Voles Microtus Research Articles

Organization of the perioral representation of the primary somatosensory cortex in prairie voles (Microtus ochrogaster).

Prairie voles (Microtus ochrogaster) are one of the few mammalian species that are monogamous and engage in the biparental rearing of their offspring. Biparental care impacts the quantity and quality of care the offspring receives. The increased attention by the father may translate to heightened tactile contact the offspring receives through licking and grooming. In the current study, we used electrophysiological multiunit recording techniques to define the organization of the perioral representation in the primary somatosensory area (S1) of prairie voles. Functional representations were related to myeloarchitectonic boundaries. Our results show that most of S1 is occupied by the representation of the contralateral mystacial whiskers and the lower and upper lips. The mystacial vibrissae representation encompassed a large portion of the caudolateral S1, while the representation of the lower and upper lips occupied a large portion of the rostrolateral aspect of S1. We found that neuronal populations representing the perioral structures tended to have small receptive fields relative to other body part representations on the head. The representation of the mystacial whiskers and perioral structures was coextensive with cytoarchitectonically defined barrel fields that extend from the caudolateral to a rostrolateral aspect of S1. We discuss our findings in the context of the magnification of behaviorally relevant sensory surfaces in other rodents, the ubiquity of the barrel systems in rodents, and behaviors associated with specialized sensory surfaces.

The effects of social loss and isolation on partner odor investigation and dopamine and oxytocin receptor expression in female prairie voles.

In humans, grief is characterized by intense sadness, intrusive thoughts of the deceased, and intense longing for reunion with the deceased. Human fMRI studies show hyperactivity in emotional pain and motivational centers of the brain when an individual is reminded of a deceased attachment figure, but the molecular underpinnings of these changes in activity are unknown. Prairie voles (Microtus ochrogaster), which establish lifelong social bonds between breeding pairs, also display distress and motivational shifts during periods of prolonged social loss, providing a model to investigate these behavioral and molecular changes at a mechanistic level. Here, a novel odor preference test was used to assess social vs non-social odor investigation, and a sucrose preference test was used to assess non-social, reward-driven motivation. Females that lost a male partner investigated partner- and food-associated cues significantly more than females that lost a female cagemate or remained intact with a male partner. However, females experiencing the loss of a male partner did not change investigation of stranger-associated cues. Western blotting revealed significant increases of dopamine receptor type 1 (DRD1) and oxytocin receptor protein content in specific brain regions in response to the loss of distinct social relationships. Such effects included an increase in DRD1 in the medial preoptic area of the hypothalamus (mPOA) in females experiencing loss of a male partner compared to all other conditions. Pharmacological antagonism of DRD1 in the mPOA blocked the loss-associated increase of investigation of the partner odor but did not affect investigation of food or stranger odors. This reveals a novel dopamine-mediated mechanism for partner-seeking behavior during periods of partner loss in female prairie voles.

Exposure to novel females increases fecundity in adult male prairie voles.

Social circuitry of the mammalian brain can influence male reproductive physiology. This often manifests as plasticity in sperm production or allocation, particularly in response to male-male competition. However, socially mediated testicular plasticity has not been investigated with respect to mating and parental strategy. Testis mass and sperm production of sexually naïve and female-exposed adult male individuals of three rodent species were compared: the socially monogamous and paternal prairie vole (Microtus ochrogaster), the promiscuous and non-paternal meadow vole (Microtus pennsylvanicus), and the promiscuous and non-paternal house mouse (Mus musculus). Monogamously paired prairie vole males exhibited significantly larger testes and greater sperm production than naïve prairie vole males. Comparatively, there were no significant differences between naïve and monogamously paired male meadow voles or mice. To investigate the role of olfactory cues for regulating this phenomenon in prairie voles, a group of naïve males exposed to soiled bedding from novel females was used. These males were more similar to paired males than to naïve males not exposed to novel female odors, demonstrating a strong role of the social olfactory system. Further, while the predictions of sperm competition theory (species with greater female promiscuity have larger testes than closely related species with less female promiscuity) are consistent between naïve meadow voles and prairie voles, the prediction does not hold for monogamously paired prairie voles and meadow voles. This demonstrates the complexity of internal social dynamics and reproductive pressures which socially monogamous paternal males face and the evolutionary adaptions that may develop in response.

Mitochondrial phylogeny of the endemic Anatolian vole (Microtus anatolicus) along with re-evaluation of the taxonomic status of Microtus socialis and Microtus irani

Mitochondrial phylogeny of the endemic Anatolian vole (Microtus anatolicus) along with re-evaluation of the taxonomic status of Microtus socialis and Microtus irani

Seasonal shifts and land-use impact: unveiling the gut microbiomes of bank voles (Myodes glareolus) and common voles (Microtus arvalis).

Gut microbial diversity influences the health and vitality of the host, yet it is itself affected by internal and external factors, including land-use. The impact of land-use practices on wild rodents' gut microbiomes remains understudied, despite their abundance and potential as reservoirs for zoonotic pathogens. We examined the bacterial and fungal gut microbiomes of bank voles (Myodes glareolus) and common voles (Microtus arvalis) across grassland and forest habitats with varying land-use intensities and types. We collected rodents seasonally and used 16S rRNA and ITS amplicon sequencing for microbe identification. We found significant differences in alpha and beta diversities between the species, with M. arvalis exhibiting higher diversity. Seasonality emerged as a prominent factor influencing microbial diversity, with significant variations between sampling months. While land-use affects the gut microbiome, its impact is subordinate to seasonal variations. Differential abundance analysis underscores the dynamic nature of microbial composition, with seasonal changes playing a predominant role. Overall, our findings highlight the significant influence of seasonality on gut microbiome diversity and composition in wild rodents, reflecting dietary shifts associated with seasonal changes. Understanding the interplay between environmental factors and microbial communities in wild rodents enahnces our knowledge of ecosystem health and resilience, warranting further investigation.

Opposite-sex pairing alters social interaction-induced GCaMP and dopamine activity in the insular cortex of male prairie voles.

The prairie vole (Microtus ochrogaster) is a monogamous rodent species which displays selective social behaviors to conspecifics after establishing a pair bonded relationship, specifically partner-directed affiliation and stranger-directed aggression. This social selectivity relies on the ability of an individual to respond appropriately to a social context and requires salience detection and valence assignment. The anterior insular cortex (aIC) has been implicated in stimulus processing and categorization across a variety of contexts and is well-situated to integrate environmental stimuli and internal affective states to modulate complex goal-directed behaviors and social decision-making. Surprisingly, the contribution of the aIC to the expression of pair bond-induced social selectivity in prairie voles has been drastically understudied. Here we examined whether neural activity and gene expression in the aIC change in response to opposite-sex pairing and/or as a function of pairing length in male prairie voles. Opposite-sex pairing was characterized by changes to calcium and dopamine (DA) transients in the aIC that corresponded with the display of social selectivity across pair bond maturation. Furthermore, D1 and D2 receptor mRNA expression was significantly higher in males after 48 hrs of cohabitation with a female partner compared to same-sex housed males, and D2 mRNA remained significantly higher in males with a female partner compared to same-sex housed males after a week of cohabitation. Together, these results implicate a role for DA and its receptors in the aIC across the transition from early- to late-phase pair bonding.

First confirmed record of Microtus rossiaemeridionalis from Iran

Abstract Microtus, the most speciose genus in the tribe Arvicolini, inhabits boreal and temperate regions. Despite recent increase in taxonomic research, the precise species diversity of Microtus voles in Iran is still unclear due to insufficient molecular work. This study aimed to identify Microtus species in West Azerbaijan province, near Turkey and Armenia, by sequencing the mitochondrial cytochrome b (cytb) gene from 20 vole specimens. Phylogenetic analyses confirmed the occurrence of Microtus rossiaemeridionalis in Iran for the first time, which has actually been predicted for the western side of Urmia (West Azerbaijan province) on the basis of habitat modelling. This study enhances the understanding of Microtus diversity in Iran, highlighting the need for comprehensive fieldwork and molecular assessments to accurately delineate rodent species composition in the country.

Extended amygdala corticotropin-releasing hormone neurons regulate sexually dimorphic changes in pair bond formation following social defeat in prairie voles (Microtus ochrogaster).

The neurobiological mechanisms underlying the connection between anxiety brought on by social stressors and the negative impact on relationship formation have remained elusive. In order to address this question, we used the social defeat model in the socially monogamous prairie vole to investigate the impact of this stress on pair bond formation. Social defeat experience inhibited partner preference formation in males but promoted preference in females. Furthermore, pair bonding increased corticotropin-releasing hormone (CRH) expression in the bed nucleus of the stria terminalis (BNST) in male prairie voles, while defeat experience increased BNST CRH expression in females. Chemogenetic excitation of BNST CRH neurons during a short cohabitation with a new partner promoted a partner preference in stress-naïve prairie voles. Interestingly, chemogenetic inhibition of BNST CRH neurons during cohabitation with a new partner blocked partner preference in stress-naïve males but promoted preference in defeated males. Inhibition of BNST CRH neurons also blocked partner preference in stress-naïve females but did not alter preference behavior in defeated females. This study revealed sexual dimorphism in not only the impact of social defeat on pair bond formation, but also in the role BNST CRHergic neurons play in regulating changes in pair bonding following social conflict.

Effects of short-term isolation on social behaviors in prairie voles.

Social isolation affects the brain and behavior in a variety of animals, including humans. Studies in traditional laboratory rodents, including mice and rats, have supported the idea that short-term social isolation promotes affiliative social behaviors, while long-term isolation promotes anti-social behaviors, including increased aggression. Whether the effects of isolation on the social behaviors of mice and rats generalize to other rodents remains understudied. In the current study, we characterized the effects of short-term (3-days) social isolation on the social behaviors of adult prairie voles (Microtus ochrogaster) during same-sex and opposite-sex social interactions. Our experiments revealed that short-term isolation did not affect rates of ultrasonic vocalizations or time spent in non-aggressive social behaviors and huddling during same-sex and opposite-sex interactions. Unexpectedly, although short-term isolation also did not affect time spent in resident-initiated and mutually-initiated aggressive behavior, we found that short-term isolation increased time spent in visitor-initiated aggression during male-male interactions. Our findings highlight the importance of comparative work across species and the consideration of social context to understand the diverse ways in which social isolation can impact social behavior.

Genomes of Microtus Rodents Highlight the Importance of Olfactory and Immune Systems in Their Fast Radiation.

The characterization of genes and biological functions underlying functional diversification and the formation of species is a major goal of evolutionary biology. In this study, we investigated the fast radiation of Microtus voles, one of the most speciose group of mammals, which shows strong genetic divergence despite few readily observable morphological differences. We produced an annotated reference genome for the common vole, Microtus arvalis, and resequenced the genomes of 10 different species and evolutionary lineages spanning the Microtus speciation continuum. Our full-genome sequences illustrate the recent and fast diversification of this group, and we identified genes in highly divergent genomic windows that have likely particular roles in their radiation. We found three biological functions enriched for highly divergent genes in most Microtus species and lineages: olfaction, immunity and metabolism. In particular, olfaction-related genes (mostly olfactory receptors and vomeronasal receptors) are fast evolving in all Microtus species indicating the exceptional importance of the olfactory system in the evolution of these rodents. Of note is e.g. the shared signature among vole species on Olfr1019 which has been associated with fear responses against predator odors in rodents. Our analyses provide a genome-wide basis for the further characterization of the ecological factors and processes of natural and sexual selection that have contributed to the fast radiation of Microtus voles.

Can repellent crops reduce the abundance of the common vole (Microtus arvalis) as a way to reduce crop damage?

The common vole is one of the most damaging rodents in agriculture. A number of methods are used to suppress its numbers and limit damage. One option is growing crops that are not suitable for voles; this might limit their numbers and distribution. Through long-term monitoring of common voles throughout the Czech Republic, their abundance in eleven crop types (annual, biennial and perennial crops) was evaluated through active burrow counts. Reference crops were selected from perennials and biennials and annual crops were selected as potential repellents. The perennial crops were clover with alfalfa, which serve as the primary habitat for voles. These crops are where voles are found in their highest densities, and from which they spread to the surrounding crops during periods of high abundance. The biennial winter rape was selected because it provides favourable conditions for voles to overwinter and multiply rapidly in the spring. Compared to perennial crops and winter rape, significantly lower numbers of voles were found in onions, poppy seeds and maize. Additionally, fewer voles were also found in mustard compared to perennial crops. Onion and poppy were the only crops to show a significantly lower abundance of voles in comparison to wheat. The annual crops tested are unattractive to voles and due to the cultivation practices used, they do not even have enough weeds as food. As profitable crops they can be cultivated over sufficiently large areas to potentially prevent the spread of voles to surrounding crops, especially during outbreak years.

Daily Diet and Temporal Variation of Swainson's Hawk Prey Deliveries

ABSTRACT The quantity and timing of prey delivery affect the development of altricial young, can induce trade-offs in parental behavior, and ultimately influence fitness of the young. We investigated the dynamics of prey deliveries in a population of Swainson’s Hawks (Buteo swainsoni) in northern California to better understand its temporal component. We installed 12 motion-triggered cameras in nests between 2013 and 2018, and documented 466 unique prey deliveries. Of those, 455 prey items were identifiable to at least class. Mammals made up the majority of prey in terms of number of deliveries (88.2%) and estimated biomass delivered to the nest (97.1%). Of those, pocket gophers (Thomomys spp.) and voles (Microtus spp.) together made up almost half of the deliveries and biomass. Most prey was delivered to the nest in midday (i.e., 1000–1459 H), suggesting these are important foraging hours for this population. However, afternoon air temperatures in the study area can be high and may require that nestlings be shaded, setting up a potential trade-off between foraging and protecting the nestlings’ thermal homeostasis. Our study provides initial insight into the daily temporal dynamics of prey delivery for one of the latest-breeding raptors in North America.

PVN-mPFC OT projections modulate pup-directed pup care or attacking in virgin mandarin voles

Many species of animals exhibit caregiving or aggression toward conspecific offspring. The neural mechanisms underlying the infanticide and pup care remain poorly understood. Here, using monogamous mandarin voles (Microtus mandarinus), we found that more oxytocin (OT) neurons in the paraventricular nucleus (PVN) were activated during pup caring than infanticide. Optogenetic activation of OT neurons in the PVN facilitated pup caring in male and female mandarin voles. In infanticide voles, optogenetic activation of PVN OT cells or PVN-medial prefrontal cortex (mPFC) OT projection fibers prolonged latency to approach and attack pups, whereas inhibition of these OT neurons or projections facilitated approach and infanticide. Optogenetic activation of PVN OT neuron projections to the mPFC in males shortened the latency to approach and retrieve pups and facilitated the initiation of pup care, but produced no effects on pup-care females. In addition, OT release in the mPFC increased upon approaching and retrieving pups, and decreased upon attacking pups. Intraperitoneal injection of OT promoted pup care and inhibited infanticide behavior. It is suggested that the OT system, especially PVN OT neurons projecting to mPFC, modulates pup-directed behaviors and OT can be used to treat abnormal behavioral responses associated with some psychological diseases such as depression and psychosis.

Habitat Suitability Assessment for Two Burrowing Rodents on the Island of Lesvos: A Niche-Based Approach.

We conducted a habitat suitability assessment for two burrowing rodents, Anatolian or Nehring's blind mole rat (Nannospalax xanthodon) and Harting's vole (Microtus hartingi), on the island of Lesvos using a niche-based approach. We collected data on the presence of the two species across the island and selected several environmental variables, including land cover, geology, and habitat topography, to assess their influence on habitat suitability. We used the Maxent species distribution modelling algorithm to predict suitable habitats. The results showed that both species preferred habitats with low slopes and specific geological substrates, i.e., alluvial deposits and volcanic rocks. M. hartingi showed a preference for open habitats such as saltmarshes and non-irrigated arable land, while N. xanthodon preferred non-irrigated arable land, pastures, and discontinuous urban fabric. The model predicted a wider area of suitable habitats for Microtus hartingi compared to N. xanthodon. Interestingly, the two species are absent from the southeastern part of the island despite our model predicting high suitability; this indicates that a natural barrier of hilly terrain, extensive pine forests, and limestone rock formations may exist that impedes dispersal. Our study provides valuable insights into the habitat preferences of these two burrowing rodents on the island of Lesvos, which can inform biodiversity conservation and ecosystem management strategies.

Early positive tactile stimulation reverses the increase of anxiety and decrease of sociality induced by early chronic mechanical pain in mandarin voles

AbstractAnimals may experience early negative (mechanical pain: being retrieved using an incisor by parents or attacked) or positive stimulation (being licked and groomed) that may affect emotional and social behaviors in adulthood. Whether positive tactile stimulation can reverse adverse consequences on emotional and social behaviors in adulthood resulting from chronic mechanical pain and underlying mechanisms remain unclear. This study used a tail‐pinching model during development to simulate mechanical pain experienced by pups in high‐social mandarin voles (Microtus mandarinus). Subsequently, brush‐like positive tactile stimuli were applied to the backs of the mandarin voles. Various behavioral tests were used to measure levels of anxiety, depression, and sociability. The results showed that early tail‐pinching delayed the eye opening of pups, increased levels of anxiety, reduced levels of sociality in male mandarin voles, and impaired social cognition in females during adulthood. Brushing on the back reversed some of these effects. While mandarin voles that were exposed to tail‐pinching during development were exposed to sub‐threshold variable stress as adults, they were more likely to show a stress‐induced increase of anxiety‐like behavior, reduction of sociability, and impairment of social cognition, displaying heightened susceptibility to stress, particularly in males. However, back‐brushing reversed some of these effects, implying that these adults display enhanced stress resilience. In addition, tail‐pinching reduced levels of serum oxytocin and increased corticosterone levels in serum, but back‐brushing reversed these effects. Overall, it was found that positive tactile stimulation reversed increases in anxiety and impairments of social behavior induced by negative stimulation in male mandarin voles via alteration of oxytocin and corticosterone levels.

Features of reproduction and postembryonic ontogenesis in voles Microtus kermanensis and M. mystacinus of the subgenus Microtus (Rodentia, Arvicolinae)

The voles Microtus kermanensis Roguin, 1988 and M. mystacinus De Filippi, 1865 are representatives of the “mystacinus” group in the subgenus Microtus (2n = 54). A three-year study of reproduction of these species was conducted in the Laboratory of Theriology of the Zoological Institute RAS. The laboratory populations of voles were created from animals caught in Iran. In this study, 38 pairs of M. kermanensis and 18 pairs of M. mystacinus were under observation. For the study of postembryonic ontogenesis, 39 litters (166 pups) of M. kermanensis voles and 14 litters (51 pups) of M. mystacinus were examined. For comparison, 10 litters (47 pups) of M. arvalis Pallas, 1978 (form “arvalis” from the Vladimir region) were used. The breeding of animals continued all year round. The average number of pups per litter was 4.3 in M. kermanensis, 3.6 in M. mystacinus, and 4.7 in M. arvalis; the sex ratio in all species was close to 1:1. High mortality was noted in the larger M. kermanensis (28.3%), with lower rates in M. mystacinus (15.7%) and M. arvalis (17.0%). In general, the development of pups of all three species during the first 12 days of life is fast. Among the distinctive features, the early separation of the auricles in M. mystacinus is of note. The large M. kermanensis is characterized by earlier divergence of the fingers on the fore and hind limbs, which ends by day 10 (in M. mystacinus and M. arvalis by day 12). Also, in M. kermanensis the timing of incisor eruption is extended; the lower incisors begin to erupt on day 3 and appear by day 9 in 100% of the pups (by day 7 in the other two species). The upper incisors begin to erupt from day 4, and 100% of the pups have them by day 10 (in M. mystacinus and M. arvalis, they completely erupt on day 8). In all three species, their eyes start opening at 9 days and open completely by day 12; such uniformity can serve as confirmation of the phylogenetic proximity of these species. All three species are characterized by the avoidance of incest.

Influence of genetic similarity and social setting on extra-pair parentage in prairie voles

Abstract Social monogamy is rare in mammals, but in those species in which it occurs, individuals frequently engage in extra-pair copulation (EPC). Numerous hypotheses have been proposed to explain EPC, but relatively few field studies have examined factors influencing EPC in socially monogamous mammals. Prairie voles (Microtus ochrogaster) are a socially monogamous rodent in which extra-pair paternity (EPP) is common. Using genetic data from a 3-year study of a natural population of prairie voles, we investigated whether the negative consequences of inbreeding and the structure of social units (male–female pairs vs. groups; adult sex ratio within groups) were factors affecting EPC. We found strong evidence that genetic similarity between males and females that produced offspring via EPC was less than that between social partners, as would be expected if avoiding inbreeding depression influenced the occurrence of EPC. Social unit structure was also a factor involved with extra-pair parentage. Contrary to our expectations, the greater the proportion of females in the group, the lower the chance of EPC resulting in offspring production by females, and, similarly, the chance of EPP by males declined as the proportion of males within the group increased. However, neither males nor females were more likely to produce offspring from EPC when they were living in pairs versus groups. One implication of these results is that EPC may be influenced more by female behaviors, such as mate choice, than male mate guarding. Overall, our results suggest that the proximate factors influencing EPC in prairie voles are complex but include the cost of inbreeding depression and the structure of social units.

Structure of the hybrid zone between allied species of the common vole, Microtus arvalis and M. obscurus: Influence of genetic factors and landscape-geographic conditions

Patterns of introgression of several genetic markers across the hybrid zone between allied species of the common vole Microtus arvalis s. str. и M. obscurus were studied in four its sections: in northwest Nizhny Novgorod region, east Vladimir and southwest of Nizhny Novgorod regions, south Lipetsk region, and northwest Voronezh region. Analysis of the clinal variability for three molecular-genetic markers (cytb, tp53, SMCY11) and for karyotypes showed a structural similarity between the “Vladimir – Nizhny Novgorod”, “Nizhny Novgorod”, and “Voronezh” sections. The maximal width was shown for the cytb cline, the minimal width – for the SMCY11 cline; the tp53 cline and chromosomal cline occupy intermediate position for this parameter. Furthermore, in these transects the centre of the cline for the cytb is shifted southeastward (into the distribution range of M. obscurus) from the centres of three other clines. The revealed asymmetric introgression of mitochondrial genome from M. arvalis to M. obscurus may be explained by the fact that the hybrid zone was formed as a result of invasion of M. obscurus into the range of M. arvalis. The “Lipetsk” transect differs from three above-mentioned transects by very narrow clines with nearly coinciding centres. Such characteristics of the “Lipetsk” transect are obviously caused by localization of the hybrid zone in this section along river Voronezh. The obtained results led us to suppose that the structure of the studied hybrid zone is determined mainly by coinciding (or non-coinciding) of its centre with local physical barriers.

Effects of DNA Methylation of HPA-Axis Genes of F1 Juvenile Induced by Maternal Density Stress on Behavior and Immune Traits in Root Voles (Microtus oeconomus)-A Field Experiment.

The literature shows that maternal stress can influence behavior and immune function in F1. Yet, most studies on these are from the laboratory, and replicated studies on the mechanisms by which maternal stress drives individual characteristics are still not fully understood in wild animals. We manipulated high- and low-density parental population density using large-scale field enclosures and examined behavior and immune traits. Within the field enclosures, we assessed anti-keyhole limpet hemocyanin immunoglobulin G (anti-KLH IgG) level, phytohemagglutinin (PHA) responses, hematology, cytokines, the depressive and anxiety-like behaviors and prevalence and intensity of coccidial infection. We then collected brain tissue from juvenile voles born at high or low density, quantified mRNA and protein expression of corticotropin-releasing hormone (CRH) and glucocorticoid receptor gene (NR3C1) and measured DNA methylation at CpG sites in a region that was highly conserved with the prairie vole CRH and NR3C1 promoter. At high density, we found that the F1 had a lower DNA methylation level of CRH and a higher DNA methylation level of NR3C1, which resulted in an increase in the expression levels of the CRH mRNA and protein expression and further reduced the expression levels of the NR3C1 mRNA and protein expression, and ultimately led to have delayed responses to acute immobilization stress. Juvenile voles born at high density also reduced anti-KLH IgG levels and PHA responses, increased cytokines, and depressive and anxiety-like behaviors, and the effects further led to higher coccidial infection. From the perspective of population density inducing the changes in behavior and immunity at the brain level, our results showed a physiological epigenetic mechanism for population self-regulation in voles. Our results indicate that altering the prenatal intrinsic stress environment can fundamentally impact behavior and immunity by DNA methylation of HPA-axis genes and can further drive population fluctuations in wild animals.

Foraging Habitat Availability and the Non-Fish Diet Composition of the Grey Heron (Ardea cinerea) at Two Spatial Scales.

Habitat structure on foraging ground is one of the crucial factors determining diet diversity in colonially breeding avian predators. Quantifying the habitat and diet composition at different spatial scales (regional and local inter-colonial) can help provide understanding of the drivers of diet composition. In this study, we examined the composition of the non-fish diet of an opportunistic predator, the Grey Heron (Ardea cinerea), based on pellets. We compared pellets from 21 colonies in two different regions of Poland that differing in composition of foraging habitats. Multivariate statistical techniques were used to analyze the relationship between diet and habitat compositions in a 20 km radius around these colonies. Significant inter-regional and inter-colony differences in Grey Heron diet and habitat composition were detected in foraging areas. However, some prey were present in the diet only from one of the regions. Around the heronries with a predominance of farmland habitats, the European water vole (Arvicola amphibius) and terrestrial invertebrates were present in relatively low frequencies, and the striped field mouse (Apodemus agrarius) was present in high frequencies. Voles (Microtus sp.) were more frequent in colonies with prevalence of non-irrigated arable land around the colony, in contrast to the bank vole (Myodes glareolus), which was less common in heronries with a higher contribution of this habitat type. Remains of aquatic invertebrates were less abundant in colonies surrounded by extensive forests. The results of our research indicate the opportunistic character of the non-fish part of the diet of the Grey Heron adapted to the local foraging habitat and prey availability.