The first description of the nymphal stages of Hoplopleura longula (Psocodea: Anoplura: Hoplopleuridae) from the harvest mouse Micromys minutus (Rodentia: Muridae)

Abstract Background Despite the widespread belief that an extensive body of knowledge exists for the sucking lice (Anoplura), some of their common, Eurasian or even cosmopolitan species still lack complete taxonomic descriptions, especially those for their nymphal stages. This applies especially to the most common rodent parasites: the lice of the genus Hoplopleura. In Europe, only two of the five most common Hoplopleura species have full taxonomic characteristics with a description of the nymphal stages. This study enriches the current state of knowledge for another species, Hoplopleura longula and presents the first description of its nymphal stages. The study includes five rare louse specimens (two nymphs I, one nymph II, two nymphs III) of H. longula collected from 63 Eurasian harvest mice Micromys minutus. The collected lice were fixed and preserved in 70% ethyl alcohol solution and then placed in polyvinyl-lactophenol to form total preparations. New information Only two of the five species found in Eurasia (H. acanthopus, H. affinis, H. captiosa, H. edentula and H. longula) have been given full taxonomic descriptions, including immature stages. This paper presents a description of the nymphal stages of H. longula (described for the first time).


Introduction
Sucking lice (Psocodea: Anoplura) are obligatory, permanent ectoparasites of mammals. Lice complete their entire lifecycle on their host and it includes: egg, three nymphal stages (nymph I, nymph II, nymph III) and an adult stage (imago). Some past research on this group has focused on their relationship with humans (e.g. Buxton 1941, Müller 1949, Piotrowski 1961, Fisher and Morton 1970, Downs et al. 1999, Roux and Raoult 1999, Levot 2000, Robinson et al. 2003, Izdebska 2014, Izdebska et al. 2018 or livestock (Piotrowski 1998, Mey 2003 and their potential for disease transmission (e.g. Piotrowski andWegner 1976, Fagir et al. 2014). In comparision, there have been relatively few studies examining the sucking lice of wild fauna. As such, only fragmentary knowledge exists about certain species.
Approximately 70% of Anoplura are associated with rodents (Rodentia), the most abundant group of mammals in terms of species (Durden and Musser 1994, Baker et al. 2002, Kim 2006. The family Hoplopleuridae (Psocodea: Anoplura) is the most speciose group parasitising the Rodentia. Amongst the family, most representatives are in the genus Hoplopleura, which contains 154 species distributed around the world. The following five are most widespread in Europe -four of them being common in Eurasia: H. acanthopus (Burmeister, 1839), H. affinis (Burmeister, 1839), H. edentula andH. longula Neumann, 1909 and one is probably cosmopolitan: H. captiosa Johnson, 1960 (Durden andMusser 1994).
Numerous publications concerning the ectoparasites of European fauna mention the presence of lice on rodents and insectivores (e.g. Krištofik 1999, Izdebska and Fryderyk 2010, Krčmar and Trilar 2017; however, concise taxonomic work concerning the genus Hoplopleura are scarce (Kim 1966, Johnson 1972, Weaver and Barton 2008 and the nymphal stages have been rarely described (Cook and Beer 1959, Johnson 1960, Kim 1966, Wegner 1966a. Of the species found in Eurasia, only two have been given full taxonomic descriptions, including their immature stages. This paper presents the first characterisation of the nymphal stages of Hoplopleura longula.

Materials and methods
Five louse specimens were included in this study (two nymphs I, one nymph II and two nymphs III). These were isolated from four (out of 63 examined individuals) Eurasian harvest mice Micromys minutus (Pallas, 1771), obtained from the Scientific Collection of the Mammal Research Institute Polish Academy of Sciences in Białowieża (Table 1). The hosts originated from the area of the Białowieża National Park and the material was collected in the period  Lice were collected from dead rodent specimens by combing the fur with tweezers and the specimens were fixed and preserved in 70% ethyl alcohol. The specimens, intended for analysis of morphological traits, were immersed in polyvinyl-lactophenol to develop total preparations for light microscope examination (Kadulski and Izdebska 2006).
Topographic preferences were observed. The entire body surface area of the test hosts was analysed and all locations were marked; this allowed for a complete (intensive) analysis of the topography.
The names and abbreviations of individual setae or body parts are provided following Kim (1966) and Kim and Ludwig (1978) (Fig. 1).
Abbreviations of head and thorax: Description Nymph I (Figs 2, 3) . Legs large in proportion to the rest of the body, rapid growth with moulting to stage II; body length 0.53 mm ( Nymph II (Figs 4, 5) . Body length: 0.78 mm (Table 2). Head. Wider than long; anterior part of the head shield square with smooth margins. Head well pronounced against the thorax (indentation visible at the contact point of both parts). Ventral side: large, concave scales around the mouth; head margins covered imbricately with scales (Ushaped); VMHS, OS and AHS present; VPHS constituting 50% of the head length. Dorsal side: AHS, DAHS and PAS present; ISHS and OSHS closely arranged; PCHS visible; ADHS and PDHS (reaching second segment of the abdomen) present; MHS minute. Thorax. Dorsal side: DPTS reaching beginning of the abdomen; pronounced border between the thorax, head and abdomen. Abdomen. Disproportionately large Table 2.
Means and ranges of different immature stages of Hoplopleura longula [in mm].
relative to the rest of the body, barrel-shaped, densely covered with U-shaped scales; traces of segmentation visible; MAS eight in number. After x-ray, structure of nymph III visible.
Nymph III (Figs 6, 7) . Body of an adult, in the final stages of moulting, individual visible through the cuticle; body size sometimes smaller than in nymph II; body length: 0.87 mm (   Hoplopleura longula nymph III, dorsal view.

present.
Thorax. Clearly visible borders between the thorax, head and abdomen. Dorsal side: DPTS reaching third segment of the thorax. Abdomen. Ovoid, elongated; MAS eight in number. After x-ray, sometimes adult individual visible (particular the plates and posterior part of the abdomen).

Biology
The lice demonstrated topographic preferences -a tendency for congregating along the sides of the host's body and on both sides of the head (between the ears and on the neck) (Fig. 8) (Table 1).

Discussion
In the present study, only H. longula was found amongst the lice. Despite the fact that there are also other species parasitising M. minutus, for example, of the genus Polyplax given in literature (Durden and Musser 1994), they were not found on the studied rodents.
The harvest mouse M. minutus is considered to be the main host of H. longula; it has not been recorded in any other hosts to date (Wegner 1966a, Krištofik andLysy 1992).
Individual nymphs of H. longula are easily identifiable. Nymph I possessed four major abdominal setae and an elongated, ovoid abdomen. Nymph II possessed eight MAS and a barrel-shaped abdomen. Like nymph II, nymph III possessed eight MAS; however, its abdomen is elongated and ovoid. In addition, like nymph I, the body of the adult individual can be seen inside the nymph.
Regarding the location of the lice on the host, no comparative data are available in previous studies regarding the H. longula-M. minutus relationship. Despite this, similarities can be found with the distribution of H. acanthopus on Microtus arvalis (Dubinin 1953): in both cases, the lice were found on the head, the posterior part of the abdomen and on both sides of the host body; however, H. longula appeared to have a wider distribution on the sides and the dorsum on its rodent hosts. Similarly to H. affinis in Apodemus agrarius (Dubinin 1953), H. longula was also found on the dorsal portion of the head and on the nape of the animal. In both of the previous studies, Dubinin did not observe lice on the ventral portion of the head; this was also confirmed in the present study.
Current research indicates that H. longula is a rare species associated with only one, also rare host, M. minutus. This is the first description of all three nymphal stages of H. longula. This characteristic will support other researchers working on lice to make positive identifications which will benefit future research examining host-parasite associations.