Endoparasitic Mites (Rhinonyssidae) on Urban Pigeons and Doves: Updating Morphological and Epidemiological Information

: Rhynonyssidae is a family of endoparasitic hematophagous mites, which are still largely unknown even though they could act as vector or reservoir of different pathogens like dermanyssids. Sampling requirements have prevented deeper analysis. Rhinonyssids have been explored in a few host specimens per species, leading to undetailed morphological descriptions and inaccurate epidemiology. We explore the relationships established between these parasites in two Columbiformes urban birds (domestic pigeon ( Columba livia domestica ) and Eurasian collared dove ( Streptopelia decaocto )), assesing 250 individuals of each type in Seville (Spain). As expected, Mesonyssus melloi (Castro, 1948) and Mesonyssus columbae (Crossley, 1950) were found in domestic pigeons, and Mesonyssus streptopeliae (Fain, 1962) in Eurasian collared doves. However, M. columbae was found for the ﬁrst time in Eurasian collared doves. This relationship could be common in nature, but sampling methodology or host switching could also account for this result. An additional unknown specimen was found in a Eurasian collared dove, which could be a new species or an aberrant individual. We also provide an epidemiological survey of the three mite species, with M. melloi being the most common one followed by M. streptopeliae and M. columbae . High variation between previous epidemiological measurements and ours highlights the importance of developing deeper studies to uncover the factors regulating mite prevalence and intensities of infection.


Introduction
Rhinonyssidae Trouessart, 1895 (Acari: Parasitiformes: Mesostigmata) is a family of hematophagous mites that inhabits the nasal cavities of various orders of birds [1]. Currently, the family Rhinonyssidae includes more than 600 species arranged in 11 genera: Larinyssus Strandtmann, 1948  . It is currently considered the most diverse family of nasal mites [2].
During their feeding activity, rhinonyssids can damage the nasal cavities of birds, leading to the development of Rhinonyssidosis avium disease [3]. Some species, like Sternostoma tracheacolum Lawrence, 1948, can reach not only the nasal passages of birds but also their tracheae, lungs and air sacs [4]. As a result, they are highly pathogenic in some host species like canaries and Gouldian finches [4,5]. Furthermore, even when not experimentally demonstrated, they could act as a potential reservoir or vector of some

Materials and Methods
In this paper, 500 urban columbiform hosts (250 Columba livia domestica and 250 Streptopelia decaocto) were examined from the municipality of Seville, Andalusia, Spain. The individuals were attracted using water and food and captured using trap cages in different urban areas of Seville throughout 2018 and 2019. From the eleven districts of Seville, eight were sampled in one or two locations (total locations = 12, Figure 1). The "Centro Zoosanitario de Sevilla", which is in charge of the bird population control program developed in the city of Seville, captured the individuals and provided them to us for parasite examination. Once the birds were captured and euthanized by institute staff, samples of each specimen were placed into individual plastic bags and refrigerated at 4 • C until examination. The procedure for bird trapping, handling and euthanizing used here amination. The procedure for bird trapping, handling and euthanizing used here was included in the animal control program of the Seville City Council and approved by the "Consejo Municipal de Protección y Bienestar Animal (COMBA)". To collect the mites, the nasal cavities were opened under an Olympus binocular stereo microscope with a scalpel and small scissors. First, the ossa mandibulae was removed together with the hyoid brush apparatus (apparatus hyobranchialis), then the scalp and feathers were removed with tweezers. Following this, a transverse section was made in the papillae pharyngeales region. A medial section was made, starting from the rima infundibuli through the choana and ending in the ruga palatina mediana. The nostrils in the area of the operculum were examined, and then the os maxillare was opened at the level of its fusion with the palate. Then, the maxillary bone was removed and the nasal cavity with its three conchae was opened: the largest of all rostral concha nasalis rostralis; the middle, concha nasalis media; and the caudal, concha, nasalis caudalis. Examination of all nasal cavities was carried out under a dissecting microscope with tweezers and a dissecting needle [14]. To avoid contamination, tweezers and needles were sterilized between host specimens by flaming over a Bunsen burner.
The mites obtained were washed in saline solution of 0.9% and fixed in 70% ethanol. They were then clarified with lactic acid and mounted on slides using Hoyer's medium. Photographs were taken using an Olympus CX 21 microscope with an attached camera To collect the mites, the nasal cavities were opened under an Olympus binocular stereo microscope with a scalpel and small scissors. First, the ossa mandibulae was removed together with the hyoid brush apparatus (apparatus hyobranchialis), then the scalp and feathers were removed with tweezers. Following this, a transverse section was made in the papillae pharyngeales region. A medial section was made, starting from the rima infundibuli through the choana and ending in the ruga palatina mediana. The nostrils in the area of the operculum were examined, and then the os maxillare was opened at the level of its fusion with the palate. Then, the maxillary bone was removed and the nasal cavity with its three conchae was opened: the largest of all rostral concha nasalis rostralis; the middle, concha nasalis media; and the caudal, concha, nasalis caudalis. Examination of all nasal cavities was carried out under a dissecting microscope with tweezers and a dissecting needle [14]. To avoid contamination, tweezers and needles were sterilized between host specimens by flaming over a Bunsen burner.
The mites obtained were washed in saline solution of 0.9% and fixed in 70% ethanol. They were then clarified with lactic acid and mounted on slides using Hoyer's medium. Photographs were taken using an Olympus CX 21 microscope with an attached camera system. Illustrations were made, and measurements were taken using a contrast phase Zeiss microscope with camera lucida.
The identification of the mites was based on morphological characteristics in accordance with previous studies [14,18,[20][21][22][23][24][25][26][27][28]. Mites were sexed based on the position of the genital pore, the presence of spermatodactyl in the chelicerae and a pair of long setae in the ventral zone, close to gnathosoma. For the epidemiological analysis, we assessed the prevalence and its confidence interval (Clopper-Pearson interval), and the mean intensities of each mite species in each host species. Fisher's exact test was used to compare the prevalence between the coexisting mite species, M. melloi and M. columbae, and bootstrap t-test (after 9999 bootstrap replicates) was used to compare the mean intensities (see [29]). Slope calculations and the Kendall correlation test were respectively employed to check for male to female mite ratios and correlations for each parasite species.

Results
A total of 1491 adult nasal mites (range 0-114) belonging to three species were collected in the analysis of 500 Columbiformes. Mesonyssus melloi and M. columbae in C. livia domestica and M. columbae and M. streptopeliae in S. decaocto ( Figure 2). One additional individual with no morphological coincidence with any species of rhinonyssids was found. Since the non-adult stages of these mites are hard to identify correctly, they were not explored in the current study.
system. Illustrations were made, and measurements were taken using a contrast phase Zeiss microscope with camera lucida.
The identification of the mites was based on morphological characteristics in accordance with previous studies [14,18,[20][21][22][23][24][25][26][27][28]. Mites were sexed based on the position of the genital pore, the presence of spermatodactyl in the chelicerae and a pair of long setae in the ventral zone, close to gnathosoma. For the epidemiological analysis, we assessed the prevalence and its confidence interval (Clopper-Pearson interval), and the mean intensities of each mite species in each host species. Fisher's exact test was used to compare the prevalence between the coexisting mite species, M. melloi and M. columbae, and bootstrap t-test (after 9999 bootstrap replicates) was used to compare the mean intensities (see [29]). Slope calculations and the Kendall correlation test were respectively employed to check for male to female mite ratios and correlations for each parasite species.

Results
A total of 1491 adult nasal mites (range 0-114) belonging to three species were collected in the analysis of 500 Columbiformes. Mesonyssus melloi and M. columbae in C. livia domestica and M. columbae and M. streptopeliae in S. decaocto ( Figure 2). One additional individual with no morphological coincidence with any species of rhinonyssids was found. Since the non-adult stages of these mites are hard to identify correctly, they were not explored in the current study.

Host Species
Parasite Species (n) The highest mean infection intensity was obtained for M. melloi, followed by M. columbae and then by M. streptopeliae ( Table 1)

Discussion
This study reveals the species of rhinonyssids harboured by the sympatric host species C. livia domestica and S. decaocto, with an updated morphological description and an accurate epidemiological survey. The morphological information published until now on rhinonyssids is abundant but not very detailed, and the illustrations are not as exhaustive as the ones we provide. Furthermore, epidemiological studies are frequently based on the

Discussion
This study reveals the species of rhinonyssids harboured by the sympatric host species C. livia domestica and S. decaocto, with an updated morphological description and an accurate epidemiological survey. The morphological information published until now on rhinonyssids is abundant but not very detailed, and the illustrations are not as exhaustive as the ones we provide. Furthermore, epidemiological studies are frequently based on the analyses of a few hosts, which makes the epidemiological survey inaccurate due to the low prevalence of the species studied [9][10][11][12][13] (but see [48]). We offer a detailed survey of the nasal mite species found in 250 C. livia domestica and 250 S. decaocto. Furthermore, we have detected M. columbae infecting S. decaocto for the first time.
The taxonomic assignment of the mite species isolated is still controversial. Since their original description in the 1950s, these species have been included in different genera, such as Neonyssus [42], Mesonyssus [23,[35][36][37][38] and Tinaminyssus [24,39,40]. Lately they have been placed in the genus Tinaminyssus by some authors [25,41] and in Mesonyssus by others [2,14,21]. As suggested by the latter, in this paper the species are arranged in the genus Mesonyssus because the genus Tinaminyssus mainly includes species associated with Tinamiformes.
The morphological features of the mite species sampled match those previously described by other authors for Messonyssus melloi [21,22,[25][26][27], M. columbae [14,[21][22][23][24][25] and M. streptopeliae [18,20,21,27,28]. Nevertheless, the published descriptions and illustrations of these species tended to be simple and only focused on few characteristics. We offer a complete re-description together with highly detailed illustrations of the species mentioned, including all the features that other authors noted (Figures 3-5). This information will help develop future investigations based on M. melloi, M. columbae and M. streptopeliae, making their identification easier and clearer.
It is noteworthy to state that a specimen of Mesonyssus was collected from S. decaocto showing the gnathosomal formula (2 + 2 + 2), completely different from the gnathosomal formula of the detected species (2 + 4 + 2) for M. melloi and M. streptopeliae, and (2 + 4 + 0) for M. columbae. Only two species worldwide follow the former formula: Mesonyssus juxtamelloi Pence et Canaris, 1976, and Mesonyssus carapachibeyus Dusbábek, 1969. However, these species and the mentioned specimen have different characteristics. M. juxtamelloi shows 32 setae on the dorsal idiosoma and 12 setae on the ventral idiosoma, and M. carapachibeyus presents three dorsal shields while the specimen collected has 21 dorsal setae, 10 ventral setae and two dorsal shields. Therefore, this individual could be a new species or an aberrant specimen. Deeper studies are needed to detect more individuals with similar characteristics.
To understand the relationship between parasites and hosts, it is crucial to obtain basic epidemiological information such as the prevalence, the intensity of infection, and the identity of the species involved. Until now, the most explored epidemiological aspect in the family Rhinonyssidae is the identification of the interacting species [48,49]. Nevertheless, in most of the species, mite information related with prevalence and intensity is almost unknown. We explored these epidemiological parameters focusing on the relationship of the nasal mite parasitic fauna of two avian species: C. livia domestica and S. decaocto, exploring 250 individuals of each one.
Traditionally, different studies on rhinonyssids have been performed throughout the world exploring prevalence; based on a scarce number of host specimens, these show low values. However, these studies include a high number of host species but a low number of specimens of each host species. For instance, Pence in 1973 [16] examined 1927 birds of 193 species and determined a total prevalence of 16.2%, Spicer in 1987 [50] studied 502 bird hosts of 103 species and found a prevalence of 17% and Knee et al. in 2008 [25] carried out a study of 450 birds of 154 species and showed a prevalence of 15%. However, each specific host-parasite relationship could have different values of prevalence, which could lead to inaccurate estimations if only a few individuals are examined. Thus, exploring specific relationships within a high number of hosts for each of these relationships is key.
We explored 250 C. livia domestica and 250 S. decaocto, finding Mesonyssus melloi and M. columbae in C. livia domestica and M. streptopeliae, and M. columbae in S. decaocoto. While most of these relationships have already been described, this is the first time that M. columbae has been detected in S. decaocoto. The sterilization of instruments prior to carrying out a new host analysis and the meticulousness during mounting, together with the fact that this species appears least frequently, leads us to think that it is not due to contamination. Thus, three possible explanations could account for this result: (i) this relationship could be common in nature but the scarce epidemiological surveys together with its low prevalence have precluded its detection; (ii) this is an incipient event of host switching, which has been found to possibly have important evolutionary consequences in other groups of avian mites [51]; (iii) this encounter is a consequence of the sampling method, which could lead to the infection of doves with mites from pigeons. Some authors have found that indirect transmission can take place via water, perches and other contaminated surfaces [15], so the sampling method of employing traps with water and food to catch individuals could lead to this event. Nevertheless, it is important to note that these two host species usually share feeding habitats, which could lead to this event in natural conditions. Studies that explore a greater number of individuals of each host species would help uncover these kinds of relationships where prevalence could be too small.
Mesonyssus melloi was significantly more prevalent and produced higher infection intensities in C. livia domestica than M. columbae. Furthermore, as previously described [14,21], they are able to coexist, infecting the same host (Table 1). Streptopelia decaocoto are almost always infected by a single mite species, M. streptopeliae (with the exception previously described). Unfortunately, the comparison of these data with other publications is difficult. First, because most studies concern morphological description where the number of host specimens considered is low, this leads to inaccurate estimates. To our knowledge, this is the first paper that explores these estimates for M. streptopeliae. Regarding M. columbae, only three publications explore this kind of data: Crossley (1952), who reported a prevalence of 20% in 60 pigeons [52]; Wilson (1964), who found a prevalence of 7% after examining 15 pigeons [23]; Rojas et al. (2020), who found a prevalence of 1.9% after examining 262 pigeons in St. Petersburg (Russia) [48]. Meanwhile, we found a prevalence of 6%. In the case of M. melloi, there is more information available: Crossley (1952) found a prevalence of 25% after examining 60 pigeons in Texas (USA) [52]; Wilson (1964) found a prevalence of 7% after examining 15 pigeons in Indiana (USA) [23]; Rózsa (1990) found a prevalence of 23% or 42%, depending on the host population, in Hungary [53]; Foronda et al. (2004) found a prevalence of 10% in 50 pigeons in Tenerife (Spain) [54]; and Rojas et al. (2020) found a prevalence of 1.1% after examining 262 pigeons in St. Petersburg (Russia) [48]. Meanwhile, we found a prevalence of 34%, one of the highest ever reported. It is noteworthy to mention, even with the scarce information available, the high variation in the prevalence reported by different authors in the same host-parasite system. Similar concerns about the abundance and intensity of infection arise from information about intensities of infections, but in this case the research is much too scarce. To our knowledge, there are two single studies focused on M. melloi that report intensity of infection: Foronda et al. (2004), who reported a mean of 218.3 ± 117.3 mites per infected host [54], and Rózsa (1990), who reported a mean of 7.9 or 10.3 mites per host [53], depending on the host population. We report an infection intensity of 14.4 mites per infected host. It is clear that infection intensities are highly variable. It is important to highlight the study performed by Rózsa (1990) that compared the prevalence and intensities between two pigeon populations located in an urban and a farmland area, finding higher values of both parameters in the urban area [53]. Prevalence and intensities are highly variable not only among host-parasite relationships but also among populations. Thus, there must be important factors behind the difference in prevalence and intensities of infection that deserve to be explored. Deeper studies at a larger spatial scale analysing a higher number of hosts must be performed in order to reveal the factors accounting for these results. The female-biased sex ratio is known by acarologists focused on Rhinonyssidae, and because of that, morphological keys are usually based on female characteristics. Biased sex ratios could be selected from different mating systems or population structures [55,56]. However, there are no studies about ratio or reproduction mode in the bibliography, even though these are key to predict and understand the mating system of these organisms. We found a relatively constant sex ratio within three specific parasite-host relationships, which could be due to similar pressures on the sex allocation. Nevertheless, specific studies on different species focused on the mating system must be developed to understand this ratio.

Conclusions
Knowledge of the relationships established between birds and their rhinonyssids parasites is scarce. The challenge of identification and studies based on a few host specimens could impede the development of further studies and lead to inaccurate results, respectively. We detailed morphological descriptions of three closely related species in order to facilitate their study. Furthermore, we estimated basic epidemiological parameters based on a high number of individuals in order to obtain accurate estimates. Deep differences between previously reported results and the ones shown here suggest that various factors could be behind these differences. Future studies will be essential to assess the mechanisms ruling the variation in prevalence and intensity.