For the first time in Russia, the intraspecific structure of the brown rat Rattus norvegicus was studied using the mitochondrial control region (D-loop) as a molecular marker. The mtDNA sequence was determined in brown rats from eight regions of European and Asian Russia. Three rat clades were identified, including one in European Russia and two in Asian Russia. The synanthropic subspecies R. n. norvegicus was found to have two, European and Asian, lineages. The European lineage included haplotypes from central and southern regions of Russia, and the Asian lineage included haplotypes from Eastern Siberia and the Russian Far East. The exoanthropic subspecies R. n. caraco from the Russian Far East formed a separate genetic lineage.

For the first time in Russia, a study of the intraspecific structure of the brown rat (Rattus norvegicus) was carried out using the mitochondrial marker of the control region (D-loop) of mtDNA. MtDNA sequences of brown rats from 8 regions of the European and Asian parts of Russia were analyzed. Three clades of rats have been identified: in the European part of Russia –1, in the Asian part – 2. Synanthropic subspecies – R. n. norvegicus is represented by european and asian lineages. The european lineage included haplotypes from the regions of central and southern Russia, and the Asian lineage included haplotypes from Eastern Siberia and the Far East. The previously described free living subspecies, R. n. caraco from the Russian Far East was represented as separate genetic lineage.

In the current research, we investigated the impact of gradually increasing inbreeding on the life span and reproductive rate of yellow steppe lemmings, Eolagurus luteus, that reproduced in the Moscow Zoo. The focal captive colony existed from 2017 to 2021. The studied animals belonged to the second to tenth generations. The founders of the colony were 5 females and 5 males originating from 3 females and 4 males livetrapped in the Zaisan basin (Kazakhstan). The degree of their descendant relatedness progressively increased. The animals intended to be used for reproduction were distributed to pairs with unfamiliar partners. We constructed the pedigree of 177 individuals and calculated their inbreeding coefficients. This parameter varied from 0 to 0.29, and the maximum values were registered in the lemmings of the seventh to tenth generations. We measured the life span of 61 individuals and used information about the reproduction or its absence in 45 pairs. A substantial decline in individual life span and reproductive parameters in the breeding pairs, along with a progressive increase in the inbreeding coefficients, was registered. The number of delivered litters, born pups, and young lived up to the age of puberty significantly depended on the level of mother inbreeding. The noticeable traits of inbreeding depression in the colony appeared in 2019-2020 when the offspring inbreeding coefficients reached approximately 0.2. Therefore, we assume that if the E. luteus population originates from a relatively small number of noninbred and unfamiliar individuals, then successful reproduction without significant inbreeding depression will continue for several generations of offspring.

Developmental plasticity is one of the main factors driving the formation of individual, sexual, and species identity. We demonstrated a modification of the activation of the hypothalamic-pituitary-gonadal axis in male house mice as a result of early postnatal experience and the maternal environment upon exposure to the odor of receptive females of their own and closely related species. To determine the effect of rearing in families of closely related species on the activation of the hypothalamic-pituitary-gonadal axis in males, in particular, to compare the hormonal response (serum levels of free testosterone, hereafter FTL) in M. m. wagneri males reared by M. spicilegus , in response to the odor of receptive conspecific females and females of the foster species. Adult male offspring of two taxa of Mus musculus s. l. species group was reared either by their biological mothers ( M. m. wagneri – 18 males, M. spicilegus – 19 males), unrelated conspecific females ( M. m. wagneri – 11 males, M. spicilegus – 4 males), or by heterospecific foster mothers ( M. m. wagneri – 13 males). We used the previously described method with modifications (Kotenkova E, Romachenko A, Ambaryan A, Maltsev A (2019) Effect of early experience on neuronal and behavioral responses to con- and heterospecific odors in closely related Mus taxa: Epigenetic contribution in formation of precopulatory isolation. BMC Evol Biol 19(l): 51. 10.1186/s12862-019-1373-8). Each male was exposed to urine twice: once from females of his own and the second time from a closely related species with an interval of 8 or 16 days. In a separate series of experiments (control), the males were exposed to saline. After a 30-minute exposure, blood samples (≤150 µl) were obtained from the retro-orbital sinus with heparinized capillaries following the standard technique (Parasuraman et al. 2010). The serum was separated by centrifugation at 3000 g for 10 minutes and stored at -20ºC until free testosterone analysis. Serum-free testosterone concentrations were measured using a commercially available ELISA kit. The level of free testosterone in the blood serum of males reared by a conspecific female was significantly lower when exposed to the urine odor of a heterospecific female in comparison with exposure to the odor of a conspecific female. When male M. m. wagneri was fostered by females of a closely related species, the testosterone response of the males to the female's chemosignals (both hetero- and conspecific) was reduced, and the FTL did not differ when the males were exposed to the odor of the female of the closely related species or their own. The data obtained indicate a significant effect of rearing conditions during ontogeny on the development of testosterone response to odor exposure of receptive con- and heterospecific females. This may be due to the modified perception of species identity of individuals of the opposite sex in male M. m. wagneri reared by M. spicilegus females.

The conditions of upbringing at an early age have a significant impact on the subsequent development, reproduction, and formation of behavioral characteristics of mammals, including rodents. Parents, primarily nursing females, are the main component of the social environment in the early period of postnatal ontogenesis in the majority of mammals. Therefore, the upbringing of mammalian pups in foster families of their own species or another one, even taxonomically distant ones, is one of the main methods for assessing the influence of early experience and the maternal environment on the formation of the behavioral characteristics and the choice of a sexual partner. Rearing of the adopted pups by females of their own or another species is used to preserve valuable species. This necessitates the development of the most effective methods of transferring offspring with maximum survival of the young. In this regard, we analyzed the survival rate and possible causes of mortality of young of a number of rodent species in foster families of mammals of their own and other species. We carried out cross-fostering (interspecific cross-upbringing) and in-fostering (intraspecific cross-upbringing) experiments using house ( Mus musculus ) and mound-building ( Mus spicilegus ) mouse pups, yellow steppe lemming ( Eolagurus luteus ) and brown rat ( Rattus norvegicus ) pups, and rearing of brown rat pups in foster families of the black polecat ( Mustela putorius ). As when feeding their own offspring, the main forms of maternal behavior (“maternal behavior complex”) of the studied species in relation to adopted young during interspecific transfer are included and manifested in a complex manner if the adoptive mother accepts the young. The survival rate of adopted offspring depends on the degree of taxonomic proximity of the parent taxa: the highest rate was observed during cross-transfer between representatives of species belonging to the supraspecific complex Mus musculus s. l., while the lowest rate is observed when the pups are transferred between representatives of different families and orders of mammals. The success of upbringing adopted young can be determined by the similar composition of milk and the duration of lactation, the similarity of parental behavior (including the degree of male participation in raising the offspring), similar features of the ontogeny of young in closely related forms, and the significant difference in these characteristics in taxonomically distant species. However, successful upbringing of foster offspring is possible if there is similarity in the maternal behavior of taxonomically distant species and taking into account the suitability of milk (for example, feeding of rat pups by female black polecats). According to the results of the analysis of our data and published material, factors influencing the survival and mortality of pups in foster families also include the age of the pups at the time of transfer, species-specific differences in odor, the level of stress of the mother and offspring, the female’s previous experience in rearing her own and other pups, and the expression of maternal instinct. All this must be taken into account when using the method of cross-fostering pups in experimental work.

For the first time, it was shown that activation of the pituitary–testicular complex in male house mice exposed to the odor of receptive females of their own and closely related species was modified under the influence of early postnatal experience and the maternal environment. We have confirmed associated formation of behavioral and physiological mechanisms of precopulatory isolation in early ontogenesis. The serum levels of free testosterone in males of closely related species M. spicilegus and M. m. wagneri differ, it is significantly lower in mound-building mice. In males fostered by a conspecific female, the level of free testosterone was significantly lower when exposed to a heterospecific female odor in comparison with a conspecific odor. The rearing of M. m. wagneri males by females of a closely related species led to a decrease in the testosterone response caused by exposure to female chemosignals (both con- and heterospecific) and to the absence of differences in the serum level of free testosterone when exposed to the odor of a female of their own or closely related species. These results indicate that the rearing conditions had a significant influence on the formation of hormonal mechanisms of reproductive isolation.

Cross-fostering of pups was used as a method for estimating the effect of early experiences and the maternal environment on the breeding success and fertility in con- and heterospecific pairs in two mouse species: house (M. m. wagneri) and mound-building (M. spicilegus). During experimental hybridization of these species, the breeding success depended both on the species identity of sexual partners themselves (con- or heterospecific) and on the species identity of the fostering female (con- or heterospecific fostering female). It was found that the intensity of reproduction (the ratio of breeding pairs to nonbreeding ones) in mixed pairs of house and mound-building mice is lower than in control ones consisting of members of the same species. It was demonstrated that in different variants of crossings (both in con- and heterospecific), the proportion of pups that did not survive to 40 days of age is significantly higher in pairs, in which at least one of the partners was fostered by a female of another species. This can be caused by a discrepancy between parental behavior and species-specific parental behavior, which disrupts the interaction between educators and pups. This can cause an increase in the mortality of pups. This study demonstrated the importance of early postnatal social experiences (postnatal social ontogenesis) and the maternal environment in the formation of species-specific patterns of behavior in adult individuals of closely related species of house mice.

Archive data were analyzed concerning the reproduction of yellow steppe lemmings ( Eolagurus luteus ) in the Moscow Zoo. We used information referring to E. luteus pairs that existed during two periods: 2008–2013 (first colony) and 2017–2020 (second colony). All breeding partners in the first colony had at least one common ancestor (they originated from two females and one male) and thus were closely related to each other. The degree of relatedness of partners in the second colony was much lower. The founders of that colony were five females and five males that had originated from four pairs of unrelated individuals live-trapped in the Zaisan Basin. We conducted a comparative assessment of the life expectancy and reproductive parameters of adult yellow steppe lemmings and the survival of their offspring in two colonies, that is, we observed the effect of different degrees of inbreeding on such parameters. We also assessed the effect of female age on those values. We assigned the females to two age categories: younger (age of 15–59 days by the time of pair establishment) and older (age of 60–701 days) category. The results of statistical analysis confirmed the significant influence of the degree of relatedness of partners on the proportion of reproducing females, the number of litters per pair, the number of survived pups per pair, the mean number of pups per litter, the inter-birth interval, the female life expectancy, and the period of pair existence. The influence of female age on these parameters was insignificant. Our data provide evidence for a pronounced inbreeding depression in the first captive colony of the yellow steppe lemming.

Genetic resistance to anticoagulants caused by mutations in the Vkorc1 gene of the most invasive rodent species—Norway rats and house mice, has not been studied in Russia. We analyzed the variability of the Vkorc1 gene in house mice and Norway rats in various settlements of Russia, and identified mutations responsible for resistance to rodenticides. Two exons of the Vkorc1 gene were analyzed in 71 Norway rats from four cities (Moscow, Tyumen, Chita, Rostov-on-Don) and 108 house mice from cities and small settlements (Moscow region, Tormosin, Nizhny Tsasuchei). Three Norway rats (15.8% of the studied individuals) in Moscow have a heterozygous state the Tyr139Ser mutation, which is responsible for resistance. House mice were not found to have mutations in the Vkorc1 gene responsible for resistance to anticoagulants of the first and second generation in the Leu128Ser and Tyr139Cys positions located in the third exon. However, in cities in the first exon, we identified two heterozygous mutations have not be described previously in the scientific literature: Lys58Arg and Ser31Trp. In Russia, the genetic resistance to rodenticides in settlements in the populations of house mice and Norway rats is significantly lower than in Western Europe.

One hundred fifty-three individuals of Mus musculus examined have been found to carry 120 haplotypes of the mtDNA control region (D-loop). Seven phylogroups have been identified: four groups in Europe, two in Asia, and one in Transcaucasia. The territory of Europe and Asia was inhabited by different phylogenetic lineages. We distinguished the “Siberian” lineage of M. musculus; it spread to the east of Asia along major transport routes. The dispersal of M. musculus in Eastern Europe could have arisen from Transcaucasia, a possible center of origin of M. musculus . No differentiation of M. musculus subspecies ( M. m. wagneri , M. m . musculus , and M. m. gansuensis ) has been found. The populations of Eastern Europe were repeatedly mixed due to the dispersal of the house mouse through human activity, while Siberia and Japan were inhabited by only one line of M. musculus . According to our data, the phylogenetic lineage of the house mouse from Transcaucasia turned out to be monophyletic, in contrast to some other identified lines.

This is the first study to analyze the variability of two exons (the 1st and 3rd) of the VCORC1 gene in Russian populations of mice and rats, namely, 125 Mus musculus and 19 Rattus norvegicus captured in 13 settlements. Previously, it has been shown that a number of mutations in these exons in the countries of Western Europe are associated with resistance to anticoagulants in synanthropic rodents. We did not find such mutations in house mice in Russia. However, in the first exon, two previously unknown mutations were identified that could potentially have such an effect (Lys58Arg and Ser31Trp). In three districts of Moscow, Norway rats were found carrying in the third exon a previously known resistance mutation (Tyr139Ser) in the heterozygous state. The results are discussed in connection with the intensity of the use of anticoagulants in the settlements of Russia and the mutation rate of the VCORC1 gene.

The resistance of house mice (M. musuclus linnaeus, 1758) and norway rats (R. norvegicus berkenhout, 1769) to anticoagulants of the 1st and 2nd generation in the VKORC1 gene in Moscow and the Moscow region was studied. House mice had two previously unknown mutations in exon 1: Lys175Arg и Ser94Trp. We assume that these mutations can increase the resistance of rodents to second-generation anticoagulants. the percentage of individuals with mutations in exon 1 in Moscow is 87.5 %, in Noginsk and Podolsk – 100 %. Of the 18 norway rats studied in Moscow, only three individuals in a heterozygous state were found to have one of the mutations (Tyr139Ser), usually associated with genetic resistance of rodents to the first generation anticoagulant warfarin. the percentage of resistant individuals in Moscow was 15.7 %. the rats in which the resistance mutation was found were captured in three districts of moscow: southwestern, northern and eastern.

To clarify genetic differences between subspecies of the house mouse Mus musculus, their distribution, and hybridization, we first conducted a comparative analysis of variability of nucleotide sequences of fragments of the nuclear gene Brca1, exon 11 (2331 bp), and mitochondrial gene Cox1 (1260 bp) in 40 house mice from West and East Europe, Transcaucasia, Siberia, and Central and South Asia. Brca1 genotypes were divided into five main groups, which differed in a number of fixed substitutions. Genotypes of each group are characteristic for the certain geographical region and the following subspecies: M. m. musculus, M. m. domesticus, M. m. castaneus, and M. m. wagneri together with M. m. gansuensis; a fifth group corresponds to an unidentified subspecies or a distinct genetic form of M. musculus from India (Sikkim State). Besides the homozygous specimens, we revealed mice, which were heterozygous for all diagnostic loci simultaneously; these specimens were determined as hybrid. Hybrid mice were mainly found in the zones of contact of subspecies, but in some cases, quite far from one of the parent subspecies (possibly, due to transportation). In two hybrid mice (from Bakhtiari Province of Iran and Transbaikalia of Russia), unique Brca1 haplotypes were detected. It cannot be ruled out that, at least partly, they may be characteristic of the M. m. bactrianus and M. m. gansuensis subspecies, respectively. Thus, the results of the study showed that the nuclear Brca1 gene is a promising molecular genetic marker for the analysis of variability, differentiation, and hybridization of house mice as well for subspecific identification of M. musculus specimens. Despite more rapid evolution of the Cox1 gene, it is not well suited for discrimination of M.m. musculus, M. m. wagneri, M. m. gansuensis specimens and Transcaucasian representatives of M.m. domesticus due to introgression and long-term maintenance of foreign mitochondrial DNA in populations. However, Cox1 gene analysis (along with the diagnostics of animals by nuclear DNA) may be useful for estimation of population differences in M. m. castaneus and M.m. domesticus subspecies.

Closely related species with different mating system may be the most suitable model taxa for studies aimed to highlight the cause formation of species-specific peculiarities of sexual behavior and behavioral mechanisms of precopulatoiy isolation. The current study aimed to clarify the role of the mating system and lifestyle, including ecology features, in patterning of behavioral activities during mating, as well as the role of behavioral patterns in the precopulatory isolation of closely related Mus taxa. Test subjects were closely related sympatric species: polygynous/promiscuous M. musculus and presumably monogamous M. spicilegus, reproductively isolated in nature. Dyadic encounters of male and receptive female were conducted in clear chambers and the behavior was recorded by means of video camera. Sexual behavior, culminating in ejaculation, was observed in conspecific dyadic encounters only; it occurred more frequently and with longer duration in males of M. spicilegus, than in males of M. musculus. In conspecific encounters, males of M. spicilegus exhibited a higher level of affiliative behavior than females. In both species total frequency and duration of aggressive behavior was higher in females compared to males. In heterospecific dyadic encounters the behavioral pattern of males and females was strictly different from those in conspecific encounters, and the elements of aggressive behavior prevailed. We demonstrated that not only the pattern of sexual behavior is important for reproductive isolation, but also all types of behavioral interactions preceded copulation. In Mus species different stereotypes of mating behavior during the encounter of potential sexual partners can prevent successful copulation and may be associated with mating system.

The fecundity, fertility, and fitness of house mice from Transcaucasian populations have been investigated in laboratory conditions. We interpret our data in accordance with Milishnikov's hypothesis (2004) that the ancestral form might have come from Transcaucasia. It is genetically close to Mus musculus s.str. and has kept a relict gene pool. The already differentiated M. musculus and M. domesticus have been introduced into this region as well. All three forms have crossed with one another, resulting in a very complicated hybrid zone formed in Transcaucasia. In our experiment, house mice from Moscow and Moscow oblast and from the hybrid zone of Transcaucasia were characterized by a similar reproduction rate. Reproduction indices weren't significantly differ in different variants of crossings of mice from Transcaucasia with M.musculus as well as in hybrids from direct and back crosses and in backcrosses. Nevertheless, these rates differed significantly from those in M. musculus and Transcaucasian mice. In some cases, the mortality of the young was relatively high. The data obtained disagree with the presumed reduced fitness of house mice in Transcaucasia. No serious evidence has been obtained for the presence of postcopulative isolating mechanisms contributing to separation of mice from Transcaucasia from M. musculus. The results of these studies are consistent with the assumed kinship of house mouse populations of Transcaucasia and M. musculus.

Our hypothesis that exoanthropic forest rodent species (e.g., bank voles) avoid the urinary odor of the synanthropic house mice was tested in field experiments in August 2016. One count of the small mammals was carried out in each of two 4-ha fields by the capture–mark–recapture technique over 12 days. Two hundred live traps (100 points, 2 traps per point) were exposed and controlled twice a day (early in the morning and in the evening) in each count. One trap in each pair served as the reference, while the second was the experimental one with 20 μL of the urine of synanthropic house mice applied to a foam rubber surface every day. Generally, bank voles chose the odorless trap more often (60.8% vs. 39.2%, χ 2 = 3.7, p = 0.054). Adult individuals had a nonsignificant tendency to avoid the odor-emitting traps. Immature (subadult and juvenile) bank voles reliably avoided the odor-emitting traps (a 2 : 1 ratio, χ 2 = 5.74, p = 0.017). A general linear model showed the influence of the age of voles on the choice of the odorless traps. The potential repellent role of sulfur-containing compounds in mouse urine is discussed.