﻿A new feather mite species of the genus Mycterialges Gaud & Atyeo, 1981 (Acari, Xolalgidae) from the Oriental Stork, Ciconiaboyciana (Ciconiiformes, Ciconiidae) in Korea

﻿Abstract A new feather mite species, Mycterialgesboycianaesp. nov. (Xolalgidae), was identified from the Oriental Stork, Ciconiaboyciana Swinhoe, 1873, in Korea. Males of M.boycianaesp. nov. are distinguished from Mycterialgesmesomorphus Gaud & Atyeo, 1981, in having a single triangular prodorsal shield, sinuous margins of the opisthosoma located between setae e2 and h2 on the hysteronotal shield, an oval-shaped epiandrum without posterior extensions, a shorter tibia + tarsus IV than femoragenu IV, and an absent ambulacral disc of leg IV. Females differ in having a prodorsal shield with a posterior margin that is blunt-angular, and a concave posterior margin of the hysteronotal shield with posterior extensions. This study presents the first record of the feather mite genus Mycterialges in birds of the genus Ciconia (Ciconiidae). Additionally, we determined the phylogenetic relationship among Ingrassiinae using the mitochondrial cytochrome c oxidase subunit (COI).


Introduction
Feather mites (Astigmata) belong to two superfamilies (Analgoidea and Pterolichoidea), most of which are permanent ectosymbionts.These mites live on specific feathers and microsites on feathers, primarily consuming fungi and bacteria (Doña et al. 2019).They are mainly transmitted vertically from parents to offspring (Doña et al. 2017), and because of these characteristics, they exhibit high host specificity (OConnor 1982;Gaud and Atyeo 1996;Dabert and Mironov 1999;Proctor 2003).Feather mites living on endangered birds could be at risk of extinction due to their high host specificity, as a decrease in the host population could affect them (Waki et al. 2023).For example, Compressalges nipponiae Dubinin, 1950 (Pterolichoidea: Caudiferidae), which is associated with the Crested Ibis Nipponia nippon (Temminck, 1835), disappeared due to the extirpation of its native host N. nippon in Japan.Consequently, this mite was registered as "Extinct" in the Red List of the Ministry of the Environment, Japan in 2020 (Waki and Shimano 2020;Waki et al. 2023).
The genus Mycterialges Gaud & Atyeo, 1981 is distinguished from other genera in the family Xolalgidae Dubinin, 1953 (Analgoidea) by the number of setae on the anterior tarsi, ambularcal disc shape, male opisthosomal lobes structure and legs IV, and the female's epigynum form (Gaud andAtyeo 1981a, 1996).This genus is associated with birds of Ciconiidae; only two species of this genus have been reported (Gaud andAtyeo 1981a, 1996;Gaud 1982).The first discovered type species was Mycterialges mesomorphus Gaud & Atyeo, 1981, which was recorded in the Wood Stork, Mycteria americana Linnaeus, 1758 in North America.Another species was found in the Saddle-billed Stork, Ephippiorhynchus senegalensis (Shaw, 1800) in Africa, but it has not yet been accurately described (Gaud 1982).
The family Ciconiidae consists of 20 species in six genera, which are found on all continents except Antarctica.In Korea, two species of the genus Ciconia occur: the Black Stork, Ciconia nigra (Linnaeus, 1758) and the Oriental Stork, Ciconia boyciana Swinhoe, 1873.Both species are observed as winter visitors and are designated as "Endangered Species Level I" by the Korea Ministry of Environment (BirdLife International 2017International , 2018;;Lee et al. 2020;Gill et al. 2023;NIBR 2023).Among them, C. boyciana has become extirpated from Korea's breeding population since the 1970s due to overfishing, habitat destruction, and food shortages (Sonobe and Izawa 1987;Chan 1991;Collar et al. 2001;Cheong 2005).In Korea, efforts have been made to restore a breeding population of C. boyciana since 1996 by importing Oriental Storks from Germany, Russia and Japan to increase their population.Since 2007, storks have been released into the wild to restore the breeding population (Park et al. 2011;Son et al. 2011;Eco-Institute for Oriental Stork 2023).
Research on the migration routes and nest selection of C. boyciana has mainly been conducted in East Asia (Shimazaki et al. 2004;Cheng et al. 2023).However, despite active research on the behavior and ecology of the Oriental Storks, research on ectosymbionts has not been actively conducted.Pelargolichus orientalis Waki, Mironov & Shimano, 2023 is the only known feather mite of C. boyciana.This mite was previously identified as Pelargolichus didactylus (Trouessart, 1885) (Dubinin 1956).However, Pérez and Atyeo (1992) identified mites in the Oriental Stork as a different species within the genus Pelargolichus, which was later described as P. orientalis by Waki et al. (2023).Except for this mite, little is known about other mites associated with Oriental Storks.Since 1996, ecological research has been conducted on the feeding behavior, reintroduction suitability, and habitat of Oriental Storks (Cheong 2005;Sung et al. 2008;Kim 2009;Ha 2019).Recently, we started research on ectosymbionts that can affect the host while interacting with storks.
This study reports a new species of feather mite of the genus Mycterialges found on captive C. boyciana in Korea.We provide information on the external morphology of the newly discovered feather mite species.In addition, we used the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) sequence information to determine the phylogenetic relationship between the new species and known closely related species.

Sampling and characterization
Feather mite sampling from three captive C. boyciana individuals was conducted in the same cage at Yesan Oriental Stork Park in Korea in October 2022, with permission from the Cultural Heritage Administration of Korea (B0030104016624).
To minimize stress during the investigation, the storks were blindfolded and immobilized by the keepers; the procedure was completed within a maximum of five minutes for each individual.A new species of feather mites was identified from the wing and body feathers of the birds.The mites were carefully removed from the feathers using tweezers, and the collected mites were preserved in 95% ethanol solution.The specimens were mounted on microscope slides using a polyvinyl alcohol mounting medium (BioQuip, California, USA) after clearing with 10% lactic acid (Downs 1943;Han et al. 2016).Specimens were examined using a Dhyana 400DC camera (TUCSEN, Fuzhou, Fujian, China), Leica DM 2000 microscope (Leica, Wetzlar, Germany) with a drawing tube.
Descriptions of a new species followed the standard formats proposed for mites of the subfamily Ingrassiinae Gaud andAtyeo 1981 (Mironov andProctor 2008;Stefan et al. 2013;Hernandes and Pedroso 2017;Han et al. 2021;Mironov and Hribar 2023).General morphological terms followed Gaud and Atyeo (1996) with minor corrections for coxal setation by Norton (1998).The classification and names of the birds followed those described by Gill et al. (2023).All measurements are in micrometres (μm).

Differential diagnosis.
The genus Mycterialges has included only the type species, Mycterialges mesomorphus, and one additional undescribed species (Gaud and Atyeo 1981a;Gaud 1982).The new species Mycterialges boycianae sp.nov.differs from M. mesomorphus by a number of characters: in males of M. boycianae, the prodorsal shield consists of a single triangular plate, the anterior part of the hysteronotal shield is widened and has a pair of narrow extensions, the paragenital apodemes are fused into a large teardrop sclerite encompassing genital apparatus, the legs IV are much shorter than legs III, and tibia + tarsus IV are shorter than femoragenu IV, tarsus IV is conical and ambulacral disc of tarsus IV is absent; in females, the posterior margin of prodorsal shield is blunt-angular and extends slightly beyond the level of setae si, the hysteronotal shield is as wide as the prodorsal shield and its posterior margin is deeply concave, setae e2 are situated on the posterior corners of the hysteroronotal shield, and setae g and 3a are situated on the same transverse level.In males of M. mesomorphus, the prodorsal shield consists of two plates (triangular anterior parts and trapezoidal posterior part), the anterior part of the hysteronotal shield is narrowed and without extensions, the paragenital apodemes are fused into ovate sclerites around genital apparatus and with a pair of posterior projections, the legs IV are almost as long as legs III, and tibia + tarsus IV are much longer than femoragenu IV, tarsus IV has a claw-like apical process, and ambulacral disc of tarsus IV is narrowly lanceolate; in females, the posterior margin of prodorsal shield is straight and does not extend to the level   of setae si, the hysteronotal shield is narrow, approximately half as wide as the prodorsal shield and its posterior margin is straight, setae e2 are situated on the soft tegument at the level of the posterior margin of the hysteronotal shield, and setae g are anterior to level of setae 3a.Actually, M. mycterialges sp.nov. is much more similar to the unnamed Mycterialges species, known only from male and illustrated but not described (Gaud 1982: fig. 6a, b), in sharing the following features: the prodorsal shield is triangular, the anterior end of the hysteronotal shield has a pair of narrow extensions, legs IV are much shorter that legs III, and tibia+tarsus IV are shorter than femoragenu IV, tarsus IV is conical, and ambulacral disc of pretarsus IV is absent.Males of M. boycianae differs from those of the unnamed Mycterialges species in the following features: setae d2 are represented by macrosetae extending beyond the posterior margin of opisthosoma, setae cp are situated on the humeral shields, and the paragenital apodemes fused into the teardrop-shaped sclerite free from epimerites IV.In the male of the unnamed Mycterialges species, setae d2 extend to midlevel between setae e2 and h2, setae cp are situated on striated tegument, and the paragenital apodemes fused into the teardrop-shaped sclerite are fused with the inner tips of epimerites IV.
Etymology.The specific name is taken from the species epithet of the type host and is a noun in the genitive case.

Phylogenetic relationships based on mtDNA COI
The ML phylogenetic tree of the COI barcode fragment showed Mycterialges boycianae sp.nov. to be grouped within the subfamily Ingrassiinae clade with three individuals of M. boycianae, exhibiting 93.7% bootstrap support for this grouping.Among the COI sequences collected from NCBI, the genus Glaucalges formed the clade closest to M. boycianae (Fig. 6).The genetic distance between M. boycianae and Glaucalges species was estimated to be ~16.1-16.2%.

Discussion
This study describes a new feather mite species, Mycterialges boycianae sp.nov.found on the wings and body of captive Ciconia boyciana at the Yesan Oriental Stork Park in Korea.The genus Mycterialges has the following features: in both sexes, tarsi I, II only have two and one ventral setae, respectively, and the ambulacral disc is inverted triangle-shaped with a concave middle; in males, the opisthosomal lobes are fused and bluntly rounded, leg IV is hypertrophied; in females, the epigynum is short and straight (Gaud andAtyeo 1981a, 1996).Mycterialges boycianae has all these main characteristics (Figs 1-5), but exhibits significant variation from the type species in the legs IV.In the case of the type species M. mesomorphus, the male legs IV are hypertrophied, but not to the terminal width, and there is an ambulacrum (Gaud and Atyeo 1981a).In contrast, legs IV of M. boycianae and M. sp.(Gaud andAtyeo 1981a, 1996), are hypertrophied to the extent of the terminal width, have a femorogenu that combines the femur and genu, and have a claw instead of an ambulacrum (Figs 1-2, 5) (Gaud and Atyeo 1996).Despite these significant differences, no other species have been identified in Mycterialges apart from the type species so far.Therefore, we have included the newly discovered feather mite in Mycterialges based on the form of M. sp., despite the lack of precise identification by Gaud andAtyeo (1981a, 1996).We observed that the mites discovered in the genus Mycterialges exhibit significant differences in legs IV, thus suggesting that there is a need to redefine this genus.
We showed the phylogenetic relationship of Ingrassiinae using the COI barcode fragment (Fig. 6).However, despite gathering all available Ingrassiinae data from the NCBI, our tree could only provide minimal information.This is due to the fact that, while the Ingrassiinae consists of 16 genera and 106 species (Gaud and Atyeo 1981a;Mironov and Galloway 2002;Mironov et al. 2005;Dabert et al. 2008;Constantinescu et al. 2013;Stefan et al. 2013;Hernandes 2014;Li and Zhang 2016;Mironov et al. 2017;Hernandes and Pedroso 2017;Han et al. 2021;Hernandes and Brito 2022), our tree data includes only seven genera and 12 species, some of which have not been accurately described.Therefore, it is necessary to collect more data on mites to obtain more accurate results (Maddison and Knowles 2006;Knowles and Klimov 2011).
Finally, we discuss the situations faced by M. boycianae.This mite's host, C. boyciana, is not only an endangered species in Korea, but is also listed as an 'Endangered' (EN) species on the International Union for Conservation Nature (IUCN) Red List (BirdLife International 2018).Moreover, the nesting sites of the Oriental Stork are extremely limited and confined to parts of Russia and China (Zhou et al. 2013;BirdLife International 2018).Given its high host specificity, this situation could be extinction-threatening for M. boycianae, a permanent ectosymbiont (OConnor 1982;Doña et al. 2019).Therefore, we believe that it is necessary to investigate the distribution, biology, ecology, and conservation status of feather mites, including M. boycianae, to confirm whether ectosymbionts are at risk of extinction.

Figure 5 .
Figure5.Mycterialges boycianae sp.nov., details, ventral view A opisthoma of male, dorsal view B leg Ι of male C leg II of male D leg III of male E leg IV of male F tibia and tarsus III of female G tibia and tarsus IV of female.

Figure 6 .
Figure 6.Maximum-likelihood phylogenetic tree of COI barcode fragment for members of the subfamily Ingrassiinae.Bootstrap percentages of more than 50% are shown.Scale bars indicate the number of substitutions per nucleotide site.The subfamily Xolalginae is the outgroup taxon.

Table 1 .
Feather mites used for the phylogenetic analysis in this study (COI barcode fragment).