animals Article Ecological Analysis of the Helminth Community of Microtus lusitanicus (Gerbe, 1879) (Rodentia) in Asturias (NW Spain) Roser Adalid 1 , Carles Feliu 1,2 , Aitor Somoano 3 , Marcos Miñarro 3 , Jacint Ventura 4,5 , Jordi Torres 1,2 , Jordi Miquel 1,2 and Màrius Vicent Fuentes 6, * 1 Secció de Parasitologia, Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Av. Joan XXIII, sn, 08028 Barcelona, Spain; roseradalid@ub.edu (R.A.); cfeliu@ub.edu (C.F.); jtorres@ub.edu (J.T.); jordimiquel@ub.edu (J.M.) 2 Institut de Recerca de la Biodiversitat (IRBio), Universitat de Barcelona, Av. Diagonal, 645, 08028 Barcelona, Spain 3 Servicio Regional de Investigación y Desarrollo Agroalimentario (SERIDA), Ctra. AS-267, PK 19, 33300 Villaviciosa, Spain; aitors@serida.org (A.S.); mminarro@serida.org (M.M.) 4 Departament de Biologia Animal, Biologia Vegetal i Ecologia, Facultat de Biociències, Campus de Bellaterra, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; jacint.ventura.queija@uab.cat 5 Àrea de Recerca en Petits Mamífers, Museu de Ciències Naturals de Granollers “La Tela”, C/Palaudàries, 102, 08402 Granollers, Spain 6 Parasites and Health Research Group, Departament de Farmàcia i Tecnologia Farmacèutica i Parasitologia, Facultat de Farmàcia, Universitat de Valencia, Av. Vicent Andrés Estellés, 46100 Burjassot, Spain * Correspondence: mario.v.fuentes@uv.es Simple Summary: The Lusitanian pine vole is an endemic rodent of the Iberian Peninsula, which  has a burrowing behaviour and prefers to live underground. It feeds on bark and roots causing  severe damage to trees. In Asturias (NW Spain), this species is considered a pest causing economic Citation: Adalid, R.; Feliu, C.; losses in apple orchards, damaging the tree, and sometimes even causing its death. With the aim to Somoano, A.; Miñarro, M.; Ventura, J.; shed light on the helminth community of this rodent pest species and to elucidate which intrinsic Torres, J.; Miquel, J.; Fuentes, M.V. and extrinsic factors affect its helminth species, a faunistic-ecological study was carried out. For Ecological Analysis of the Helminth this purpose, our own collection of 710 voles from several orchards of various locations in Asturias Community of Microtus lusitanicus was used. The results of the ecological study revealed the influence of climate variables, the year (Gerbe, 1879) (Rodentia) in Asturias and season of capture, as well as the host age, on the diversity of the helminth community and the (NW Spain). Animals 2021, 11, 3055. infection parameters of helminth species, underlining the importance of their life cycles. Our findings https://doi.org/10.3390/ani11113055 on the helminth community of the Lusitanian pine vole in Asturias could be used to improve the biological methods applied to control the population of this rodent pest. Academic Editor: Jacques Cabaret Received: 29 September 2021 Abstract: The Lusitanian pine vole, Microtus lusitanicus, an endemic fossorial rodent of the Iberian Accepted: 22 October 2021 Peninsula, has a burrowing behaviour and prefers to live underground. It feeds on bark and roots Published: 26 October 2021 causing severe damage to trees. In Asturias (NW Spain), where M. lusitanicus is considered a pest in several orchards, a faunistic-ecological study was carried out to describe the helminth community Publisher’s Note: MDPI stays neutral of this species and the main factors that could influence its helminth component species. For this with regard to jurisdictional claims in purpose, our own collection of 710 voles from several orchards of various locations in Asturias published maps and institutional affil- was used. Eight helminth species, four cestodes and four nematodes, were found. Statistical non- iations. parametric tests were used to analyse the effects of extrinsic and intrinsic factors on the diversity of the helminth community and species prevalence and abundance. The results show the influence of climate variables, the year and season of capture, as well as host age, on the diversity of the helminth community and the infection parameters of some helminth species, underlining the importance Copyright: © 2021 by the authors. of their life cycles. In addition to shedding light on the helminth community of this rodent in Licensee MDPI, Basel, Switzerland. Asturias, the results obtained could be used to improve the biological methods applied to fight the This article is an open access article M. lusitanicus pest. distributed under the terms and conditions of the Creative Commons Keywords: helminth community; Lusitanian pine vole; Microtus lusitanicus; Asturias; Spain Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2021, 11, 3055. https://doi.org/10.3390/ani11113055 https://www.mdpi.com/journal/animals Animals 2021, 11, 3055 2 of 22 1. Introduction The Lusitanian pine vole, Microtus lusitanicus (Gerbe, 1879), is an endemic fossorial rodent, which inhabits the northwestern quadrant of the Iberian Peninsula and the south- western tip of France [1]. It can be found from sea level to up to 2000 m, in both natural and agricultural habitats [1,2]. In all cases, the ground must be soft and humid with a high vegetal cover, which are the necessary conditions for the voles to build their gallery systems [1]. This rodent has a burrowing behaviour and prefers to live underground, although surface movements under abundant vegetation may be frequent. Vole galleries remain open as a small hole in the ground [3] with superficial galleries used for feeding and escaping and deep ones used for storing food and nesting [1]. Microtus lusitanicus is a monogamous species: a couple of voles and their offspring (juvenile and sub-adult individuals) share the same gallery system [4–6]. Lusitanian pine voles are small sized rodents (body length: 77.5–105.0 mm; body mass; 14.0–19.0 g) with big head, blunt snout, small eyes and cylindrical body [1]. Microtus lusitanicus can cause severe damage to fruit trees, carrots or potatoes [1]. In Asturias (northwest Spain), this rodent species is, along with the montane water vole, Arvicola scherman (Shaw, 1801), one of the main causes of economic losses in apple or- chards [3,7,8]. Both species feed on bark and roots, damaging the tree, and sometimes even causing its death [3]. In Spain, M. lusitanicus is officially considered a pest, and the control of its populations is recommended [9]. Nowadays, rodenticides should comply with current legislation and not involve adverse environmental consequences, produce resistance in the target species, unnecessary pain or to be a threat to non-target animals and humans (EU Regulation 528/2012) [10], making the examination of other potential strategies necessary. Among specific and environmentally benign control practices, increas- ing landscape heterogeneity, fertility control, the use of repellents or biocontrol (predators, parasites and other pathogens) have been proposed as promising tools [11,12]. Parasites may have a negative effect on host fitness [13]. Host–parasite models predict that parasites can regulate the population dynamics of the hosts [14,15]. Regulation is the process through which high population numbers are lowered to a normal level, but also increased up to this level when they reach low values [16]. The aim of pest control is to reduce the population size to lower levels than the natural equilibrium. It must be considered that many pest species cause damage in spite of their low population densities. To reach this goal, it is necessary that the negative impact of the parasite on the host must exceed the population’s intrinsic growth rate and infection, and that this impact needs to be persistent. Parasites can play two roles in rodent pest management: as bio-rodenticides or as vehicles of immunocontraception causing sterility to infected animals [17]. The helminth fauna of M. lusitanicus in the Iberian Peninsula was previously anal- ysed [18,19]. These studies, carried out on voles from “damaged vegetable gardens” in several localities of the Iberian Peninsula, especially in the central-western part, reported the detection of a total of 14 helminth species: 1 trematode, 7 cestodes, and 6 nematodes. The structure and dynamics of the vole’s helminth community and the environmental factors influencing it have also been analysed. Environmental factors, such as temperature and rainfall, as well as intrinsic factors, such as host age and sex, were found to influence the helminth community of the Lusitanian pine vole. The main aims of the present study are to shed light on the helminth community of M. lusitanicus, to elucidate the potential effect extrinsic and intrinsic factors have on it, and to update and expand previously reported data. Moreover, some new proposed hypotheses could be confirmed: (1) pesticides used in pest control in areas of intensive agricultural use are responsible for the absence of some helminth species found in other localities belonging to the geographical distribution of the Lusitanian pine vole, due to the loss of invertebrate intermediate hosts; (2) the parasitological patterns of those helminth species that are influenced by climate variables will be affected by global warming and/or climate change; (3) the results can be used to improve the biological control methods against this vole pest in agro-ecosystems. Animals 2021,11, Animals2021, 11,3055 x FOR PEER REVIEW 33of of22 22 2. Materials and Methods Asturias is located on the northern coast of Spain (43 ◦ 300 N, (43°30′ 5◦ 300 W) N, 5°30′ W) (Figure (Figure 1). 1). The The climate is istemperate temperateoceanic oceanicwith abundant with abundant rainfall spread rainfall fairly spread throughout fairly the year throughout the and year mildmild and temperatures both both temperatures in winter and summer. in winter This area and summer. This isarea characterized by small is characterized agri- by small cultural plotsplots agricultural separated by hedgerows separated by hedgerows andand woodlands, woodlands, withwith an irregular topography an irregular topography of smooth of smoothhills and hills valleys. and The valleys. fertile The fertilesoil soiladded addedtotothese theseweather weatherconditions conditionsfavours favoursthethe establishment of an evergreen and dense dense vegetal vegetal cover coverininorchards orchardsall allyear yearround round[3,20,21]. [3,20,21]. Figure 1. Figure 1. Distribution Distribution of of Microtus Microtus lusitanicus lusitanicus in in the the Iberian Iberian Peninsula Peninsula(striped (stripedgreen) green)and andmap mapof of Asturias (dark green) showing the trapping sites of the individuals analysed (black points). Asturias (dark green) showing the trapping sites of the individuals analysed (black points). 2.1. Zoological and Helminthological Helminthological Procedures Procedures A total of 710 M. lusitanicus (Figure 2) individuals individuals obtained obtained from from thethe collection collection of of SERIDA (Servicio Regional de Investigación y Desarrollo Agroalimentario, Asturias) Investigación y Desarrollo Agroalimentario, Asturias) was was helminthologically analysed. The sample was was collected collected during during aa population populationpeak,peak,for fortwo two consecutive years (January 2011 to January 2013), 2013), inin apple apple orchards orchards of of99Asturian Asturianlocalities localities (Figure 1). The recommendations recommendations of of the the Directive Directive ofof the the European European Parliament Parliamentand andof ofthe the Council on the Protection of Animals Animals UsedUsed for for Scientific Scientific Purposes Purposes [22][22] were were followed followedin in the field work. individuals analysed, Of the total individuals analysed, 343343 were were captured captured in in 2011, 2011,354354inin 2012 2012andand13 13in in 2013. Considering Considering sex,sex, 363 363 were were male male and and 347 347 were were female. female. Animals Animalswere wereclassified classifiedby by body mass in three three age age groups: juveniles, ≤ groups: juveniles, 10 g; ≤10 g; sub-adults sub-adults 10.1–14.9 10.1–14.9 g:g: adults, ≥15gg[2]. adults, ≥15 [2]. According According to tothis thiscriterion, criterion,6363 specimens specimens were werejuveniles, 334334 juveniles, were sub-adults were and 313 sub-adults andwere 313 adults. Seasons were adults. werewere Seasons defined as: winter defined (January–March), as: winter (January–March), spring (April–June), spring (April–June), summer sum- (July–September) mer (July–September) and autumn (October–December). and autumn (October–December). Following this distribution Following by months, this distribution by 139 months, 139 captures took place in winter, 187 in spring, 196 in summer and 188 in au-A captures took place in winter, 187 in spring, 196 in summer and 188 in autumn. comprehensive description tumn. A comprehensive of the host description of sample the hostanalysed is shownisinshown sample analysed Tablesin1 Tables and 2. 1 and 2. In the laboratory, specimens were dissected, and all viscera were extracted for the parasitological study. All helminths found were preserved in ethanol 70%. Cestodes were stained with alcoholic chlorhydric carmine, differentiated with acidified ethanol, dehydrated in alcohol series, cleared with xylene and mounted in Canada balsam. Some cestode scolices and some nematodes were cleared in Amann lactophenol. The helminth specimens were identified at specific level, based on their morphology and morphometry according to the most relevant descriptions and findings in the scientific literature. Animals 2021, 11, 3055 4 of 22 Animals 2021, 11, x FOR PEER REVIEW 4 of 22 Figure 2. Figure 2. An An individual individual of of Microtus Microtus lusitanicus lusitanicus in in its its natural naturalhabitat. habitat. Table1.1.Distribution Table Distributionof ofMicrotus Microtuslusitanicus lusitanicusanalysed analysedby byhost hostsex sexand andage ageand andseason seasonofofcapture. capture. HostAge Host Age Host Sex Season Host Sex Season Juveniles Sub-Adults Adults Total Juveniles Sub-Adults Adults Total Males Winter 3 26 39 68 Males Winter 3 26 39 68 Spring Spring 1010 35 35 51 51 96 96 Summer Summer 66 63 63 36 36 105105 Autumn Autumn 55 63 63 26 26 94 94 Total Total 2424 187 187 152152 363363 Females Females Winter Winter 33 33 33 35 35 71 71 Spring Spring 88 38 38 45 45 91 91 Summer 18 36 37 91 Summer Autumn 1810 36 40 44 37 94 91 Autumn Total 1039 40 147 161 44 34794 Total 39 147 161 347 Table 2. Distribution of Microtus lusitanicus analysed by year and season of capture. Table 2. Distribution of Microtus lusitanicus analysed by year and season of capture. Season Year Season Year Winter Spring Summer Autumn Total Winter Spring Summer Autumn Total 2011 2011 2525 9292 110 110 116116 343 343 2012 101 95 86 72 354 2012 101 95 86 72 354 2013 13 0 0 0 13 2013 Total 13913 1870 196 0 188 0 71013 Total 139 187 196 188 710 2.2. Helminth Community Analysis In the laboratory, specimens were dissected, and all viscera were extracted for the The helminth parasitological community study. composition All helminths and structure found were preservedof in theethanol Lusitanian 70%.pine vole were Cestodes were analysed considering each particular life cycle as well as prevalence, mean abundance, stained with alcoholic chlorhydric carmine, differentiated with acidified ethanol, dehy- range and total number of helminths calculated [23]. drated in alcohol series, cleared with xylene and mounted in Canada balsam. Some ces- The analysis of helminth community components was made by means of calculating tode scolices and some nematodes were cleared in Amann lactophenol. The helminth the frequency of occurrence of the number of helminth species, the abundance index specimens were identified at specific level, based on their morphology and morphometry and the frequency distribution of helminths. The abundance index (AI) (excluding those according to the most relevant descriptions and findings in the scientific literature. parasite species for which M. lusitanicus acts as an intermediate host) was calculated [24,25]. Animals 2021, 11, 3055 5 of 22 The helminth community of a host species is characterized by the presence of the following categories of species: dominant species (AI > 1); co-dominant species (0.1 ≤ AI ≤ 1); successful immigrant species (0 < AI < 0.1); unsuccessful immigrant species (AI = 0). The frequency distribution of helminth species was calculated by means of the Lefkovitch index (L) [26], where L = (1/45)tang−1 (variance/mean) − 1 (1) ranging from −1 (positive binomial or uniform distribution), 0 (Poisson or random distri- bution) to +1 (negative binomial or aggregated distribution). The diversity/uniformity analysis of the helminth community was carried out using the Shannon index (H’) [27,28], Simpson index (D’) [29] expressed as 1-D’ [28], Berger- Parker index (d) [30,31], expressed as 1-d [28] and Shannon evenness index (E) [28,32]. The helminth infracommunity structure study was established using the analysis of the number of helminths, number of helminth species, the Brillouin index (HB) [27,28], Brillouin index for infected hosts only and percentage of infected hosts. The role played by intrinsic (host sex and age) and extrinsic (climate data, year and season of capture) factors (independent variables) in determining the helminth community dependent variables (species richness − the number of helminth species; the helminth community diversity—Brillouin index; the prevalence—% of parasitation; and the worm burden − helminth abundance) was statistically analysed. Climate data, related to temper- ature (mean daily temperature calculated for each season) and precipitation (mean daily rainfall calculated for each season and cumulative rainfall during each season) (Table 3), were obtained from the nearest climate stations (AEMET—Spanish Meteorological Agency). Values of climate variables, belonging to the year before capture (which are related to both the helminth and vole life cycles), were correlated with the annual values of the dependent variables by means of the Spearman correlation coefficient (rs ); prevalence was previously transformed logarithmically, log (x/(1 − x)). The influence of year and season of capture, and host sex and age on prevalence was analysed using binary logistic regression (BLR), while their influence on the other dependent variables was analysed by means of standard non-parametric tests, i.e., the Mann–Whitney (U) and Kruskal–Wallis (H) tests. Table 3. Number of Microtus lusitanicus analysed (n) and seasonal values of climate variables corresponding to the year prior to host capture. Mean Temperature Mean Rainfall Cumulative n (◦ C) (mm) Rainfall (mm) Winter 2010 25 10.1 3.0 269.0 Spring 2010 92 15.0 4.0 338.4 Summer 2010 110 19.9 0.7 65.9 Autumn 2010 116 12.6 5.5 503.4 Winter 2011 101 11.1 2.9 246.0 Spring 2011 95 16.2 1.0 88.5 Summer 2011 86 19.7 1.3 116.2 Autumn 2011 72 14.6 2.2 199.0 Winter 2012 13 10.2 1.4 125.1 SPSS 26.0—IBM for Windows was the software package used for statistical analysis. Statistical significance was established at p < 0.05. 3. Results 3.1. Helminth Species A total of 594 Lusitanian pine voles (83.67%) were found to be infected with a to- tal of eight helminth species: four cestodes and four nematodes (Table 4). Among the helminth species observed, although the hymenolepidid Rodentolepis asymmetrica and the heligmosomid Carolinensis minutus have been previously reported in other Arvicolinae 3.1. Helminth Species A total of 594 Lusitanian pine voles (83.67%) were found to be infected with a total of eight helminth species: four cestodes and four nematodes (Table 4). Among the hel- Animals 2021, 11, 3055 minth species observed, although the hymenolepidid Rodentolepis asymmetrica and the 6 of 22 heligmosomid Carolinensis minutus have been previously reported in other Arvicolinae host species [18], both helminth species are now reported for the first time parasitizing M. lusitanicus. host species [18], both helminth species are now reported for the first time parasitizing M. characteristics Table 4. Selected lusitanicus. of the helminth community of 710 Microtus lusitanicus analysed. Prevalence Table 4. Selected characteristics of the helminth community Meanlusitanicus of 710 Microtus Abundance Median Intensity analysed. Helminth Species Site LC n (95% CI) (SE) (range) Prevalence Mean Abundance Median Intensity CESTODA Helminth Species Site LC n (95% CI) (SE) (Range) Cysticercoid sp. larvae I F 1 0.15 (0.1–0.5) <0.00 (<0.00) 1 (1) CESTODA Hydatigera taeniaeformis larvae L F 59 8 (6–10) 0.10 (0.01) 1 (1–5) Cysticercoid sp. larvae I F 1 0.15 (0.1–0.5) <0.00 (<0.00) 1 (1) Paranoplocephala Hydatigera taeniaeformis omphalodes larvae L I FNF 2 59 0.3 (0.1–0.7) 8 (6–10) <0.00 0.10(<0.00) (0.01) 1 1(1–5) (1) Rodentolpeis Paranoplocephala asymmetrica I omphalodes I NF NF 52 0.70.3(0.4–2) (0.1–0.7) 0.01 <0.00(<0.00) (<0.00) 11(1–2) (1) Rodentolpeis asymmetrica NEMATODA I NF 5 0.7 (0.4–2) 0.01 (<0.00) 1 (1–2) Trichuris arvicolae NEMATODA C F 36 5 (4–7) 0.06 (0.01) 1 (1–4) Trichuris arvicolaeminutus Carolinensis C I FF 30836 5 (4–7) 44 (39–47) 0.06(0.91) 5.80 (0.01) 31(1–417) (1–4) Carolinensis minutus I F 308 44 (39–47) 5.80 (0.91) 3 (1–417) Heligmosomum costellatum I F 25 4 (3–6) 0.16 (0.05) 3 (1–22) Heligmosomum costellatum I F 25 4 (3–6) 0.16 (0.05) 3 (1–22) Syphacia Syphacia nigeriana nigeriana C C F F 158158 22 (19–25) 22 (19–25) 4.54 (0.81) 4.54 (0.81) 5 (1–262) 5 (1–262) LC, life cycle; N, number of voles parasitized; CI, confidence interval; SE, standard error; I, intestine; L, liver; C, caecum; LC, life cycle; N, number of voles parasitized; CI, confidence interval; SE, standard error; I, intestine; L, liver; C, caecum; FES, helminths FES, have which helminths which have ainfectious a free-environmental free-environmental infectious stage for the vole; stage for the vole; NF, No-FES. NF, No-FES. Themorphology The morphologyand andmorphometry morphometryofofsome somestages stagesofofthe thehelminth helminthspecimens specimensfound found parasitizingthe parasitizing thevoles voles were were analysed analysed under under the microscope the microscope and some and some specific specific measure- measurements ments were wereand made made and compared compared withreported with those those reported in descriptions in descriptions of the of the scientific scientific liter- literature, ature, making making it possible it possible to identify to identify all of all of them at them at level, specific specific level, with the with only the only exception exception being a being a cysticercoid cysticercoid found in found in the intestine the intestine of a vole.of a vole. 3.1.1.Hydatigera 3.1.1. Hydatigerataeniaeformis taeniaeformis(Batsch, (Batsch,1786) 1786)Larvae Larvae The Thelarval larval stage stage of of this this taenid tapeworm, aa strobilocercoid taenid tapeworm, strobilocercoidenglobed englobedininaacyst cystofofa a5- 5–12 mm diameter, was found on the liver. The scolex (Figures 3 and 4) has a rostellum 12 mm diameter, was found on the liver. The scolex (Figures 3 and 4) has a rostellum with with 30-3630–36 hookshooks of twoof different two different sizessizes [33]. [33]. Figure3.3.Rostellar Figure Rostellarhooks hooksofofHydatigera Hydatigerataeniaeformis taeniaeformislarvae. larvae.LH, LH,large largehook; hook;SH, SH,small smallhook. hook.Scale Scale bar = 300 µm. bar = 300 µm. Animals 2021, 11, x FOR PEER REVIEW 7 of 22 Animals 2021, 11, 3055 7 of 22 Animals 2021, 11, x FOR PEER REVIEW 7 of 22 Figure 4. Large (A) and small (B) hooks of Hydatigera taeniaeformis larvae. Scale bar = 200 µm. 3.1.2. Paranoplocephala omphalodes (Hermann, 1783) Figure4. Figure 4. Large Large (A) (A)and andsmall small(B) (B)hooks hooksof ofHydatigera Hydatigerataeniaeformis taeniaeformislarvae. larvae.Scale Scalebar bar== 200 200 µm. µm. This anoplocephalan tapeworm was found in the small intestine. The unarmed sco- lex, 3.1.2.the morphology and 3.1.2. Paranoplocephala Paranoplocephala disposition omphalodes omphalodes of reproductive (Hermann, (Hermann, 1783) organs, as well as the size and mor- 1783) phology This of the eggs obtained Thisanoplocephalan anoplocephalan from was tapeworm tapeworm gravid proglottids wasfound foundinin thethe allow small small its identification intestine. The intestine. unarmed The at specific unarmedscolex, sco- level the [34]. morphology and disposition of reproductive organs, as well as the size and morphology lex, the morphology and disposition of reproductive organs, as well as the size and mor- of the eggs phology ofobtained the eggsfrom gravid obtained proglottids from allow its identification gravid proglottids at specific level allow its identification [34]. at specific 3.1.3. Rodentolepis asymmetrica (Janicki, 1904) level [34]. Rodentolepis 3.1.3. The asymmetrica adult stage (Janicki, 1904) cestode was found in the small intestine. The of this hymenolepidid armed 3.1.3.The scolex adulthas a rostellum stage Rodentolepis with 18–21 of this hymenolepidid asymmetrica (Janicki, hooks 1904) of 18–20 cestode µm. The was found eggs in the are spherical, small with intestine. The armed scolex a diameter ofhas a 45–67 rostellum µm, with containing 18–21 an hooks of oncosphere 18–20 of a The 20–30 µm. µmeggs are spherical, diameter, The adult stage of this hymenolepidid cestode was found in the small intestine. The with with embry- a diameter onic hooks of 45–67 of has 8.5–9µm, containing µm lengthwith an oncosphere of a 20–30 µm diameter, with embryonic [34].18–21 hooks of 18–20 µm. The eggs are spherical, with armed scolex a rostellum hooks of 8.5–9 µm length [34]. a diameter of 45–67 µm, containing an oncosphere of a 20–30 µm diameter, with embry- 3.1.4. Trichuris arvicolae Feliu et al., 2000 onic hooks 3.1.4. of 8.5–9 Trichuris µmFeliu arvicolae lengthet [34]. al., 2000 This Trichurinae nematode was found in the caecum. The morphology of the oesoph- agus,This Trichurinae as well nematode was asarvicolae the morphology and found in the caecum. morphometry The and of female morphology male sexualof the oesoph- organs and 3.1.4. Trichuris Feliu et al., 2000 agus, as eggsThis well (Figuresas 5the and morphology 6) allowed and the morphometry recent descriptionof female of and this male nematode sexual as a organs new and species eggs Trichurinae nematode thewas found in the caecum. of this The morphology ofspecies the oesoph- [35].(Figures agus, 5 and 6) allowed recent description nematode as a new as well as the morphology and morphometry of female and male sexual organs and [35]. eggs (Figures 5 and 6) allowed the recent description of this nematode as a new species [35]. Figure 5.5. Vulvar Figure Vulvar region region of ofaaTrichuris Trichurisarvicolae arvicolaefemale. female.U, U, uterus; V, vulva; uterus; Va, Va, V, vulva; vagina. ScaleScale vagina. bar = 200 µm. bar = 200 µm. Figure 5. Vulvar region of a Trichuris arvicolae female. U, uterus; V, vulva; Va, vagina. Scale bar = 200 µm. Animals 2021, 11, 3055 8 of 22 Animals 2021, Animals 2021, 11, 11, xx FOR FOR PEER PEER REVIEW REVIEW 88 of of 22 22 Figure6.6. Figure Figure 6.Egg Eggofof Egg ofTrichuris Trichurisarvicolae. Trichuris arvicolae.ES, arvicolae. ES,eggshell; ES, eggshell;OP, eggshell; OP,opercular OP, opercularplugs. opercular plugs.Scale plugs. Scalebar Scale bar===2525 bar 25µm. µm. µm. 3.1.5.Carolinensis 3.1.5. 3.1.5. Carolinensisminutus Carolinensis minutus(Dujardin, minutus (Dujardin,1845) (Dujardin, 1845) 1845) This This heligmosomid nematode This heligmosomid nematode wasfound heligmosomid nematode was was foundinin found inthe thesmall the smallintestine. small intestine.ItIt intestine. Itisis ischaracterized characterizedby characterized by by its itsvery its verysmall very smallsize, small size,the size, thepresence the presenceofof presence oflongitudinal longitudinalridges longitudinal ridgesthat ridges thatcover that coverits cover itsbody, its body,the body, thevesicle the vesiclethat vesicle that that covers covers its itshead, head,and and the thecaudal caudalbursa bursa of ofmales [36] males [36] (Figure (Figure covers its head, and the caudal bursa of males [36] (Figure 7). 7). 7). Figure 7. Figure 7. Caudal Caudal bursa bursa of a Carolinensis Carolinensis minutus minutus male. male. D, D, dorsal dorsal ray; ray; G, G, gubernaculum; gubernaculum; S, spicules. spicules. Figure 7. Caudal bursa ofofaaCarolinensis minutus male. D, dorsal ray; G, gubernaculum; S,S,spicules. Scale bar Scalebar = 1 µm. bar==11µm. µm. Scale 3.1.6.Heligmosomum 3.1.6. 3.1.6. Heligmosomumcostellatum Heligmosomum costellatum(Dujardin, costellatum (Dujardin,1845) (Dujardin, 1845) 1845) Thisother This This otherheligmosomid other heligmosomidnematode heligmosomid nematodewas nematode wasfound was foundinin found inthe thesmall the smallintestine. small intestine.ItIt intestine. Itpresents presents presents transversal transversal stretch transversalstretch marks stretchmarks marksthatthat cover thatcover its coverits cuticle, itscuticle, and cuticle,and a typical andaatypical caudal typicalcaudal bursa caudalbursa of males bursaofofmales [36] males[36] [36] (Figures888and (Figures (Figures and9). and 9). 9). Animals 2021, 11, 3055 9 of 22 Animals 2021, 11, x FOR PEER REVIEW 9 of 22 Animals 2021, 11, x FOR PEER REVIEW 9 of 22 Figure8.8. Cephalic Figure Cephalic extremity extremityofofa aHeligmosomum Heligmosomumcostellatum female. costellatum Arrows female. point Arrows at cuticular point stria- at cuticular tions. FigureScale bar = 100 8. Cephalic µm. extremity of a Heligmosomum costellatum female. Arrows point at cuticular stria- striations. Scale bar = 100 µm. tions. Scale bar = 100 µm. Figure 9. Caudal bursa of a Heligmosomum costellatum male. EDR, externodorsal rays. Scale bar = 100 µm. Figure9.9. Caudal Figure Caudalbursa bursaofof a Heligmosomum costellatum a Heligmosomum male. costellatum EDR, EDR, male. externodorsal rays. Scale externodorsal rays.barScale = 100 µm. bar = 100 µm. 3.1.7. Syphacia nigeriana Baylis, 1928 3.1.7.This 3.1.7. Syphacia Syphacia nigeriana nigeriana oxyurid Baylis, Baylis, nematode 1928 1928 was found in the caecum. The oxyuriform oesophagus is one of the This This oxyurid oxyurid most nematode nematode relevant wasfound was found characteristics ininthe at genus thelevel. caecum. caecum. The TheThe oxyuriformoesophagus oxyuriform morphological oesophagus and isisone morphometric one ofthe of themost mostrelevant relevant characterization characteristics characteristics of its sexual organsatatgenus isgenus level.The level. necessary The to morphological morphological enable andmorphometric and identification atmorphometric species level characterization (Figures 10of characterization [36] ofitsits and sexual organs sexual 11). is necessary organs is necessary to enable identification to enable at species identification level [36] at species level (Figures 10 and [36] (Figures 1011). and 11). Animals 2021, 11, 3055 10 of 22 Animals 2021, 11, x FOR PEER REVIEW 10 of 22 Animals 2021, 11, x FOR PEER REVIEW 10 of 22 Figure10. Figure 10.Cephalic Cephalicextremity extremityofofaaSyphacia Syphacianigeriana nigerianafemale. female.O, O,oesophagus. oesophagus.Scale Scalebar bar==100 100µm. µm. Figure 10. Cephalic extremity of a Syphacia nigeriana female. O, oesophagus. Scale bar = 100 µm. Figure 11. Caudal extremity of a Syphacia nigeriana male. CS, ventral cuticular swellings; G, guber- Figure 11. Caudal naculum; Figure 11. Caudal S, extremity spicule. of=aa100 Scale bar extremity of Syphacia nigeriana male. µm. nigeriana Syphacia male. CS, CS, ventral ventral cuticular cuticular swellings; swellings; G, G, guber- guber- naculum; S, spicule. Scale bar = 100 µm. naculum; S, spicule. Scale bar = 100 µm. 3.2. Helminth Community Analysis 3.2. Helminth Community 3.2. Community Analysis Analysis Carolinensis minutus showed the highest prevalence of infection (43.38%) and was the Carolinensis mostCarolinensis minutus abundant minutus (5.80), showed showed while the highest the highest S. nigeriana prevalence prevalence presented the ofofinfection infection highest mean (43.38%) (43.38%) andvalue and intensity wasthe was the (5). most most abundant abundant (5.80), (5.80), while while S. S. nigeriana nigeriana presented presented the highest highest mean mean The five most prevalent species, C. minutus (43.38%), S. nigeriana (22.25%), H. taeniaeformis intensity value (5). The The five(8.31%), five larvae most prevalent most prevalent species, species, T. arvicolae C. C. minutus (5.07%) minutus (43.38%),S. and H. (43.38%), costellatum nigeriana S.(3.52%), nigeriana (22.25%), (22.25%), were H. H. taeniaeformis analysed taeniaeformis considering larvae larvae (8.31%), (8.31%), T. T. arvicolae arvicolae (5.07%) (5.07%) and and H. H. costellatum costellatum sex, age, season and year of capture of the host (Tables 5 and 6). (3.52%), (3.52%), were were analysed analysed considering considering sex, sex, age, age, season season andand year year ofof capture captureof ofthe thehost host(Tables (Tables55and and6).6). Table 5. Selected characteristics More of the than 60%prevalent five most of the hosts presented helminth specieseither by host onesexor two and helminth age. species, CI—confidence but infra- interval, Table 5. Selected SE—standard characteristics error. of the five of communities mostupprevalent to 4 specieshelminth werespecies by host also found sex and (Table 7).age. TheCI—confidence abundance index interval, values SE—standard error. (Table 8) make it possible to establish the following community structure: C. minutus and S. nigeriana as dominant species; H. costellatum as Species co-dominant species; and T. arvicolae Species H. taeniaeformis as successful immigrant species. T. arvicolae C. minutus Paranoplocephala omphalodes H.and costellatum S. nigeriana R. asymmetrica are con- Sex Males Prevalence (CI 95%) H. taeniaeformis 9 (6–12) T. arvicolae C.21minutus H. costellatum S.13 nigeriana sidered as non-successful immigrant3 species. (2–5) The (17–26) two larval stages 2 (1–3) (10–17) (H. taeniaeformis and Sex Males Prevalence (CI Mean Abundance (SE) 95%) Cysticercoid sp.) are 9 (6–12) not included 0.10 (0.02) in 3 (2–5) this 21 classification 0.07 (0.02) 4.60 (0.93)(17–26) since the 2 (1–3) Lusitanian 0.18 (0.07) pine 13 (10–17) 5.28 (1.22)as vole acts Mean Abundance intermediate Median Intensity (SE) (range) 0.10 host. 1 (1–2)(0.02) 0.07 (0.02) 1 (1–2) 4.60 (0.93) 3 (1–239) 0.18 (0.07) 3 (1–21) 5.28 (1.22) 6 (1–262) Females Median Intensity Prevalence (CI 95%)(range) 1 (1–2) 7 (5–10) 1 (1–2) 4 (2–6) 3 (1–239) 47 (42–52) 3 (1–21) 3 (2–5) 6 (1–262) 19 (15–23) Females Prevalence Mean (CI 95%) Abundance (SE) 7 (5–10) 0.10 (0.02) 4 (2–6) 0.06 (0.02) 47 7.07 (42–52) (1.58) 3 (2–5) 0.13 (0.07) 19 (15–23) 3.76 (1.04) Mean Abundance Median Intensity (range) (SE) 0.10 (0.02) 1 (1–5) 0.06 (0.02) 1 (1–4) 7.07 (1.58) 3 (1–417) 0.13 (0.07) 2 (1–22) 3.76 (1.04) 4 (1–191) Median Intensity (range) 1 (1–5) 1 (1–4) 3 (1–417) 2 (1–22) 4 (1–191) Animals 2021, 11, 3055 11 of 22 Table 5. Selected characteristics of the five most prevalent helminth species by host sex and age. CI—confidence interval, SE—standard error. Species H. taeniaeformis T. arvicolae C. minutus H. costellatum S. nigeriana Sex Males Prevalence (CI 95%) 9 (6–12) 3 (2–5) 21 (17–26) 2 (1–3) 13 (10–17) Mean Abundance (SE) 0.10 (0.02) 0.07 (0.02) 4.60 (0.93) 0.18 (0.07) 5.28 (1.22) Median Intensity 1 (1–2) 1 (1–2) 3 (1–239) 3 (1–21) 6 (1–262) (range) Females Prevalence (CI 95%) 7 (5–10) 4 (2–6) 47 (42–52) 3 (2–5) 19 (15–23) Mean Abundance (SE) 0.10 (0.02) 0.06 (0.02) 7.07 (1.58) 0.13 (0.07) 3.76 (1.04) Median Intensity 1 (1–5) 1 (1–4) 3 (1–417) 2 (1–22) 4 (1–191) (range) Age Juveniles Prevalence (CI 95%) - - 62 (50–74) 3 (0.4–11) 14 (7–25) Mean Abundance (SE) - - 20.19 (7.21) 0.11 (0.09) 2.51 (1.08) Median Intensity - - 9 (1–417) 3.5 (2–5) 13 (1–41) (range) Sub- Prevalence (CI 95%) 5 (3–8) 3 (2–5) 45 (40–50) 3 (2–5) 23 (19–28) adults Mean Abundance (SE) 0.05 (0.01) 0.04 (0.01) 5.56 (1.06) 0.22 (0.10) 5.71 (1.42) Median Intensity 1 (1–2) 1 (1–2) 3 (1–239) 4 (1–22) 4 (1–262) (range) Adults Prevalence (CI 95%) 14 (10–18) 8 (5–12) 38 (33–43) 4 (2–7) 23 (19–28) Mean Abundance (SE) 0.17 (0.03) 0.10 (0.02) 3.16 (0.87) 0.10 (0.04) 3.70 (1.00) Median Intensity 1 (1–5) 1 (1–4) 2 (1–238) 1 (1–8) 5 (1–191) (range) Table 6. Selected characteristics of the five most prevalent helminth species by season and year of capture. CI—confidence interval, SE—standard error. Species H. taeniaeformis T. arvicolae C. minutus H. costellatum S. nigeriana Season Winter Prevalence (CI 95%) 10 (6–16) 2 (0.4–6) 28 (21-36) 6 (3–11) 30 (23–38) Mean Abundance (SE) 0.12 (0.03) 0.02 (0.01) 0.78 (0.21) 030 (0.17) 4.15 (1.40) Median Intensity 1 (1–2) 1 (1) 1 (1–22) 2.5 (1–22) 3 (1–128) (range) Spring Prevalence (CI 95%) 10 (6–15) 7 (4–12) 51 (44–59) 5 (2–9) 24 (18–31) Mean Abundance (SE) 0.10 (0.02) 0.08 (0.02) 6.14 (0.99) 0.22 (0.12) 9.65 (2.71) Median Intensity 1 (1) 1 (1–2) 4.5 (1–80) 2 (1–21) 11 (1–262) (range) Summer Prevalence (CI 95%) 6 (3–10) 7 (4–12) 59 (52–66) 3 (1–6) 17 (12–23) Mean Abundance (SE) 0.07 (0.02) 0.09 (0.03) 13.30 (3.07) 0.12 (0.06) 1.60 (0.43) Median Intensity 1 (1–3) 1 (1–2) 5 (1–417) 4 (1–7) 4.5 (1–54) (range) Autumn Prevalence (CI 95%) 8 (5–13) 3 (1–7) 31 (25–38) 0.5 (0.1–4) 20 (15–26) Mean Abundance (SE) 0.11 (0.04) 0.05 (0.02) 1.36 (0.29) 0.02 (0.02) 2.80 (0.73) Median Intensity 1 (1–5) 1 (1–4) 2 (1–31) 3 (3) 6 (1–75) (range) Year 2011 Prevalence (CI 95%) 8 (5–11) 5 (3–8) 51 (46–57) 3 (2–5) 16 (12–20) Mean Abundance (SE) 0.10 (0.02) 0.06 (0.01) 8.11 (1.64) 0.17 (0.07) 2.37 (0.61) Median Intensity 1 (1–5) 1 (1–2) 3.5 (1–417) 4.5 (1–21) 4 (1–130) (range) Animals 2021, 11, 3055 12 of 22 Table 6. Cont. Species H. taeniaeformis T. arvicolae C. minutus H. costellatum S. nigeriana 2012 Prevalence (CI 95%) 9 (6–12) 5 (3–8) 36 (31–41) 4 (2–7) 29 (24–34) Mean Abundance (SE) 0.10 (0.02) 0.07 (0.20) 3.76 (0.88) 0.15 (0.07) 6.81 (1.50) Median Intensity 1 (1–3) 1 (1–4) 2 (1–238) 2 (1–22) 5 (1–262) (range) 2013 Prevalence (CI 95%) 8 (0.2–34) - 31 (9–57) - - Mean Abundance (SE) 0.08 (0.08) - 0.54 (0.27) - - Median Intensity 1 (1) - 1.5 (1–3) - - (range) Table 7. Frequency of occurrence of the number of helminth species present in infracommunities of Microtus lusitanicus from Asturias. No. of Helminth Species n % 0 260 36.6 1 320 45.1 2 117 16.5 3 12 1.7 4 1 0.1 Table 8. Abundance Index (AI) and Lefkovitch Index (L) of the most prevalent helminth species of Microtus lusitanicus from Asturias. Helminth Species AI L Hydatigera taeniaeformis larvae - 0.23 Trichuris arvicolae 0.06 0.24 Carolinensis minutus 5.80 0.99 Heligmosomum costellatum 0.16 0.88 Syphacia nigeriana 4.54 0.99 The frequency distribution of the most prevalent helminth species (Table 8) showed that all species analysed presented a negative binomial distribution, but H. taeniaeformis and T. arvicolae were close to a Poisson or random distribution. The values of the Shannon, Simpson, Berger–Parker and Shannon evenness indices reflect the diversity/uniformity of the helminth community. They were analysed consid- ering the total host population, host age and sex, as well as season and year of capture (Tables 9 and 10). Males presented a slightly lower helminth diversity than females. More- over, helminth diversity increases with the age of the host being similar between sub-adult and adult subpopulations. Hosts captured during spring and autumn and in 2012 pre- sented the highest diversity, while those captured during summer and in 2013 presented the lowest. Table 9. Diversity characteristics of the helminth community of Microtus lusitanicus from Asturias by host sex and age. Sex Age Males Females Juveniles Sub-Adults Adults Total Shannon index 0.86 0.78 0.38 0.82 0.93 0.84 Simpson index 0.53 0.48 0.20 0.53 0.55 0.52 Berger–Parker index 0.48 0.36 0.11 0.51 0.49 0.46 Shannon evenness index 0.44 0.40 0.34 0.46 0.45 0.40 Animals 2021, 11, 3055 13 of 22 Table 10. Diversity characteristics of the helminth community of Microtus lusitanicus from Asturias by season and year of capture. Season Year Winter Spring Summer Autumn 2011 2012 2013 Shannon index 0.76 0.81 0.45 0.81 0.68 0.81 0.38 Simpson index 0.38 0.50 0.22 0.48 0.39 0.49 0.22 Berger–Parker index 0.23 0.41 0.12 0.35 0.25 0.38 0.13 Shannon evenness index 0.39 0.41 0.25 0.50 0.38 0.39 0.54 Tables 11 and 12 show the diversity characteristics of the helminth infracommunities, including the Brillouin index, according to host sex and age, and season and year of capture, which follow a similar trend as the helminth community diversity. Table 11. Diversity characteristics of helminth infracommunities of Microtus lusitanicus (M.l.) from Asturias by host sex and age. SE—standard error. Sex Age Male Female Juveniles Sub-Adults Adults Total Mean species richness X 0.86 0.81 0.79 0.80 0.88 0.84 SE 0.04 0.04 0.08 0.04 0.05 0.03 Brillouin index 0.06 0.06 0.05 0.06 0.07 0.06 SE 0.01 0.01 0.02 0.01 0.01 0.01 Max 0.60 0.75 0.62 0.65 0.75 0.75 BI infected M.l. only X 0.07 0.06 0.04 0.06 0.06 0.06 SE 0.01 0.01 0.02 0.01 0.01 0.01 % of M.l. infected 62.5 64.3 68.3 61.1 64.9 63.38 Table 12. Diversity characteristics of helminth infracommunities of Microtus lusitanicus (M.l.) from Asturias by season and year of capture. SE—standard error. Season Year Winter Spring Summer Autumn 2011 2012 2013 Mean species richness X 0.78 1.01 0.92 0.62 0.83 0.86 0.38 SE 0.07 0.06 0.05 0.05 0.04 0.04 0.14 Brillouin index X 0.06 0.07 0.05 0.05 0.06 0.06 0.02 SE 0.01 0.01 0.01 0.01 0.01 0.01 0.02 Max 0.65 0.62 0.55 0.75 0.75 0.65 0.28 BI infected M.l. only X 0.07 0.08 0.06 0.04 0.06 0.06 0.06 SE 0.02 0.01 0.01 0.01 0.01 0.01 0.01 % of M.l. infected 56.8 73.8 71.4 49.5 65.0 62.7 38.5 The role extrinsic (season and year of capture) and intrinsic (host age and sex) factors play in the component species of the helminth community of M. lusitanicus is shown in Table 13 in relation to helminth prevalence, and in Table 14 with respect to helminth abundance. The analysis of these results shows that, in general, the helminth component species of the Lusitanian pine voles were significantly affected by host age, season and year of capture. Host sex did not have any important effect on helminth prevalence and abundance. Animals 2021, 11, 3055 14 of 22 Table 13. Binary logistic regression models for the prevalence of the helminth component species of Microtus lusitanicus from Asturias by year and season of capture, host sex and age, expressed by χ2 values with associated probabilities (p) for the model created, including independent variables. Only statistically significant models are reported. Helminth Species/Independent df χ2 p Variables Included in the Model Global M. lusitanicus parasitation Season of capture 3 32.471 <0.0001 Hydatigera taeniaeformis larvae Host age 2 27.540 <0.0001 Trichuris arvicolae Host age 2 13.789 0.001 Carolinensis minutus Host age 2 13.333 0.001 Host age and sex 2 7.189 0.027 Season of capture 3 49.574 <0.0001 Year of capture 2 17.216 <0.0001 Heligmosomum costellatum Season of capture 3 11.074 0.011 Syphacia nigeriana Season of capture 3 8.711 0.033 Year of capture 2 23.797 <0.0001 Df—degree of freedom. Table 14. Values of Mann–Whitney (U) and Kruskal–Wallis (χ2 ) tests, with associated probabilities (p), applied in the analyses of the helminth component species abundances of Microtus lusitanicus from Asturias. The Mann–Whitney test was applied to host sex and the Kruskal–Wallis test was applied to host age, season and year of capture. Only significant results are reported. Helminth Species/Independent df U/χ2 p Variables Included in the Model Hydatigera taeniaeformis larvae Host age 2 23.327 <0.0001 Trichuris arvicolae Host age 2 10.992 0.004 Carolinensis minutus Host age 2 23.558 <0.0001 Season of capture 3 68.329 <0.0001 Year of capture 2 21.101 <0.0001 Heligmosomum costellatum Season of capture 3 8.868 0.031 Syphacia nigeriana Season of capture 3 8.386 0.039 Year of capture 2 20.706 <0.0001 df—degree of freedom. Spearman’s correlation values between climate factors (temperature and rainfall) and diversity parameters (species richness and Brillouin index), prevalence and abundance of parasitation (Table 15) showed significantly positive correlations between prevalence and mean temperature (T. arvicolae) and abundance and mean temperature (T. arvicolae and C. minutus). However, the absence of any correlation between climate factors and diversity parameters stands out. Animals 2021, 11, 3055 15 of 22 Table 15. Values of Spearman Correlation Coefficient (rs ), with associated probabilities (p), between climate variables (temperature and rainfall) and diversity (species richness and Brillouin index), prevalence and abundance. Only significant results are reported. Correlations rs p Trichuris arvicolae prevalence and mean temperature 0.691 0.009 Trichuris arvicolae abundance and mean temperature 0.663 0.014 Carolinensis minutus abundance and mean temperature 0.698 0.008 4. Discussion 4.1. Remarks on the Helminth Species 4.1.1. Hydatigera taeniaeformis Larvae This taenid tapeworm of cosmopolitan distribution has a diheteroxenous life cy- cle, having rodents as the intermediate host, with carnivores, such as felids, canids and mustelids, even domestic ones, as its definitive host [37]. 4.1.2. Paranoplocephala omphalodes This anoplocephalan tapeworm has a Holarctic distribution with its adult stage para- sitizing the small intestine of several species of the genus Microtus, which act as its definitive host, while some mite species act as its intermediate host [38]. 4.1.3. Rodentolepis asymmetrica Adults of this hymenolepidid cestode parasitizes mainly the small intestine of arvi- colines, also having a Holarctic distribution. Its life cycle is diheteroxenous, with some arthropod species acting as its intermediate host [34]. 4.1.4. Trichuris arvicolae This Trichurinae nematode has been reported in some Arvicolinae species, having a Holarctic distribution, similar to their hosts. Its life cycle is monoxenous, a so-called pseudogeohelminth (the infective form for the wood mouse is not a free larva but an egg embryonating in the soil) [35,39,40]. 4.1.5. Carolinensis minutus This heligmosomid nematode also has a Holarctic distribution and parasitizes the small intestine of some Arvicolinae species. Its life cycle is monoxenous, a so-called geohelminth (the infective form is a free larval stage, which must be ingested by the host) [36,41]. 4.1.6. Heligmosomum costellatum This other heligmosomid nematode mainly parasitizes the small intestine of some Microtus species, also having a Holarctic distribution and a monoxenous geohelminth life cycle [36,42]. 4.1.7. Syphacia nigeriana This oxyurid nematode mainly parasitizes the caecum of some Arvicolinae species, also having a Holarctic distribution. Its life cycle is monoxenous, a so-called ageohelminth, with eggs shed being infective after only a few hours. 4.2. Helminth Community Analysis The helminthfauna of the M. lusitanicus population from Asturias shows a similar structure to other Arvicolinae, i.e., helminth species with low specificity at is host level (eurixens and oligoxens) are dominant [18,43]. Moreover, the species richness at infracom- munity level is poor. The semi-fossorial life and the vegetarian diet could explain the low Animals 2021, 11, 3055 16 of 22 parasite diversity found in M. lusitanicus when compared to other arvicolines of the Iberian Peninsula [18,44]. Comparing the current results with previous studies [18,19,43], some differences in the helminth community composition have been found. The absence of Notocotylus neyrai (Trematoda), other adult cestodes, such as Anoplocephaloides dentata and Arostrilepis horrida), and Heligmosomoides laevis (Nematoda), as well as the presence of R. asymmetrica (Cestoda) and C. minutus (Nematoda) are the main differences observed. The lack of helminths with an indirect life cycle, which have M. lusitanicus as definitive host, could be explained by the use of pesticides in some crops that negatively affect the survival of intermediate hosts (arthropods and snails). Another fact to be considered is the small size of the crop fields that may increase the stochastic extinction of helminth species, while recolonization is difficult due to the poor connectivity of the habitats [45]. Indeed, a recent landscape genetics study showed that this agricultural landscape affects successful dispersal and colonization of voles at local scale [46]. The low values of helminth prevalence and mean species richness agree with the values of the diversity indices. Previous studies also obtained low prevalence [19], with S. nigeriana being the most prevalent helminth species. The semi-hypogea life and the herbivore diet of the Lusitanian pine vole may explain its low helminth diversity when compared to other epigean Arvicolinae from the Iberian Peninsula [18,44]. Six of the eight helminth species have an FES biological cycle, helminths which have a free-environmental infectious stage for the vole. Moreover, only five voles were parasitized after the ingestion of an infected arthropod. The analysis of the frequency distribution of helminths revealed that dominant and co- dominant species, namely C. minuts, S. nigeriana and H. costellatum, showed a high level of aggregation. However, T. arvicolae and H. taeniaeformis were close to a random distribution. A high-level aggregation of parasites is associated with the stability of the host–parasite relationship, whereas low-level aggregation is associated with destabilization [47–49]. Consequently, according to the current results, only T. arvicolae and H. taeniaeformis might be responsible for the destabilization of the Lusitanian pine vole population in the study area. The high aggregation of all dominant and co-dominant helminth species may be caused by various factors, such as heterogeneities in host populations and/or infection pressure [50]. The aggregation level does not seem to be affected by the helminth transmis- sion mode or route, revealing the optimal distribution of helminths, and the co-evolutionary adaptative processes between parasites and their hosts [51]. Hidatygera taeniaeformis and T. arvicolae showed the lowest aggregation among the main helminth species found. In both cases, the infection of voles occurs after the ingestion of the egg, after its embryonation in the case of T. arvicolae. Both species have an FES biological cycle, the same as the other three helminth species with a high level of aggregation. Diversity and species richness increase from younger to older hosts. According to the Kruskal–Wallis test and binary logistic regression, the species showing an age effect are H. taeniaeformis, T. arvicolae and C. minutus. For H. taeniaeformis and T. arvicolae, the likelihood of infection increases with age, although in C. minutus it decreases. In H. taeniaeformis, the results are the expected ones: juveniles are protected from the eggs by the IgA they ingest by the colostrum and the transplacental IgA and IgG. Adults present a partial protection from them, i.e., some eggs develop into cysts in the host liver [52]. Moreover, older animals are more likely to be infected given their age and having covered more territory to come into contact with infective forms [53]. Prevalence and intensity of helminth parasites increase with host age in other studies with wild rodents [19,54–68]. In C. minutus, the results were those to be expected. Survival of parasites decreases with age through the development of an acquired partial immunity in hosts [58]. According to the life cycle of M. lusitanicus, juveniles spend more time in galleries, so they are more likely to come into contact with larval forms of C. minutus. The acquired immunity developed against this parasite reduces the number of parasites in older individuals. Unexpected results were obtained in the case of T. arvicolae. This helminth is a pseudogeohelminth, Animals 2021, 11, 3055 17 of 22 whose eggs develop outside the body where they become infective forms. Younger hosts spend more time in galleries than older ones, so they have a greater likelihood to be infected. Younger animals would be expected to be more parasitized than older ones that have undergone the development of acquired immunity. However, our results showed a clear increase of infection with host age, with the absence of this helminth species in the juvenile host subpopulation. No differences by host sex were detected in diversity and richness values. No effect in any helminth species was detected by binary logistic regression and the Mann–Whitney test. However, previous studies documented the host sex effect. Usually, males are more infected than females [69], which can be explained by different reasons [70]. Males are usually larger than females and, hence, not only provide more resources to parasites but are also more susceptible to them. Immunocompetence against infections in males is reduced as a consequence of steroid hormones, but this mechanism is not clear. Furthermore, behavioural aspects benefit infection, such as fights, reduced grooming in the mating season, covering larger territories, are all factors that increase their exposure to infective forms. However, some studies found a female bias [70]. In these cases, the reproductive state and hormone levels can affect their immunity levels and they are more susceptible to parasitic diseases. This effect increases during pregnancy and lactation. Additionally, the fact that females share nests with other females could increase the probability of infection by parasites [69]. Nevertheless, the lack of a sex bias has been reported in various small mammal species [52,59,61,68,70,71]. Some results support the idea that the influence of sex on the parasite load may depend largely on the parasite taxa and/or host-parasite associations [72–75]. Another study [70] suggests the hypothesis that a sex bias is related to a seasonally dependent sexual dichotomy in reproductive behaviour. Analysing the effects of season, the lowest values of diversity indices are reached in summer and the highest in spring and autumn, while species richness reaches its lowest values in winter and its highest in spring. As winter is likely the season with the lowest outside activity of Lusitanian pine voles, they have fewer occasions to come into contact with parasite infective forms, which is then the reason to explain these values. According to binary logistic regression and Kruskal–Wallis test results, prevalence and abundances of C. minutus, H. costellatum and S. nigeriana were significantly influenced by the season of capture. Heligmosomum costellatum and C. minutus are geohelminths requiring high humidity conditions for larval survival in the environment [76]. In H. costellatum, higher prevalence values are linked with the reproductive stage in hosts [42]; as M. lusitanicus reproduces along the entire year [3], the low prevalence and intensity values in autumn are more related to environmental conditions that influence the survival of dispersal stages [77]. Summer could affect larvae survival negatively and, hence, fewer hosts are infected in autumn. So far, no data concerning the seasonal effect on C. minutus have been reported in the scientific literature. As with other geohelminth species, external conditions are essential for infective forms to survive. According to our results, summer and spring are the most favourable seasons for C. minutus. In the case of S. nigeriana females, when they are gravid, they migrate from the intestine to the anus. Then, they emerge and leave eggs in the perianal region, which, as an ageohelminth, become infective in a short time. These eggs can infect the same individual or other ones by grooming or sharing nests and helminths can spread among the rest of the group [77]. As M. lusitanicus reproduces along the year [3], the high values of S. nigeriana in winter could be due to the higher aggregation between individual hosts as a consequence of adverse climate conditions, spending more time in the tunnels, increasing their density and, consequently, the likelihood to be parasitized. These results agree with previous studies, which also reported positive correlations between helminth prevalence and abundances and host densities [68,78–80]. Concerning the year of capture, diversity index and species richness values are sim- ilar between both years, showing the medium-term stability of the Lusitanian pine vole helminth community. Among the helminth species, only the prevalence and abundance Animals 2021, 11, 3055 18 of 22 of C. minutus and S. nigeriana are influenced by the year of capture, due to the annual variability of the environmental conditions and the abundance of the host. Although the absence of correlations between climate variables and diversity (species richness and Brillouin index) stands out, the prevalence and/or the abundance of some helminth species, such as T. arvicolae and C. minutus, with a pseudogeohelminth, respec- tively, and a geohelminth life cycle, correlate positively with mean temperature values of the year before capture, which influence the survival of the infective forms. 4.3. The Use of the Helminths of Microtus lusitanicus in Pest Control Nowadays, the impact of parasites on reproduction and survival of wild animal populations has been recognised [81–85]. Moreover, parasites may play a key role in driving population regulation in vertebrates [81,86,87]. Concerning helminth species present in the helminth community of M. lusitanicus, Deter et al. [88,89] demonstrated that T. arvicolae affects the fecundity of its host, and proposed that this intestinal nematode could control population growth in several rodent species, representing an in-built pest control [90]. Likewise, it has been demonstrated that rodents infected with H. taeniaeformis have lower levels of leptin and higher levels of neuropeptide-Y in plasma than uninfected ones [91]. Accordingly, parasites stimulate appetite in their hosts and they become hungry. Models predict that hungry animals tend to be more willing to take risks than satiated individuals [92,93]. Therefore, infected animals as intermediate hosts are likely to be preyed upon by the final hosts of this parasite [91]. Hydatigera taeniaeformis larvae could play a role in population control resulting in a decrease in the number of rodents. According to our results, T. arvicolae and H. taeniaeformis might have a potential destabilization effect on the M. lusitanicus population, and considering their role in host population control, both species can be proposed as potential pest control in the Lusitanian pine vole population in Asturias and elsewhere in this geographic distribution. 5. Conclusions The current analysis on the helminth community of the Lusitanian pine vole in As- turias provides further information on this vole pest reported in previous studies through- out its Iberian distribution, with the most remarkable difference being the near complete absence of adult cestodes parasitizing the population studied. The influence of extrinsic factors, such as climate variables and the season and year of capture, confirms the more fragile life cycles of psedudogeohelminths, i.e., T. arvicolae, and geohelminths, i.e., C. minutus, species, while the influence of the host population density seems to affect the ageohelminth species, S. nigeriana, more significantly. The influence of host age was expected as it has been reported in several previous studies in various host populations, but the absence of host sex influence is surprising and remains without any convincing explanation. An increase in the mean temperature seems to favour the transmission of these psedu- dogeohelminth and geohelminth species, a fact that could be of great interest considering the potential increase of mean temperatures due to global warming and climate change. Moreover, the potential destabilization effect that H. taeniaeformis and T. arvicolae could exercise on the Lusitanian pine vole population, due to their low-level aggregation, should be considered in the control programs of this studied rodent pest, even in the case of the cestode introducing its definitive hosts, carnivores which spread infective eggs and also predate the voles. Animals 2021, 11, 3055 19 of 22 Author Contributions: Conceptualization, C.F., A.S., M.M., J.V., J.T., J.M.; Methodology, R.A., C.F., A.S., M.M., J.V., J.T., J.M., M.V.F.; Formal Analysis, R.A., C.F., A.S., M.M., J.V., J.T., J.M., M.V.F.; Investigation, R.A., C.F., J.T., J.M., M.V.F.; Data Curation, R.A., J.T., J.M.; Statistical Analy- sis, R.A., M.V.F. Writing—Original Draft Preparation, R.A., C.F., A.S., M.M., J.V., J.T., J.M., M.V.F.; Writing—Review & Editing, R.A., C.F., J.M., M.V.F.; Project Administration, C.F., J.T., J.M. All authors have read and agreed to the published version of the manuscript. Funding: This study has been supported by Gobierno del Principado de Asturias, FEDER, Caja Rural de Gijón, CADAE, and AACOMASI (Project FICYT PC10-52). DOC-INIA Program granted to M.M.; FPI-INIA Program granted to A.S. Institutional Review Board Statement: All vole specimens included in the study were obtained from the collection of SERIDA (Servicio Regional de Investigación y Desarrollo Agroalimentario, Asturias). The sample was collected during a population peak, for two consecutive years (January 2011 to January 2013), in apple orchards of Asturian localities where the Lusitanian pine vole was a great pest. The recommendations of the Directive of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes were followed in the field work. 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