Shrews Under-Represented in Fruit Farms and Homesteads

Simple Summary Between 2018 and 2022, we surveyed small mammals at 23 sites in Lithuania—meadows, commercial orchards and berry farms, kitchen gardens, homesteads and farms—with the aim to assess the proportion of shrews in the community and their diet using stable isotope analysis. We found that in these natural, agricultural, and commensal habitats, common (Sorex araneus) and pygmy (Sorex minutus) shrews were under-represented—having a proportion of 3.1%, less than a half that of the long-term average in the country. The diet of these two species was similar in both agricultural and commensal habitats. On farms and in orchards with intensive farming, there were no catches of shrews. Contamination by plant protection products and a lack of invertebrates, which are the main food of shrews, may be factors limiting their numbers in the agriculturally managed habitats. Two species of water shrews, Neomys fodiens and Neomys anomalus, were found for the first time in homesteads, including in outbuildings, and their diet requires further investigation. Abstract Shrews are a less studied group of small mammals than rodents. Between 2018 and 2022, we surveyed 23 sites in Lithuania, including natural and anthropogenic habitats, with the aim to assess the proportion of Soricidae in small mammal communities and their diet based on stable isotope analysis. The average representation of Soricidae was 3.1%, about half the long-term average in other habitats in the country. The highest proportions were in meadows and farmsteads, at 4.9% and 5.0% respectively. Shrews were not trapped on farms or in young orchards, and their relative abundance was very low in intensively managed orchards (0.006 individuals per 100 trap days). Neomys fodiens and N. anomalus were unexpectedly found in homesteads, including in outbuildings. Sorex araneus and S. minutus had similar diets. The trophic carbon/nitrogen discrimination factor between invertebrates and shrew hair was 2.74‰/3.98‰ for S. araneus, 1.90‰/3.78‰ for S. minutus in the orchards. The diet of N. fodiens and N. anomalus at the homesteads requires further investigation. We propose that the under-abundance of shrews may be due to contamination by plant protection products and a lack of invertebrates under intensive agricultural practices.


Introduction
The family Soricidae is divided into two subfamilies, Soricinae and Crocidurinae [1], the latter not currently found in Lithuania. With more than 370 species, the family Soricidae is a very diverse family of mammals [2], exhibiting genetic diversity [3,4]. The family Soricidae originated in Eurasia [5] and is still very abundant in boreal forests [6]. Moisture is thought to be a key factor in the evolution of the Soricidae [5] and is of great importance for the ecology of shrews [7,8]. According to Sheftel and Hanska [6], wet and vegetationrich habitats in the Eurasian boreal forests are characterized by the highest abundance and species richness of shrew communities.
Four shrew species occur in Lithuania: the common shrew (Sorex araneus), the pygmy shrew (S. minutus), the water shrew (Neomys fodiens) [9], and the Mediterranean water shrew (N. anomalus), the last one identified only in 2012 [10]. As for the habitat preferences, Diet composition and resource overlap among species is key to understanding ecological communities, including the adaptation to different environments [36]. Differences in the diet of S. araneus and S. minutus in grassland habitats have been shown to reduce competition between these species, as shown by studies of the digestive tract contents [37]. In contrast, an almost complete overlap of trophic niches between these species was found in a mountainous area with limited food supply, suggesting competition [38]. In marshland in the Bialowieza Forest in eastern Poland, abundant invertebrate prey allowed four species of Soricidae to coexist. In this case, the diets of S. araneus, S. minutus, N. fodiens, and N. anomalus overlapped considerably, despite signs of prey selectivity [39]. Using isotopic analysis, an overlapping diet was also found between S. araneus and S. minutus in a resource-rich floodplain meadow in western Lithuania [40]. The importance of abundant invertebrates as key micro-environmental features for shrews [41,42] was thus confirmed.
Insectivores are not listed as pests; therefore, they are rarely monitored [43]. As with other mammals, climate change [44] is an important factor influencing the distribution and biology of shrews [45,46]. In agricultural habitats, shrews are very important as model species, as they are more likely to be contaminated by various compounds used as insecticides, plant protection products, etc., and, consequently, they show higher contamination levels [47]. Shrews also respond positively to habitat protection measures [17,48] and can therefore demonstrate the effectiveness of the measures.
The aim of this study was to assess the proportions of Soricidae in small mammal communities in meadows, commercial fruit and berry farms, and commensal habitats (homesteads, kitchen gardens, and farms) of Lithuania, as well as their trophic niche, as determined by stable isotope analyses of their hair.

Study Sites
Between 2018 and 2022, small mammals were surveyed at 23 sites in various regions of Lithuania: 10 commercial apple orchards (AO), two plum orchards (PO), three raspberry plantations (RP), three currant plantations (CP), one highbush blueberry plantation (B), two homesteads (H), one kitchen garden (KG), and two farms (F) (Figure 1). The average size of apple orchards was 63.7 ha, of the plum orchards was 0.81 ha, of the currant plantations was 22.0 ha, of the raspberry plantations was 2.3 ha, and of the blueberry plantation was 3.80 ha. Agricultural study sites were old fruit orchards (AO2-9), middle-aged fruit orchards, and berry plantations (AO10; PO2; CP1-3; RP1, 2; B1), young orchards, and plantations (AO1, 6; PO1; RP3). The intensity of on-site agricultural measures was also different, from high (AO1, 2, 4, 6, 8, 10; RP2, 3; B1), to medium (AO3; PO1; CP3; RP2), and to low (AO5, 7, 9; PO2; CP1, 2; RP1). We characterized three levels of intensity depending on soil scarification, grass mowing, mulching of the plant interlines, and the usage of rodenticides and plant protection agents. Sites with only grass mowing once or several times per season were classified as low intensity, while usage of two measures from those listed above, once or twice per season, was defined as medium intensity. The application of several measures or frequent application of two measures per season was defined as high intensity. The nearest non-agricultural habitat was used as a control in every study site, these being mowed or unmowed meadows, or forest (Table A1).
The kitchen garden was characterized by a limited number of buildings and a limited period of availability of human food. Both homesteads had a variety of buildings, some of which contained human food that was readily available for most of the year. The farms had the most complex building structures and unlimited access to human food and fodder for livestock, poultry, or rabbits throughout the year.

Small Mammal Trapping
In orchards and plantations, small mammals were snap-trapped using a standard method-rows of 25 traps at 5 m spacing, set for three days and checked once per day in the morning. Depending on the size of the orchard, two to four trapping lines were set, while in the meadows, the number of lines was one or two.
Inside the building structures of the kitchen garden, homesteadss and farms, small mammals were trapped opportunistically using 5 to 20 traps. Opportunistic trapping was also performed around all accessible buildings. Thus, the term "commensal habitats" was used sensu lato. More details of trapping are given in [49,50].
In 2018-2021, we trapped small mammals in the orchards and their control meadows twice a year: in June and September-October. In 2022, small mammals were trapped in the orchards only in autumn. Trapping effort in 2018-2022 amounted to 36,978 trap days. Details regarding the trapping effort in orchards and plantations are given in Table A2. Differences in trapping efforts were inevitable due to differences in habitat availability, but, as shown in our previous publications [49][50][51], they did not affect the trappability of the more abundant species and the diversity of the small mammal community. This was tested using species accumulation curves ( Figure A1) under different trapping efforts. Using long-term data, it was shown S. araneus was the fourth-most abundant species in Lithuania [27]. The species accumulation curves show that it should be trapped with the minimum trapping effort, or a very low number of trapped individuals ( Figure A1); therefore the absence of this species cannot be related to under-trapping.
Trapping on the homesteads, farms, and kitchen gardens took place throughout the year, with at least several trapping sessions each season. The trapping efforts on farm F1 amounted to 1530 trapping days and on homestead H2, to 210 trapping days. On homestead H1, the trapping effort in 2019-2020 was 1480 trapping days. In the other commensal habitats, the precise trapping effort was not calculated due to the use of the opportunistic trapping method.
Therefore, the relative abundance of small mammals, including shrews, expressed as the number of individuals per 100 trap days, was not always available for habitats other than orchards.
Trapped shrews were identified by external features, such as the base of the tail and the teeth in the Sorex species, using an identification key [9], and the hairy tail keel in the Neomys species [10].

Stable Isotope Analysis
In addition to stomach analysis, pellet analysis and laboratory feeding trials were used earlier to investigate shrew diets [37][38][39], and stable carbon and nitrogen isotope signatures in the hair may be used as a diet proxy [40]. Isotopic niches are substitutes for trophic or dietary niches, with a higher ratio of nitrogen ( 15 N/ 14 N) indicating consumption of animal foods [51].
We analyzed carbon and nitrogen isotopic ratios in the hair of shrews, using an elemental analyzer (Flash EA1112) coupled to an isotope ratio mass spectrometer (Thermo Delta V Advantage) via a ConFlo III interface. About 5 mm of hair was clipped from the back of individuals between the shoulders. Before analysis, dirty hair samples were washed in deionized water and methanol, then desiccated. Dry samples were weighted (0.5-1 mg) into tin capsules and stored in the sample plate. The stable isotope ( 13 C/ 12 C and 15 N/ 14 N) ratios were expressed relative to international standards, Vienna Pee Dee Belemnite, and atmospheric air, respectively. More details of sample preparation and analyses are given in [40,51].
Prior to preparation and analysis, samples (n = 10) of small mollusks and arthropods from orchards were stored in a freezer at below -20 • C. After drying in an oven at 60 • C to a constant weight for 24-48 h, invertebrate samples were homogenized to a fine powder, using mortar and pestle and a Retsch mixer mill MM 400 [51].

Data Analyses
We estimated the proportion of Soricidae and the two most abundant species, S. araneus and S. minutus, in the total number of small mammals trapped (Table 1), presenting it as mean and 95% CI for each habitat type. Differences in proportions were assessed using the G test from an online calculator [52]. Table 1. Characteristics of small mammal communities: N-number of trapped small mammal individuals; P%-proportion of all Soricidae species (95% CI); n1-number of S. araneus; n2-number of S. minutus; RA1-relative abundance of S. araneus, RA2-relative abundance of S. minutus (individuals per 100 trap days). Superscript letters denote differences, significant at p < 0.05.

Habitat
Year The δ 13 C and δ 15 N values of the hair samples were expressed as the arithmetic mean ± 1 SE and the range, from the minimum to maximum observed value. The isotopic values of species and trophic groups, including those with sample size n < 5, were visualized in isotopic biplots.
We used ANOVA to determine the influence of the habitat, the age of the orchard, and the intensity of the agricultural measures on the dependent parameters: the relative abundance and the hair δ 15 N and δ 13 C values in S. araneus and S. minutus. Differences between groups were evaluated with the post-hoc Tukey's test, and pairwise comparisons were completed using the Student's t test. The normality of the distributions of the hair δ 15 N and δ 13 C values were tested using the Kolmogorov-Smirnov's D test online [53]. Homogeneity of variances of the hair δ 15 N and δ 13 C values in S. araneus and S. minutus was assessed using the Levene test (Table A3).
The minimum confidence level was set as p < 0.05. However, the small sample size of two Neomys species may result in a low power for statistical analysis. Calculations were performed in Statistica for Windows, version 6.0 (StatSoft, Inc., Tulsa, OK, USA); biplots were drawn in SigmaPlot ver. 12.5 (Systat Software Inc., San Jose, CA, USA).
The relative abundance of all species was low, with S. araneus being most abundant in meadows, followed by berry plantations. S. minutus was most abundant in meadows ( Table 1). The highest relative abundance of S. araneus and S. minutus was recorded in the control meadows, at 2.67 and 4.00 ind. per 100 trap days, respectively.
Agricultural treatment intensity had no effect on the relative abundance of S. minutus (ANOVA, F 2,322 = 0.92, p = 0.43) and a weak effect on the relative abundance of S. araneus (F 2,322 = 3.03, p < 0.05). The relative abundance of the latter species was 0.006 ind. per 100 trap days in intensively managed crops, 0.09 in moderately managed crops, 0.19 in low-managed crops, and 0.28 ind. per 100 trap days in control habitats, with a difference of more than ten times.
The relative abundance of S. araneus and S. minutus was not affected by the age of the orchard or plantation (F = 0.96, p = 0.46); however, in the young crops, shrews were not trapped at all.

Stable Isotope Ratios of Shrews
Statistics for the stable isotope ratios of the insectivorous species in agricultural and commensal habitats are given in Table 2. In the commercial orchards, the trophic niche of S. araneus was wider than that of S. minutus, with a range of δ 13 C values of 1.9-fold, and δ 15 N values of 1.5-fold. The two species were fully separated according δ 13 C and did not differ according δ 15 N (Figure 2). The δ 15 N value of N. fodiens was about 2.4 times higher than those of Sorex shrews. We calculated the trophic discrimination factor, TDF, of invertebrates (the most likely food for shrews) and the shrew hairs ( Figure 2). The TDF of carbon and nitrogen was 2.74‰/3.98‰ for S. araneus, 1.90‰/3.78‰ for S. minutus, and 3.03‰/13.50‰ for the single N. fodiens individual in the orchards. The TDF of nitrogen in the latter species indicates that N. fodiens can use not only invertebrates, but also other food sources of animal origin.
In the commensal habitats, namely the kitchen garden and the homestead, the four Soricidae species did not exhibit a distinct separation in dietary space ( Table 2), but the number of trapped individuals was minimal. Lower δ 15 N values were observed for both Neomys species compared to those of S. araneus and S. minutus; however, a larger sample is required for statistical analysis. In Lithuania, N. fodiens and N. anomalus were trapped for the first time in commensal habitats such as outbuildings.

Trophic Position of Insectivores in Relation to Other Groups of Small Mammals
In commercial orchards, the positions of the small mammal trophic groups were well separated (Figure 3a) in terms of both δ 13 C (F = 184.3, p < 0.001) and δ 15 N (F = 28.8, p < 0.001).
Four herbivore species, the common vole (Microtus arvalis), short-tailed vole (M. agrestis), root vole (M. oeconomus), and water vole (Arvicola amphibius), had the lowest δ 13 C and δ 15 N values, which were different from all other trophic groups (HSD, p < 0.001). The three granivore species, the striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), and harvest mouse (M. minutus), showed similar δ 13 C values, with their mean different from other trophic groups at p < 0.001. In terms of mean δ 15 N, they exhibited higher values than did herbivores (p < 0.001) and lower than did the insectivores (p < 0.05). Insectivores and omnivores, these being the bank vole (Clethrionomys glareolus), house mouse (Mus musculus) and Northern birch mouse (S. betulina), did not differ in terms of either δ 13 C or δ 15 N means (Figure 3a). The separation of small mammal trophic groups by δ 13 C (F = 16.7, p < 0.001) and δ 15 N (F = 34.1, p < 0.001) was also significant in commensal habitats (Figure 3b). However, intergroup differences were not as pronounced as those in orchards. In commensal habitats, insectivores had higher mean δ 13 C values than herbivores (HSD, p < 0.01), but did not differ from those of granivores and omnivores.
The mean δ 15 N value of insectivores was only higher than that of granivores (HSD, p < 0.005). Other small mammal trophic groups were heterogeneous, with herbivorous M. arvalis and omnivorous C. glareolus having higher δ 15 N values than Neomys shrews. The granivore A. agrarius was at the same level as both Sorex species, while the omnivore M. musculus was characterized by the highest δ 15 N (Figure 3b).

Discussion
The results of the 2018-2022 small mammal trapping indicate that shrews were an under-represented group in the agricultural and commensal habitats of Lithuania, and their proportion was approximately half the long-term average proportion of Soricidae in other habitats [27]. However, two Neomys species were unexpectedly trapped in commensal habitats, including outbuildings far from the nearest water source.
As for shrews living in commensal habitats, this group can be expected to be underrepresented, as this is a general problem of simplified diversity, especially in urban areas [77]. The hostility of anthropogenic environments, such as urban habitats, is compensated by changes in Soricidae behavior, such as increased boldness and high individual variation in aggression [78]. The presence of suitable habitats is of primary importance in the anthropization gradient, but these habitats in urbanized areas create opportunities for contact and interaction between humans or domestic animals with wild animals [79]. Shrews can be found in very unusual habitats, including airport fields: at Chisinau airport in Moldova, one shrew species was recorded from three species found in the adjacent area [80].
We propose the hypothesis that one of the reasons for the low numbers of shrews in agricultural habitats is the biomagnification of pollutants. Shrews have a higher contamination rate than mice [47], and these authors argue that the effectiveness of so-called organic or ecological farming to avoid insectivore pollution is limited. This is partly contradicted by Pelosi et al. [81], who found higher concentrations of pesticide residues in the soil and earthworms of recently treated areas. The higher contamination of shrews than rodents is based on their position in the food chain, and even new "safe" insecticides concentrate in shrews [82]. This is consistent with general patterns of terrestrial vertebrate exposure to pollutants [83]. We will not speculate further on this point, but in our study, in the orchards with the most intensive agricultural treatments (including the use of plant protection products), shrews were not trapped. The susceptibility of shrews to pesticide exposure, "that can be oral via direct consumption and watering or grooming, trophic transfer, inhalation, and/or dermal contact" [84], justified the suitability of shrews as a focal species for risk assessment of plant protection products.
Another reason for their low numbers may be the limited trophic resources available to shrews, these absent due to agricultural treatment in orchards, berry plantations, and homesteads. Such agricultural activity was not present in the other commensal habitats. The diet of shrews is based exclusively on invertebrates [37,39,41,42]. S. araneus mainly consumes coleopterans, insect larvae, araneids, opilionids, and isopods [85], whereas araneids, lumbricids, and coleopterans are also common foods for S. minutus [38]. These prey groups should be affected in orchards, but are unlikely to be affected in commensal habitats. Recent observations of S. minutus in commensal habitats (roof cavities) have been attributed to possible avoidance of harsher climates, resulting in a change in diet [86]. In our study, S. araneus and S. minutus did not differ in stable isotope values between commensal and agro-habitats (see Figure 2).
With regard to the diet of Neomys shrews, there are differences in the prey related to the hunting method, as only N. fodiens can hunt underwater [87]. As all of our trappings for both N. fodiens and N. anomalus were in atypical agricultural or commensal habitats, the dietary characteristics are unknown. Two possible reasons for the high δ 15 N value in N. fodiens from orchards can be suggested as the influence of fertilization or preying/scavenging on vertebrate food. Occasional vertebrates in the diet of N. fodiens have been previously recorded [88]. An increase in δ 15 N values in the hair of small mammals was detected under the influence of guano from a colony of great cormorants, Phalacrocorax carbo [89]. Therefore, both factors are possible. It is also known that pesticide use can alter the diets of shrews and rodents [90].
The dispersal of shrews between control areas and orchards is possible, as the migration distance of S. minutus ranges from 475 to 2570 m [91]. The migration distance of S. araneus is unknown, but it has a very limited home range of about 500 sq. m and an activity radius of 13 m [92]. Therefore, the diet of the shrews in our study probably corresponded to the habitat in which they were trapped.
Finally, we suggest that there was no possible influence of removal trapping on shrew presence and abundance sensu Sullivan et al. [93]. Long-term trapping of similar intensity has been used in several studies in Lithuania and has not shown any resultant significant changes in the proportions of any small mammal species, including shrews [27,40,94,95]. Therefore, the reasons given for the low abundance or absence of shrews in intensively managed orchards must be correct and not influenced by trapping.

Conclusions
Based on the results of the small mammal survey in agricultural and commensal habitats, it can be concluded that Soricidae (S. araneus and S. minutus) were an underrepresented group of small mammals in orchards and homesteads, their proportion being less than the average in the other habitats. The diets of these two species in both habitat groups were similar, as determined by stable isotope analysis. We hypothesize that the main reasons for their limited abundance are intensive agricultural practices, contamination with plant protection products, and a lack of invertebrates, which are the main food of shrews. The presence of N. fodiens and N. anomalus was not expected in the homesteads, and their diet requires further investigation. Data Availability Statement: Due to ongoing investigation and preparation of PhD thesis, the data of this study are available from the second author upon reasonable request.
Acknowledgments: Ida Šaltenienė, Sigitas Podėnas, and Vilius Vitkauskas helped with trapping of small mammals in their property. We thank all the owners and managers of the commercial orchards used in this study for the permission to work on their properties.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Table A1. Characteristics of study sites in orchards and plantations; site numbers as in Figure 1.       [96]. Calculated in PAST version 4.01 (Paleontological Museum, University of Oslo, Oslo, Norway) [97].