of Oestrids, a broad survey was designed for updating the situation of nasal myiasis in roe deer from Central Spain and to con rm whether it was an accidental infestation or a spillover to a new host.

Discussion
Our results reveal that the prevalence and intensity of C. stimulator in roe deer from this area is much lower (16.3%)than that previously detected by Martínez-Calabuig et al. (2022b) in northern Spain.Nevertheless, our data show an irregular distribution between provinces that ranged from 0% in Guadalajara and Segovia to 57.1% in Zaragoza (Fig. 1).In addition, noticeable prevalence found for the sheep bot y, O. ovis suggests that its presence in roe deer was not accidental as it had been hypothesized after its rst identi cation.
Oestrosis is extensively distributed in domestic and wild sheep and goats in Spain (Alcaide et al. 2003(Alcaide et al. , 2005;;Gracia et al. 2010).Nevertheless, there have been widespread reports of accidental infestations in other species, including humans and dogs (Lucientes et al. 1997;Moreno et al. 1999;Lobato et al. 2011), suggesting that gravid O. ovis females are not strictly host speci c (Colwell 2001).According to our ndings, the occurrence of late larval stages (L2-L3) in roe deer suggests that O. ovis could complete its life cycle in Cervidae.Alcaide et al. (2005) indicated that both high O. ovis prevalences and densities of its main hosts (domestic small ruminants), may potentially increase the risk of spillover of this parasite to new hosts.
High roe deer densities and the existence of shared habitats with abundant sheep ocks in the area of study may have favoured the interspeci c spillover of O. ovis.A similar situation has been previously described for Hypoderma actaeon (Diptera: Oestridae), considered speci c for red deer, which has become an emerging myiasis in roe deer in Central Spain (Panadero et al. 2017).Changes in the pattern of distribution of potentially susceptible hosts (red deer/roe deer) in Central Spain may have favoured the spreading of this myiasis to other hosts (Panadero et al. 2020).It is worth noting that some authors (Moreno et al. 1999) consider oestrid ies as opportunistic so that their speci city would be very in uenced by the availability of adequate host species (Price 1980).Additional studies to reveal a hypothetical spillover of C. stimulator to domestic sheep and goats in this area are needed.
Our results also con rm a strong predominance of rst instars for both Oestrinae species in winter and spring months (January-April), indicating the existence of a hypobiotical period where larvae delay their development while waiting for the optimal breeding season (Alcaide et al. 2003).Additionally, the existence of a low proportion of L2 and L3 during this period reveals the end of the quiescence period and the beginning of larval reactivation.
Larval burdens, mainly represented by rst instars, were much higher for C. stimulator than for O. ovis.Taking into consideration that L1 represent the insurance of the survival of the parasitic population (Tabouret et al. 2001), those differences could be attributed to the major suitability of roe deer as a host for C. stimulator than for O. ovis.In this sense, several factors promoting larviposition and subsequent larval survival have been related to the suitability of a host for Oestrinae, e.g.host related odour (Poddighe et al. 2010), moistness (Cepeda-Palacios et al. 2000) and structure of the host's muzzle (Cogley and Anderson, 1981), host immune reaction (Tabouret et al., 2003), as well as behavioural responses to avoid larviposition (Anderson 1975).
The oestrids have very different life cycles and adaptations for survival at different sites into the host (Colwell et al. 2006).Early larval stages (L1) of C. stimulator are found in the nasal cavity of roe deer, whereas late stages (L2-L3) are mainly located in the pharyngeal pharyngeal pouches or diverticula (Angulo-Valadez et al. 2010, Martínez-Calabuig 2020).In this study, all O. ovis larvae were only found in the nasal passages of roe deer, whereas in its natural hosts, sheep and goats, larvae are found in the nasal cavity as well as in nasal and frontal sinuses (Angulo-Valadez et al. 2010).
Statistical analysis only allowed to identify the sex and the month of roe deer capture as factors related to the prevalence and intensity of infestation by C. stimulator, respectively.It is very likely that the low number of roe deer infested by O. ovis did not allow the appearance of signi cant differences when considering the different factors.
The highest occurrence of Oestrinae in males could be associated to sex-related odour differences (Poddighe et al. 2010).Moreover, the territorial behaviour of bucks may favour the encounter with bot ies, because they are often forced to leave the forest and wander in open spaces where they are more vulnerable to larviposition (Dudziňski 1970).
Our results also reveal that the geographical distribution of both Oestrinae in roe deer from Central Spain is not homogeneous.Cephenemyia stimulator is a high prevalent myasis which is actually present in three out of the ve provinces surveyed in this study, whereas O. ovis was only sporadically found in three provinces.auribarbis and its faster larval development compared to P. picta in southern Spain may re ect asynchronous life cycles of both oestrids decreasing the co-occurrence of both sympatric species (Buenode la Fuente et al. 1998).In addition, it was reported that the intensity of P. picta in concomitant infections with C. auribarbis was lower than in pure infections (Vicente et al. 2004), providing good evidence of interspeci c competence, which could be dealt with by parasites by means of asynchronous life cycles and different maturation periods.

Conclusion
Our data reveal the interspeci c transmission of O. ovis from domestic ruminants to roe deer in central Spain; this may be due to the high density of this wild ungulate and free ranging sheep which usually share habitats in this area.Thus, this Spanish region may be considered a hotspot for cross-transmission of different Oestridae between Cervidade and Bovidae, as has been previously reported for H. actaeon.Since wildlife may contribute to the reinfestation of domestic ocks with O. ovis, the e cacy of oestrosis control programs can be compromised.Thus, monitoring strategies must include sheep and goat ocks together with sympatric wild ruminants.Phylogenetic tree clustering of the partial COI of Oestridae.The tree was obtained using a General Time Reversible substitution model with gamma-shaped rate variation with a proportion of invariable sites (GTR+G+I) with MrBayes software 3.2.7 (Ronquist et al., 2012), using Bayesian inference with Markov Chain Monte Carlo sampling (10,000,000 generations, sampling every 1,000 generations).This analysis involved 39 nucleotide sequences.The nucleotide sequence of Lucillia caesar was used as an outgroup.Isolates obtained in this study or identical to those obtained in the present study are highlighted in bold.

Figure 1 Map
Figure 1

Figure 3
Figure 3 ; Pérez et al. 2016).Cephenemyia (Latreille, 1818) and Pharyngomyia (Schiner 1861) are the main genera infecting Cervidae in Holarctic regions.Cephenemyia exclusively parasitize deer from the Cervinae and Odocoileinae subfamilies (Bueno de la Fuente et al. 1998).Four species in this genus (C.ulrichii, C. auribarbis, C. stimulator and C. trompe) infest cervids in the Paleartic region (Morrondo et al. 2021).The roe deer nasal bot, Cephenemyia stimulator is very prevalent in the European roe deer (Capreolus capreolus), especially in Central Europe (Király and Egri 2007; Morrondo et al. 2021).In Spain, cephenemyiosis is a recent myiasis in roe deer that is well established in the North of the country, with prevalences ranging from 31.6-43.2%and mean parasite burdens of 16.9-19.7larvae/animal (Arias et al. 2016; Martínez-Calabuig 2020).Pharyngomyia picta, commonly known as deer throat bot y has been reported in deer from Europe and Asia (Colwell 2001).In Spain has frequently been found simultaneously with Cephenemyia auribarbis in the Iberian red deer (Cervus elaphus hispanicus) (Ruiz and Palomares 1993; Bueno-de la Fuente et al. 1998; de la Fuente et al. 2000; Vicente et al. 2004) and they can also infest other sympatric wild cervids as fallow deer (Dama dama) (Ruiz & Palomares, 1993).However, mixed infections by Oestrinae were not reported in roe deer, and only a simultaneous infection by C. stimulator and Lucilia caesar (Diptera: Calliphoridae) has been detected in the nasal cavity of one roe deer in northern Spain (Martínez-Calabuig et al. 2022a). ).

Table 2
Monthly distribution of larval stages of C. stimulator and O. ovis in roe deer from Central Spain.
(Darriba et al. 20122 et al. 2012) by Bayesian approach with Markov Chain Monte Carlo sampling (10,000,000 generations sampling every 1,000 steps).A General Time Reversible substitution model with gamma-shaped rate variation with a proportion of invariable sites (GTR + G + I), was used.The model was selected based on AIC value (Akaike Information Criterion) using the free software jModelTest v.2.1.10(Darribaetal. 2012).The tree was visualized and edited using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/ gtree/).
1 and AF497767.1 of O. ovis larvae recovered from sheep and goat from Spain and Italy (Otranto et al. 2003; Aleix-Mata et al. 2021).The phylogenetic analysis revealed that our sequences clustered with other O. ovis sequences obtained in Spain, Turkey, and Kyrgyzstan, being clearly separated from a novel Oestrus species detected in an Iberian ibex from Spain (Moreno et al. 2015) (Fig. 3).In addition, the COI sequences of C. stimulator were identical to those (MG763915.1 and NC_059850.1)recoveredfromroe deer from Spain (Aleix-Mata et al. 2021; Fidalgo et al. 2021); the phylogenetic tree showed that all these sequences clustered with other sequences of C. stimulator recovered from roe deer in our country (de la Fuente et al. 2021).The partial sequences obtained in the present study were deposited under the accession numbers PP078924.1-PP078925.1.No mixed infestations by both Oestrinae were found in any animal.Table2shows a strong predominance of rst instars (> 65%) for both species, especially in January and February.
(Martínez-Calabuig et al. 2023, at the beginning of 2023, O. ovis larvae were identi ed in the nasal turbinates of a roe deer from Central Spain(Martínez-Calabuig et al. 2023 (Notario and Castresana 2001) by C. stimulator is relatively frequent in roe deer from Spain (Martínez-Calabuig 2020;Morrondo et al. 2021).This species was rstly detected in a roe deer in Central Spain which was imported from France in 2001(Notario and Castresana 2001).Since them, cephenemyiosis has experienced a rapid expansion throughout the northern half of the Iberian Peninsula.Thus, recent data demonstrated high prevalences (62.2%) and intensities (41.2 ± 52.7) of infestation(Martínez- Anderson (1975)ing that the infestation by O. ovis was more prevalent in the province of Guadalajara, where C. stimulator is absent, than in Burgos and Soria; furthermore, in those provinces where both species coexist, no roe deer was simultaneously parasitized by both species.Further studies to elucidate the in uence some biotic (host density) and abiotic (climate, altitude, etc) factors in the distribution of nasal myiasis are needed.The absence of co-occurrence within the same individual could be attributed to behavioural changes of roe deer after being attacked by C. stimulator ies.In this senseAnderson (1975)evidenced that, after a rst infection by Cephenemyia spp., experienced deer try to evade larviposition by oestrid females, also reducing the infection success by O. ovis.Although co-occurrence of different oestrids such as P. picta and C. auribarbis are commonly reported in red deer from Europe (Ruiz and Palomares, 1993; de la Fuente et al. 2000; Vicente et al. 2004; Leitner et al. 2016; Miranda et al. 2022), the earlier larviposition by C.