Description of the Nymphal Stages of Hoplopleura affinis (Anoplura: Hoplopleuridae) from the Striped Field Mouse Apodemus agrarius with a Global Checklist of Hoplopleura Species from the Genus Apodemus

Simple Summary Sucking lice are blood-feeding, external parasites of mammals. To date, approximately 540 species have been discovered, occurring in 830 hosts. New species are discovered every year, and the total number is estimated at 1500. The discovery of a species is associated with a detailed description of morphological characteristics. Most descriptions concern only adult specimens. The present study adds to the knowledge by characterizing nymphs of Hoplopleura affinis, a species that parasites the striped field mouse Apodemus agrarius, a common rodent in Europe and Africa. In addition, a checklist of Hoplopleura species parasitizing members of the genus Apodemus was compiled. Abstract The genus Hoplopleura is the most speciose genus of sucking lice observed parasitizing rodents and lagomorphs (pikas). Despite the fact that the majority of Anoplura are believed to be monoxenic, many species within Hoplopleura may be oligoxenic. In addition, the occurrence of more than one parasite species per host species on individuals has been confirmed. As such, a precise species identification of the parasite, especially of the nymphs, is of high significance. The study is based on the material of 245 sucking louse specimens taken from 179 individuals of the striped field mouse Apodemus agrarius collected between 2008 and 2017. The study employs scanning microscopy to provide superior quality resolution of the studied traits. The study presents the first record of the characters of the nymphal stages of H. affinis, one of the common Eurasian species of the genus. Additional aspects of the biology and the host–parasite relationship of H. affinis are presented, e.g., female, male and nymphs of lice, showing different preferences in the choice of location (topography) on the host body. In addition, a global checklist has been made of all the species of Hoplopleura found parasitizing rodents of the genus Apodemus. Generally, the ranges of the occurrence of lice of this genus coincide with the geographic distribution of typical hosts, although this has not always been confirmed by local studies.


Introduction
Sucking lice (Psocodea: Anoplura) are hematophagous and wingless parasites of placental mammals (Mammalia: Eutheria) [1]. Most of the species have only one host throughout their life cycle, which represents the habitat for all developmental stages (eggs, three stages of nymphs, and adults). Thus far [2], 532 sucking lice species have been described, and these are known to be parasites of 830 mammal species. However, the
Where coparasitism of two sucking lice species of the genus Hoplopleura was observed, the individuals were not taken into account when creating nymphal stage characters due to risk of error. However, as the nymphs of Hoplopleura and Polyplax differ markedly, in cases where H. affinis and Polyplax serrata co-occurred, the nymphs were included in the identification. Table 1. Host and lice specimen data from Poland (physico-geographical regions follow [14]). The lice were collected with tweezers from ethanol-preserved rodent specimens (UGDIZP; Collection of Extant Invertebrates, University of Gdańsk, Department of Invertebrate Zoology and Parasitology, Gdańsk, Poland), by combing the coat. The lice present were kept in labeled individual vials (separately for each host) and preserved in 70% ethyl alcohol. Next, morphological structures and body surfaces of specimens were analyzed (measurements are given in mm), using two microscopic techniques:

1.
Specimens intended for analysis under a light microscope were prepared by slidemounting in polyvinyl-lactophenol [15].

2.
Individuals intended for analysis with scanning electron microscopy were subjected to a series of alcohols (80-100%) and then dried in a mix of ethyl alcohol and hexamethyldisilazane (HMDS) in 1:3, 1:1, and 3:1 proportions. Finally, the specimens were transferred to pure HMDS and placed in an incubator for 24 h (37 • C) [16]. The specimens were stuck to double-sided copper tape (by Mierzejewski Materiały Samoprzylepne) and fixed on the table of a scanning electron microscope. Observations and photographs were performed with the use of Field Emission Scanning Electron Microscope JSM-7800F (manufacturer JEOL; stocked in Department of Materials Engineering and Bonding-Faculty of Mechanical Engineering, Gdańsk University of Technology, Gdańsk, Poland).
The louse specimens were deposited in the Collection of Extant Invertebrates, University of Gdańsk, Department of Invertebrate Zoology and Parasitology, Gdańsk, Poland [17].
The names and abbreviations of individual setae or body parts are provided mostly following [1] (Figures 1 and 2).
The lice were collected with tweezers from ethanol-preserved rodent specimens (UG-DIZP; Collection of Extant Invertebrates, University of Gdańsk, Department of Invertebrate Zoology and Parasitology, Gdańsk, Poland), by combing the coat. The lice present were kept in labeled individual vials (separately for each host) and preserved in 70% ethyl alcohol. Next, morphological structures and body surfaces of specimens were analyzed (measurements are given in mm), using two microscopic techniques: 1. Specimens intended for analysis under a light microscope were prepared by slidemounting in polyvinyl-lactophenol [15]. 2. Individuals intended for analysis with scanning electron microscopy were subjected to a series of alcohols (80-100%) and then dried in a mix of ethyl alcohol and hexamethyldisilazane (HMDS) in 1:3, 1:1, and 3:1 proportions. Finally, the specimens were transferred to pure HMDS and placed in an incubator for 24 h (37 °C) [16]. The specimens were stuck to double-sided copper tape (by Mierzejewski Materiały Samoprzylepne) and fixed on the table of a scanning electron microscope. Observations and photographs were performed with the use of Field Emission Scanning Electron Microscope JSM-7800F (manufacturer JEOL; stocked in Department of Materials Engineering and Bonding-Faculty of Mechanical Engineering, Gdańsk University of Technology, Gdańsk, Poland).
The louse specimens were deposited in the Collection of Extant Invertebrates, University of Gdańsk, Department of Invertebrate Zoology and Parasitology, Gdańsk, Poland [17].
The names and abbreviations of individual setae or body parts are provided mostly following [1] (Figures 1 and 2). Using the scanning microscopy (SEM), three variants of ornamentation in the form of scales and setae shape are known to exist: smoothly-ended (U-shaped), sharp-ended (V-shaped), and needle-shaped setae ( Figure 3).

The Checklist
The checklist of Hoplopleura species parasitizing the genus Apodemus has been compiled on the basis of data published during the period 1956-2019 (12 items: [4-7,10,18-24]. Apodemus species are ordered alphabetically. The scientific names, common names, and

Description of Nymphal Stages
Nymph I (Figure 4; measurements n = 1), Body length: 0.44. Head: length 0.10, width 0.13.  Ventral: no GP; in its place, a large number of tubercles (they form an oval field), some reminiscent of small setae in shape; three tubercles (looking like a small setae) at the base of the antennae. Head posteriorly slightly concave on both ventral and dorsal surfaces. AHS 4 in number; OS almost as long as VMHS, but thinner; above OS, there are two short bristles with a tubercle between them; VMHS measuring approx. 1/3 VPHS. Tubercles (2)(3)(4) are also on the 2nd-4th segment of antennae.
Dorsal: DAHS, PCHS, AS present; OSHS and ISHS minor, at a considerable distance from one another; MHS present, but MMHS closer to the mid-portion of the body; PDHS reaches the first thoracic segment; ADHS above the preceding one; ACHS minute. Thorax: length 0.10, width 0.21.
Abdomen heart-shaped, heavily wrinkled. U and V-shaped scales covering the abdomen in a tile-like manner; spiracles visible on both ventral and dorsal surfaces, heavily depressed. VCAS one pair on segment 1 (difficult to detect); MAS 2; AcS 2.
Nymph II ( Figure 5; measurements n = 9), Body length: 0.52 (0.37-0.72). Head: length Ventral: no GP; in its place, a large number of tubercles (they form an oval field), some reminiscent of small setae in shape; three tubercles (looking like a small setae) at the base of the antennae. Head posteriorly slightly concave on both ventral and dorsal surfaces. AHS 4 in number; OS almost as long as VMHS, but thinner; above OS, there are two short bristles with a tubercle between them; VMHS measuring approx. 1/3 VPHS. Tubercles (2)(3)(4) are also on the 2nd-4th segment of antennae.
Dorsal: DAHS, PCHS, AS present; OSHS and ISHS minor, at a considerable distance from one another; MHS present, but MMHS closer to the mid-portion of the body; PDHS reaches the first thoracic segment; ADHS above the preceding one; ACHS minute.
Abdomen heart-shaped, heavily wrinkled. U and V-shaped scales covering the abdomen in a tile-like manner; spiracles visible on both ventral and dorsal surfaces, heavily depressed. VCAS one pair on segment 1 (difficult to detect); MAS 2; AcS 2.
Abdomen very broadly heart-shaped; VCAS one pair on segment 1 (difficult to detect); MAS 4 in number; AcS 2.
Ventral: VPHS not as long; AHS, OS, and VPHS present; tubercles similar to first instar nymph; in place of GP heavily convex V-shaped tubercles present (they are arranged in the shape of a GP plate seen in adults); on the lateral corners the tubercles are flat, U-shaped.
Dorsal: DAHS, PAS, AS, and PCHS present; OSHS and ISHS not as close to one another, very short; MMHS closer to AMHS than PMHS; PDHS barely reaches the first segment of thorax; ADHS very short and thick; whole surface covered by wide U-shaped scales (apart from the occiput); ACHS minute. Thorax: length 0.12, width 0.22.
Dorsal: DPTS reaches the first abdominal segment; DPtS, DMsS, and DMtS present; first part of the first segment covered thickly by scales as on the head; around the thoracic spiracle two horn-like processes, the upper larger. Abdomen: length 0.39, width 0.34. Ventral: convex U-shaped scales covering almost the entire surface; tubercles more flat on head corners. Dorsal: DAHS present; OSHS and ISHS at a minor distance from one another; MHS present; PDHS reaches the first thorax segment; ADHS equal to the previous setae; ACHS minute.
Ventral: VPHS not as long; AHS, OS, and VPHS present; tubercles similar to first instar nymph; in place of GP heavily convex V-shaped tubercles present (they are arranged in the shape of a GP plate seen in adults); on the lateral corners the tubercles are flat, U-shaped.
Dorsal: DAHS, PAS, AS, and PCHS present; OSHS and ISHS not as close to one another, very short; MMHS closer to AMHS than PMHS; PDHS barely reaches the first segment of thorax; ADHS very short and thick; whole surface covered by wide U-shaped scales (apart from the occiput); ACHS minute.
Thorax: length 0.12, width 0.22. Dorsal: DPTS reaches the first abdominal segment; DPtS, DMsS, and DMtS present; first part of the first segment covered thickly by scales as on the head; around the thoracic spiracle two horn-like processes, the upper larger.
Abdomen heavily wrinkled, strongly covered with differently shaped scales, with well-visible spiracles (forming two lines separating the abdomen into three parts); anal area well marked, entrance heavily indented; VCAS one pair on segment 1 (difficult to detect); MAS 8 in number; AcS 2; AnS 2.
No morphological anomalies were observed in the study nymphs. The parasite completes its entire life cycle on the host (eggs, nymphs, and adults), and feeds actively: most individuals were engorged with blood. This is confirmed by the prevalence of 36.3% and the mean intensity of 3.8 specimens (in 65 infested hosts) was recorded.
Co-occurrence of different louse species was found on one host (Apodemus agrarius). Hoplopleura affinis and H. acanthopus were observed in one case, and H. affinis and Polyplax serrata (Anoplura: Polyplacidae) in 16 cases.
Topographic preferences have been observed for H. affinis ( Figure 6). Females (n = 119) were observed on the abdominal surface, only near the right rear leg groin. On the dorsum, however, they formed three bands, i.e., along the spine and on the lateral parts of the body, which joined on the nape of the animal. In addition, the females were observed on the head of the host animal, forming a band from the nape, between the ears up to the nose. Males (n = 27) covered the entire right side of the host animal, from the dorsum to the venter. They were found along the dorsum; they formed two bands before the forelimbs and three bands on the head, i.e., on the mid-point and on the sides of the dorsal portion, as well as between the vibrissae (upper and lower labial and jugal). Nymphs and eggs were numerous on both sides of the animal (dorsal and ventral), forming two clear bands joining on the mid-point of the dorsum. In addition, they formed three bands on the head, joining between the eyes and ears. Nymphs and eggs were rarely observed on the dorsal surface of the neck. Abdomen heavily wrinkled, strongly covered with differently shaped scales, with well-visible spiracles (forming two lines separating the abdomen into three parts); anal area well marked, entrance heavily indented; VCAS one pair on segment 1 (difficult to detect); MAS 8 in number; AcS 2; AnS 2.
No morphological anomalies were observed in the study nymphs. The parasite completes its entire life cycle on the host (eggs, nymphs, and adults), and feeds actively: most individuals were engorged with blood. This is confirmed by the prevalence of 36.3% and the mean intensity of 3.8 specimens (in 65 infested hosts) was recorded.
Co-occurrence of different louse species was found on one host (Apodemus agrarius). Hoplopleura affinis and H. acanthopus were observed in one case, and H. affinis and Polyplax serrata (Anoplura: Polyplacidae) in 16 cases.
Topographic preferences have been observed for H. affinis ( Figure 6). Females (n = 119) were observed on the abdominal surface, only near the right rear leg groin. On the dorsum, however, they formed three bands, i.e., along the spine and on the lateral parts of the body, which joined on the nape of the animal. In addition, the females were observed on the head of the host animal, forming a band from the nape, between the ears up to the nose. Males (n = 27) covered the entire right side of the host animal, from the dorsum to the venter. They were found along the dorsum; they formed two bands before the forelimbs and three bands on the head, i.e., on the mid-point and on the sides of the dorsal portion, as well as between the vibrissae (upper and lower labial and jugal). Nymphs and eggs were numerous on both sides of the animal (dorsal and ventral), forming two clear bands joining on the mid-point of the dorsum. In addition, they formed three bands on the head, joining between the eyes and ears. Nymphs and eggs were rarely observed on the dorsal surface of the neck.

Checklist of Hoplopleura spp. on Murid Rodents of the Genus Apodemus
In 12 species of Apodemus mice (out of 20 known species), lice of the genus Hoplopleura (10 species) were recorded. Their distribution is consistent with the geographic ranges of the hosts, mainly in Eurasia and Africa (Table 2).

Discussion
The morphology of H. affinis nymphs considerably differs from other European species (H. acanthopus-Holarctic parasite on voles, H. captiosa-cosmopolitan on M. musculus, H. edentula-Eurasia, H. longula-Eurasia) ( Table 2). No morphological characters of H. edentula nymphs exists. The characteristic, heart-shaped body, is a feature that distinguishes the nymphs of H. affinis from the other three mentioned above. H. affinis has a heart-shaped, more or less broad, corrugated abdomen, while H. acanthopus, H. captiosa, and H. longula have an elongated, egg-shaped or barrel-shaped abdomen with poorly or without corrugation [11][12][13].
Nymphs of H. longula have very long posterior dorsal head setae (PDHS), extending to the second abdominal segment. PDHS of H. affinis never reach the second abdominal segment [13].
The tubercles cover the upper part of the head of H. acanthopus, on H. affinis they additionally form a field along the middle part of the head (in place of the gular plate GP), while on the head of H. longula they form a cross-shape. The tubercles on ventral surface of head and antennae H. captiosa are blunt, H. affinisline small setae in shape (V-shaped) [11][12][13].
Nymphs of H. affinis can be easily distinguished from one another: nymph I has two major abdominal setae (MAS) and two accessory setae (AcS); nymph II has four major abdominal setae and two accessory setae; nymph III-eight major abdominal setae, two accessory setae and two anal setae (AnS).
The nymphal stages of H. affinis demonstrate a similar heart-shaped body to H. malabarica and H. sicata, but these parasites were not found on Apodemus [26].
Anomalies in morphology (differences in the number and size of the setae on the sternal plates of the abdomen and irregularities in the structure of the plate itself) were observed for the adult examined individuals [27].
Hoplopleura affinis appears to be a typical parasite of A. agrarius in Europe; despite extensive research, it has not been found on Myodes glareolus, Microtus agrestis, Mus musculus, Apodemus sylvaticus, or Sorex araneus [4,25,[28][29][30]. Other results indicate this as well: occurrence of all life stages on the host, observation of blood-filled individuals (this study).
Few studies provide the locations of the sucking lice on the host body. However, Dubinin [31] notes that for H. affinis parasitizing A. agrarius, the lice were located only on the dorsal head and a small neighboring part of the neck. The current study provides more extensive knowledge, with the louse being found to cover a larger preferred area, reaching the posterior limbs of the host. There are also no data on the occurrence of H. affinis nymphs and eggs present on A. agrarius in the available literature. The present study adds to the knowledge, with multiple occurrences on the dorsal and ventral parts of the animal. No differences were observed in topographic preferences for different life stages of H. affinis. One common feature is evident: none of the stages occurred on the ventral head. Perhaps this is due to the structure of the host's coat, the hairs are sparser and shorter there [32] and specific behavior patterns, e.g., self-grooming or self-cleaning behaviors, when rodents self-groom by scratching to clean or groom the fur [33,34].
Analyzing the global checklist of Hoplopleura species of the genus Apodemus (Table 2), it can be seen that the absence of sucking lice findings in a given host is mainly due to its status: endemic, difficult to access or recently described species. This applies, for example, to A. alpicola, a poorly understood species discovered in the mid-twentieth century, endemic to the alpine region, and often inhabiting hard-to-reach protected areas. Similarly, poorly known is A. hyrcanicus, described only in 1992, limited in range to forest regions from the southern Caucasus to Central Asia, moreover, usually not sympatric with either Apodemus [9], which also limits the transmission of parasites. In contrast, in widely distributed and much better-studied species, such as A. agrarius, A. flavicollis, A. sylvaticus, and A. uralensis, there are usually several, also usually widespread, Hoplopleura species.

Conclusions
Research on sucking lice (Anoplura) has so far been carried out selectively, focusing mainly on their pathogenicity and related aspects. Descriptions of new species are usually based only on adults. The lack of knowledge of the juvenile stages generate false data on the host specificity of individual parasite species, their host circle, transfer possibilities between hosts, geographical distribution, or habitat preferences. Consequently, it leads to false conclusions regarding various aspects of the functioning of the parasite-host systems, the spread and transmission possibilities of parasites, and in the case of particularly pathogenic parasites or pathogens vectors, their health significance. The issue of the correct identification of the stages of juvenile parasites is currently one of the universal problems of parasitology. This especially applies to the phases of the life cycles, where juvenile stages are the dominant group in the structure of the parasite population, or mature stages, which are usually the basis for species identification, do not occur at all. An example is the parasitic nematodes of the Anisakidae family with complex life cycles where only larval stages are present in intermediate/paratenic hosts (fish). They are of zoonotic importance as they can be invasive and pathogenic to humans [35][36][37]. Problems with identifying the larval stages may also concern various ticks important as vectors of pathogens [38]. The lack of descriptions for nymphs causes them to be mistakenly assigned to species and consequently to host species. It also causes a lack of recognition the structure and dynamics of the population development of the lice species within the host, as well as the seasonal dynamics. Hence, it is important to describe the nymphal stages as well. The current study adds to the knowledge on this subject by providing descriptions for the nymphs of H. affinis. It should be kept in mind that assigning nymphs to a given species is extremely difficult and requires the consideration of a number of inter-related criteria, including the presence of adult stages and exclusion of coparasitism with similar species.
Author Contributions: Conceptualization, P.K. and J.N.I.; microscopy analysis, P.K. and J.N.I.; data analysis, P.K. and J.N.I.; sampling, P.K., J.N.I. and R.Ł.; writing-original draft preparation, P.K. and J.N.I.; writing-review and editing, P.K., J.N.I. and R.Ł.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.